Delivery vehicles comprising proglucagon derived polypeptides and anabolic polypeptides and uses thereof

Abstract

Delivery vehicles, such as liposomes, that display combinations for fat loss and muscle enhancing peptides for treating obesity and / or diabetes are provided. The delivery vehicles can be administered in combinatorial regimens, and administration in rotational combinatorial regimens.

Description

[0001] DELIVERY VEHICLES COMPRISING PROGLUCAGON DERIVED POLYPEPTIDES AND ANABOLIC POLYPEPTIDES AND USES THEREOF Related applications

[0002] Benefit of priority is claimed to U.S. provisional application Serial No. 63 / 638,893, filed on April 25, 2024, entitled “DELIVERY VEHICLES COMPRISING PROGLUCAGON DERIVED POLYPEPTIDES AND ANABOLIC POLYPEPTIDES AND USES THEREOF,” to inventors Jorge Luis Cabrera and Jorma A. Virtanen, and Applicant StarRock Pharma Inc.

[0003] Benefit of priority is claimed to U.S. provisional application Serial No. 63 / 675,214, filed on July 24, 2024, entitled “DELIVERY VEHICLES COMPRISING PROGLUCAGON DERIVED POLYPEPTIDES AND ANABOLIC POLYPEPTIDES AND USES THEREOF,” to inventors Jorge Luis Cabrera and Jorma A. Virtanen, and Applicant StarRock Pharma Inc.

[0004] This application is related to International PCT application No. PCT / US23 / 77508, filed on October 23, 2023, and published as International PCT publication No. WO2024 / 091863, published on May 02, 2024, entitled “COMBINATORIAL, AND ROTATIONAL COMBINATORIAL THERAPIES,” to inventor Jorge Luis Cabrera, and Applicant StarRock Pharma Inc.

[0005] Where permitted, the subject matter of each of these applications is incorporated by reference in its entirety.

[0006] INCORPORATION BY REFERENCE OF SEQUENCE LISTING FILED ELECTRONICALLY

[0007] An electronic version of the Sequence Listing is filed herewith, the contents of which are incorporated by reference in their entirety. The electronic file was created on April 19, 2025, is 64,041 bytes in size, and is titled 2702PCSEQ001.xml.

[0008] FIELD

[0009] Delivery vehicles comprising polypeptides for treating obesity are provided. Therapies include administration combinatorial regimens, and administration of rotational combinatorial regimens. The regimens comprise combinations of drugs and / or non-drug treatments. BACKGROUND

[0010] In the United States, there has been an unprecedented rise in overweight and obesity in the last decade. There are more obese US adults than those who are just overweight (Deng etal., Annual Review of Pathology (2016) 11(1): 421- 49). In 2008, the Journal of the American Medical Association (JAMA), reported that the obesity rate among adult Americans was estimated at 32.2% for men and 35.5% for women, which was confirmed by the CDC for 2009-2010. According to the CDC, “obesity is higher among middle-aged adults, 40-59 years old (39.5%) than among younger adults, age 20-39 (30.3%) or adults over 60 or above (35.4%) adults” (“Adult Obesity Facts,” published by the Centers for Disease Control and Prevention. Retrieved November 22, 2015). Projections indicate an increase in obesity prevalence to 60% in adult men, 40% in adult women, and 25% in children by 2050 (CDC Data Brief, 2014). The overweight and obesity epidemic undermines health, was formally classified as a “disease” in June 2013 by the American Medical Association, with much controversy.

[0011] Obesity is not confined to the United States. It is a global epidemic with serious medical and financial consequences. In 1997, the WHO formally recognized obesity as a global epidemic and viewed it as one of the most serious public health problems of the 21stcentury. Based on global trend data from 1975 to 2014, and an estimated world population of 8-9 billion, with the prevalence of obesity reaching 18% in men and 21% in women, almost a three-fold increase compared to prior to 1975. The Organization for Economic and Co-operation Development has projected an increase in obesity rates world-wide until at least 2030, especially in the United States, Mexico and England with rates reaching 47%, 39% and 35%, respectively (“Obesity Update 2017,” published by the Organisation for Economic Co-operation and Development. Retrieved 6 October 2018). Once considered a problem only of high-income countries, obesity rates are rising worldwide. Globally, there are now more people who are obese than who are underweight, a trend observed in every region over the world except parts of sub-Saharan Africa and Asia (World Health Organization, Obesity and overweight. Fact sheet updated June 2016. Geneva. Retrieved 22 Sept 2017).

[0012] There are treatments for obesity, but few that result in sustained weight loss and few that result in a loss of weight of more than 10% body weight. Hence, there is a need for treatments for obesity that result in sustained and continued weight loss to eliminate obesity.

[0013] SUMMARY

[0014] Provided are delivery vehicles for treating obesity and / or for treating type 2 diabetes. The delivery vehicles display combinations of polypeptides for weight loss, such as proglucagon polypeptides, and a polypeptide that prevents or reduces muscle loss associated with weight loss or promotes muscle growth (referred to as a muscle enhancing polypeptide). The delivery vehicles can contain or display other drugs, such as small molecule drugs, and they can contain the peptide drugs. Thus, the vehicles are for delivery of combinations of drugs, including small molecules and peptides. Also provided are combinations of delivery vehicles where the delivery vehicles each display a polypeptide and / or contain a small molecule drug, and a plurality of delivery vehicles are co-formulated or administered together so that the combination of at least two weight loss promoting peptides and a muscle enhancing and optionally a small molecule drug, such as an appetite inhibiting drug, are administered together. Devices for administering the delivery vehicles, such as syringes or pens, that contain single or multiple dosage amounts of the delivery vehicles also provided. The devices can contain multiple chambers, one containing a lyophilized composition containing the delivery vehicles or mixtures thereof, and a second containing a vehicle, such as PBS, for mixing with the delivery vehicles, for administration. A variety of the types of polypeptides linked to or embedded in the delivery vehicles can provided so that the combinations of polypeptides can be rotated, such as in regimens described in copending International PCT application No. PCT / US23 / 77508 (International PCT publication No. WO2024 / 091863). Provided are delivery vehicles for effecting treatment of obesity and / or diabetes, and for implementing the regimens and methods described in the copending application. The polypeptides described therein can be linked to or embedded in delivery vehicles, such as liposomes or extracellular vesicles or vehicles, such as exosomes, for administration. Two or three different polypeptides can be linked to or embedded in the delivery vehicle and administered, such as by injection, such as subcutaneous injection, and, depending upon the selected delivery vehicle by inhalation into the lungs or nose or by mucosal delivery, including orally. Most liposomal preparations and exosomal preparations cannot be formulated for oral administration, but there are some that can be so-administered.

[0015] The delivery vehicles or combinations for delivery vehicles contain or display a polypeptide for weight loss and a polypeptide for muscle enhancement (prevention or reduction of muscle loss associated with weight loss and / or increasing muscle mass). The delivery vehicles and composition include uses for treating obesity and / or diabetes, and / or other comorbidities, such as high cholesterol, heart disease, and hypertension, and other diseases, disorders, and conditions associated with metabolic syndrome.

[0016] All the methods and regimens and uses can provide the drug, generally a peptide drug, displayed on a delivery vehicle, such as a liposome or exosome. The peptides can be linked, by standard well-known methods, to the delivery vehicle or the delivery vehicle can be produced with the peptide incorporated into the surface. The delivery vehicles as detailed herein can display a plurality of peptides, or can display one or more and mixtures thereof can be administered or used together to provide a desired combination of peptides. In general, the methods, uses, and regimens herein are for treating obesity and / or diabetes. In general, the methods, regimens, and uses comprise a mixture of peptides for weight loss and for muscle enhancement for promoting or sustaining muscle growth, such as an anabolic peptide).

[0017] Provided are delivery vehicles, comprising a combination of therapeutic peptides, where: the peptides are linked to the surface directly or indirectly via a linker or are part of the surface of delivery vehicle; the surface of the delivery optionally is modified for linkage of the polypeptides; the combination of peptides comprises at least three different peptides; at least two of the peptides target different pathways and / or have different activities; and the therapeutic peptides target pathways involved in obesity and / or diabetes, or have activity for treating obesity and / or diabetes. Also provided are compositions, comprising a mixture of delivery vehicles, where: each delivery displays at least one therapeutic peptide on the surface; and the composition comprises delivery vehicles selected so that the composition comprises at least three different displayed peptides. The composition can be formulated for any suitable route of administration, including, for example, intramuscular, intravenous, mucosal, parenteral, subcutaneous administration, oral, intranasal, inhalation, and other route. For example, the delivery vehicles and compositions are provided for subcutaneous administration. They can be provided as liquids or powders for reconstitution as a liquid.

[0018] Delivery vehicles include, but are not limited to liposomes, lipid nanoparticles, exosomes, and extracellular vesicles. The peptides displayed in or on the delivery vehicles comprise fat loss and muscle enhancement peptides; as noted muscle enhancement includes muscle loss reduction or prevention and / or increased muscle mass, such as an anabolic peptide. Exemplary of muscle enhancing peptides are sermorelin, tesamorelin, and IGF-1. The muscle enhancement polypeptide by enhancing muscle growth prevents or reduces the loss of muscle that is a problem with administration of GLP-1 agonists. For example, at least two of the peptides are fat loss peptides, and one is a muscle enhancement polypeptide. Polypeptides for fat loss can be selected from among: GLP-1, adiponectin, leptin, oxyntomodulin, PYY (peptide YY), amylin, pancreatic peptide, enterostatin / GIP (Gastroinhibitory Polypeptide), glicentin, glucagon, GRPP (glicentin-related pancreatic polypeptide), HGH (human growth hormone), CCK (cholecystokinin), neurotensin, secretin, IIP (myo-inositol 1 -phosphate), and MPGF (major proglucagon fragment). Peptides for muscle enhancement, include, but are not limited to, sermorelin, tesamorelin; and IGF1 (or HGH). For example, the combinations of drugs comprise peptides that comprise GLP-1, Oxyntomodulin, enterostatin / GIP (Gastroinhibitory Peptide); and Sermorelin. The liposomes and other delivery vehicles also can contain or display small molecule weight loss drugs, such as phentermine.

[0019] Delivery vehicles include liposomes, such as large multilamellar vesicles (LMV) and SMVs (small MVs). Liposomes include those that comprise phospholipids, such as, for example, one or more of phosphatidyl choline (PC), phosphatidyl ethanol amine (PE), and phosphatidyl serine (PS), and phosphatidic acid (PA). Phospholipids include synthetic and phospholipids from natural sources, such as egg yolks. Cholesterol can be added to the liposomes to improve properties, such as permeability. The molar percentage of cholesterol in the liposome is less than 60%, 50%, 40%, 30%, 20%, 10%, or less. Provided are delivery vehicles and compositions including liposomes comprising modified lipids. The liposome can include lipids modified with a reactive group for coupling with a peptide or with a peptide modified with a reactive group. In some liposome fabrication method and product embodiments, the reactive group for the coupling reaction is selected from among amino, thiol, maleimide, bromo- or iodoacetyl, pyridyl di thio, carboxylic, hydrazide, p-nitrophenyl carbonate, azide, and / or alkyne reactive groups. In some embodiments, the reactive group is an amino group that forms an amide bond with an activated carboxylic ester, or is a thiol group that binds with maleimide, bromo- or iodo acetyl, pyridyldithio groups, or is a hydrazide that bind with carbonyl groups, or is p-nitrophenyl carbonate that reacts with amines forming an amide bond, or comprise azide and alkyne group that bind with each other in the presence of a copper ion catalyst, to attach the peptide or protein the liposome.

[0020] Provided are delivery vehicles and compositions where the peptides are linked to the peptides via bonds formed by reaction of the reactive groups. For example, the delivery vehicle is a liposome and the peptide and / or liposome is / are PEGylated for linking the peptide to the liposome. The peptide and / or liposome can be PEGylated for linkage, or linked to or coated with streptavidin for reaction with biotin, such as biotin linked to the peptide. Provided are delivery vehicles and compositions, where the peptide and liposome are linked via an amide / peptide bond or linker, a thioester bond or linker, a disulfide bond or linker, a hydrazone bond or linker, a carbamate bond or linker, and a 1,2,3-triazole linker.

[0021] The linkage between the delivery vehicle, such as liposome, can comprise a spacer, such as, but are not limited to, a spacer that comprises polyethylene glycol (PEG) and / or and an oligonucleotides bound to the liposome and to the peptide linked to a complementary oligonucleotide. The delivery vehicle, such as a liposome, can comprise streptavidin bound to biotin-linked peptide or the streptavidin is bound to the liposome and to biotin-linked peptide. For example, the delivery vehicles, such as liposomes, are coated with a monolayer of streptavidin and linked to peptides functionalized with biotin-PEG-NHS to form liposomes that display the peptides upon mixing these peptide derivatives with streptavidin liposomes. The linkage can include a spacer, such as a PEG spacer or oligonucleotide; one end of the spacer can be attached biotin, and the other comprises a reactive group, such as an NHS active ester, that easily forms an amide bond with the PEG.

[0022] Provided are containers comprising the delivery vehicles or compositions provided herein. Containers include, for example, pens and syringes for administering the delivery vehicle.

[0023] Provided are pharmaceutical compositions comprising the delivery vehicles provided herein.

[0024] Methods of treatment of obesity and diabetes are provided. The methods comprise administering a delivery vehicle or composition provided herein. The methods can include the rotational combinatorial and rotational methods described herein, where the peptides are provided displayed on delivery vehicles, such as liposomes. The disease, disorder, or condition contemplated for treatment is / are obesity and / or diabetes. One combination of peptides can be administered, where the combination is displayed on the delivery vehicle. A plurality of combinations can be administered where the different combinations are provided on delivery vehicles. Mixtures of delivery vehicles displaying different peptides can be combined and administered. Corresponding regimens, as described herein are provided. Provided are methods and regiments and delivery vehicles that comprise combinations of peptides mimic effects of gastric bypass. For example, the methods and regimens include a combination of peptides on the delivery vehicle comprises at least three selected from among: a peptide that inhibits gastric emptying selected from among one or more of GLP1, Amylin, and Pancreatic Polypeptide Therapeutic; a peptide drug that enhances satiety comprising one or more drugs selected from among glucagon-like peptide-1 (GLP-1), peptide YY (PYY), amylin, enterostatin / gastric inhibitory peptide (GIP), cholecystokinin (CCK), and glicentin; a peptide that increases insulin release and / or sensitivity comprising one or both of GLP1 and adiponectin; and a peptide that modulates energy expenditure comprising leptin, oxyntomodulin, and glicentin. The methods and regimens can further comprise a delivery vehicle that comprises a peptide that results in muscle enhancement, such as, for example, one or more of sermorelin, tesamorelin and / or growth hormone, and testosterone. By virtue of muscle enhancement, the loss of muscle associated with weight loss treatment that accompanies weight loss is reduced or prevented. As a result, treated subjects, whether or not they exercise, have little or no muscle loss. The methods and regimens can comprise administration of a peptide that promotes intestinal smooth muscle relaxation, such as vasoactive intestinal peptide (VIP). Peptides in the methods and regimens and linked to delivery vehicles, such as liposomes, for example can be selected from among: GLP-1, Adiponectin, leptin, oxyntomodulin, peptide tyrosinetyrosine (PYY), amylin, pancreatic peptide, enterostatin / gastric inhibitory polypeptide (GIP), cholecystokinin (CCK), vasoactive intestinal peptide (VIP), glicentin, human growth hormone or an active portion thereof or an analog of human growth hormone or an active portion thereof, ephedrine, caffeine, aspirin (ECA), oxyntomodulin, neuropeptide Y (NPY), antimicrobial peptide 2 (LEAP2), vaccine CYT009-GhrQb, the peptide-binding compound Nox-Bl 1, and the ghrelin analog AZP-531 (SEQ ID NO: 15); and / or the peptides are a GLP-1 agonist, an appetite suppressant, a thyroid hormone, a carbonic anhydrase inhibitor, an alpha-glucosidase inhibitor, a dipeptidyl peptidase-R (DPP -4) inhibitor, a sodium-glucose co-transporter 2 (SGLT2) inhibitor, a muscle enhancer, drugs that modulate energy expenditure, a GLP-1 agonist, peptides that increase gastric inhibitory polypeptide (GIP), drugs that modulate GIP2, and mitochondrial uncouplers; and the peptides are combined by displaying a plurality on each delivery vehicle or by mixing delivery vehicles that display different peptides. The delivery vehicles also can contain and / or display a small molecule weight loss drug, such as an amphetamine or appetite suppressant, such as phentermine. Exemplary of peptides that are displayed and small molecules that are incorporated into delivery vehicles are those selected from among: dulaglutide, bydureon, semaglutide, exenatide, liraglutide, phentermine, liothyronine, topiramate (carbonic anhydrase inhibitor), acarbose (alpha-glucosidase inhibitor), sitagliptin (dipeptidyl peptidase-4 (DPP-4) inhibitor), canagliflozin (sodium-glucose cotransporter 2 (SGLT2) inhibitor), dapagliflozin ( SGLT2 inhibitor), sermorelin, mirabegron (beta-3 adrenergic agonist), and amylin. For example, the peptides can be selected from among: a GLP-1 agonist, phentermine, thyroid hormone, carbonic anhydrase inhibitor, carbonic anhydrase inhibitor, alpha-glucosidase inhibitor, DPP -4 inhibitor, SGL2 inhibitor, muscle enhancer, and an appetite suppressant.

[0025] The methods and regimens and / or delivery vehicles further can comprise a mitochondrial uncoupler linked to a delivery vehicle or mixed in the composition or administered as a free molecule not bound to a delivery vehicle. Exemplary of mitochondrial uncoupler include, for example, uncoupling protein 1 (UCP1), a catecholamine, and a small molecule uncoupler, such as 2,4-dinitrophenol (DNP) and BAM15 (N5,N6-bis(2-Fluorophenyl)[l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine). The small molecule drugs, which are available in oral dosage forms, such as phentermine, can be co-administered with the liposomes containing or displaying the polypeptides.

[0026] The methods and regimens herein include administering a plurality of different combinations of drugs, wherein: each combination of drugs is administered for a predetermined time; each combination of drugs is rotated until all combinations are administered at least once to comprise a cycle; a cycle comprises at least two different combinations; a combination comprises at least two different drugs that target different pathways or targets involved in the disease, disorder, or condition; each combination of drugs is unique among the combinations administered in a cycle, but a drug can be part of a plurality of combinations as long as the resulting combinations are unique; the disease, disorder, or condition is a chronic disease, disorder, or condition that requires treatment for at least 6 months; and the disease, disorder, or condition is not a cancer.

[0027] Delivery vehicles containing / displaying a combinations of the peptides, and optionally a small molecule(s), such as phentermine or a drug to treat a comorbidity of obesity, such as metabolic syndrome, diabetes, high cholesterol, heart disease, and hypertension, are provided. Examples of combinations of polypeptides for displaying on or delivering in a delivery vehicle, such as a liposome or an exosome include combinations of at least two or three weight loss drugs and a muscle enhancer, and can further include a small drug, such as an appetite suppressor, an amphetamine family drug, and / or drugs for treating obesity co-morbidities. The small molecules can be fabricated in association with the liposomes, or can be separately administered. Examples of fabrication of liposomes is detailed in the above examples, and known and / or apparent to those skill in the art. Exemplary combinations of peptides and sequences are as follow:

[0028] Drug 1 : GLPl / GIPl / Oxyntomodulin + ME* (Sermorelin or Tesamorelin or IGF1) SEQ ID NOs: 1, 10, 4 or 47 + SEQ ID NOs: 5, 8, 44 Drug 2: GLPl / GIPl / Amylin + ME (Sermorelin or Tesamorelin or IGF1) SEQ ID NOs: 1, 10, 7 + SEQ ID NOs: 5, 8, 44 Drug 3: GLP1 / GIP1 / Glucagon + ME (Sermorelin or Tesamorelin or IGF1) SEQ ID NOs: 1, 10, 27 + SEQ ID NOs: 5, 8, 44 Drug 4: GLP1 / GIP1 / CCK+ ME (Sermorelin or Tesamorelin or IGF1) SEQ ID NOs: 1, 10, 11 + SEQ ID NOs: 5, 8, 44 Drug 5: GLP1 / GIP1 / PYY + ME (Sermorelin or Tesamorelin or IGF1) SEQ ID NOs: 1, 10, 6 or 36 + SEQ ID NOs: 5, 8, 44 Drug 6: GLP1 / GIP1 / Leptin + ME (Sermorelin or Tesamorelin or IGF1) SEQ ID NOs: 1, 10, 3 + SEQ ID NOs: 5, 8, 44 *ME= A muscle enhancer such as Sermorelin or Tesamorelin or IGF1 or other muscle enhancing peptide or drug that ameliorates the loss of muscle accompanying weight loss by promoting muscle growth

[0029] Different delivery vehicles can be rotated to provide rotational combinatorial treatment.

[0030] Provided are rotational combinatorial regimens for treating a disease, disorder, or condition, comprising a plurality of combinations of drugs and / or treatments for a disease, disorder, or condition, wherein: the disease, disorder, or condition has more than one therapeutic intervention target or pathway for therapeutic intervention; the disease, disorder, or condition is a chronic condition that requires treatment for at least 6 months; the rotational combinatorial therapy comprises at least two different combinations of drugs and / or treatment; the drugs and / or treatments in each combination target different pathways or targets involved in the disease, disorder, or condition; each combination is administered at least once a cycle; each cycle comprises administration of each combination at least once; a cycle comprises at least two different combinations; a cycle can be repeated a plurality of times; the cycle for each combination can be the same or a different length of time; and at least one of the combinations comprises at least two different drugs that target different targets or pathways.

[0031] Provided are methods of treating a disease, disorder, or condition, comprising administering a drug regimen comprising serially administering two or more combinations of drugs and / or treatments, by administering the delivery vehicles provided herein, wherein: the disease, disorder, or condition is a disease, disorder, or condition that has more than one therapeutic intervention target or pathway for therapeutic intervention; the disease, disorder, or condition is a chronic condition that requires treatment for at least 6 months; the drug regimen comprises at least two combinations; at least one combination in the regimen includes at least two drugs that treat at least one target or pathway involved in the disease, disorder, or condition; the at least two drugs treat different targets, pathways, and / or have different activities.

[0032] Provided are combinatorial therapeutic methods of treating a disease, disorder, or condition, comprising administering a combination of at least 2 or 3 different drugs selected from among a plurality of drugs, and generally including a muscle enhancer; wherein the disease, disorder, or condition is a chronic disease, disorder, or condition; the disease, disorder, or condition is a disease, disorder, or condition that has more than one therapeutic intervention target or pathway for therapeutic intervention; each of the plurality of drugs can treat a target or pathway involved in the disease, disorder, or condition; each of the selected drugs has a different activity from the other drugs in the combination; and the combination reduces or eliminates desensitization to one or more of the selected drugs. Included are methods and regimens where desensitization results from downregulation of a receptor agonized by a drug in a combination, when it is administered as a monotherapy, or upregulation of a receptor antagonized by a drug in a combination, when it is administered as a monotherapy. In some embodiments, the methods and regimens are those where the drugs and / or treatments in each combination are administered simultaneously, sequentially, or intermittently. In administering the drugs / treatments in a combination they can be administered together, simultaneously, serially, or intermittently. They can be administered within a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or longer, such as up to 2 or 3 days. Generally, the drugs and treatments in each combination are administered within 24 hours. The methods and regimens include those where the disease, disorder, or condition is selected from a disease, disorder, or condition that includes treatments that when used as a monotherapy, the subject becomes desensitized to the drug. The rotational methods herein can avoid desensitization. The methods and regimens can comprise at least 2, 3, or more cycles, whereby treatment lasts at least 6 months, 9 months, a year, or longer.

[0033] Among the embodiments provided herein are rotational combinatorial methods and regimens that comprise at least 3 different combinations in a cycle. Included are methods and regimens where each combination of drugs and / or treatments comprises at least 2 different drugs or treatments that target different pathways or targets for intervention. In some embodiments, the drugs and treatments in a combination or in more than one combination can target the same pathway or target for intervention.

[0034] In all embodiments, the cancer is not among the disease, disorder, or condition that is treated for which the regimen is employed.

[0035] Among the diseases, disorders, and conditions is obesity. Provided are methods and regimens for treating obesity. Provided are combinatorial methods and regimens for treating obesity, comprising administering a combination of at least three different drugs, wherein each targets a different pathway or different target for intervention for treatment of obesity. The methods and regimens can comprise rotating combinations of drugs, to thereby provide rotational combinatorial therapy. The methods and regimens include those where the combination(s) of drugs that is / are selected mimic the effects of gastric bypass. Provided are combinatorial weight loss regimens, comprising a combination of at least three different drugs, where the activity of the drugs mimics or has the activity of a peptide whose activity is altered following gastric bypass surgery. In general, in all embodiments or in at least one cycle of administration, a delivery vehicle comprising a muscle enhancing polypeptide, is administered to prevent or reduce loss of muscle associated with muscle or to enhance muscle growth.

[0036] Regimens for treating obesity comprising a combination or combinations of drugs whose effects or activities mimic the biological effects of gastric bypass surgery, such as methods and regimens where biological effects of gastric bypass comprise reduced absorption and / or malabsorption of food, decreased appetite, increased satiety, increased glycogenolysis and / or lipolysis, increased insulin sensitivity, modulation of energy expenditure, and inhibition of gastric emptying. Such methods and regimens can comprise or further comprise drugs that increase growth hormone, and / or promote or result in muscle enhancement so that at least the loss of muscle associated with weight loss is reduced or eliminated.

[0037] In accord with all of the methods and regimens provided herein, they can comprise combinations of drugs / treatments or selecting combinations of drugs and treatments, where the combinations of drugs and / or treatments are administered for a predetermined time of at least 1 week followed by administration of a different combination of drugs for a second predetermined time of at least a week, until all selected combinations of drugs are administered to complete a cycle; and repeating the same or a different cycle of combinations of drugs. For example, each combination can be administered for at least 1 week, or at least 2 weeks, or at least 3 weeks, or at least 4 weeks, or at least one month, or at least two months, or at least 3 months. Each combination can be administered for the same predetermined length of time, or each combination can be administered for a different length of time, or at least one of the combinations can be administered for a different length of time from the other combinations.

[0038] Provided are drug regimens for use for treating obesity, comprising a combination or combinations of drugs or drugs and treatments whose combined effects mimic gastric bypass, and optionally comprising additional drugs that promote or result in weight loss, wherein each combination comprises at least three different drugs that target a different pathway or intervention target involved in the etiology of obesity. The methods and regimens and uses can comprise one or more of a drug that inhibits gastric emptying selected from among one or more of GLP1, Amylin, and Pancreatic Polypeptide Therapeutic; a drug that enhances satiety comprising one or more drugs selected from among glucagon-like peptide- 1 (GLP-1), peptide YY (PYY), amylin, enterostatin / gastric inhibitory peptide (GIP), cholecystokinin (CCK), and Glicentin; a drug that increases insulin release and / or sensitivity comprising one or both of GLP1 and adiponectin; a drug that modulates energy expenditure comprising a drug selected from among leptin, oxyntomodulin, and glicentin; a drug that results in muscle enhancement comprising one or more of sermorelin, tesamorelin and / or growth hormone, and testosterone; and a drug that promotes intestinal smooth muscle relaxation comprising vasoactive intestinal peptide (VIP).

[0039] The combinatorial methods, regimens and uses can be those where the drugs are selected from among drugs that have activities or effects selected from among: drugs that inhibit gastric motility, increase insulin sensitivity, accelerate glycogenolysis and / or lipolysis, reduce eating or appetite, inhibit or modulate gastric acid secretion, limit or decrease the rate of gastric emptying, enhance muscles, increase glycogenolysis, increase insulin sensitivity, enhance the body weightlowering and / or glucose-lowering efficacy of GLP-1, medication(s) or therapy that decrease ghrelin or ghrelin-associated activation pathways, and drugs and treatments that reduce or antagonize ghrelin. Exemplary combinations of drugs include combinations of drugs that are selected from among GLP-1, Adiponectin, leptin, oxyntomodulin, peptide tyrosine-tyrosine (PYY), amylin, pancreatic peptide, enterostatin / glucose-dependent insulinotropic peptide or gastric inhibitory polypeptide (GIP,) cholecystokinin (CCK), vasoactive intestinal peptide (VIP), glicentin, human growth hormone or an active portion thereof or an analog of human growth hormone or an active portion thereof, ephedrine, caffeine, aspirin (ECA), oxyntomodulin, neuropeptide Y (NPY), antimicrobial peptide 2 (LEAP2), vaccine CYT009-GhrQb, the peptide-binding compound Nox-Bl 1, and the ghrelin analog AZP-531 (SEQ ID NO: 15). Other exemplary combinatorial methods, regimens, uses include those where the drugs that are combined comprise a GLP-1 agonist, an appetite suppressant, a thyroid hormone, a carbonic anhydrase inhibitor, an alpha-glucosidase inhibitor, a dipeptidyl peptidase-R (DPP-4) inhibitor, a sodium-glucose co-transporter 2 (SGLT2) inhibitor, a muscle enhancer, drugs that modulate energy expenditure, a GLP-1 agonist, drugs that increase gastric inhibitory polypeptide (GIP2), and mitochondrial uncouplers, such as, but not limited to those in which the drugs are selected from among one or more of dulaglutide, bydureon, semaglutide, exenatide, liraglutide, phentermine, liothyronine, topiramate (carbonic anhydrase inhibitor), acarbose (alphaglucosidase inhibitor), sitagliptin (dipeptidyl peptidase-4 (DPP -4) inhibitor), canagliflozin (sodium-glucose co-transporter 2 (SGLT2) inhibitor), dapagliflozin ( SGLT2 inhibitor), sermorelin, mirabegron (beta-3 adrenergic agonist), and amylin; those where drugs in a combination or the combinations are selected from among a GLP-1 agonist, phentermine, thyroid hormone, carbonic anhydrase inhibitor, carbonic anhydrase inhibitor, alpha-glucosidase inhibitor, DPP -4 inhibitor, SGL2 inhibitor, muscle enhancer, and an appetite suppressant. The methods, regimens, and uses include those comprising a mitochondrial uncoupler, such as, for example, where the mitochondrial uncoupler is selected from among uncoupling protein 1 (UCP1), a catecholamine, and a small molecule uncoupler, such as 2,4-dinitrophenol (DNP) and BAM 15 ((2 -fluorophenyl) { 6- [(2-fluorophenyl)amino]( 1 ,2,5 -oxadi azolo [3 ,4e] pyrazin-5-yl)} amine). The small molecule drugs, which are available in oral dosage forms, such as phentermine, can be co-administered with the liposomes containing or displaying the polypeptides.

[0040] For all of the methods, regimens, and uses the combination or each combination of drugs comprises at least three drugs that target different pathways or targets for intervention. These include those in which the disease, disorder, or condition requires treatment for at least 6 months, 9 months, or 1 year, or longer, or longer and indefinitely, or for life.

[0041] Provided are methods for preventing (or reducing the risk) of desensitization to treatments for a disease, disorder, or condition, comprising administering a rotational combinatorial therapeutic regimen. Such methods employ the methods and regimens and uses described above and elsewhere herein. As described the combinations of drugs and treatments can be administered together, sequentially, intermittently, and / or within a predetermined time period, such as within up to a 24- hour period, or up to a 12-hour period, or up to a 6-hour period or less.

[0042] Exemplary of the methods, regimens and uses is a rotational combinatorial regimen for treating obesity, comprising administering a rotational combinatorial regimen comprising the following combinations: a) a first combination comprising drugs that suppress appetite, increase insulin sensitivity, accelerate glycogenolysis and / or lipolysis, enhance muscles, and inhibit gastric emptying; b) a second combination comprising drugs that inhibit gastric motility, increase insulin sensitivity, accelerate glycogenolysis and / or lipolysis, reduce eating, reduce gastric acid secretion, limit the rate of gastric emptying, promote muscle enhancement, and increase glycogenolysis; and c) a third combination comprising drugs that increase insulin sensitivity, accelerate glycogenolysis and / or lipolysis, enhance the body weight-lowering and glucose-lowering efficacy of GLP-1, and promote muscle enhancement.

[0043] Provided are rotational combinatorial regimens and methods and uses for treatment of obesity comprising at least three different combinations per cycle, wherein: a) a first combination comprises drugs that suppress appetite, increase insulin sensitivity, accelerate glycogenolysis and / or lipolysis, enhance muscles, and inhibit gastric emptying; b) a second combination comprises drugs that inhibit gastric motility, increase insulin sensitivity, accelerate glycogenolysis and / or lipolysis, reduce eating, reduce gastric acid secretion, limit the rate of gastric emptying, promote muscle enhancement, and increase glycogenolysis; and c) a third combination comprises drugs that increase insulin sensitivity, accelerate glycogenolysis and / or lipolysis, enhance the body weight-lowering and glucose- lowering efficacy of GLP-1, and promote muscle enhancement. For example, any of the methods, regimens, and uses herein can be a rotational combinatorial regimen for treating obesity, comprising administering a rotational combinatorial regimen comprising the following combinations: a) a first combination comprising drugs that inhibit gastric motility, increase insulin sensitivity, accelerate glycogenolysis and / or lipolysis, reduce eating, reduce gastric acid secretion, limit the rate of gastric emptying, promote or result in muscle enhancement, and inhibit gastric emptying; b) a second combination comprising drugs that suppress appetite, increase insulin sensitivity, accelerate glycogenolysis and lipolysis, decrease food intake and body weight, promote or result in muscle enhancement, and increase glycogenolysis; and c) a third combination comprising drugs that increase insulin sensitivity, accelerate glycogenolysis and lipolysis, enhance the body weight-lowering and glucose-lowering efficacy of GLP-1, and enhance or promote muscle. For example, a method, use, or rotational combinatorial regimen for treatment of obesity comprises at least three different combinations per cycle, wherein: a) a first combination comprises drugs that inhibit gastric motility, increase insulin sensitivity, accelerate glycogenolysis and / or lipolysis, reduce eating, reduce gastric acid secretion, limit the rate of gastric emptying, promote or result in muscle enhancement, and inhibit gastric emptying; b) a second combination that comprises drugs that suppress appetite, increase insulin sensitivity, accelerate glycogenolysis and lipolysis, decrease food intake and body weight, promote or result in muscle enhancement, and increase glycogenolysis; and c) a third combination that comprises drugs that increase insulin sensitivity, accelerate glycogenolysis and lipolysis, enhance the body weight-and lowering efficacy of GLP- 1, and enhance or promote muscles. In another embodiment that is a rotational combinatorial regimen for treating obesity, the regimen, method, or use comprises the following combinations or administering a rotational combinatorial regimen comprising the following combinations: a) a first combination comprising drugs that increase insulin sensitivity, accelerate glycogenolysis and lipolysis, enhance the body weight-lowering and glucose-lowering efficacy of GLP-1, promote or result in muscle enhancement, and inhibit gastric emptying; b) a second combination comprising drugs that suppress appetite, increase insulin sensitivity, accelerate glycogenolysis and lipolysis, decrease food intake and body weight, promote or result in muscle enhancement, and increase glycogenolysis; and c) a third combination comprising drugs that inhibit gastric motility, increase insulin sensitivity, accelerate glycogenolysis and lipolysis, reduce eating and gastric acid secretion and limit the rate of gastric emptying, and promote or result in muscle enhancement. In other embodiments, the rotational combinatorial regimen, method, or use for treatment of obesity comprises at least three different combinations per cycle, wherein: a) a first combination comprises drugs that increase insulin sensitivity, accelerate glycogenolysis and lipolysis, enhance the body weight-lowering and glucose-lowering efficacy of GLP-1, promote or result in muscle enhancement, and inhibit gastric emptying; b) a second combination comprises drugs that suppress appetite, increase insulin sensitivity, accelerate glycogenolysis and lipolysis, decrease food intake and body weight, promote or result in muscle enhancement, and increase glycogenolysis; and c) a third combination comprises drugs that inhibit gastric motility, increase insulin sensitivity, accelerate glycogenolysis and lipolysis, reduce eating and gastric acid secretion and limit the rate of gastric emptying, and promote or result in muscle enhancement.

[0044] In all embodiments herein, the method or regimen or use can comprise combinations in each combination or in each cycle administered for 2-, 3-, 4-, 5-, or 6-month intervals. The intervals between or for each administration of each combination are the same or vary. In accord with the methods, regimens, and uses, at least one combination in a cycle can comprise at least three different drugs. For example, at least one combination in a cycle comprises at least four different drugs; and optionally at least one combination includes only a single drug.

[0045] In particular, provided are delivery vehicles, including liposomes and exosomes, compositions, regimens, methods, uses, combinations, kits, and articles of manufacture as follows and / or including variations thereof within the skill in the art. Provided are delivery vehicles, comprising a combination of therapeutic peptides, where: the peptides are linked to the surface directly or indirectly via a linker or are part of the surface of delivery vehicle or in the delivery vehicle; the surface of the delivery vehicle optionally is modified for linkage of the polypeptides; the combination of peptides comprises at least three different peptides; at least two of the peptides target different pathways and / or have different activities; and the peptides target pathways involved in obesity and / or diabetes, or have activity for treating obesity and / or diabetes or other obesity comorbidity. These vehicles can further comprise a small molecule drug for treatment of obesity or an associated comorbidity.

[0046] Provided are compositions, comprising a mixture of delivery vehicles, where: each delivery vehicle displays at least one of the weight loss or fat loss peptides on the surface and / or contains the at least one peptide; and the composition comprises delivery vehicles that are selected so that the composition comprises at least three different peptides. The compositions can be formulated for a suitable route of administration, including injection, oral, and intravenous. For, example, the delivery vehicles are administered by subcutaneous (SC) injection in a volume of less than about 5 mL or 3 mL, or in a volume of 10 mL or more, particularly if formulated with an excipient the facilitates administration, such as a hyaluronidase.

[0047] Delivery vehicles include, but are not limited to, liposomes, lipid nanoparticles (LPNs), exosomes, and other extracellular vesicles.

[0048] The peptides in or on the delivery vehicles or in compositions or for combination therapy or for administration with the delivery vehicles comprise fat loss and muscle enhancement peptides, wherein muscle enhancement polypeptides reduce or eliminate loss of muscle mass or increase muscle mass. Additional drugs for administration include small molecule drugs, which can be incorporated in or on or associated with the delivery vehicle (such as by non-covalent interaction), for weight loss, for treatment of comorbidities, such as hypertension, high cholesterol, and diabetes, associated with obesity, and or for treating other diseases, disorders, and conditions.

[0049] Exemplary of the delivery vehicles are those where two of the peptides are fat loss peptides, and one is a muscle enhancement polypeptide. For example, the polypeptides for fat loss (PeptideFL) are selected from among: PeptideFL 1=GLP-1,

[0050] PeptideFL 2=Adiponectin,

[0051] PeptideFL3=Leptin,

[0052] PeptideFL4= Oxyntomodulin,

[0053] PeptideFL5=PYY,

[0054] PeptideFL6= Amylin,

[0055] PeptideFL7=Pancreatic peptide,

[0056] PeptideFL8=Enterostatin / GIP (Gastroinhibitory Polypeptide), PeptideFL9= Glicentin, PeptideFL 10 = Glucagon, PeptideFL 11=GRPP, PeptideFL 12=HGH 176-191, Peptide FL13= CCK, PeptideFL 14= Neurotensin, PeptideFL15= Secretin, PeptideFL 16= IP1, and

[0057] PeptideFL17= MPGF (major proglucagon fragment); and optionally a muscle enhancing polypeptide and / or a drug such as a myostatin pathway inhibitor, such as an antibody, such as bimagrumab, that inhibits muscle wasting. In embodiments, the peptides for muscle enhancement (PeptideME) are selected from among: PeptideMEl=Sermorelin;

[0058] PeptideME2=Tesamorelin; and PeptideME2=IGFlor human growth hormone to induce IGF1. Alternatively, in place of or in addition to the ME drugs, a myostatin pathway inhibitor can be included, such as for example, an antibody or an antigenbinding fragment thereof that competes with apitegromab for antigen binding. Other myostatin pathway inhibitors include, but are not limited to, a ligand trap (e.g., ACE- 031, ACE-083, and BIIB-110 / ALG-801); an anti-ActRIIb antibody (e.g., bimagrumab); a neutralizing anti-myostatin antibody (e.g., stamulumab (MYO-029), domagrozumab (PF-06252616), or Landogrozumab (LY2495655)), a myostatin peptibody (e.g., AMG-745 / PINTA-745), or an anti -myostatin adnectin (e.g., RG6206 or BMS-986089 (also known as taldefgrobep alfa)); wherein, further optionally, the non-selective myostatin pathway inhibitor also inhibits Activin A and / or GDF11 (see, US20240368262, which describes myostatin pathway inhibitors).

[0059] In other embodiments, the peptides for delivery and / or administration are peptides for fat loss (FL) and optionally peptides for ME, where: a) the polypeptides for fat loss are selected from among:

[0060] PeptideFL 1=GLP-1,

[0061] PeptideFL 2=Adiponectin,

[0062] PeptideFL3=Leptin,

[0063] PeptideFL4= Oxyntomodulin,

[0064] PeptideFL5=PYY,

[0065] PeptideFL6= Amylin,

[0066] PeptideFL7=Pancreatic peptide,

[0067] PeptideFL8=Enterostatin / GIP (Gastroinhibitory Polypeptide),

[0068] PeptideFL9= Glicentin,

[0069] PeptideFL 10 = Glucagon,

[0070] PeptideFL 11=GRPP,

[0071] PeptideFL 12=HGH 176-191,

[0072] Peptide FL13= CCK,

[0073] PeptideFL 14= Neurotensin,

[0074] PeptideFL15= Secretin,

[0075] PeptideFL 16= IP1, and

[0076] PeptideFL 17= MPGF (major proglucagon fragment); and b) the peptides for muscle enhancement are selected from among:

[0077] PeptideMEl=Sermorelin;

[0078] PeptideME2=Tesamorelin; and

[0079] PeptideME2=IGFl (or human growth hormone to induce IGF1).

[0080] Exemplary combinations include those, for example, where the peptides comprise GLP-1, Oxyntomodulin, enterostatin / GIP (Gastroinhibitory Peptide); and Sermorelin, and other combinations as detailed herein.

[0081] In all embodiment, including liposomes, exosomes, and LPN, and extracellular vesicles, the peptides and other drugs can be exogenously introduced into and / or onto the vehicle or fabricated with the vehicle so that the peptide / drug is associated with or in the vehicle by any suitable interaction, including covalent bonding and non-covalent interactions.

[0082] Delivery vehicles, include, but are not limited to, liposomes, including liposomes that are large multilamellar vesicles (LMVs). For example, the liposome can comprise phospholipids, such as, for example, one or more of phosphatidyl choline (PC), phosphatidyl ethanol amine (PE), and phosphatidyl serine (PS), and phosphatidic acid (PA). Liposomes can be from the phospholipid is from a natural source and loaded with the peptides and drugs or reacted to become associated with the peptides and other drugs, such as by covalent and / or non-covalent interactions. The liposomes can cholesterol, particularly so that the amount of cholesterol is sufficient to increase the permeability of the liposome compared to the liposome that does not contain the cholesterol, such as for example, where the molar percentage of cholesterol in the liposome is less than 60%, 50%, 40%, 30%, 20%, 10%, or less. The liposome or other vehicle can lipids or other molecules modified with a reactive group for coupling with a peptide or peptide modified with a reactive group, such as for coupling with a peptide or peptide modified with a reactive group. Exemplar reactive groups for the coupling reaction include, but are not limited to, amino, thiol, maleimide, bromo- or iodoacetyl, pyridyl di thio, carboxylic, hydrazide, p-nitrophenyl carbonate, azide, and alkyne groups. The liposome can comprise lipids modified with a reactive group for coupling with a peptide or peptide modified with a reactive group. Reactive groups include, but are not limited to, an amino group that forms an amide bond with an activated carboxylic ester, or a thiol group that binds with maleimide, bromo- or iodo acetyl, pyridyldithio groups, or a hydrazide that binds with carbonyl groups, or a is p-nitrophenyl carbonate that reacts with amines forming an amide bond, or comprises azide and / or alkyne group that bind with each other in the presence of a copper ion catalyst, to attach the protein to the liposome. Other delivery vehicles and peptides can be similarly modified as appropriate. In the resulting delivery vehicles, the peptides can be linked to the vehicle via the bonds formed by reaction of the reactive groups. For example, provided are liposomes or other vehicle, where the peptide and liposome (or other vehicle) are linked via an amide / peptide bond or linker, a thioester bond or linker, a disulfide bond or linker, a hydrazone bond or linker, a carbamate bond or linker, and a 1,2, 3 -triazole linker. In other embodiments the peptide and / or vehicle, such as the liposome, is / are PEGylated for linking the peptide to the liposome. In some embodiments that the linkage of the peptide to the delivery vehicle, such as the liposome, comprises a spacer, such as, but are not limited to, polyethylene glycol (PEG) and / or and an oligonucleotides bound to the vehicle, such as a liposome, and to the peptide linked to a complementary oligonucleotide. In other embodiments that vehicle, such as the liposome, comprises streptavidin bound to biotin-linked peptide or the streptavidin is bound to the liposome and to biotin-linked peptide. In other embodiments, the vehicle, such as the liposomes, are coated with a monolayer of streptavidin and linked to peptides functionalized with biotin-PEG-NHS to form vehicles, such as liposomes, that display the peptides upon mixing these peptide derivatives with streptavidin liposomes. The linkage can comprise a PEG spacer, where one end of the spacer is attached biotin, and the other comprises a reactive group, such as an NHS active ester, that easily forms an amide bond with the PEG.

[0083] Also provided are combination of the delivery vehicle or compositions provided herein that comprises one or more small molecule drug(s), where: the small molecule drug enhances weight loss or treats a comorbidity associated with obesity; and the small molecule is formulated in or with the delivery vehicle or is for administrations separately from the delivery vehicle. Exemplary small molecule drug(s) is / are selected from among one or more of: Phentermine, Topiramate, Metformin, Empagliflozin, Dapagliflozin, Bexagliflozin, Ertugliflozin, Linagliptin, Canagliflozin, NS-2330, Liothyronine, Diethylpropion, Zonisamide, Albuterol, Clenbuterol, Levothyroxine, Naltrexone, Orlistat, Testosterone Cypionate, and Testosterone Enanthate. The delivery vehicle and small molecule drugs can be for administration together in a single composition or for administration in separate compositions for administration at substantially the same time, sequentially, or intermittently, or in accord with a particular combination therapy regimen, in which the drugs are administered in a particular order and / or according to a particular schedule. Regimens for administration of the combinations of the drugs / peptides are provided.

[0084] Also are provided are containers that comprise the delivery vehicle or compositions or combinations provided herein. Exemplary of containers are vials, pens, and syringes, including injectors, such as autoinjectors. Autoinjectors are known that provide for self-administration. Included are autoinjectors are multicompartment containers, where one compartment contains the delivery vehicles or a mixture of different delivery vehicles. They can be provided in a pharmaceutically acceptable vehicle or can be lyophilized. In some embodiments the peptides / drugs are provided as lyophilized powders, and there is a separate compartment that contains the pharmaceutically accept vehicle for solubilizing or dissolving the powders to produce a solution or suspension, such as microemulsion, suitable for injection, particularly subcutaneous injection.

[0085] A pharmaceutical compositions containing any of the delivery vehicles and mixtures there in a pharmaceutically acceptable vehicle for administration are provided. These compositions for administration or for use in methods for treating obesity and / or associated diseases, disorders, and conditions. Hence, methods for treating obesity as well as other comorbidities and uses for the vehicles, compositions, and combinations are provided. For example, provided are methods and uses for treatment of obesity or diabetes in which a delivery vehicle, and / or composition and / or combination provided herein is / are administered. This includes combinatorial and rotational methods, such as those, where the drugs include peptides and optionally small molecule drugs; and the disease, disorder, or condition is obesity and / or diabetes; where at least two combinations are rotated for each cycle of treatment; and treatment comprises at least two cycles.

[0086] Regiments for treating a disease, disorder, or condition, comprising combinations of peptides for use in a rotational combinatorial regimen are provided, such as where the disease, disorder, or condition is obesity or diabetes, or other obesity comorbidity, or other chronic disease, disorder, or condition that requires treatment for at least 6 months; each combination comprises at least two different peptides that target different pathways or intervention targets; the combinations of peptides comprise a delivery vehicle or composition provided herein. The regimen can include a small molecule drug or drugs for weight loss and / or for treating a comorbidity associated with obesity or other chronic condition. For example, the small molecule drug can be selected from one or more of Phentermine, Topiramate, Metformin, Empagliflozin, Dapagliflozin, Bexagliflozin, Ertugliflozin, Linagliptin, Canagliflozin, NS-2330, Liothyronine, Diethylpropion, Zonisamide, Albuterol, Clenbuterol, Levothyroxine, Naltrexone, Orlistat, Testosterone Cypionate, and Testosterone Enanthate, and a statin. In accord with the regimen, the small molecule drug can be incorporated in or on the delivery vehicle and / or the small molecule drug is for co-administration, simultaneously, sequentially, or intermittently with the delivery vehicle(s). The methods can be a rotational combinatorial method in which combinations of peptides and / or drugs are rotated. Such methods and regimens can comprise a plurality of cycles of administration of different combinations of peptides and optionally the small molecules, where each combination is administered at least once a cycle; a cycle comprises administration of each combination at least once; a cycle comprises at least two different combinations; a cycle can be repeated a plurality of times; and at least one of the combinations comprises at least two different drugs that target different targets for intervention or pathways. For example, each cycle can comprise one delivery vehicle that comprises at least 3 different peptides and optionally small molecule drugs, or mixtures of delivery vehicles that comprise different peptides and / or small molecule drugs, each delivery vehicle comprises at least peptide, whereby at least three different peptides are administered in each cycle; and the different delivery vehicles are administered together or serially. The small molecule drugs include, for example, one or more of Phentermine, Topiramate, Metformin, Empagliflozin, Dapagliflozin, Bexagliflozin, Ertugliflozin, Linagliptin, Canagliflozin, NS-2330, Liothyronine, Diethylpropion, Zonisamide, Albuterol, Clenbuterol, Levothyroxine, Naltrexone, Orlistat, Testosterone Cypionate, and Testosterone Enanthate, and a statin. Different vehicles or combinations of vehicles and / or drugs can be administered within 24 hours or other suitable time period determined by the skilled person, of each other.

[0087] In the delivery vehicles, methods, and / or regimens, each peptide can be selected to targets a different pathway or different target for intervention for treatment of obesity. For example, the combinations of peptides mimic all or some of the effects of gastric bypass. Exemplary combinations of peptides include, but are not limited to, combination of peptides on the delivery vehicle comprises at least three selected from among: a peptide that inhibits gastric emptying selected from among one or more of GLP1, Amylin, and Pancreatic Polypeptide Therapeutic; a peptide drug that enhances satiety comprising one or more drugs selected from among glucagon-like peptide-1 (GLP-1), peptide YY (PYY), amylin, enterostatin / gastric inhibitory peptide (GIP), cholecystokinin (CCK), and glicentin; a peptide that increases insulin release and / or sensitivity comprising one or both of GLP1 and adiponectin; and a peptide that modulates energy expenditure comprising leptin, oxyntomodulin, and glicentin.

[0088] In accord with the descriptions of the delivery vehicles, methods and regimens provided herein, the vehicle can comprise and / or display and / or contain combinations of peptides selected from among combinations of drugs for fat loss + a drug for muscle enhancement as follows: a) GLP1 / GIP 1 / Oxyntomodulin + Sermorelin or Tesamorelin or IGF1; b) GLPl / GIPl / Amylin + Sermorelin or Tesamorelin or IGF1; c) GLP1 / GIP 1 / Glucagon + Sermorelin or Tesamorelin or IGF1; d) GLP1 / GIP1 / CCK+ Sermorelin or Tesamorelin or IGF1; e) GLP1 / GIP1 / PYY + Sermorelin or Tesamorelin or IGF1; and f) GLP1 / GIP 1 / Leptin + Sermorelin or Tesamorelin or IGF1. Exemplary amino acid sequences of each of the peptides for each of a)-f) are as set forth set forth in the following SEQ IDs, or are portions, or are variants thereof that have at least 90% or at least 95% sequence identity thereto and retain activity for effecting fat loss or muscle enhancement: a) SEQ ID NOs: 1, 10, 4 or 47 + SEQ ID NOs: 5, 8, 44; b) SEQ ID NOs: 1, 10, 7 + SEQ ID NOs: 5, 8, 44; c) SEQ ID NOs: 1, 10, 27 + SEQ ID NOs: 5, 8, 44; d) SEQ ID NOs: 1, 10, 11 + SEQ ID NOs: 5, 8, 44; e) SEQ ID NOs: 1, 10, 6 or 36 + SEQ ID NOs: 5, 8, 44; and f) SEQ ID NOs: 1, 10, 3 + SEQ ID NOs: 5, 8, 44.

[0089] In embodiments herein the delivery vehicle, composition, combination, method, and / or regimen includes a peptide that results in muscle enhancement or can include a peptide or other molecule that inhibits or reduces muscle loss, such as a myostatin pathway inhibitor. Exemplary muscle enhancement peptides and drugs include, but are not limited to, one or more of sermorelin, tesamorelin and / or growth hormone, and testosterone. The herein the delivery vehicle, composition, combination, method, and / or regimen can comprise a peptide that promotes intestinal smooth muscle relaxation, such as vasoactive intestinal peptide (VIP).

[0090] Peptides can be selected from among: a) GLP-1, Adiponectin, leptin, oxyntomodulin, peptide tyrosine-tyrosine (PYY), amylin, pancreatic peptide, enterostatin / gastric inhibitory polypeptide (GIP), cholecystokinin (CCK), vasoactive intestinal peptide (VIP), glicentin, human growth hormone or an active portion thereof or an analog of human growth hormone or an active portion thereof, ephedrine, caffeine, aspirin (EC A), oxyntomodulin, neuropeptide Y (NPY), antimicrobial peptide 2 (LEAP2), vaccine CYT009-GhrQb, the peptide-binding compound Nox-Bl 1, and the ghrelin analog AZP-531 (SEQ ID NO: 15); and / or b) the peptides are a GLP-1 agonist, an appetite suppressant, a thyroid hormone, a carbonic anhydrase inhibitor, an alpha-glucosidase inhibitor, a dipeptidyl peptidase-R (DPP -4) inhibitor, a sodium-glucose co-transporter 2 (SGLT2) inhibitor, a muscle enhancer, drugs that modulate energy expenditure, a GLP-1 agonist, peptides that increase gastric inhibitory polypeptide (GIP), drugs that modulate GIP2, and mitochondrial uncouplers; and the peptides are combined by displaying a plurality on each delivery vehicle or by mixing delivery vehicles that display different peptides.

[0091] Exemplary of embodiments herein are those in which the peptides and drugs are selected from among: dulaglutide, bydureon, semaglutide, exenatide, liraglutide, phentermine, liothyronine, topiramate (carbonic anhydrase inhibitor), acarbose (alphaglucosidase inhibitor), sitagliptin (dipeptidyl peptidase-4 (DPP -4) inhibitor), canagliflozin (sodium-glucose co-transporter 2 (SGLT2) inhibitor), dapagliflozin (SGLT2 inhibitor), sermorelin, mirabegron (beta-3 adrenergic agonist), and amylin. In other embodiments of the delivery vehicles, methods, regimens, compositions, and combinations, are those where the peptides and drugs are selected from among: a GLP-1 agonist, phentermine, thyroid hormone, carbonic anhydrase inhibitor, carbonic anhydrase inhibitor, alpha-glucosidase inhibitor, DPP -4 inhibitor, SGL2 inhibitor, muscle enhancer, and an appetite suppressant. In all embodiments herein, the delivery vehicle, composition, method, use, or regimen can include or further comprise a mitochondrial uncoupler, wherein the mitochondrial uncoupler is provided linked to a delivery vehicle or mixed in the composition or administered as a free molecule not bound to a delivery vehicle, such as a mitochondrial uncoupler is selected from among uncoupling protein 1 (UCP1), a catecholamine, and a small molecule uncoupler, such as 2,4-dinitrophenol (DNP) and BAM15 (N5,N6-bis(2- Fluorophenyl)[l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine).

[0092] It is understood that those of skill in the art can fabricate or obtain the peptides, and delivery vehicles, and / or fabricate the delivery vehicles by known methods. Nevertheless, methods for preparing or fabricating the delivery vehicles and / or components thereof are provided. Included are methods for preparing a PEGylated peptide, comprising preparing a PEGylated lysine and adding it to peptide during solid phase synthesis, where optionally the PEGylated lysine is prepared by reacting the epsilon-amino group of the lysine with a carboxyl terminated PEG moiety via an amide group to produce a PEGylated lysine. The peptides can be modified by replacing lysines that are not intended to be PEGylated with a conservative amino acid. The methods of synthesis can include adding the PEGylated lysine to a peptide during solid phase synthesis of the peptide. The PEG moiety comprises a functional group for conjugation with a reactive group, such as one on a liposome. Functional groups include, but are not limited to, acetylene that is reacted with azide in the liposome (click chemistry). Other methods of conjugating a polypeptide to a liposome or other delivery vehicle, comprise: adding a lysine to a polypeptide during solid phase synthesis by reacting the epsilon-amino group of lysine with a carboxyl terminated PEG moiety via an amide group to produce a peptide comprising a lysine comprising the PEG moiety, which comprises a functional group for conjugation with the liposome; and conjugating the PEGylated peptide to the liposome. The functional group, such as acetylene, can be reacted, for example, with azide fabricated in or added to the delivery vehicle, such a liposome, thereby employing click chemistry. Any of the delivery vehicles provided herein can be so-prepared, including where the peptide is a GLP-1 pathway agonist and / or a muscle enhancer peptide, or any other combinations as described herein or that are apparent to the skilled artisan from the description herein. The method can include prepared at least three pegylated peptides and linking the peptides to one delivery vehicle, such as a liposome, or each to a different delivery vehicle, such as a liposome, or two can be lined to one delivery vehicle, such as a liposome, and the third on a separate delivery vehicle, such as a liposome, or other combinations, or all can be linked or introduced into or onto a single delivery vehicle, such as a liposome. Exemplary thereof are wherein the peptides linked to the delivery vehicles, such as a liposome, or to different delivery vehicles, such as liposomes, wherein a least two of the peptides are for fat loss, and one is for muscle enhancement. For example, peptides can be selected where: a) the peptides for fat loss (FL) are selected from among:

[0093] PeptideFL 1=GLP-1,

[0094] PeptideFL 2=Adiponectin,

[0095] PeptideFL=Leptin,

[0096] PeptideFL4= Oxyntomodulin,

[0097] PeptideFL5=PYY,

[0098] PeptideFL6= Amylin,

[0099] PeptideFL7=Pancreatic peptide,

[0100] PeptideFL8=Enterostatin / GIP (Gastroinhibitory Polypeptide),

[0101] PeptideFL9= Glicentin,

[0102] PeptideFL 10 = Glucagon,

[0103] PeptideFL 11=GRPP,

[0104] PeptideFL 12=HGH 176-191,

[0105] Peptide FL13= CCK,

[0106] PeptideFL 14= Neurotensin,

[0107] PeptideFL15= Secretin,

[0108] PeptideFL 16= IP1, and

[0109] PeptideFL 17= MPGF (major proglucagon fragment); and b) the peptides for muscle enhancement (ME) are selected from among: PeptideMEl=Sermorelin;

[0110] PeptideME2=Tesamorelin; and

[0111] PeptideME2=IGFl (or human growth hormone to induce IGF1).

[0112] Provided are delivery vehicles, such as liposomes that comprise one or more of a fat loss peptide and a muscle enhancement peptide produced by any of the above methods or methods that in light of the disclosure herein are known to the skilled artisan.

[0113] In all embodiments herein, the delivery vehicle, composition, use, method, regimen, or liposome, can include a small molecule drug, such as where the drug is for weight loss and / or a co-morbidity associated with obesity. Comorbidities include, but are not limited to, diabetes, hypertension, high cholesterol and / or other elevated lipids (dyslipidemia), metabolic syndrome, and heart disease. Small molecule drugs include, but are not limited to, Phentermine, Topiramate, Metformin, Empagliflozin, Dapagliflozin, Bexagliflozin, Ertugliflozin, Linagliptin, Canagliflozin, NS-2330, Liothyronine, Diethylpropion, Zonisamide, Albuterol, Clenbuterol, Levothyroxine, Naltrexone, Orlistat, Testosterone Cypionate, and Testosterone Enanthate, and a statin.

[0114] In all embodiments herein, the products and methods can include phentermine, including regiments and methods where the phentermine is provided in or on the delivery vehicle, or wherein phentermine is provided as a separate composition, such as in a tablet, capsule, gel, or other form for oral administration, for combination therapy. In all embodiments, or the delivery vehicles, compositions, combinations, uses, method, regimen, the delivery vehicle can be an extracellular vesicle or an LPN, or an exosome, or a liposome.

[0115] Also provided are kits and articles of manufacture containing the delivery vehicles and containers and compositions and combinations.

[0116] Brief Description of Drawings

[0117] Figure 1 depicts bioactive members of the PGDP family include glucagon-like peptides -1 and -2 (GLP-1 and GLP-2), oxyntomodulin, glicentin and glicentin-related pancreatic peptide, which are produced via tissue-specific processing of proglucagon by the prohormone convertase (PC) enzymes, PC 1 / 3 and PC2.

[0118] Figures 2A and 2B depict a liposome with linkages and linked moieties. Figure 3 depicts PEG (X) with an N-hydroxy succinimide active ester on one end. Figure 4 depicts an exemplary coupling chemistry in which X is Br or I.

[0119] Figures 5A-C depict a polypeptide with free lysines and Pegylation thereof. Figure 5A depicts a short segment of a polypeptide, such as any of the GLP-1 agonist polypeptides and / or a muscle enhancement polypeptide, which is Pegylated as shown herein, and Figure 5B depicts an exemplary resulting product in which a lysine is PEGylated. As an example, the PEG linked to the lysine has an azide group at the end for coupling to a liposome that has an acetylene group on the surface. Figure 5C depicts the product.

[0120] OUTLINE

[0121] A. DEFINITIONS

[0122] B. OVERVIEW

[0123] C. Delivery vehicle -components

[0124] 1. Component -Proglucagon derived Peptides (PGDP) and muscle enhancement peptides a. Proglucagon Derived Peptides and tissue specific secretion b. Unimolecular multi-agonists c. Enhancing fat loss efficacy via proglucagon derived peptides

[0125] (PGDP) and co-secretory molecules released after a RYGBP

[0126] 2. Delivery vehicles

[0127] 3. Conjugation / Binding of peptides on the liposomes

[0128] 4. Spacers

[0129] 5. Linkage of peptides to the Delivery vehicles a. PEGylation of Peptides for linkage to lipid particles, such as liposomes b. Synthesis of Pegylated peptides for conjugation to liposomes c. Liposome preparation for linking to the peptides

[0130] D. FORMULATIONS AND ROUTES AND MODES OF DELIVERY

[0131] E. COMBINATORIAL THERAPY AND ROTATIONAL COMBI NA-'TORIAL THERAPY

[0132] 1. Identification of diseases, disorders, or conditions for treatment a. Disease, Disorder, or Condition with a Plurality of Known Treatments b. Chronic Conditions c. Conditions where Patients Develop a Tolerance to Treatments

[0133] 2. Development of a Combinatorial Rotational Therapy Regimen

[0134] 1) Identify known treatments / therapies for each disease state or pathways associated with each disease state

[0135] 2) Identify the pathways, mechanism of actions or targets

[0136] 3) Select treatments / therapies that activate different pathways, have different mechanism of actions or targets, and / or that are compatible with a rotational therapy

[0137] 4) Identify combinations that include at least 2 therapeutics known to activate different molecular and / or cellular pathways and design a regimen for administration of the combinations 5) Create a regimen to administer multiple rounds of treatment, with combinations of therapeutics

[0138] F. METHODS OF TREATMENT AND USES

[0139] 1. Therapeutic Uses of the Combinatorial Therapy a. Combination Therapies in Cancer b. Combination Therapies in Pain Management c. Combination Therapies in Oral Contraception d. Combination Therapies to Treat Pathogens e. Combination Therapies to Treat Alzheimer’s Disease f. Combination Therapies to Treat Hypertension g. Combination Therapies to Treat Parkinson’s Disease h. Combination Therapies to Treat Chronic Obstructive Pulmonary Disease (COPD) i. Combination Therapies to Treat Obesity-Associated Diseases and Conditions j. Combination Therapies to Treat Overweight and Obesity

[0140] G. COMBINATORIAL AND ROTATIONAE COMBINATORIAL THERAPY FOR WEIGHT EOSS

[0141] 1. Limitations of Existing Treatments for Weight Loss

[0142] 2. Obesity and the Challenges of Treatment

[0143] 3. Pharmacological Treatments a. Amphetamines (e.g., phentermine-topiramate) b. Lipase inhibitors (e.g., Orlistat) c. Serotonergic agonists - Neuromodulators (e.g., lorcaserin) d. Bupropion / Naltrexone (such as the product sold under the trademark Contrave®) e. Glucagon-like peptide-1 receptor (GLP1R) agonists f. Mitochondrial uncouplers g. Thyroid hormones h. Drug cocktails i. Cannabinoid receptor antagonists j. GIPR agonists and GIPR / GLP1R combination agonists k. GLPIR / glucagon dual agonists l. Summary

[0144] 4. Surgical Treatments

[0145] H. MODIFICATIONS AND ENHANCEMENTS OF PHARMACOLOGICAL WEIGHT LOSS TREATMENTS TO IMPROVE CLINICAL OUTCOMES

[0146] 1. Developing Combination and Rotational Combinatorial Therapies for Weight Loss

[0147] 2. Pathways to Target for Weight Loss and Exemplary Polypeptides a. Therapeutic Combinations and Regimens b. Combination Drug Therapy to Mimic Gastric Bypass I. PHARMACEUTICAL PRODUCTION, COMPOSITIONS, AND FORMULATIONS

[0148] 1. Formulation and Administration of the Combinatorial Therapy

[0149] 2. Dosage Forms

[0150] 3. Dosage and Administration

[0151] 4. Dosage and Administration for Treating Obesity and Overweight

[0152] 5. Routes of Administration of the Combinations a. Administration of Combinatorial Treatments for Improved Weight Loss

[0153] 6. Articles of Manufacture and Kits

[0154] J. METHODS OF ASSESSING ACTIVITY, BIOAVAILABILITY AND PHARMACOKINETICS

[0155] K. SEQUENCE SUMMARY

[0156] L. EXAMPLES

[0157] A. DEFINITIONS

[0158] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong(s). All patents, patent applications, published applications and publications, GenBank sequences, databases, websites, and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. If there is / are a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0159] As used herein, a delivery vehicle refers to macro-molecular structures in an emulsion, such as micelles, inverse micelles, lipid bilayers (liposomes) and cubosomes or a mixture thereof, as well as lipid nanoparticles and other lipid-based vehicles. Delivery vehicles include, but are not limited to, liposomes, lipid nanoparticles, extracellular vehicles, such as exosomes. The polypeptides are displayed on the surface of the vehicles such as by linkage to moieties on the surface. In other embodiments, the polypeptides can be embedded in the surface when the delivery vehicle is formed; and / or can be expresses in a membrane protein in vivo, such as during formation or preparation of an extracellular vehicle. As used herein, therapies used interchangeably with treatments include drugs and can include other non-drug treatments. Thus, treatments include drugs (or medications) and non-drug treatments, such as, for example, surgery.

[0160] As used herein, drugs include any administered therapeutic and is used interchangeably with medications.

[0161] As used herein, a therapeutic agent is used interchangeably with therapeutic and includes, but is not limited to: conventional drugs and drug therapies; vaccines; pharmaceutical medications; pharmaceuticals; homeopathic medications; peptides; protein therapeutics; radio-protectants; radiation therapy; and chemotherapeutics. A therapeutic agent can effect amelioration of symptoms of a disease, disorder, and / or condition or can prevent or reduce the risk of developing a disease, disorder, or condition, or reduce the severity of disease, disorder, or condition. The combinations when used for the combinatorial therapy as described herein include at least 3 treatments, such as three different therapeutics. For combinatorial rotational therapy combinations that are rotated, the combinations can include 2 treatments in a combination or combinations of treatments. Some of the rotations can include a single drug or therapy, as long as one or more others rotational combinations include at least two. Generally, each rotated combination includes at least two different drugs or treatments.

[0162] As used herein, combinatorial therapy or combinatorial protocol or regimen refers to combinations of treatments for a disease, disorder, or condition from among a plurality of treatments, at least 2 and generally at least 3, where each treatment acts on or interacts with or modulates a different target molecule and / or target pathway involved in or that mediates the disease, disorder, or condition.

[0163] As used herein, a rotational combinatorial therapy is used interchangeably with combinatorial rotational treatment, or rotational combinatorial therapy, or rotational combination therapy, or rotational combination treatment or CRT, or grammatical variations thereof. A rotational combinatorial therapy or protocol or regimen includes at least two different therapeutic combinations at least one of which, and generally each of which, includes at least two therapeutics or therapies known to activate different molecular and / or cellular pathways or targets involved in a disease, disorder, or condition. Combinatorial rotational therapy, thus, is a combinatorial therapy protocol that includes a plurality of rounds of treatment with different combinations of treatments or with the same combination but not successively. The different combinations are therapeutics are administered for a predetermined time, and then rotated for another predetermined time. The number of rotations of combinations can be two, three, four, five or more, and can be administered for weeks, months, years, and indefinitely depending upon the disease, disorder, or condition. The predetermined time can be days or weeks or months, generally is about 2-6 weeks. Each predetermined time is not necessarily the same.

[0164] As used herein, a rotational combinatorial pharmacological treatment (also referred to a rotational combinatorial therapy refers to a combinatorial therapy or treatment with pharmaceuticals or medications or drugs or treatments in the combinations where combination are rotated. The rotational combinatorial pharmacological treatment includes a plurality of combinations of a plurality of pharmaceuticals or medications or drugs that are rotated in accord with a rotational regimen or protocol. The rotational regimen generally is employed to prevent desensitization to a particular drug. A rotational combinatorial regimen herein includes two or more different therapies or therapeutics (e.g., combinations of therapies and / or therapeutics) that are administered such that the combinations of the therapies are rotated for a plurality of rounds of treatment. Where there is a plurality of rounds of treatment, at least one round can include only a single treatment. Generally, for rotational combinatorial protocols, the rounds include combinations of at least two different treatments, where each targets a different pathway involved in the disease, disorder, or condition.

[0165] As used herein, rotate or rotational or grammatical variations thereof include an exchange of one treatment for another. A combinatorial rotation refers to changing one therapeutic combination to another, which has the effect of improving the therapeutic response, such as by avoiding desensitization to a therapeutic of combination thereof. This also can reduce adverse side effects, and / or the severity of adverse side effects. In examples herein, a rotation includes a regimen in which a first combination of more than one therapeutics is administered, and the therapy is rotated (or switched) to a second combination one or more one therapeutic, and, optionally, the therapy is rotated (or switched) to a third combination of more than one therapeutics. Generally, the rotations occur according to a predetermined time schedule, or in view of a physician’s judgement, such as based on an observation that a combination has reduced effect. In some examples, the therapies are rotated more than three times, such as more than four times, more than five times, more than six times, more than seven times, more than eight times, more than nine times, or more. In some instances, such as chronic diseases, disorders, and conditions, the rotation of therapies continues for up to life.

[0166] As used herein, a regimen is used interchangeably with protocol and refers to a course of medical treatment. In some examples the regimen is designed to improve or preserve the health of the patient or to attain a particular result. A therapeutic regimen or protocol includes the timing for administration of the therapeutics, timing for cessation of therapeutics, therapeutic dosage(s), the particular therapeutics that are included in particular combinations, and other components of experimental design, such as how therapeutics are administered. A protocol can include provisions for how the protocol can be modified, such as, for example, in the case of receptor downregulation or an increase in adverse side effects.

[0167] As used herein, a cycle is generally a series of events that are repeated regularly in the same order. A cycle, with reference to a rotational combinatorial regimen, refers to the repeated administration of combinations of drugs, where a combination of drugs (or a drug) is administered for a period of time followed by another combination of drugs, and so on until all combinations in a cycle of treatment are administered. The cycle then can be repeated; generally, the same combinations of drugs or treatments are administered. It, however, is within the discretion of the physician to change the drugs / treatments in accord response(s) of the treated subject, such as to improve therapeutic effectiveness, eliminate or reduce desensitization, and / or for other reasons, such as to reduce adverse side effects. In accord with a rotational regimen, a combination can include a single drug, as long as at least one combination in the cycle includes two or more drugs or treatments.

[0168] As used herein, disease or disorder or condition refers to a pathological or undesirable or undesired state in an organism resulting from a cause or condition including, but not limited to, infections, acquired conditions, and genetic conditions, and that is characterized by identifiable symptoms. Obesity and overweight are conditions characterized by excess body weight.

[0169] As used herein, mucosal delivery refers to delivery of an agent in which the agent is introduced to the body across a mucous membrane which allows for the avoidance of the gastrointestinal tract and first pass liver metabolism and consequently allows the agent to directly enter circulation. This can include passage through the gastrointestinal tract as by oral ingestion, but refers to delivery through the mucosa of such locus.

[0170] As used herein, a chronic condition is one that is expected, based on physician’s experience and knowledge in the art to last more than 6 months, and generally more than a year. It is a condition that requires treatment for at least 6 months, and can require at 1 year or more, including for life.

[0171] As used herein, treating a subject with a disease, disorder, or condition means that a drug, therapeutic, composition, combination or other therapy is administered to the subject and the subject’s symptoms or manifestations of the disease or conditions are partially or totally ameliorated, or remain static (do not worsen) following treatment. In examples, treating a subject with a disease, disorder, or condition includes treating a subject with a rotational combinatorial therapy described herein.

[0172] As used herein, a pathway refers to a biological pathway that is involved in or mediates disease, disorder, or condition. A pathway can be targeted via a component of the pathway, such as a receptor or ligand involved in the pathway. Depending upon the disease, disorder, or condition and the role of the pathway in the disease, disorder, or condition, the targets can be antagonized or agonized.

[0173] As used herein, a “combination” refers to any association between two or among more items. The association can be spatial or refer to the use of the two or more items for a common purpose. A combination for therapeutic use(s), such as a rotational combinatorial therapy provided herein, includes more than one therapeutic (z.e., medication), such as two, three, four, five, or more therapeutics, but generally includes up to five therapeutics in the combination. The rotational combinatorial therapy provided herein includes a regimen in which two, three, four, five or more combinations are rotated. The combinations for rotation in the regimen provided herein can contain different therapeutics or can contain some of the same therapeutics, but do not contain all of the same therapeutics in the combinations. A “combination” is used herein interchangeably with a cluster, which includes two or more treatments, such as two or more medications. Each combination can be administered as a therapy, or can be rotated and administered for a predetermined time, which can be shortened or lengthened according to the judgement of a physician. When not rotated each combination generally includes at least 3 different treatments

[0174] As used herein, treatment means any manner in which the symptoms of a condition, disorder or disease are ameliorated or otherwise beneficially altered. Hence, treatment encompasses prophylaxis, therapy and / or cure. Treatment also encompasses any pharmaceutical use of the compositions and combinations herein. Treatment also encompasses any pharmaceutical use of a combination of pharmaceutical and non-pharmaceutical therapeutics and compositions provided herein. Treatment encompasses a rotational combinatorial therapy described herein, in which a combination of therapeutics is administered in accord with any regimen described herein.

[0175] As used herein, amelioration of the symptoms of a particular disease or disorder by a treatment, such as by administration of a pharmaceutical composition or other therapeutic(s), refers to any lessening, whether permanent or temporary, lasting or transient, of the symptoms that can be attributed to or associated with administration of the composition or therapeutic(s). In some examples, amelioration of symptoms includes amelioration of side effects of the composition or therapeutic(s). In general, amelioration of adverse symptoms of a disease or disorder is a decrease in number or severity of the symptoms of the disease compared to the number or severity of the symptoms of the disease prior to treatment. In some examples, amelioration of a disease or condition can include a decrease in levels of a biomarker of the disease, such that the biomarker level is decreased by at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to the biomarker level prior to treatment. In other examples, symptoms of the disease are decreased, such as the amount or severity of symptoms that are decreased by at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to the amount or severity of symptoms level prior to treatment.

[0176] As used herein, incidence refers to how often an event occurs. For example, adverse side effect incidence refers to how often adverse side effects occur after administration of a therapeutic, for example the frequency of adverse side effects after administration of a combination therapy provided herein. For example, an adverse side effect(s) can occur 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the time after administration of a rotational combinatorial therapy provided herein. In examples herein, adverse side effects incidence decreases when multiple therapeutics are formulated in a combination and administered in accord with a rotational regimen provided herein compared to a monotherapy or compared to a combination that is not rotated (z.e., is continuously administered or is administered over a longer period of time). For example, the incidence of an adverse side effect(s) can decrease 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% multiple therapeutics are formulated in a combination and administered in accord with a rotational regimen provided herein, compared to a monotherapy or compared to a combination that is not rotated.

[0177] As used herein, severity refers to degree of illness or symptoms of the illness or side effects of the illness or side effects of treatment s) for the illness manifested by a patient. The skilled physician can assess the severity of an illness or progression of the severity of an illness or the severity of side effects of the illness or side effects of treatments with therapeutics for the illness based on the knowledge in the field of medicine and pharmacology. The skilled physician or scientist can assess the adverse side effects associated with administration of therapeutics for a disease, disorder, or condition; for example, as severe e.g., adverse side effects requiring medical care), or as mild (e.g., adverse side effects not requiring medical intervention or treatment). For example, adverse side effects can be classified by severity using medical diagnosis codes used in clinical assessment and tracking, such as classification of severity as minor, moderate, major, and extreme. In some examples herein, adverse side effects following administration of the rotational combinatorial therapy provided herein are less severe than adverse side effects following administration of monotherapy or following administration of a combination therapy that is not rotated.

[0178] As used herein, “adverse effect” or “adverse side effect” refers to a harmful, deleterious and / or undesired effect of administering a medication or drug. Side effects or adverse effects are graded based on toxicity and various toxicity scales exist providing definitions for each grade. Exemplary of such scales are toxicity scales of the National Cancer Institute Common Toxicity Criteria version 2.0, the World Health Organization or Common Terminology Criteria for Adverse Events (CTCAE) scale. Generally, the scale is as follows: Grade 1 = mild side effects; Grade 2= moderate side effects; Grade 3= Severe side effects; Grade 4= Life Threatening or Disabling side-effects; Grade 5= Fatal. Assigning grades of severity is within the experience of a physician or other health care professional.

[0179] As used herein, a dose-limiting toxicity (DLT) refers to the dose of a drug that produces side effects severe enough to prevent larger doses being given. It is within the level of skill of a skilled physician to assign or determine a DLT depending on the treatment protocol, the administered treatment, the disease to be treated, the dosage regime, and the particular patient to be treated. Generally, for the treatments, protocols, and regimens provided herein, a DLT is the dose of a drug results in an adverse event or side effect that on the toxicity scale of at least an ongoing or persistent Grade 2 toxicity that fails to resolve over the course of treatment and that limits the patient’s ability to comply with the protocol therapy. As part of rotational therapy, a drug that results in such side effects can be administered at a lower dose, since the combinations of drugs / treatments can act synergistically, or the drug can be discontinued, and optionally replaced with another drug with similar activity or effect.

[0180] As used herein, prevention or prophylaxis refers to methods in which the risk of developing disease or condition is reduced. Prophylaxis includes reduction in the risk of developing a disease or condition and / or a prevention of worsening of symptoms or progression of a disease or reduction in the risk of worsening of symptoms or progression of a disease and / or a prevention of worsening of symptoms or progression of a disease or reduction in the risk of worsening of symptoms or progression of a disease. Prevention includes inhibition or avoidance of a disease, disorder, or condition by administration of a rotational combination therapy provided herein.

[0181] As used herein, a “prophylactically effective amount” or a “prophylactically effective dose” refers to the quantity of an agent, compound, material, or composition containing a compound, which, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset, or reoccurrence, of disease or symptoms, reducing the likelihood of the onset, or reoccurrence, of disease or symptoms, or reducing the incidence of viral infection. The full prophylactic effect does not necessarily occur by administration of one dose, and can occur only after administration of a series of doses, such as a rotation of combinations of therapeutics. Thus, a prophylactically effective amount can be administered in one or more administrations. A prophylactically effective amount of a combination therapy as described herein can be lower than a prophylactically effective amount of a monotherapy. A prophylactically effective amount of a rotational combinatorial therapy as described herein can be lower than a prophylactically effective amount of a monotherapy or a combination therapy that is not rotated.

[0182] As used herein, an “effective amount” of a compound or composition for treating a particular disease is an amount that is sufficient to ameliorate, or in some manner reduce the symptoms associated with the disease. Such amount can be administered as a single dosage or can be administered according to a regimen, whereby it is effective. The amount can cure the disease but, typically, is administered in order to ameliorate the symptoms of the disease. A measure of effective amount or dosage is the median effective dose (ED50), which is the dose that produces a response that is fifty percent of the maximum obtainable response. As described herein, an effective amount of an individual therapeutic or a therapeutic combination is the amount required to achieve a desired amelioration of symptoms. A rotational combination therapy as can employ a lower effective amount of drug compared to a than a monotherapy or a combination therapy that is not rotated.

[0183] As used herein, a therapeutically effective amount or a therapeutically effective dose refers to the quantity of an agent, compound, material, or composition containing a compound that is at least sufficient to produce a therapeutic effect following administration to a subject. Hence, it is the quantity necessary for preventing, curing, ameliorating, arresting, or partially arresting a symptom of a disease or disorder. A therapeutically effective amount or dose can reference the amount or dose of a particular therapeutic in a combination, and / or can reference the amount or dose of the combination of therapeutics in a combination.

[0184] As used herein, “therapeutic efficacy” refers to the ability of an agent, compound, material, composition, or combination of agents, compounds, materials, or compositions containing a compound to produce a therapeutic effect in a subject to whom the agent, compound, material, composition, or combination of agents, compounds, materials or compositions containing a compound has been administered. For example, the therapeutic efficacy can refer to the therapeutic efficacy of a combination of therapeutics or a plurality of combinations, such as the combination(s) for use in a rotational combinatorial therapy.

[0185] As used herein, a standard dosage of a therapeutic is the dosage for formulation or administration that is approved by applicable regulatory agencies in the field, for example the U.S. Food and Drug Administration (FDA), such as the FDA Data Standards Advisory Board. In some examples a standard dosage includes an amount (z.e., mass) of a therapeutic (ie., drug) for formulation. A standard dosage also can include a dosage regimen for administering the standard dosage (z.e., biweekly dosing). A standard dosage as approved by a regulatory agency or body also can be a range of dosages or the dosage can vary based on characteristics of the patient to whom the therapeutic is administered. For example, the dosage can vary depending on the weight of the patient, or the severity of the disease or condition for which the therapeutic(s) is / are administered. A standard dosage also can be defined by pooled analysis using scientific studies of real-world data, where efficacy is established for various doses, and the dosage is established as a dosage with a particular efficacy. When standard dosage is established, efficacy, adverse side effects, and other factors are considered. An individual therapeutic (ie., drug) in a combination for a rotational combinatorial therapy provided herein can be formulated in a standard dosage. In some examples, an individual therapeutic (z.e., drug) in a combination for a rotational combinatorial therapy provided herein is initially formulated in a standard dosage and the dosage is modified from the standard dosage for subsequent administrations. For example, the dosage of the individual therapeutic is formulated in a lower or higher dosage compared to a standard dosage.

[0186] As used herein, polypeptide, peptide, and protein refer to polymers of amino acids of any length. Where not used interchangeably other characteristics, such as size, and structure, are contemplated, as defined below. The polymer can be linear or branched, and can contain amino acids, including modified amino acids, and it can be interrupted by non-amino acids. Also included are amino acid polymers that include sequence modifications including, replacements, insertions, deletions, and transpositions. Also included are amino acid polymers that contain post-translational modifications, such as disulfide bonds, glycosylation, sialylation, conjugation to other proteins, peptides, and polypeptides, such, but not limited to, conjugation to a detectable marker, or reporter.

[0187] As used herein the term "amino acid" includes natural and / or unnatural or synthetic amino acids, including glycine and the D or L optical isomers, and amino acid analogs and peptidomimetics.

[0188] As used herein, suitable conservative substitutions of amino acids are known to those of skill in this art and can be made generally without altering the biological activity of the resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene,4th Edition, 1987, The Benjamin / Cummings Pub. Co., p.224). Such substitutions can be made in accordance with those set forth as follows:

[0189] Other substitutions also are permissible and can be determined empirically or in accord with known conservative substitutions. Amino acid substitutions contemplated include conservative substitutions, such as those set forth in the table below. Substitutions that alter properties of the peptides cam be effected; such substitutions are generally non-conservative, but can be readily effected by those of skill in the art. Conservative amino acid substitutions generally can be effected without altering the activity of a peptide. Variants of the weight loss peptides are known and / or can be used herein.

[0190] As used herein, a protein is a polypeptide that has a three-dimensional structure and can include bonds in addition to peptide bonds, such as disulfide bonds and other interactions, that participate in forming the two- and three-dimensional structure.

[0191] As used herein, a peptide refers to a polypeptide that is from 2 to 300, but generally shorter than 100, amino acids in length. Peptides include therapeutic peptides that are administered for treatment of a disease, disorder, or condition. Peptides that are administered for therapeutic treatment are administered in an amount effective to elicit a therapeutic effect. Peptides for treatment can be administered at a dosage wherein the resulting circulating or accumulating levels of the peptide mimic normal levels or the peptide levels of a person who is not afflicted by the disease or condition. A therapeutic peptide or peptides also can be administered at a dosage so that the circulating level of the peptide is similar or the same of an endogenous peptide that elicits a therapeutic effect. For example, as described herein, gastric bypass is the most effective treatment to effect sustained and significant weight loss. It is known in the art that gastric bypass, not only reduces the size of the stomach to thereby limit food intake; it also results in changes in peptide hormones involved in regulating food intake and effects (see, e.g., Beckman et al. (2010) J Am Diet Assoc. 110:571-584, doi: 10.1016 / j.jada.2009.12.023). In accord with the combinatorial therapeutic methods and regimens provided herein, peptides (or agonists), such as glucagon-like peptide-1 (GLP-1) (SEQ ID NO: 1), peptide tyrosine-tyrosine (PYY) (SEQ ID NO:6), and leptin (SEQ ID NO:3) that increase after gastric bypass can be exogenously administered to overweight or obese patients to mimic peptide levels and patterns of expression observed after gastric bypass to effect weight loss. Antagonists of peptides that decrease after bypass can be administered.

[0192] As used herein, a polypeptide is an amino acid chain that contains a plurality of peptides, and is generally 100 amino acids or longer. For purposes herein, polypeptides and peptides can be used interchangeably to refer to the therapeutics, such as those for weight loss.

[0193] As used herein, parenteral and parenterally refer to administration of an agent via any route other than oral administration. Parenteral includes the injection of a dosage form into the body by a sterile syringe or some other mechanical device, such as, for example an infusion pump. For example, parenteral administration includes injection (z.e., subcutaneous, intramuscular, and / or intravenous injection), infusion, implantation, intraperitoneal routes of administration, and any other mode of delivery other than ingestion to any site in or on the body of a subject.

[0194] As used herein, monotherapy refers to the use of a single therapeutic (z.e., drug or medication) to treat a particular disorder or disease. A monotherapy can include continued treatment, such as for months or years, with a single therapeutic.

[0195] As used herein, combination therapy refers to the administration of two or more different therapeutics or other treatment(s), such as radiation and surgery. Multiple therapeutic agents or treatments in the combination therapy can be provided and / or administered separately, sequentially, intermittently, simultaneously, or provided in a single composition. Generally, the two or more different therapeutics or other treatment(s) in a combination therapy are administered together or separately, or intermittently. For example, the treatments can be within 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 72 hours, 4 days, 5 days, 6 days, or 1 week of the other treatments in the combination. The timing and order of administration depends upon the disease, disorder, or condition, and is within the judgement of a physician.

[0196] As used herein, an orexigenic pathway is one that promotes appetite. Orexigenic pathways can be activated by increased AMP-activated protein kinase (AMPK) activity by ghrelin to promote appetite (orexia). The peptides orexin A (SEQ ID NO:36), neuropeptide Y (NPY) (SEQ ID NO: 35) and ghrelin (SEQ ID NO:23) stimulate appetite and act through orexigenic pathway(s).

[0197] As used herein, a receptor is a molecule that has an affinity for a particular ligand. For example, a receptor is a protein that specifically binds a signal molecule and then initiates a response. Receptors can be naturally-occurring or synthetic molecules. Receptors also can be referred to in the art as anti-ligands. In some examples, multiple receptor types are involved in a signaling pathway. In the context of pharmacology, receptors include macromolecules in the membrane or inside the cell that specifically (chemically) bind a ligand (drug).

[0198] As used herein, downregulate or downregulation refers to reducing or suppressing the body’s response to specific stimuli. Receptor downregulation is characterized by a decrease in total receptor number in the cell. Receptor downregulation can be caused by long-term exposure to agonists. Cells undergo receptor downregulation as a reversible process of adaptation, or desensitization, whereby a prolonged exposure to a stimulus decreases the cellular response to that level of exposure. In response to continuous drug exposure, receptor downregulation occurs and can decrease the drug efficacy due. Rotational and / or combinatorial drug therapy described herein decreases receptor downregulation due to the activation of multiple pathways as opposed to the one activated pathways in monotherapy. Rotational and / or combinatorial drug therapy described herein also can overcome the decreased therapeutic response due to receptor downregulation by rotating to a next combination with therapeutics that activate a pathway that is not downregulated.

[0199] As used herein, body mass index (BMI) is a value derived from the mass (weight) and height of a person. The BMI is defined as the body mass divided by the square of the body height, and is expressed in units of kg / m2, resulting from mass in kilograms and height in meters. BMI value is used to categorize a person as underweight (under 18.5 kg / m2), normal weight (18.5 to 24.9), overweight (25 to 29.9), or obese (30 or more) based on tissue mass (muscle, fat, and bone) and height. BMI under 20 and over 25 have been associated with higher all-causes mortality, with the risk increasing with distance from the 20-25 range.

[0200] As used herein, weight loss means a decrease in body mass of a person. Weight loss includes overall loss of fat, muscle and water compared to a pre-set time point. In examples herein, weight loss is assessed after treatment with a rotational combinatorial therapy compared to weight prior to treatment.

[0201] As used herein, off label use means the U.S. Food and Drug Administration (FDA) has approved the drug for a particular use and the approved drug is used for a different therapeutic use. The FDA has determined the benefits of using the drug for a particular use outweigh the potential risks, but has not assessed the risk or benefits of the drug for the off-label use.

[0202] As used herein, potency is the drug concentration required to produce an effect of a specified intensity. Potency generally is calculated as the concentration (or dose) required to produce 50% of the drug’s maximal effect (EO50). EC50 is used to express the potency in in vitro studies and also is the dose required for an individual to experience 50% of the maximum effect, and median effective dose or ED50 is used to measure a drug’s potency in a population (z.e., animal studies or human populations). ED50 is the dose that produces the desired effect in 50% of the population. As applied to clinical settings, potency can indicate the dose of the drug, whereas efficacy can indicate the magnitude of the response (regardless of the dose).

[0203] As used herein, an “adverse effect,” or “side effect,” or “adverse event,” or “adverse side effect,” refers to a harmful, deleterious and / or undesired effect associated with administering a therapeutic agent. For example, side effects associated with the administration of a monotherapy, such as continuous administration of a monotherapy over an extended period of time. Such adverse side effects include, for example, headaches, nausea, diarrhea, heartburn, gas, constipation, dry mouth, dizziness, increased blood pressure, increased heart rate, restlessness, drug dependence, abuse, and withdrawal symptoms. Other serious adverse effects include infections, such as tuberculosis, and other infections caused by viruses, fungi and bacteria, including upper respiratory infections, as well as dermatological and dermal toxicity, such as rash. Thus, “adverse effect” or “side effect” refers to a harmful, deleterious and / or undesired effect of administering a therapeutic agent. Side effects or adverse effects are graded on toxicity, and various toxicity scales exist, providing definitions for each grade. Examples of such scales are toxicity scales of the National Cancer Institute Common Toxicity Criteria version 2.0, and the World Health Organization or Common Terminology Criteria for Adverse Events (CTCAE) scale. Assigning grades of severity is within the skill of an experienced physician or other health care professional. The severity of symptoms can be quantified using the NCI Common Terminology Criteria for Adverse Events (CTCAE) grading system. The CTCAE is a descriptive terminology used for Adverse Event (AE) reporting. The grading (severity) scale is provided for each AE term. The CTCAE displays Grades 1 through 5, with clinical descriptions for severity for each adverse event based on the following general guideline: Grade 1 (Mild AE); Grade 2 (Moderate AE); Grade 3 (Severe AE); Grade 4 (Life-threatening or disabling AE); and Grade 5 (Death related to AE / fatal).

[0204] As used herein, serum level refers to the amount of a therapeutic in blood plasma. Serum level can be used to assess whether the amount of the therapeutic administered is safe and / or effective. Therapeutic drug monitoring of the concentration of mediation(s) in body fluids such as blood serum can be used during treatment and / or for diagnostic purposes. Assessing drug serum levels can be used, for example, to avoid drug toxicity, to determine if drug serum level is toxically high; to adjust dose, such as, for example, after reaching a steady state, to determine if the loading dose was adequate, and / or to predict a patient’s dosing requirements; and for monitoring patient compliance, diagnosing undertreatment (ie., when drug dose can be increased), and diagnosing ineffective treatment.

[0205] As used herein, comorbidity refers to the presence of one or more additional conditions co-occurring (that is, concomitant or concurrent) with a primary condition. Comorbidity indicates that the one or more conditions occur simultaneously with the primary condition or as a result of the primary condition. For example, hypertension can be a comorbidity of obesity, where a subject has hypertension as a result of the obese state. In some examples, complications of the primary condition and the comorbidity are the same or overlap.

[0206] As used herein, disease or disorder refers to a pathological condition in an organism resulting from cause or condition including, but not limited to, infections, acquired conditions, genetic conditions, and characterized by identifiable symptoms. Diseases and disorders of interest herein are those that can be treated with a rotation of pharmaceutical agents. Diseases and disorders also include those where treatment of the disease or disorder is impaired due to downregulation of receptors or drug targets, leading to a decreased efficacy of the therapeutics. Of particular interest herein are those disorders where treatment is not effected due to resistance to the expected efficacy of the therapeutic molecule or drug or peptide.

[0207] As used herein, “chronic” is used to characterize the duration of a disease, disorder, or condition. For example, a chronic condition is a condition that is prolonged and requires ongoing medical intervention or limit activities of daily living or both. A chronic condition generally is a condition that lasts more than 3 months, 6 months, 1 year or more, or that is terminal. A chronic condition can persist when the affected individual receives treatment or therapeutics, and during the amelioration of signs or symptoms of the condition. Chronic conditions include chronic diseases or disorders. Chronic conditions can be characterized by one or more of etiology, duration, onset, recurrence / pattem, prognosis, sequelae, diagnosis, severity and prevalence (see e.g., O’Halloran et al., Family Practice, (2004) 21(4): 381-386).

[0208] As used herein, “obesity” refers to a condition in which the subject has abnormal or excessive fat accumulation, such as a body mass index of greater than 30. Obesity can be a health risk. Obesity can be caused by a multitude of factors, including genetic and environmental factors. Patients with obesity can have other comorbidities, such as, for example, high blood pressure, type 2 diabetes, cardiovascular disease, high cholesterol, and others.

[0209] As used herein, “overweight” refers to a condition in which the subject has extra fat accumulation or weight that is higher than typical, with a body mass index that is, for example, greater than 25. Overweight also can be assessed by other methods known in the art, such as by assessing waist and / or hip circumference, subcutaneous fat thickness, percent of fat or muscle compared to total body composition, overall body weight, and similar assessments. Other metrics can be used to determine whether a subject is overweight, such as a waist to hip ratio of greater than, for example, 1.0. Overweight subjects are at increased risk various comorbidities, including high blood pressure, high cholesterol, COPD, type II diabetes, and others. Overweight can be caused by a multitude of factors, including genetic and environmental factors.

[0210] As used herein, a “pharmacokinetic property” refers to a property related to the action of a drug or agent, such as a therapeutic peptide, in the body and in particular the rate at which drugs are absorbed, distributed, metabolized, and eliminated by the body. Pharmacokinetics can be assessed by various parameters. These include, but are not limited to, clearance, volume of distribution and serum half-life. Pharmacokinetic properties of peptides can be assessed using methods well known in the art, such as, for example, administering the peptide to a human or animal model and assessing the amount of the peptide in the body (e.g., in the bloodstream) at various time points. The various parameters, such as clearance, volume of distribution and serum half-life, are assessed using calculations well known in the art and described herein.

[0211] As used herein, “improved pharmacokinetic properties” refers to a desirable change in a pharmacokinetic property of a peptide or combination of peptides or combination of therapeutics, such as peptides in a pharmaceutical composition for administration with the timing or regimen set forth herein, compared to, for example, a peptide or therapeutic administered continuously or once. The change can be an increase or a decrease.

[0212] As used herein, “synergistic effect” or “synergy” or grammatical versions thereof refers to a larger therapeutic effect of the combined treatment compared to the effect predicted from the sum of each therapeutic alone. For example, when the action of one drug is increased when administered in the presence of another drug.

[0213] As used herein, plasma half-life (t 1 / 2) refers the elimination half-life of a peptide(s) or therapeutic(s) or combinations thereof or the time at which the plasma concentration of the administered peptide(s) or therapeutic(s) or combinations thereof has reached one half of its initial or maximal concentration following administration. Reference to plasma half-life includes plasma half-life during the a-, P-, and / or y- phase. Plasma half-life can be assessed using methods well known in the art. For example, assays in which a peptide(s) or therapeutic(s) or combinations thereof is administered to subjects can be performed, and the plasma half-life of the peptide(s) or therapeutic(s) or combinations thereof assessed by measuring the amount of the peptide(s) or therapeutic(s) or combinations thereof in the plasma at various time points. The t’AB, for example, is calculated as -ln2 divided by the negative slope during the terminal phase of the log-linear plot of the plasma concentration-versus- time curve. In some examples, the plasma half-life can aid the skilled artisan in determining when a secondary peptide or therapeutic or combinations thereof can be administered to a subject, such as, for example, in a rotational therapy regimen described herein.

[0214] As used herein, clearance refers to the removal of an agent, such as a peptide, from the body of a subject following administration. Clearance can be assessed using methods well known in the art. For example, assessment of peptide levels in blood or serum or another fluid from a patient administered a peptide or combination of peptides or therapeutics can be performed, and the clearance of the peptides from the body assessed by measuring the amount of the peptide(s) in the plasma at various time points and calculating the clearance as Dose / AUC 0-inf. In some examples, clearance of a peptide results in a decrease in the circulating peptide levels in serum compared to circulating levels directly or recently after peptide administration. The clearance of administered peptide(s) or therapeutic(s) or combinations thereof can result in a decrease in circulating levels by at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, compared to circulating levels directly or recently after peptide administration.

[0215] As used herein, the term assess or assesses and grammatical variations thereof, is intended to include quantitative and qualitative determination in the sense of obtaining an absolute value for the activity of a pharmaceutical(s) or therapeutic(s) or phenotype(s), and of obtaining an index, ratio, percentage, visual or other value indicative of the level of the activity. Assessment can be direct or indirect. For example, assessing the effect of administration of a combination therapy provided in accord with a rotational regimen described herein on a subject for weight loss can be evaluated by a variety of metrics, including fat, weight, water, and muscle loss or gain. Assessing the effect of administration of a combination therapy provided in accord with a rotational regimen described herein also can be evaluated by measuring levels of the therapeutics or other molecules in the bloodstream. In some examples, a decrease in the amount of the therapeutic(s) such as, for example, a therapeutic peptide or combinations of peptides in the bloodstream or serum can indicate that another peptide(s) or therapeutic(s) or combinations thereof can be administered to the subject. For example, a secondary therapeutic(s) (z.e., peptide e.g., therapeutic peptide) or combinations thereof that is different from the initially administered therapeutic(s) ) (z.e., peptide(s) e.g., therapeutic peptide(s)) or combinations thereof can be administered when the concentration of the primary is decreased by at least or about or about at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% compared to the level or amount of the therapeutic(s) directly after administration, where the level of amount of the therapeutic(s) directly after administration is assessed at least or at least about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours or more after administration.

[0216] As used herein, the term “subject” refers to an animal, including a mammal, such as a human being. Subjects include human patients.

[0217] As used herein, a “patient” refers to a human subject. In some examples “patients” or “subjects” are humans who participated in a therapeutic regimen, such as a therapeutic regimen described in the examples herein. In some examples a “patient” is a human who has a chronic condition, disorder, or disease, such as overweight or obesity. Patients or subjects can be treated with the rotational combinatorial therapy herein.

[0218] As used herein, “animal” includes any animal, such as, but not limited to, primates including humans, gorillas, and monkeys; rodents, such as mice and rats; fowl, such as chickens; ruminants, such as goats, cows, deer, and sheep; pigs; and other animals. Non-human animals exclude humans as the contemplated animal.

[0219] As used herein, a “control” refers to a sample that is substantially identical to the test sample, except that it is not treated with a test parameter, or, if it is a plasma sample, it can be from a normal volunteer not affected with the condition of interest. A control also can be a subject, such as a subject that is not treated with rotational combinatorial therapy provided herein, or is treated with a monotherapy, or combination therapy that is not rotated, or a placebo. A control also can be an internal control.

[0220] As used herein, a “composition” refers to any mixture of two or more products or compounds, for example, but not limited to, peptides, therapeutic molecules, agents, modulators, and regulators. A composition can be, for example, a solution, a suspension, an emulsion, a liquid, a powder, a paste, aqueous or non-aqueous formulations, and any combination thereof.

[0221] As used herein, an “article of manufacture” is a product that is made and sold. As used throughout this application, the term is intended to encompass peptides or drugs, or therapeutics and combinations thereof contained in articles of packaging.

[0222] As used herein, “fluid” refers to any composition that can flow. Fluids thus encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams and other such compositions.

[0223] As used herein, a “kit” refers to a packaged combination, optionally including reagents and other products and / or components for practicing methods using the elements of the combination. For example, kits containing a combination of pharmaceuticals provided herein and another item for a purpose including, but not limited to, administration, diagnosis, and assessment of a biological activity or property are provided. Kits optionally include instructions for use.

[0224] As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a compound, comprising “a therapeutic” includes combinations with one or a plurality of therapeutics.

[0225] As used herein, ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence, “about 5 hours” means “about 5 hours” and also “5 hours.”

[0226] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally included therapeutic means that the therapeutic is included or is not included.

[0227] B. OVERVIEW

[0228] Provided are delivery vehicles that display peptides for weight loss and anabolic peptides. Provided herein are delivery vehicles, such as liposomes, exosomes, extracellular vesicles, lipid nanoparticles (LPNs), for displaying or containing peptides for treating obesity and / or diabetes, and optionally containing small molecule drugs in or on the vehicles.

[0229] Exemplary proglucagon derived peptides are provided, and anabolic peptides and other peptides and drugs that that promotes muscle growth and / or prevents or inhibits muscle wasting are described. The delivery vehicles display at least one of the proglucagon peptides and a muscle wasting / promoting peptide or a peptide that inhibits muscle loss, such as a myostatin pathway inhibitor, or a plurality thereof. Alternatively, or in combination, the delivery vehicles display one the peptides and combinations of at least three different delivery vehicles are administered together, such as in a co-formulation, or administered sequentially, or simultaneously. Generally, the delivery vehicles are formulated for administration by injection, such as intravenous injection or subcutaneous injection. The delivery vehicles also can be formulated for mucosal administration via contacting with oral mucosa, intestinal mucosa, and / or intranasal mucosa.

[0230] The delivery vehicles, include, but are not limited to, liposomes, such as those exemplified herein, or any suitable liposome, or a lipid nanoparticle (LNP), or an extracellular vesicle, including an animal or plant extracellular exosome, such as a milk-derived, or a microalgal extracellular vesicle (see, International PCT Publication No. WO2023 / 144127; International PCT application No. WO2023 / 076418;and International PCT application No. WO2023 / 076418). The peptides drugs can be displayed on or contained in or both using any suitable delivery vehicle, including extracellular vesicles, exosomes, LPNs, and liposomes. Additionally, small molecule drugs, such as phentermine, can be combined with the delivery vehicle, such as introducing it into extracellular vehicle or fabricating them together. Thus, delivery vehicles are provided that deliver combinations of drugs to treat obesity and reduce or eliminate the concomitant muscle loss that accompanies weight loss by enhancing muscle growth.

[0231] Suitable peptides and combinations thereof and regimens are described herein and in commonly owned International PCT application No. PCT / US23 / 77508 (now subsequently published International PCT publication No. WO2024 / 091863). Hence, provided are combinatorial treatment protocols and rotational combinatorial treatment protocols in which diseases, disorders, and conditions are treated by targeting a plurality of molecules and / or pathways involved in the disease, disorder, or condition, to reduce or avoid desensitization to a particular treatment, and also can reduce toxicity and adverse side effects. The diseases, disorders, and conditions include obesity, diabetes, and associated diseases, disorders, and conditions, such as obesity comorbidities.

[0232] The combination of treatments can be rotated so that a subject is treated with different combinations of treatments for limited periods of time. Also provided are methods for developing such protocols, including selecting a disease, disorder, or condition for treatment with a combinatorial protocol and / or a rotational combinatorial protocol. These protocols and methods are exemplified herein with respect to obesity, and several other diseases, disorders, and conditions that are difficult to treat and that become resistant to treatment over time. In accord with the disclosure herein, the peptides and other drugs are displayed on or provided in delivery vehicles, such as liposomes and exosomes, and formulated for administration to a subject, such as by injection, including subcutaneous injection, such as by autoinjector.

[0233] Obesity is exemplar of a disease, disorder, or condition, as shown herein that can be treated with a combinatorial, particularly a rotational combinatorial treatment protocol. Obesity is a medical condition in which excess body fat has accumulated. The excess body fat has adverse effects on health (see, e.g., “Obesity and overweight Fact sheet N°311,” published by the World Health Organization (WHO) in January 2015. Retrieved 2 February 2016). Obesity increases the likelihood of various diseases and physical and mental conditions. For example, these increases are manifested in metabolic syndrome (Haslam etal., (2005) Lancet 366 (9492): 1197- 209), and include cardiovascular diseases, high blood pressure, high blood cholesterol, high triglyceride levels, diabetes mellitus type 2, obstructive sleep apnea, certain types of cancer, osteoarthritis, and depression (Haslam et al., (2005) Lancet 366 (9492): 1197— 209). Obesity reduces life expectancy. (Jura etal., (2016) Age (Dordr) 38(1):23; Peeters etal., Ann lnternMed (2003) 138(l):24-32).

[0234] These health complications either are directly caused by obesity or indirectly related to obesity through mechanisms sharing a common cause such as a poor diet, a sedentary lifestyle, and / or genetics. The strength of the link between obesity and specific conditions varies; one of the strongest is the link with type 2 diabetes; approximately 64% and 77% of cases of diabetes in men and women, respectively, can be attributed to excess body fat (Maggio et al., Endocrinol Metab Clin North Am. (2003) 32(4):805-22).

[0235] The biochemical link between the development of obesity and health consequences fall into two broad categories: those attributable to the effects of increased fat mass, such as osteoarthritis, obstructive sleep apnea, and social stigmatization, and those due to the increased number of fat cells, such as diabetes, cancer, cardiovascular disease, non-alcoholic fatty liver disease (Stenkula etal., Am J Physiol Regul Integr Comp Physiol. (2018) 315(2):R284-R295). Increases in body fat alter the body's response to insulin, potentially leading to insulin resistance. Increased fat also creates a proinflammatory state and a prothrombotic state (Kawai et al., Am J Physiol Cell Physiol. (2021) 320(3):C375-C391; Moghbeli et al., Adv Clin Chem (2021) 101 : 135-168; Samad et al., Blood(2013) 122(20):3415-22; Bovolini et al., IntJ Sports Med. (2021) 42(3): 199-214).

[0236] Combinatorial protocols involve the administration of a plurality of treatments, each of which targets different pathways involved in the disease, disorder, or condition. The combinatorial protocols involve combination of at least three different treatments, such as at least two peptides that promote or result in weight loss and one that prevents or reduces muscle loss associated with weight loss. The combinatorial protocol(s) can be integrated into a rotational combinatorial protocol in which different combinations of treatment are rotated. Rotational protocols can be selected where monotherapy or combination of treatments become less effective or require higher doses to be effective. Provided herein are exemplary rotational combinatorial protocols and methods of developing such protocols.

[0237] Provided herein is platform in which combinations of weight loss and associated drugs are provided in or displayed on a delivery vehicle, such as an exosome or extracellular vesicle or LPN. Liposomes are exemplary of such vehicles. The peptide drugs can be displayed on the vehicle or incorporated into it; the small molecule drugs can be incorporated or associated with the surface so that they can be delivered together. The stoichiometry of the amounts of each drug can be adjusted to deliver the proper dosage, adjust, and provide relative amounts or ratios of the drugs for dosing the proper amount of each. The platform provides the ability to adjust various parameters and deliver combinations of drugs for treating obesity and concomitantly avoiding or reducing muscle loss that accompanies weight loss. The can be provided in or on separate vehicles, which can be mixed before administration to provide combinations.

[0238] In particular embodiments, the delivery vehicles, such as liposomes, provide more than a plurality of combinations (combinatorial) of the peptides (such as 3 weight loss peptides and 1 anabolic) providing an array of combinations to rotate and / or select the best for treating a particular subject. For example, each of the unique combinations can be rotated, such as every three months to prevent drug desensitization and reduce or avoid adverse side effects. The peptide stoichiometry can be adjusted, such as by modifying the surface of the liposome to reflect a dominant population of the more efficacious peptide or to increase the dose of one or more relative to others. The size of the delivery vehicle can be adjusted or selected to affect the amount of drug(s) in or on the liposome and to control binding kinetics of the surface peptides.

[0239] Liposome are among the delivery vehicles. Liposomes and other vehicles provide several advanced pharmacokinetic features, including: a. protection of the peptides from endogenous peptidases in serum, thus extending half life b. a delivery system within the liposome itself, of which we will describe below.

[0240] As detailed herein, combinational, combinatorial and rotational therapies provide advantages compared to monotherapy. They permits treatment via multiple pathways or targets involved in obesity or other disease, disorder, or condition.

[0241] The delivery vehicles also can for fabricated or formulated to deliver small molecule drugs including, but are not limited to: Phentermine, Topiramate, Metformin, Empagliflozin, Dapagliflozin, Bexagliflozin, Ertugliflozin, Linagliptin, Canagliflozin, NS-2330, Liothyronine, Diethylpropion, Zonisamide, Albuterol, Clenbuterol, Levothyroxine, Naltrexone, Orlistat, Testosterone Cypionate, and Testosterone Enanthate, in addition to the peptides. These are known drugs; dosages can be established when used in combination with the other drugs and peptides. The combinations, thus, include at least two or three known peptides for treatment of obesity and additionally one or more peptides (or other drugs) that reduce or eliminates muscle loss that accompanies weight loss. Generally, the drugs that reduce or eliminate muscle loss re muscle enhancing anabolic peptides, such as sermorelin, tesamorelin, IGF-1, or other growth hormone or peptide derived therefrom. Other drugs that inhibit the causes of muscle loss also are known, and, optionally, can be included in the combinations. These include myostatin pathway inhibitors, such as the antibody bimagrumab, which inhibit the activity of myostatin. Myostatin also is known as growth differentiation factor 8 or GDF-8; it is a member of the transforming growth factor-P (TGF-P) superfamily. Myostatin is a negative regulator of skeletal muscle growth, so its inhibition can contribute preservation of lean muscle mass. Inhibitors of myostatin, thus, are distinct from muscle enhancers promote muscle growth; they have a different target and mode of action. Myostatin inhibitors are not muscle enhancers. It has been reported that myostatin pathway inhibitors used in combination with a GLP-1 pathway agonist can enhance overall weight loss (see, e.g., US patent application publication US 20240368262).

[0242] Advantages of the delivery vehicles, such as liposomes, provided herein include, but are not limited to:

[0243] 1. Synergistic weight loss to the peptides (should, for example, exceed Tirzapeptide’s claimed efficacy of over 20% in 12 months).

[0244] 2. Numerous combinations can be made with these molecules, in addition to surface PDP’s.

[0245] 3. Resistance to drug sensitization.

[0246] 4. Safety and profiles and dosing are known.

[0247] 5. The delivery vehicles, such as the liposomes, can provide “dual peak” activity. They display peptides, which can have an immediate pharmacological effect, and then, once the vehicle, such as the liposome ruptures, there is a second time release of any weight loss push. There can be a further peak or release of any drug / peptide from the ruptured vehicle .

[0248] 6. Since these are well-known drugs, serum levels and other parameters can be calculated easily from known prescribing doses (for example, phentermine is prescribed at 37.5mg daily) and then packaged into each vehicle in an amount to mimic that to achieve therapeutic serum levels and match other parameters.

[0249] 7. The delivery vehicles can concurrently deliver the small molecule drugs to treat other diseases, such as diabetes (metformin), hypertension (ACEi / ARBS), dyslipidemia (Statins), and others for obesity co-morbidities.

[0250] Depending on the selected delivery vehicle, they can be administered by various routes, including subcutaneous injection, and oral, inhalation, and intramuscular. As described herein and known in the art, auto-injectors for selfadministration are routinely used for administration. Volumes up to 3 mL generally are administered. Higher volumes can be administered. For example, high speed autoinjectors can deliver higher doses, as can adding excipients, such as a hyaluronidase, to the compositions. The high speed autoinjectors inject fairly large volumes quickly, and the formulations also contain excipients, such as a soluble hyaluronidase, to facilitate administration of large volumes Typical GLP-l / GIP injectors, for example, deliver from 0.5ml to 3ml.

[0251] As described herein, small molecule drugs, such as phentermine can be incorporated into or onto the liposomes. Small molecules inside the liposome or other vehicle provide unique pharmacokinetic properties. For example, for liposomes exemplified herein, the peptides act immediately since they are on the outside of the liposome and actuate a chain of molecular events for weight loss synergistically (First phase). As the liposome dissolves, there are some small molecules inside the liposome that are then released (2nd Phase), and eventually any small molecules attached to the inner surface of the liposome will be available for binding to its receptor (third phase).

[0252] In exemplary embodiments, the vehicles are liposomes or exosomes that contain at least 2, generally at least three, peptide drugs, and additionally muscle enhancer, such as an anabolic hormone. Among the possible peptides and drugs, there are at least 12 different peptides, which are known to produce or facilitate weight loss, and at 2 or 3 anabolic peptides, exemplified herein. These include, for example: GLP- 1 (SEQ ID NO: 1), Leptin (SEQ ID NO:3), Oxyntomodulin (OXM; SEQ ID NO:4); PYY (SEQ ID NO: 6), Amylin (SEQ ID NO: 7); Tesamorelin (SEQ ID NO: 8), GIP, SEQ ID NO: 10; CCK (SEQ ID NO: 11); Glucagon (SEQ ID NO:27); Sermorelin (SEQ ID NO:5); and IGF1 (SEQ ID NO:44), and variants or biologically active portions of each as well known in the art and / or detailed herein. Other known peptides and small molecule weight loss drugs can be included.

[0253] As noted above, there are a variety of peptides and small molecule drugs that can be combined. There are more than 200 different combinations based on the lists herein. For example, among these are the following exemplary combinations:

[0254] Drugl : GLPl / GIPl / Oxyntomodulin + GH (Sermorelin or Tesamorelin or IGF1) SEQ ID Nos: 1, 10, 4 or 47 + SEQ ID Nos: 5, 8, 44

[0255] Drug2 : GLPl / GIPl / Amylin + GH (Sermorelin or Tesamorelin or IGF 1) SEQ ID Nos: 1, 10, 7 + SEQ ID Nos: 5, 8, 44

[0256] Drug3: GLP1 / GIP1 / Glucagon + GH (Sermorelin or Tesamorelin or IGF 1) SEQ ID Nos: 1, 10, 27 + SEQ ID Nos: 5, 8, 44

[0257] Drug4: GLP1 / GIP1 / CCK+ GH (Sermorelin or Tesamorelin or IGF1)

[0258] SEQ ID Nos: 1, 10, 11 + SEQ ID Nos: 5, 8, 44

[0259] Drug5: GLP1 / GIP1 / PYY + GH (Sermorelin or Tesamorelin or IGF 1) SEQ ID Nos: 1, 10, 6 or 37 + SEQ ID Nos: 5, 8, 44

[0260] Drug6: GLP1 / GIP1 / Leptin + GH (Sermorelin or Tesamorelin or IGF 1) SEQ ID Nos: 1, 10, 3 + SEQ ID Nos: 5, 8, 44.

[0261] For displaying on delivery vehicles, the polypeptides can be pegylated or modified by group, such azido for click chemistry. Delivery vehicles are prepared by known methods, including those exemplified herein, for linkage of the polypeptides. In some embodiments, Lysine (K) residues, for which pegylation reduces or eliminate activity of the peptide when displayed, are replaced with a conservative amino acid replacement, such as Arg, Glu, Gin, His, so that only a lysine or lysine residues that do not substantially alter activity are available for pegylation. For example, Peptides can be linked to pre-formed liposomes or linked to components of the liposomes during preparation of the liposomes. A goal is to preserve as much of the tertiary conformation of the native peptides need to retain activity, reduce steric hinderance between or among the up to 4 peptides on a single liposome, protect the N-terminus from serum proteases, and / or, for example, set up an azio-PEG24 linker for copper- catalyzed azide-alkyne cycloaddition (CuAAC) and bioconjugation to preformed liposomes.

[0262] The stoichiometry can be assessed, for example, in vitro via cAMP assays as described below. As an example, liposomes are fabricated as exemplified. The peptides can be acetylated at the N-terminus to protect them from in vivo degradation. The activity of the peptides displayed on the resulting delivery vehicles can be assessed by known assays, including commercially available kits. These kits are based on the use of antibodies that specifically recognize both intracellular cAMP and an exogenous labeled cAMP conjugate that acts as a competitor. This is followed by detection of the labeled cAMP conjugate using a variety of detection technologies, including fluorescence resonance energy transfer (FRET) or enzymatic reactions. Based on known properties of our quad-peptide liposome, each activating cAMP messenger pathway, we expect a significant increase in intracellular cAMP over baseline and single, double, or triple peptide liposome. Adipose-derived stem cells (ADSCs) cell line can be used with any of these kits for assaying in vivo cAMP. Several kits available on the market that can be used to measure intracellular cAMP levels, including, for example:

[0263] 1. HTRF cAMP kit from Cisbio,

[0264] 2. LANCE cAMP kit from PerkinElmer,

[0265] 3. HitHunter cAMP kit, 4 DiscoverX cAMP kit,

[0266] 5. Abeam and BioVision cAMP Direct Immunoassay Kit,

[0267] 6. GloSensor cAMP assay from Promega.

[0268] C. Delivery vehicle -components

[0269] The peptides are provided linked to or incorporated into the surface of delivery vehicle. The following sections describe that the vehicles contain three components or aspects: 1) the delivery vehicle; 2) linkers or linkages; 3) the peptides. Peptides listed herein can be incorporated in and / or on the delivery vehicles.

[0270] 1. Component -Proglucagon derived Peptides (PGDP) and muscle enhancement peptides

[0271] In obesity, several organs are involved in the orchestration of body weight homeostasis. These include the pancreas (a-cells of the islets of Langerhans), gut (intestinal enteroendocrine L-cells), and brain (caudal brainstem and hypothalamus). The proglucagon gene is expressed in these organs and produces several key hormones that regulate satiety; these hormones are referred to as proglucagon-derived peptide (PGDP). Bioactive members of the PGDP family include glucagon-like peptides -1 and -2 (GLP-1 and GLP-2), oxyntomodulin, glicentin and glicentin-related pancreatic peptide, which are produced via tissue-specific processing of proglucagon by the prohormone convertase (PC) enzymes, PC 1 / 3 and PC2 (Figure 1). PGDP peptides exert unique physiological effects that influence metabolism and energy regulation; several of these peptides have been exploited in the form of long-acting, enzymatically resistant analogues for treatment of various pathologies. These can be linked to delivery vehicles (or incorporated in the surface thereof) as described herein for use in combination with other such peptides and / or delivery vehicles. a. Proglucagon Derived Peptides and tissue specific secretion

[0272] Proglucagon is expressed in both alpha-cells of the pancreatic islets as well as neuroendocrine L-cells primarily located in the distal ileum and colon. However, the PGDP profile is not identical in the pancreas and gut, due to differential post- translational processing of proglucagon by tissue-specific enzymes termed prohormone convertases (PC). It is accepted that pancreatic alpha-cells mainly possess PC2, which cleaves proglucagon to generate glicentin-related pancreatic peptide (GRPP), glucagon, intervening peptide- 1 (IP-1) and major proglucagon fragment (MPGF). In contrast, in the L-cell, proglucagon is cleaved by PC 1 / 3 yielding glicentin, GRPP, oxyntomodulin (OXM), GLP-1, intervening peptide-2 (IP- 2) and GLP-2. Some degree of crossover exists.

[0273] The gut and brain are extra-pancreatic sources of glucagon, while local intraislet GLP-1 production has also been established in alpha cells, particularly in times of beta-cell stress. In the brain the solitary nucleus of the medulla oblongata which utilizes PC 1 / 3 in a similar fashion to the gut generates PGDP’s in the central nervous system (CNS) b. Unimolecular multi-agonists

[0274] Employing combinations of single gut hormones or analogues provided a sound basis for the application of multi-agonism in T2DM. With the combination of liraglutide plus an acylated GIP analogue, synergy was demonstrated leading to improved glucose-lowering and insulinotropic actions in obese-diabetic mice compared to either of the individual incretin analogues alone. Based on this, we conceive that unimolecular multi-agonists represent the next step in future in the therapeutic application of PGDPs, with increasingly complex and experimental molecules being developed. This is evidenced by the observation that secretion and action of a number of gut hormones, including the PGDPs GLP-1, GLP-2, OXM and glicentin, together with PYY, GIP, cholecystokinin (CCK), neurotensin (NT) and secretin, are positively modulated in concert following Roux-en-Y gastric bypass (RYGBP or RYGB). These are major determinants in the improvements of appetite, body weight, glucose tolerance and insulin sensitivity demonstrated post-surgery. In view of the costs, limited availability, risks associated with surgical procedures, methods and regimens and products provided herein are designed to emulate the post- surgical, hormonal mechanisms of RYGB. The combination therapies provided herein can evoke an array of positive actions within various organs, thereby surpassing advantages observed with individual peptides. c. Enhancing fat loss efficacy via proglucagon derived peptides (PGDP) and co-secretory molecules released after a RYGBP

[0275] Adipose tissue plays a central role in various disease states such as metabolic syndrome, diabetes, coronary' artery disease etc. Focusing on fat loss ignores skeletal muscle, another relevant tissue that affects general health, skeletal muscle (SM) is the body’s largest non-fat component and can contribute up to 40% of the adult human body weight and be responsible for 30% of energy expenditure. It is also an established independent predictor of cardiometabolic diseases and mortality. Previous studies have reported that low levels of muscle mass are associated with an increased risk of insulin resistance, and CVD. Low muscle-to-fat ratios were associated with an increased risk of metabolic syndrome, diabetes, and cardiovascul r mortality.

[0276] Based on peptide changes after a RYGBP, and the concept of enhancing muscle-to-fat ratio, we propose a system that can increase fat loss and enhance muscle mass using liposomes or exosomes with at least one or more peptides for fat loss, and 1 or more peptides for muscle enhancement. We also consider overcoming the pharmacological phenomena of tolerance and desensitization to chronic exposure to the same drug by adjusting the combination of the fat loss (FL) and (ME) peptides and rotating them over intervals of 3 months. This multi-agonist approach uniquely combined and rotated would maintain weight loss over longer periods as compared to monotherapy or combined therapy without rotation of peptides. As detailed in the disclosure herein, a goal is to provide regimens that mimic secretory and pulsatile response of these endogenous peptides that occur following RYGBP and other bariatric weight loss surgeries. Provided are delivery vehicles for effecting and providing the combination therapies. Combinations of peptides are described throughout the disclosure herein. It is to be understood that the mixtures of peptide therapeutics can be linked to or incorporated into a delivery vehicle, such as a liposome or extracellular vehicle, such as an exosome, or a synthetic lipid-based nanoparticle. The delivery vehicles can be prepared to deliver a single peptide or a plurality thereof, mixed to provide complementary activities to mimic hormonal and other changes that occur following bariatric surgery. The general approach is to administer one or two peptides for fat / weight loss and one or more to prevent or inhibit muscle loss associated with fat loss or weight loss, or a peptide that promotes muscle growth. For example, two or three proglucagon peptides can be linked to a liposome, and a peptide that inhibits or prevents muscle wasting or that enhances muscle production can be prepared. Alternatively, few than all of the peptides can be linked to a liposome, and mixtures of liposomes delivering the combination of peptides can be co-formulated or administered at the same time or serially. Included among the peptides for linkage to or conjugation to or incorporation into a delivery vehicle, such as a liposome, are the following:

[0277] Peptides for fat loss include (FL=fat loss):

[0278] PeptideFLl=GLP-l

[0279] PeptideFL2=Adiponectin

[0280] PeptideFL3=Leptin

[0281] PeptideFL4= Oxyntomodulin

[0282] PeptideFL5=PYY

[0283] PeptideFL6= Amylin

[0284] PeptideFL7=Pancreatic peptide

[0285] PeptideFL8=Enterostatin / GIP (Gastroinhibitory Polypeptide)

[0286] PeptideFL9= Glicentin PeptideFLIO = Glucagon

[0287] PeptideFLl 1=GRPP

[0288] PeptideFLl 2=HGH 176-191

[0289] Peptide FL13= CCK

[0290] PeptideFLl 4= Neurotensin

[0291] PeptideFLl 5= Secretin

[0292] PeptideFLl 6= IP1

[0293] PeptideFLl 7= MPGF (major proglucagon fragment)

[0294] Peptides for muscle enhancement ME:

[0295] PeptideMEl=Sermorelin

[0296] PeptideME2=Tesamorelin

[0297] PeptideME2=IGFL

[0298] 2. Delivery vehicles

[0299] Delivery vehicles include any lipid nanoparticles on which peptides can be displayed on the surface and that are biologically compatible so that they can be administered to a human subject. Delivery vehicles include, but are not limited to, lipid nanoparticles, extracellular vesicles, liposomes, and other such delivery vehicles known to those of skill in the art. Exemplary of lipid-based delivery vehicles are exosomes, such as extracellular delivery vehicles, and liposomes. The peptides can be incorporated into the surface of the vehicles, such as by recombinant expression in a surface protein, or by chemical linkage to a moiety on the surface. The surface can be modified for linking peptides by methods known to those of skill in the art, including those described herein.

[0300] Liposomes are exemplary delivery vehicles

[0301] Liposomes are versatile drug delivery vehicles. They have unique properties, which include, for example, site-targeting, sustained or controlled release, protection of drugs from degradation and clearance, superior therapeutic effects, and lower toxic side effects. Liposomes include large multilamellar vesicles (LMV), and small unilamellar vesicles (SUV). Lamellas are phospholipid bilayers, in which the fatty acids are inside the bilayer, and polar head groups are outside in contact with water. LMVs can have diameters in micrometer range, while SUVs can be classified as nanoparticles, because diameter is typically under 100 nm. SUVs can traffic in vivo in the blood and go anywhere blood goes.

[0302] Liposome preparation include any of the following techniques, assuming the smallest possible particle to effectively carry at least 3 or 4 total (2 o3 fat loss (FL) peptides, 1 muscle enhancing (ME) peptide peptide) peptides without causing steric hinderance for each of their respective targets. Film hydration methods, double emulsification method, solvent injection techniques, sonification, and in situ preparation of liposomes are well-established methods for preparing the liposome.

[0303] To ensure stability and for performance of the liposomes, size reduction techniques as briefly described are employed. This includes, for example, (ultra)sonication either by bath or probe, French press Barenholtz, extrusion, homogenization, or combination methods, such as freeze-thaw extrusion, freeze-thaw sonication, and a high-pressure homogenization-extrusion technique [Pupo], Among these techniques, extrusion, and high-pressure homogenization (HPH) are among the most frequently employed in pharmaceutical manufacturing.

[0304] Liposomes can be prepared using phospholipids, such as phosphatidyl choline (PC), ethanol amine (PE), and serine (PS), or phosphatidic acid (PA). Phospholipids include those of natural origin, such as egg yolk, and also cholesterol, such as synthetic cholesterol, Cholesterol, which makes the bilayer tighter and less permeable, can be added. The molar percentage of cholesterol should be less than 60%, 50%, 40% or less. To prepare liposomes, the phospholipids, and other components first are dissolved into a volatile solvent, such as chloroform, methanol, t-butanol, cyclohexane, or a mixture thereof, so that components are evenly distributed. The Concentration of the phospholipids and other component can be low, such as less than 30 mg / ml, less than or equal to 20 mg / ml, less than 15 mg / ml or lower. Solvent then is evaporated, for example, by a rotary evaporator. For in vivo applications the solvent should be completely removed by keeping the lipid mixture under high vacuum for several hours. Dried lipid mixture is hydrated, such as with physiological salt solution (0.9 % NaCl), or other suitable buffer. Hydration can take several hours. At this point phospholipids are in thick multilamellar film or cake. Mechanical mixing leads to the formation of the LMVs. More powerful methods such as sonification, or high pressure microfluidic spraying break LMVs into SUVs. If hydration solution contains some drug molecules they are partially incorporated inside liposomes. For purposes herein, the biologically active peptides are conjugated to the outer surface of the liposomes.

[0305] 3. Conjugation / Binding of peptides on the liposomes

[0306] Method for linking peptides to liposomes are well known (see, e.g., Frisch et a / .(1996) Synthesis of short polyoxyethelene-based heterobifunctional cross-linking reagents. Application to the coupling of peptides to liposomes, Bioconjugate Chem. 7: 180). Any of several methods can be used to attach peptides and proteins to the liposomes. First liposomes that are prepared that some lipids that have a reactive group that can be used for the coupling reaction. Exemplary groups include, but are not limited to, amino, thiol, maleimide, bromo- or iodoacetyl, pyridyl di thio, carboxylic, hydrazide, p-nitrophenyl carbonate, azide, and alkyne. Amino groups form amide bond with an activated carboxylic ester, such as N-hydroxy succinimide (NHS; see. e.g., Redford etal., (1991) Cholesterylsuccinyl-N-hydroxysuccinimide as a cross linking agent for the attachment of protein to liposomes, Biochem Pharmacol. 77:307)). Thiols bind with maleimide, bromo- or iodo acetyl, pyridyldithio groups. Amines form an amide bond with carboxylate in the presence of water soluble carbodiimide (EDS), and N-hydroxy succinimide (NHS).

[0307] Most amino couplings involve the lysine amino group but coupling also can occur at the terminal amino group. If one of these amino groups is at the active binding site, conjugation, such as PEG conjugation leads to reduction of the activity. Hence, for purposes herein, pegylation is effect whereby lysines in the active are not pegylated. A method for preparing peptides in which selected lysines are pegylated is described below. This is effected by PEGylating the lysine residue prior to synthesis and employing the pegylated residue during peptide synthesis. This method can be used in general for preparing pegylated peptides and polypeptides.

[0308] Other methods include modification of the peptides to replace lysine with a conservative residue and then PEGylating the modified polypeptide, selecting amounts of PEG moiety so that not all lysines residues are pegylated. For peptides in which the lysines are not accessible or not in an active site or such that pegylation does not affect secondary and tertiary structure, the peptides are pegylated by standard methods.

[0309] Hydrazides bind with carbonyl groups. P-Nitrocarbonate reacts with amines forming an amide bond. Azide and alkyne groups bind with each other very fast in the presence of a copper ion catalyst (click chemistry). Site specific attachment of PEG can be achieved using a transglutaminase enzyme to couple succinimide activated PEG to glutamine.

[0310] 4. Spacers

[0311] Because of the diversity of the size of peptides including a linker is a strategy for loading a drug, such as a polypeptides on the liposome. Spacers can be included between the liposome, and peptide. Spacers allow peptides to orientate better for the binding with the receptor. Exemplary of spacers are water soluble polymer chains. For example, polyethylene glycol (PEG) is a commonly used spacer. Oligonucleotides also can be used as spacers. Oligonucleotides allow specific binding of selected peptides so that the amount of peptides can be better controlled. To employ oligonucleotides as spacers, first, selected oligonucleotides are bound with liposomes. Typically, oligonucleotides are bound in the same ratio as they are in the reaction mixture. The actual ratio of oligonucleotides can be measured by using a set of complementary oligonucleotides that are conjugated with fluorescent labels. If the ratio of oligonucleotides on the liposomes is equal to the desired ratio, the liposomes can be used as such. If correction is need, some additional oligonucleotides can be used. If there is a deficiency of some specific oligonucleotide, one or more of these additional oligonucleotides can be used to supplement this deficiency Peptides are conjugated with complementary oligonucleotides and mixed with the liposomes in a hybridization buffer, whereby that peptides are bound in a desired ratio.

[0312] Somewhat different methods of attaching peptides on the liposomes employ biotin-streptavidin binding. One streptavidin has four biotin binding sites. One, two, or even three can be used to bind streptavidin with a liposome, and at least one is still available for the binding of peptides. PEG spacers can again be used. On one end is attached biotin, and the other end can be NHS active ester that easily forms an amide bond with PE, when pH is right (slightly basic). The liposomes can be coated with a monolayer of streptavidin. Peptides similarly can be functionalized with biotin-PEG- NHS. The final product is obtained by mixing these peptide derivatives with streptavidin liposomes. This approach has certain merits. Both components are stable, and can be purified, for instance, by size exclusion chromatography. Because a small number of unbound peptides would not be harmful to the final product, further purification is not required. Oligonucleotide spacers for attaching peptides also are compatible with streptavidin liposomes.

[0313] 5. Linkage of peptides to the Delivery vehicles a. PEGylation of Peptides for linkage to lipid particles, such as liposomes

[0314] Polyethylene glycol (PEG) has been widely used in biomaterials, biotechnology and medicine primarily because PEG is a biocompatible, nontoxic, water-soluble polymer that is typically nonimmunogenic (Zhao and Harris, ACS Symposium Series 680: 458-72, 1997). In the area of drug delivery, PEG derivatives are used in covalent attachment (z.e., "PEGylation") to proteins to reduce immunogenicity, proteolysis and kidney clearance and to enhance solubility (Zalipsky, Adv. Drug Del. Rev. 16: 157-82, 1995). Similarly, PEG has been attached to low molecular weight, relatively hydrophobic drugs to enhance solubility, reduce toxicity and alter biodistribution. Typically, PEGylated drugs are injected as solutions.

[0315] Numerous reagents for PEGylation have been described in the art. Such reagents include, but are not limited to, N-hydroxysuccinimidyl (NHS) activated PEG, succinimidyl mPEG, mPEG2-N-hydroxysuccinimide, mPEG succinimidyl alpha-methylbutanoate, mPEG succinimidyl propionate, mPEG succinimidyl butanoate, mPEG carboxymethyl 3 -hydroxybutanoic acid succinimidyl ester, homobifunctional PEG-succinimidyl propionate, homobifunctional PEG propionaldehyde, homobifunctional PEG butyraldehyde, PEG maleimide, PEG hydrazide, p-nitrophenyl-carbonate PEG, mPEG-benzotri azole carbonate, propionaldehyde PEG, mPEG butryaldehyde, branched mPEG? butyraldehyde, mPEG acetyl, mPEG piperidone, mPEG methylketone, mPEG “linkerless” maleimide, mPEG vinyl sulfone, mPEG thiol, mPEG orthopyridylthioester, mPEG orthopyridyl disulfide, Fmoc-PEG-NHS, Boc-PEG-NHS, vinylsulfone PEG-NHS, acrylate PEG- NHS, fluorescein PEG-NHS, and biotin PEG-NHS (see e.g., Monfardini el al., Bioconjugate Chem. 6:62-69, 1995; Veronese et aL, J. Bioactive Compatible Polymers 12: 197-207, 1997; U.S. 5,672,662; U.S. 5,932,462; U.S. 6,495,659; U.S. 6,737,505; U.S. 4,002,531; U.S. 4,179,337; U.S. 5,122,614; U.S. 5,324, 844; U.S. 5,446,090; U.S. 5,612,460; U.S. 5,643,575; U.S. 5,766,581; U.S. 5,795, 569; U.S. 5,808,096; U.S. 5,900,461; U.S. 5,919,455; U.S. 5,985,263; U.S. 5,990, 237; U.S. 6,113,906; U.S. 6,214,966; U.S. 6,258,351; U.S. 6,340,742; U.S. 6,413,507; U.S. 6,420,339; U.S. 6,437,025; U.S. 6,448,369; U.S. 6,461,802; U.S. 6,828,401; U.S. 6,858,736; U.S. 2001 / 0021763; U.S. 2001 / 0044526; U.S. 2001 / 0046481; U.S. 2002 / 0052430; U.S. 2002 / 0072573; U.S. 2002 / 0156047; U.S. 2003 / 0114647; U.S. 2003 / 0143596; U.S. 2003 / 0158333; U.S. 2003 / 0220447; U.S. 2004 / 0013637; US 2004 / 0235734; W00500360; U.S. 2005 / 0114037; U.S. 2005 / 0171328; U.S. 2005 / 0209416; EP 1064951; EP 0822199; WO 01076640; WO 0002017; WO 0249673; WO 9428024; and WO 0187925).

[0316] In one example, the polyethylene glycol has a molecular weight ranging from about 3 kD to about 50 kD, and typically from about 5 kD to about 30 kD. Covalent attachment of the PEG to the drug (known as "PEGylation") can be accomplished by known chemical synthesis techniques. For example, the PEGylation of protein can be accomplished by reacting NHS-activated PEG with the protein under suitable reaction conditions.

[0317] While numerous reactions have been described for PEGylation, those that are most generally applicable confer directionality, use mild reaction conditions, and do not necessitate extensive downstream processing to remove toxic catalysts or biproducts. For instance, monomethoxy PEG (mPEG) has only one reactive terminal hydroxyl, and thus its use limits some of the heterogeneity of the resulting PEG- protein product mixture. Activation of the hydroxyl group at the end of the polymer opposite to the terminal methoxy group is generally necessary to accomplish efficient protein PEGylation, with the aim being to make the derivatized PEG more susceptible to nucleophilic attack. The attacking nucleophile is usually the epsilon-amino group of a lysyl residue, but other amines also can react (e.g., the N-terminal alpha-amine or the ring amines of histidine) if local conditions are favorable. A more directed attachment is possible in proteins containing a single lysine or cysteine. The latter residue can be targeted by PEG-maleimide for thiol-specific modification. Alternatively, PEG hydrazide can be reacted with a periodate peptide and reduced in the presence of NaCNBEE. PEGylated CMP sugars can be reacted with a peptide in the presence of appropriate glycosyl-transferases. One such technique is the “PEGylation” technique where a number of polymeric molecules are coupled to the peptide. When using this technique, the immune system has difficulties in recognizing the epitopes on the peptide's surface responsible for the formation of antibodies, thereby reducing the immune response. For peptides introduced directly into the circulatory system of the human body to give a particular physiological effect (i.e., pharmaceuticals) the typical potential immune response is an IgG and / or IgM response, while peptides which are inhaled through the respiratory system (i.e., industrial peptide) potentially can cause an IgE response (i.e., allergic response). The polymeric molecule(s) can shield epitope) on the surface of the peptide responsible for the immune response leading to antibody formation. The heavier the conjugate is, the more reduced immune response is obtained.

[0318] PEG moieties are conjugated, via covalent attachment, to the peptides. Techniques for PEGylation include, but are not limited to, specialized linkers and coupling chemistries (see e.g., Roberts et al., Adv. Drug Deliv. Rev. 54:459-476, 2002), attachment of multiple PEG moieties to a single conjugation site (such as via use of branched PEGs; see e.g., Guiotto et al., Bioorg. Med. Chem. Lett. 12:177-180, 2002), site-specific PEGylation and / or mono-PEGylation (see e.g., Chapman et al., Nature Biotech. 17:780-783, 1999), and site-directed enzymatic PEGylation (see e.g., Sato, Adv. Drug Deliv. Rev., 54:487-504, 2002). Methods and techniques described in the art can produce proteins having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 PEG or PEG derivatives attached to a single protein molecule (see e.g., U.S. Pub. No. 2006 / 0104968).

[0319] As an exemplary illustration of the PEGylation, PEG aldehydes, succinimides and carbonates have each been applied to conjugate PEG moieties, Succinimidyl PEGs (as above) comprising either linear or branched PEGs can be conjugated peptides. PEGylated peptides have been generated using NHS chemistries, as well as carbonates, and aldehydes, using each of the following reagents: mPEG2-NHS-40K branched, mPEG-NHS-lOK branched, mPEG-NHS-20K branched, mPEG2-NHS- 60K branched; mPEG-SBA-5K, mPEG-SBA-20K, mPEG-SBA-30K; mPEG-SMB- 20K, mPEG-SMB-30K; mPEG-butyraldehyde; mPEG-SPA-20K, mPEG-SPA-30K; and PEG-NHS-5K-biotin. PEGylated peptides have been prepared using PEG reagents available from Dowpharma, a division of Dow Chemical Corporation; including peptides PEGylated with Dowpharma's p-nitrophenyl-carbonate PEG (30 kDa) and with propionaldehyde PEG (30 kDa). Schemes for effecting PEGylation are well known to those of skill in the art. b. Synthesis of Pegylated peptides for conjugation to liposomes

[0320] A drawback with many peptide drugs is a short half-life, because they are degraded by proteases that are in the blood. To increase half-life, peptides are chemically coupled to polyethylene glycol (PEG). For purposes herein, the polypeptides can be Pegylated to provide a group for conjugation to a liposome. One end of the PEG moiety is linked to the peptide, and the other end includes a group for conjugation to the liposome. One or more different peptides can be conjugated with one liposome. Typically, PEG is conjugated with an amino group. Amino group can be terminal or in a lysine residue. If the amino group occurs in the active site of the peptide or in a site such that conjugation alters activity, conjugation can reduce or inhibit peptide activity. It is important to select the site for PEGylation so that activity of the peptide is not altered. Detailed herein is a method to achieve this, such that polypeptide is synthesized with PEGylated lysine(s) at the site(s) desired for PEGylation, and any other lysines have conventional protective groups.

[0321] In the method herein, the peptide is synthesized in PEGylated form. The lysines are PEGylated, and employed during synthesis of the polypeptide, such as by solid phase synthesis so that a PEG-lysine is incorporated in the peptide chain at a selected lysine residue. Figure 5A schematically depicts a short segment of a polypeptide. Amino acids k, 1, and m are lysines, and that can be separated from each other by several amino acids. In accord with normal peptide synthesis schemes, the terminal carboxylic group is protected by a methyl group that is removed by basic hydrolysis before the next step. The methyl ester of next amino acid is added together with a condensing agent that often is dicyclohexyl carbodiimide (DCCI). The polypeptide often is synthesized on a solid phase surface onto which the growing peptide chain is chemically attached. This allows the washing of the unused reagents and soluble reaction products off. Figure 5B shows a product, in which lysine, residue 1, is PEGylated, and the PEG has an azide group at the end. Azide can be used to couple the peptide to a liposome that has acetylene group on the surface (Click chemistry). If all lysines in a polypeptide occur in the active site or other site for which pegylation affects activity or binding of the peptide, PEG-lysines can be added either in amino or carboxylic end of the polypeptide. This kind of addition can be done to make the binding of the polypeptide with liposome stronger.

[0322] When lysine is added into a polypeptide chain, the epsilon amino group must be protected. Protective groups include allyloxycarbonyl and trityl groups. These are orthogonal to other protective groups. For purposes herein in which selected lysines are to be Pegylated for conjugation to a liposome, the epsilon-amino group of lysine is reacted with carboxyl terminated PEG. The bonding is effected via an amide bond that renders the amino group totally inert. As a result, a peptide that contains a PEG moiety in a desired site is produced. The other end of PEG can have a functional group that can be used for the conjugation with a liposome. For example, the functional group can be acetylene that can be reacted with azide that is in the liposome (click chemistry). Depending upon the number of PEG moi eties to be included, other lysines can have conventional protective groups. Thus, no separate PEGylation step is needed; the peptide is synthesized in PEGylated form. Figure 5B depicts a product, in which lysine 1 is PEGylated, and the PEG has an azide group at the end. Azide can be used to couple the peptide to a liposome that has acetylene group on the surface (Click chemistry). Figure 5C shows a PEGylated lysine. c. Liposome preparation for linking the peptides

[0323] Figures 2A and 2B depicts a liposome 201 (only the outer atomic layer is schematically shown). The liposome contains 1 - 5 % phosphatidyl ethanolamine that has two fatty acids containing at least 16 carbon atoms, generally 18 - 24 carbon atoms each. A PEG spacer 202 having 10 - 200 ethylene glycol moi eties can bound to each amino group.

[0324] These liposomes have one biotin moiety chemically bound per at least 20, 50, 100, or 120, generally about 100 phospholipids. A calculated amount of streptavidin 203 is added. Streptavidin can bind four biotins very strongly; only one or two of these binding sites should be used to bind streptavidin with a liposome, so that two or three binding sites can be used to bind the peptides 205 - 207 (Fig. 2B).

[0325] The PEG can contain an N-hydroxy succinimide active ester on one end (Fig. 3), and biotin moiety on the other end. Reagents are chosen so that they are water soluble, somewhat stable in water, and react at room temperature.

[0326] Peptides have also amino groups, and PEG spacers can be connected to these amino groups, such as one PEG spacer 204 having a biotin moiety on the other end. Additional PEGs 208 can be added subsequently to provide stealth property against the body’s immune system that tends to remove foreign particles.

[0327] Streptavidin coated liposomes provide certain advantages. For example, once liposomes and PEG-peptides are prepared, any and all combinations can be fabricated by mixing the components. The skilled person, such as the physician, can personalize the treatment for the needs and particulars of the treated subject. Personalized regimens of combinations of the therapeutics can be designed and implemented.

[0328] Figure 4 depicts another exemplary coupling chemistry in which X is Br or I.

[0329] Carbonyl activates the nearby halogen so that it is easily substituted by a strong nucleophile such as a negative sulfur atom. In this case the PEG has a terminal thiol group, and the other end has biotin. The liposome has 1 - 5 % bromo-, or iodoacetyl cardiolipin.

[0330] Cardiolipin (For example, see structure below) is an exemplary phospholipid for anchoring the peptides to liposomes and other lipid-based delivery vehicles. Although cardiolipin has been used in liposomes for drug delivery, it generally is not used for anchoring purposes. Cardiolipin liposomes tend to go to the heart, because the heart has more cardiolipin than other organs. To avoid this, a small amount of cardiolipin that is chemically modified is used. Cardiolipin has one free hydroxyl group that will be is esterified with bromo-, or iodoacetyl moiety for the binding of peptides via PEG.

[0331] Thiol-PEG is added under slightly basic conditions to form a thioether bond. Streptavidin can be bound as described above. If the PEG has a carboxyl group instead of biotin on the other end, EDC can be used to couple peptides directly with PEG. Cardiolipin has four fatty acids instead of two like all the other phospholipids. This provides stronger anchoring of the peptides to the liposome.

[0332] Exemplary of a delivery vehicle, produced as described above and in the Examples, are liposomes displaying a plurality of different peptides including at least two proglucagon peptides and an anabolic peptide. For example, provided are liposomes that display:

[0333] PeptideFLl=GLP-l;

[0334] PeptideFL4= Oxyntomodulin;

[0335] PeptideFL8=Enterostatin / GIP (Gastroinhibitory Peptide); and PeptideMEl=Sermorelin

[0336] D. FORMULATIONS AND ROUTES AND MODES OF DELIVERY

[0337] The delivery vehicles with linked peptides can be formulated for any suitable route of delivery, including, but not limited to, injection, inhalation, mucosal, and other routes. The route includes the indication treated or tissue targeted and the particular delivery vehicle. Many delivery vehicles cannot be orally administered; some such as certain exosomes can be orally administered. In general, the contemplated route of administration is injection. Pens and syringes containing formulations of the delivery vehicles, as a single dose or multiple doses, are provided. As described herein, the delivery vehicles, such as liposomes, can display a plurality of peptides, such as two or three fat loss peptides and one muscle enhancing peptide, or they can display fewer and mixtures of the liposomes with different peptides can be administered. They can be provided with single peptides linked thereto, and the physician can select combinations of peptides according to a rotational or combinatorial or both regimen, such as any described herein.

[0338] E. COMBINATORIAL THERAPY AND ROTATIONAL COMBINATORIAL THERAPY

[0339] It is understood that for purposes herein, the peptides are provided on delivery vehicles, such as liposomes. For weight loss and also for diabetes, the delivery vehicles can display at least two peptides for weight loss, and at least one for muscle enhancement, or combinations of delivery vehicles each displaying one peptide are administered together. The regimens as described below administer that therapeutics displayed on or linked to or embedded in delivery vehicles.

[0340] Individuals treated with therapeutic agents for conditions, disorders, or diseases, such as chronic conditions, disorders, and diseases, are at risk of developing a tolerance or resistance to the therapeutic effects of the medications. In the context of biological systems and pharmacology, the activation of cell surface receptors can trigger regulatory processes that restrict signaling duration and / or strength. Downregulation is a process by which a particular cellular component, such as a protein (e.g., receptor), decreases in abundance or decreases activity responsive to an external stimulus (z.e., long term administration of an agonist). For example, the expression of a specific receptor or decrease in total receptor number in the cell can decrease (z.e., by enhanced receptor degradation or decreased receptor synthesis or ligand-induced internalization of receptors (endocytic downregulation)). In response to increased or long-term exposure to an agonist, such as a hormone or neurotransmitter or other signaling molecule, the decrease in receptor activation or number can reduce cell sensitivity to the agonist. In the presence of sustained ligand stimuli, the receptor system enters a refractory state thereby preventing the cell from over-responding to the ligand.

[0341] This negative feedback mechanism is important in drug treatment, where chronic exposure to a drug and / or therapeutic molecule can become less effective upon long-term or chronic exposure. An example of this negative feedback mechanism are hormone receptors feedback systems. When receptors have been chronically exposed to an excessive amount of a ligand or exposed to a ligand for a prolonged period of time, either from endogenous mediators or from exogenous drugs, this can result in ligand-induced desensitization or internalization of that receptor. Downregulation, therefore, effectively minimizes overstimulation of a pathway to prevent disruption of an organism’s internal processes as a result of the increased or chronic activation.

[0342] Downregulation is a cellular mechanism characteristic of long-term administration of therapeutic agents. Understanding downregulation and cellular responses to chronic drug exposure is fundamental to developing an effective drug regimen for treating a complex disease state. Targeted therapeutics that are rotated to decrease receptor or cellular downregulation, or other cellular responses that decrease efficacy or activity of a therapeutic agent can be used to treat diseases, disorders, and conditions, such as obesity, and have wide reaching implications across medical specialties for drug development for chronic conditions in which there are a plurality of targets for therapeutic intervention for in which desensitization to treatments occurs.

[0343] Combinatorial treatments involve the use of two or more, generally at least three, different treatments that target different pathways for treating a disease, disorder, or condition. The combinatorial treatments, particularly if they start to lose effectiveness for treatment of chronic diseases, disorders, and conditions, can be rotated, which avoids the problems, particularly desensitization, which occurs with long-term therapies for diseases, disorders, and conditions, such as obesity, Parkinson’s disease, and other chronic diseases, disorders, and conditions that require treatment for many months, and generally for life.

[0344] Rotational combinatorial therapy involves protocols / regimens for treatment of a disease, disorder, and / or condition, in which two or more different therapies (or therapeutics) in combination are administered such that the combinations of the -n- therapies are rotated for predetermined or multiple rounds of treatment. Rotational combinatorial therapy or pharmacology is a protocol for treating a disease, disorder, and / or condition, in which a number of therapeutics or treatments, at least two, different therapeutics / treatments for a disease are administered according to a schedule in which different therapeutics and combinations thereof are administered. Each therapeutic / treatment can be different, having a different target or mode of action. There also can be overlap of the therapeutics in the combinations, especially if there is a time period (gap) between therapies (z.e., giving the pathway time to ‘recover’ or regain the ability to become activated). As set forth below, a combination in the rotational protocol can contain more than one therapeutic, wherein each targets the same pathway to effect a therapeutic effect, which can, in some instances, achieve a synergistic effect such that lower doses, compared to monotherapy, of each therapeutic can be administered. In these examples the timing for rotation can be decreased. Because the medications are used in a therapeutic combination, in some examples a medication or medications in the combination can be used at lower dosages than treatment with a monotherapy, to reduce the risk or severity of adverse side effects.

[0345] In accord with rotational combinatorial protocols, each combination of treatments is administered for a predetermined time, generally at least a week, and then replaced by a different combination, which is administered for a predetermined period of time. Each combination can include a drug in common, and / or one of the rotations can include only a single therapeutic, but each combination is different from the other combinations. A rotational protocol can include at least two different combinations that are rotated, and generally includes at least three combinations of therapeutics and treatments that are rotated. The protocol can be repeated a plurality of times.

[0346] The predetermined rounds for treatment and timing for treatment can be modified if the subject demonstrates signs or signals that a pathway is downregulated, or the subject becomes less responsive to the therapeutic effects of the therapy or the subject exhibits an increase in adverse side effects. For example, the combinations can be switched prior to the predetermined time. In other examples, if the subject exhibits a decrease in the side effects of the medication, this can indicate that downregulation of the pathway has occurred or is occurring, and the dosage can be increased or another therapeutic that activates a different pathway can be substituted. For example, if administration of a medication results in a side effect of jitteriness, and after a set period (z.e., 2 months) the jitteriness diminishes, the diminution can indicate that the therapeutic pathway is downregulated and medication that acts through a different pathway can be substituted.

[0347] The regimen can involve a first round in which one, two, or more of the therapeutics / treatments are administered for a period of time, followed by second round in which a different therapeutic / treatment or different combination of therapeutics is / are administered for another period of time, and repeating round one and round two, or administering a third round of pharmaceuticals for a predetermined period of time.

[0348] Depending on the number of therapeutics that are part of the treatment, as well as the disease, disorder, or condition, and particulars of the treated subject, different combinations of therapeutics are administered for each time period, providing different combinations of therapeutics rotated for predetermined periods of time. In some examples, the time periods for treatment are not predetermined; the different combinations are administered for each period of time, and the combinations of therapeutics are rotated when the patient is showing reduced therapeutic benefit(s), or the patient is showing increased or intolerable side effects from the therapeutics. The therapy involves at least two different therapeutics, administered sequentially, and then together, or administered together, and typically involves at least three different therapeutics, usually drugs, administered separately or in combinations. Generally, all combinations and orders of administration can be included in the protocol. Hence, the name rotational combinatorial pharmacology.

[0349] A regimen of a rotational combinatorial therapy described herein comprises two or more different therapeutic combinations (also referred to as clusters), where each combination includes two or more medications or treatments. Each combination is rotated for a predetermined time of treatment. Rotation can revert to combination one or any other combination in the regimen.

[0350] In some examples, a rotational combinatorial treatment or therapy includes the administration of a first therapeutic agent or combination of therapeutic agents and, after a set time point and in fixed intervals, administration of a different therapeutic agent or combination (cluster) of therapeutic agents, where the second combination (cluster) of agent(s) target a receptor / and or pathway distinct from at least one of the initial therapeutic agent(s), and, optionally, any subsequent combinations are administered after a set time point and in fixed intervals. In exemplary regimens described herein, the first therapeutic agent(s) combination is / are administered and then, such as three months later, a second therapeutic agent(s) combination is / are administered. In examples, there is a third agent(s) combination administered after the second agent(s), at a set time point and after the fixed interval. In some examples, there are multiple rounds of administration of agent(s) at fixed time points after the fixed interval(s). Treatment can proceed for years, and can, if necessary be administered for life. Any of the therapeutic agents used in the rotational therapy herein can be a pharmaceutical or non-pharmaceutical therapy.

[0351] In some examples, the combinations can be administered in accord with any of the regimens set forth in the any of following tables:

[0352] Table 1. Exemplary Regimen for a Rotational Combinatorial Therapy Rotated at three-month intervals

[0353] Table 2. Exemplary Regimen for a Rotational Combinatorial Therapy Rotated at three-month intervals

[0354] Table 3. Exemplary Regimen for a Rotational Combinatorial Therapy Rotated at One Month Intervals

[0355] Tables 4 and 5, below, set forth rotational combinatorial therapy regimens that are rotated at 4-month and 6-month intervals, respectively. In some examples herein, downregulation generally occurs after at least 3 months and, thus, rotations of combinations generally occur at or about 3 months or later. In some examples, such as where downregulation of particular pathways is slow or takes longer than 3 months, regimens such as those set forth in Tables 4 and 5 are employed.

[0356] Table 4. Exemplary Regimen for a Rotational Combinatorial Therapy Rotated at Four Month Intervals

[0357] Table 5. Exemplary Regimen for a Rotational Combinatorial Therapy Rotated at Six Month Intervals

[0358] Regimens for rotating and administering therapeutic combinations can be developed in accord with the description provided herein, and by modifying regimens described herein and those set forth in the following tables:

[0359] Table 6.1: Exemplary Regimen for a Rotational Combinatorial Therapy Rotated at three-month intervals

[0360] Table 6.2: Exemplary Regimen for a Rotational Combinatorial Therapy Rotated at three-month intervals

[0361] Table 6.3: Exemplary Regimen for a Rotational Combinatorial Therapy Rotated at three-month intervals

[0362] Exemplary of therapeutics in Tables 6 are the following:

[0363] Therapeutic #1=GLP-1

[0364] Therapeutic #2=Adiponectin

[0365] Therapeutic #3=Leptin Therapeutic #4= Oxyntomodulin

[0366] Therapeutic #5=Sermorelin

[0367] Therapeutic #6= Peptide YY (PYY)

[0368] Therapeutic #7=Amylin

[0369] Therapeutic #8=tesamorelin Therapeutic #9=Pancreatic polypeptide (PP)

[0370] Therapeutic #10=Enterostatin / GIP

[0371] Therapeutic # 11 = Cholecystokinin (CCK)

[0372] Therapeutic # 12= Vasoactive Intestinal Therapeutic

[0373] Therapeutic # 13=Glicentin Table 6.4

[0374] The tables above and description herein provide exemplary of regimens for administration of therapeutic combinations, and rotational combinations for weight loss as exemplary of a condition that can be treated by combinatorial and rotational combinatorial methods and regimens provided herein. The combinations, therapeutics in the combinations, dosages, timing and other aspects of the regimens can be modified as required to achieve a therapeutic benefit and / or decrease adverse side effects. The skilled person can select from among medications that have the recited function and combine them to achieve additive and synergistic effects.

[0375] The combinatorial treatment or therapy and rotational combinatorial treatment or therapy provides an improved treatment of diseases and disorders compared to treatment with monotherapies, such as disease states that result from derangements of multiple molecular or cellular pathways or a that show a decrease or plateau in the efficacy of the monotherapy; the combinatorial therapy and / or rotational combinatorial therapy described herein modulates the multiple pathways derangements to ameliorate negative effects of the disease or disorder. Modulation of multiple pathways provides improved therapeutic benefits compared to monotherapy or even dual therapy, which target fewer molecular or cellular targets or pathways. Rotating multiple therapeutics (z.e., pharmaceuticals) is an effective method of preventing molecular and cellular adaptation that occurs after continued (z.e., longterm) treatment with monotherapy. Rotating multiple therapeutics or therapies also can prevent cellular adaptation that occurs after therapy with combination therapy (z.e., treatment with more than one therapeutic) that is not rotated. For example, rotating multiple therapeutics or therapies also can prevent cellular adaptation that occurs after therapy with a monotherapy or combination therapy that is administered for a prolonged period (e.g., longer than 3 months).

[0376] In some examples the therapeutics in the combination therapy and / or rotational combinatorial therapy are synergistic; one therapeutic (z.e., drug) enhances the clinical activity of another therapeutic (z.e., drug) when used in the combination, and the cumulative effects of the therapeutics exceed the expected clinical benefit of the sum of the multiple drugs in the combination. In other examples, increased efficacy of the combination works through independent drug action rather than a synergistic effect of the therapeutics in combination; the therapeutic benefit is attributable to a single therapeutic in the combination and the benefits over monotherapy are due to increasing the odds that the combination includes a drug that is effective for a particular patient.

[0377] Provided herein are combination therapies for use in a rotational regimen for use in the treatment of chronic conditions, such as conditions lasting more than three months, and conditions where patients develop a tolerance to treatment. The rotational combinatorial therapy is for treating chronic conditions with a plurality of known treatments or known pathways associated with disease progression or pathology. A rotational combinatorial therapy regimen can be developed by identifying known treatments / therapies the disease(s), disorder(s), and / or condition(s) for treatment; identifying the pathways, mechanism of actions or targets for treatment; selecting treatment(s) and / or therapies that include treatment(s) and / or therapies that activate different pathways or that have different mechanisms of action; creating combinations that include at least two therapeutics known to activate different molecular and / or cellular pathways and design a regimen for administration of the combinations; and creating a regimen to administer multiple rounds of treatment, with different combinations. In some embodiments, combinations used in one or more rounds of rotation can include a single therapeutic. Generally, all combinations that are rotated include two or more therapeutics or treatments.

[0378] 1. Identification of diseases, disorders, or conditions for treatment

[0379] The combinatorial rotational therapy provided herein can be used in methods of therapy for treating diseases, disorders and / or conditions where the affected patients are at risk of developing a tolerance or resistance to the therapeutic effects of the therapeutic(s) (z.e., medications). The diseases, disorders, and / or conditions have more than one target / pathway for therapeutic intervention. Generally, the disease, disorder, or condition is chronic or requires extended or life-long treatment. Other diseases, disorders and / or conditions are those where the afflicted individuals are at risk of developing a tolerance or resistance to the therapeutic effects of the medications and include chronic conditions, where the disorder or its effects are persistent or long-lasting, or is a disease that develops over time. Patients with a variety of diseases or conditions (e.g., chronic conditions) can benefit from a rotational combinatorial therapy described herein. These conditions include, for example those that can be treated by: 1) a combination of more than one medication and 2) a rotational aspect, such as conditions where the prescribed therapeutics have the potential to decrease efficacy over time. Thus, the methods herein, address the problems of inadequate effectiveness of a monotherapy, and the desensitization. Combinations of a plurality of drugs can improve effectiveness; combining this with rotation of the combinations of the drugs (or of the drugs), leads to sustained weight loss.

[0380] A disease, disorder, or condition for treatment with a rotational combinatorial therapy described herein is one where 1) there are a plurality of different therapeutics / treatments known or that can be developed for treating the disease, disorder, or condition, such as where there are a plurality of therapeutic intervention pathways or targets; and / or 2) the disease, disorder, or condition generally is chronic; and / or 3) the disease, disorder, or condition is one for which treatment often fails because tolerance to the therapeutics / treatments develops and / or the therapeutics / treatments become ineffective over time. a. Disease, Disorder, or Condition with a Plurality of Known Treatments

[0381] The rotational combinatorial therapy provided herein can be used for treatment of any condition where there is an effective treatment or a plurality of different known therapeutics and / or treatments. For example, the rotational combinatorial therapy provided herein can be used for treatment of any condition where there is a known pharmaceutical or plurality of pharmaceuticals that ameliorate(s) symptoms of the condition. The rotational combinatorial therapy provided herein also can be used for treatment of any condition with a previously characterized mechanism of action or pathway, such that a therapeutic can be developed for treating the disease or disorder or condition For example, a disease, disorder, or condition in which there are a plurality of different known molecular targets for treatment or multiple known molecular or cellular pathways that are involved in disease progression can be treated with the rotational combinatorial therapy described herein. For example, the rotational combinatorial therapy described herein can be used to target multiple pathways that are associated with a disease or disorder, but where a pharmacotherapy has not yet been developed.

[0382] In disorders involving a host of cellular receptor(s), activation of the receptor(s) can lead to activation of an array of cellular pathways. Administration of a monotherapy, which activates or effects activation of a single pathway can have limited efficacy for improving the disorder. An approach for reducing the symptoms or effects of the disorder can require administering a combination of compounds targeting the different pathways and processes.

[0383] The combination therapies described herein can include treatments and / or therapies that target multiple pathways, and diseases or conditions for treatment where targeting a single pathway or mechanism will not confer significant or complete amelioration of the disease state or symptoms. For example, the rotational combinatorial therapy provided herein can be used to diseases where administration of a single therapeutic in animal models or in human patients has a limited therapeutic effect.

[0384] In some examples, the disease, disorder, and / or condition is a multifactorial process where the therapeutic benefit from a combination of different therapeutics exceeds the therapeutic benefit of a monotherapy. For example, the disease, disorder, or condition is influenced by multiple genes (polygenic) and resultant genetic pathways, and generally in combination with lifestyle and environmental factors, such as exercise, diet, or pollutant exposures. The combination of genetic and environmental factors act together in concert to trigger the development and progression of the multifactorial disease.

[0385] The concept of rotational and / or combinatorial pharmacology provides a method for treating complex disease processes, such as obesity, which involve multiple pathways, in which monotherapy and dual therapy have shown limited success. The combinatorial pharmacology for weight loss as exemplified herein is exemplary of a condition that can be treated with a rotational therapy. Other conditions that involve multiple molecular and / or cellular pathways also can be treated by rotational combinatorial pharmacology, such as conditions where monotherapies are inadequate. Combination therapy also can overcome limitations of monotherapies, where multiple medications, by virtue of the number of medications in the combination, increases the chance the subject is responsive to a particular therapeutic. b. Chronic Conditions

[0386] Chronic conditions require ongoing therapeutic intervention and can negatively impact or limit the daily activities or the quality of life of the affected individual. Chronic conditions include conditions in which known therapeutics are ineffective or that do not significantly or completely ameliorate the symptoms or characteristics of the condition. Chronic conditions also include conditions where patients treated with monotherapies are not responsive to treatment or who have not improved on the monotherapy or who continue to have adverse symptoms of the condition despite the therapeutic intervention. Chronic conditions also include conditions where patients treated with combination therapies (z.e., continuous and / or prolonged administration of more than one therapeutic) are not responsive to treatment or who have not improved on the combination therapies or who continue to have adverse symptoms of the condition despite the therapeutic intervention.

[0387] Chronic conditions are conditions that persist over time. In some examples, chronic conditions last more than 3 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years or more. The rotational combinatorial therapy provided herein is for treating chronic conditions. In some examples, the rotational combinatorial therapy described herein is for treating a chronic condition, disease, or disorder in which the course of the condition, disease or disorder lasts more than about three months. A chronic condition also can be characterized by the amount of time the therapeutics for treating the condition are administered. For example, chronic conditions can be conditions in which therapeutic(s) for treating the condition is / are administered for an extended period of time (ie., about 3 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years or more).

[0388] Patients with chronic conditions can be administered a medication or combination of medications for months or years, during which time they can develop a decreased response to the medication(s), such as decreased response due to signal downregulation or desensitization to the medication, and can suffer from side effects from prolonged use of the medication(s). Subjects with chronic conditions can benefit from treatment with combinations of therapeutics that are rotated, which can increase the likelihood of a prolonged therapeutic response compared to treatment with a monotherapy or combination of therapeutics that are not rotated.

[0389] Provided herein are combination therapies for use in a rotational regimen for use in the treatment of chronic conditions. In some examples of chronic conditions, patients are administered therapeutic treatment(s) for extended periods of time and can develop a tolerance to treatments, such that the therapeutics for treating the conditions become or are less effective. For example, provided herein are combination therapies for use in a rotational regimen for use in the treatment of chronic conditions, for use in the treatment of overweight or obesity, and / or for weight loss. The combination therapies for use in a rotational regimen also are for use for treating or ameliorating the symptoms of comorbidities of overweight and / or obesity. Exemplary of such comorbidities include diabetes (e.g., diabetes mellitus type 2), cardiovascular disease (i.e., cardiovascular disease leading to heart attack or stroke), high blood pressure, high blood cholesterol, high triglyceride levels, persisting neurodegenerative disorders, metabolic syndrome, obstructive sleep apnea, depression, non-alcoholic fatty liver disease, and cancer, such as but not limited to, pancreatic cancer, breast cancer, prostate cancer, gastric cancer, colon cancer, ovarian cancer, head and neck cancer and others. The combination therapy and rotational regime can be modified to decrease the amount or severity of adverse side effects associated with administration of the therapeutic(s). For example, the dosage of the therapeutic(s) can be lowered, or the therapeutics or therapeutic combinations can be rotated with greater frequency.

[0390] Rotating combinations of therapeutics can expose patients to each of the particular therapeutics for a shorter amount of time or to a lower dosage, which can each decrease the chance of developing side effects or the severity of side effects from the particular medication. The rotational combinatorial therapy provided herein can be used to minimize side effects during treatment or therapy for diseases or disorders, compared to treatment with a monotherapy or dual therapy or combination therapy with two or more therapeutics. In some examples, the combination therapy described herein can be used and / or administered at a lower dosage than the dosage of the therapeutic used for monotherapy. For example, the combination therapy is used at a lower dosage than the monotherapy which results in fewer or less severe adverse side effects during treatment.

[0391] Patients with a chronic condition who previously were treated with a monotherapy, where the monotherapy did not decrease disease signs or symptoms or where the monotherapy did not inhibit or stop disease progression, can be administered a combination therapy or rotational combinatorial therapy provided herein to improve the therapeutic response or to decrease the adverse symptoms associated with the disease state. In other examples, patients with a chronic condition who were previously treated with a combination therapy, where the combination therapy did not decrease disease signs or symptoms or where the combination therapy did not inhibit or stop disease progression, can be administered a rotational combinatorial therapy provided herein.

[0392] A rotational combinatorial therapy also can be used for treating subjects with chronic conditions who have previously been treated with a monotherapy or a combination therapy and who are responsive to the treatment, but where the treatment did not completely ameliorate symptoms of the condition or did not cure the disease. In these examples, a patient can benefit from a combination therapy targeting multiple molecular and / or cellular to target pathways to which the subject is more responsive. A combination of therapeutics administered in a rotation or regimen described herein can show improved amelioration of symptoms of a chronic condition compared to a monotherapy or combination of therapeutics that are not rotated by decreasing downregulation of therapeutic pathways and activating molecular pathways that were not previously activated (z.e., not previously subject to downregulation). c. Conditions where Patients Develop a Tolerance to Treatments

[0393] Patients with chronic conditions can develop a decreased response (tolerance) to the medication(s) prescribed for treating the condition or for ameliorating symptoms of the condition. Such decreased therapeutic response over time can be related or due to signal downregulation or desensitization to the medication(s). Patients that develop a tolerance to a monotherapy or combination therapy can benefit from a rotational combinatorial therapy to overcome the desensitization, such as desensitization due to receptor downregulation.

[0394] A rotational combinatorial therapy, thus, can be used for treating conditions where patients develop a tolerance to treatments. For example, subjects with chronic conditions who have previously been treated with a monotherapy or a combination therapy who are responsive to the treatment (z.e., showed an amelioration of symptoms) for a limited time period but where the treatment was less effective or the subject failed to respond to the treatment after a time period can be treated with a combinatorial rotational therapy. For example, a subject who initially showed amelioration of symptoms of the disease or condition, but where the symptoms increased after prolonged treatment, such as, for example, a subject who showed amelioration of symptoms but where symptoms increased after at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months or more of treatment.

[0395] 2. Development of a Combinatorial Rotational Therapy Regimen

[0396] Rotational combinatorial therapies provided herein are for treating a disease, disorder, and / or conditions where there are a plurality of different therapeutics / treatments known or that can be developed for treating the disease, disorder, or condition, and the disease, disorder, or condition generally is chronic and / or is one for which treatment fails because tolerance to the therapeutics / treatments develops or the therapeutics / treatments become ineffective over time so that ultimately treatment of the disease, disorder, or condition fails or becomes increasingly ineffective.

[0397] In examples, development of a rotational combinatorial therapy can include a protocol including:

[0398] 1) identify known treatments / therapies for each disease state or pathways associated with each disease state;

[0399] 2) identify the pathways, mechanism of actions or targets;

[0400] 3) select treatments / therapies that activate different pathways, have different mechanism of actions or targets, and / or that are compatible with a rotational therapy; 4) create combinations that include at least 2 therapeutics known to activate different molecular and / or cellular pathways and design a regimen for administration of the combinations; and

[0401] 5) create a regimen to administer multiple rounds of treatment, with combinations of therapeutics.

[0402] Each of these aspects is discussed in turn as follows.

[0403] 1) Identify known treatments / therapies for each disease state or pathways associated with each disease state

[0404] A protocol for developing a rotational combination therapy can include identification of treatments or therapies that are known and / or have previously been characterized for treating a disease, disorder, or condition. For example, a protocol for developing a rotational combination therapy described herein can include identifying treatments or therapeutics that have previously demonstrated activity or efficacy for treating the disease, disorder, or condition or for treating or ameliorating secondary effects or symptoms associated with the disease, disorder, or condition. For example, a therapy or therapeutic is a drug that is approved by the Food and Drug Administration (FDA) for human use in the United States. In other examples, the therapeutic can show efficacy for treating the disease, disorder, or condition or treating or ameliorating secondary effects or symptoms associated with the disease, disorder, or condition in animal models.

[0405] In other examples, the therapeutic (z.e., pharmaceutical) for inclusion in the rotational combination therapy described herein can be one that was previously identified as a therapeutic that alters (z.e., activates or inhibits) a pathway associated with the disease, disorder, or condition or symptoms thereof in vitro, or in in vivo and / or ex vivo model systems. The in vitro results or results from model systems can indicate that targeting a particular pathway or pathways or use of a particular therapeutic (i.e., drug / pharmaceutical) or treatment will be effective for treating subjects (i.e., human patients) in which the pathway or pathways is / are implicated or use of a particular therapeutic is predicted to be effective. Studies or demonstrations that a therapy or therapies is / are effective for treating or ameliorating a disease, disorder, or condition can be used to select therapeutics for inclusion in the rotational combination therapy described herein. In other examples, the therapeutic(s) (z.e., pharmaceutical) for inclusion in the rotational combination therapy described herein can be identified in screens for effective therapeutics for treating a disease or disorder. For example, in vitro screens in model systems that replicate disease characteristics can be conducted for new substances showing activity and additionally for assessing effectiveness for treating a disorder. In some examples, the therapeutic(s) (z.e., pharmaceutical) for inclusion in the rotational combination therapy described herein are identified in vitro in screens conducted in cell culture, in a Boyden chamber, in three-dimensional cultures, in microfluidic systems, using 3D bioprinting, or in other systems that can be used to identify therapeutics that can be for treating a disease, disorder, or condition with the rotational combinatorial therapy described herein.

[0406] In examples of a rotational combinatorial therapy for treating obesity or overweight or for effecting weight loss, known weight loss medications, such as weight loss medications described herein can be included in the combination. In some examples, a previously identified weight loss medication can be included, including one or more of mitochondrial uncouplers, amphetamines, thyroid hormones, drug cocktails, neuromodulators, lipase inhibitors, cannabinoid receptor antagonists, gastrointestinal-derived peptides chemically optimized for pharmaceutical use, and others. In some examples, one or more FDA approved medications can be included in the combination therapy and / or incorporated into a liposome, generally into a liposome that displays one or more of the peptides. For example, one or more of orlistat, phentermine-topiramate, naltrexone-bupropion, liraglutide, tirzepatide (is a GIP analog that acts on GIP and GLP-1 receptors; sold under the trademark Mounjaro®) dual agonist, and semaglutide can be included in the combinations described herein for treating obesity or overweight or for effecting weight loss. In other examples, FDA approved medications that are for use for a disease or disorder where the subjects exhibit weight loss as a result of the therapeutic regimen, but for which weight loss is not the primary objective, can be included in the combination therapy provided herein, for effecting weight loss.

[0407] 2) Identify the pathways, mechanism of actions or targets

[0408] A protocol for developing a rotational combinatorial therapy can also include identification of pathways, such as, for example, molecular or cellular pathways, associated with the disease, disorder, or condition. The molecular and / or cellular pathways can be used to determine the therapeutic(s) for inclusion in the combinatorial therapy.

[0409] Treatments or therapeutics for inclusion in the rotational combinatorial therapy described herein can be selected from compounds that target a pathway associated with the disease, disorder, or condition; or symptoms, or secondary or side effects of symptoms associated with the disease, disorder, or condition. Treatments or therapeutics can activate a pathway associated with the disease, disorder, or condition; or symptoms, or secondary or side effects of symptoms associated with the disease, disorder, or condition, such that activation of the pathway ameliorates symptoms of the disease, disorder, or condition or secondary side effects. Alternately, treatments or therapeutics can inhibit a pathway associated with the disease, disorder, or condition; or symptoms, or secondary or side effects of symptoms associated with the disease, disorder, or condition, such that inhibition of the pathway (z.e., inhibition of an overactive pathway or an off-target pathway) ameliorates symptoms of the disease, disorder, or condition or secondary side effects.

[0410] When identifying a pathway for targeting in the rotational combinatorial therapy described herein, biomarkers can signal an abnormal process or a condition of the disease. Biomarkers can be used to identify targets for treatment and potential responses to therapeutics and therapeutic combinations. For example, biomarkers can be used to predict a patient’s response to individual therapeutics (z.e., drugs) in a combination. For example, higher receptor expression in a patient can correspond to a better response to a corresponding therapy, and lower expression of receptor activators can indicate a higher or lower chance of responsiveness to a particular therapeutic(s) (z.e., drugs). Thus, biomarker identification and characterization, and use of validated biomarkers (z.e., biomarkers of a particular disease state or status) can help predict patient response and therapy -related side effects and inform selection of therapeutics for inclusion in the combination and / or rotational combinatorial therapy herein.

[0411] The skilled artisan can evaluate the literature, for example articles published in the scientific literature, such as, for example, in the US National Library of Medicine and the National Center for Biotechnology Information, which assembles biomedical literature from MEDLINE, life science journals, and online books. The scientific literature can identify and assess molecular and cellular pathways for treating diseases, disorders, and / or conditions described herein. Molecular targets for treating a disease, disorder, or condition can be identified and / or evaluated in the scientific literature and used to determine molecular targets for therapeutics for treating the conditions identified and described herein.

[0412] In other examples, a pathway associated with the disease, disorder, or condition or symptoms thereof for treatment with a combination or rotational combinatorial therapy described herein can be identified using in vitro, in vivo, and / or ex vivo model systems. The in vitro results or results from model systems can indicate that targeting a particular pathway or pathways or use of a particular therapeutic (i.e., drug / pharmaceutical) or treatment will be effective for treating subjects (i.e., human patients) in which the pathway or pathways is implicated or use of a particular therapeutic is predicted to be effective. Studies or demonstrations that a molecular and / or cellular pathway plays a role in pathogenesis or progression of a disease or condition can be used to determine therapeutics for including in the combination and rotational combination therapy described herein. The therapeutic (i.e., pharmaceutical) for inclusion in the rotational combination therapy described herein can be one that alters (i.e., activate or inhibit) a pathway associated with the disease, disorder, or condition or symptoms thereof in vitro, or in in vivo and / or ex vivo model systems.

[0413] In examples of a rotational combinatorial therapy for treating obesity or overweight or for effecting weight loss, molecular and / or cellular pathways that are altered in gastric bypass patients can be targeted in the therapy by combining therapeutics that target each pathway. For example, molecular and / or cellular pathways that are altered following gastric bypass or other surgical weight loss procedures can be used to formulate a pharmacotherapy for inclusion in the combination and / or rotational combination therapy described herein. For example, neuro-hormonal gut peptides that are altered following bariatric surgery; or exogenous (i.e. rationally designed) peptides that mimic gut peptides or that are gut peptide receptor agonists; or therapeutics that alter expression and / or activation and / or activity of neuro-hormonal gut peptides that are altered following bariatric surgery, such as, for example, therapeutics that increase expression and / or activation and / or activity of neuro-hormonal gut peptides that are increased following bariatric surgery, can be included in the combination therapy described herein. The combinatorial regimens and the rotational combinatorial regimens can mimic the effects of bariatric surgery, particularly gastric bypass surgery, such as the Roux-en-Y surgery. Gastric bypass surgery has effects that include decreased absorption of food, including intestinal absorption, on glucose homeostasis, and results in hormonal changes that alter appetite / satiety and energy consumption and other physiological processes. Rerouting food through the gastrointestinal tract leads to changes in gut hormone secretion. Changes in gut hormone levels after RYGB, include increased anorectic hormones, such as GLP-1 and PYY, which induce satiety, and decreased levels of orexigens, such as ghrelin, an appetite-stimulating hormone. The rotational combinatorial methods herein allow a variety of combinations of drugs / treatment to reduce desensitization, and adverse effects, while providing the advantages of gastric bypass surgery by combining administration of various drugs that reduce appetite, and hormones that alter appetite. Combinations of such drugs and treatments are detailed herein and are exemplified in the working examples and throughout the description.

[0414] Provided are combinatorial methods and combinatorial rotational methods in which combinations of medications, such as peptide hormones or agonists thereof and / or antagonists of peptide hormones that are reduced following bariatric surgery, are administered. Particular combinations are described herein and exemplified in the working examples.

[0415] In other examples of a rotational combinatorial therapy for treating obesity or overweight or for effecting weight loss, molecular and / or cellular pathways associated with satiation, metabolism, hunger and weight gain or loss can be identified and used to formulate targets and or therapeutics for the combination therapy.

[0416] 3) Select treatments / therapies that activate different pathways, have different mechanism of actions or targets, and / or that are compatible with a rotational therapy

[0417] Combination therapies containing at least two, and generally at least three, combinations of a plurality of therapeutics and rotated to prevent desensitization of activated pathways exhibit efficacy that is greater than a monotherapy or combination therapy that is not rotated. The combinations contain therapeutics that activate or are known to activate different molecular or cellular pathways. For example, the combinations contain therapeutics that activate or are known to activate a total of at least two, three, four, five or more different molecular or cellular pathways. By virtue of rotating the therapeutic combinations at set intervals, where the combinations contain therapeutics that target different molecular pathways, there is a decreased probability of downregulation of the pathways and, thus, a decreased chance of lower therapeutic effect.

[0418] Therapeutics that decrease in efficacy over time can be included in the combinations herein, including, for example, pharmaceutical medications that activate molecular pathways that are downregulated, leading to decreased efficacy of the pharmaceutical. The combinatorial therapy herein includes rotating from one combination comprising a plurality of therapeutics to a next combination comprising a plurality of therapeutics. The rotational aspect overcomes limitations associated with therapeutics that decrease in efficacy over time; the switch to a new combination results in a continuous therapeutic effect that does not wane, or wanes less than treatment with a monotherapy containing one of the therapeutics in the combination.

[0419] In examples, therapeutics for inclusion in the combination for the rotational combinatorial therapy herein are those that do not have severe adverse side effects upon ceasing use of the particular therapeutic. For example, therapeutics that do not induce withdrawal symptoms as a result of ceasing or reducing use of the therapeutic (z.e., medication). When selecting medications for inclusion in the combinations, medications that induce withdrawal syndromes and rebound upon discontinuation can be avoided. Therapeutics for inclusion in the rotational combinatorial therapy are those with a low chance of withdrawal or adverse events upon switching to the next combination of medications or those that do not induce adverse events upon switching to the next combination of medications.

[0420] In examples, therapeutics for inclusion in the combinatorial therapy are those that are not delayed in producing a therapeutic effect or are not delayed in ameliorating symptoms of the disease or condition to be treated. The therapeutics included in the rotational combinatorial therapy are administered in rotation, for predetermined periods, such as for at least 1 month, 2 months, 3 months or 4 months or 5 months or 6 months or more, and in some examples rotated at least every 3 months. In these examples, the therapeutic(s) takes effect (z.e., has activity) or the therapeutic effect(s) is / are produced or amelioration of symptoms begins near to the time of initial administration, such as within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 72 hours, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months or more of treatment. For example, the therapeutic(s) takes effect (ie., has activity) or the therapeutic effect(s) is / are produced or amelioration of symptoms begins within at least 1 week of initial administration. The therapeutic activity can begin within the first half, third, quarter, or tenth of the rotational time period. For example, for a rotational combinatorial therapy where the combinations are rotated at 3-month intervals, the therapeutics in the combinations can show therapeutic activity within 1 month, and more generally within at least 2 weeks or 1 week or less than 1 week, such as two or three days after initial administration.

[0421] Medications that are toxic if administered at high dosages can be included in the rotational combinatorial therapy described herein. Therapeutics (ie., drugs) that show toxicity or adverse side effects when administered in therapeutic amounts can be included in the combination therapy described herein; the therapeutics used in combination with other, complementary, therapeutics can be administered in lower dosages such that when they are administered in combination with other therapeutics, and in a rotational regimen as described herein, they effect amelioration of symptoms or treatment of the disease, disorder, or condition without the adverse effects. Administration of therapeutics in a rotational regimen described herein can reduce drug toxicity, and adverse side effects, while maintaining or improving clinical efficacy. In some examples, a particular medication in the combination is administered at a lower dosage than the medication would be administered in a monotherapy; administration of the medication at the lower dosage decreases drug associated toxicity and adverse side effects.

[0422] Previous treatment (z.e., with a monotherapy or combination therapy) can be used to predict a patient’s response to a therapeutic or class of therapeutics or to similar therapeutics (ie., therapeutics that act through the same molecular pathway). Previous treatment (z.e., with a monotherapy or combination therapy) also can be used to predict whether targeting a particular molecular pathway will be effective in that patient. Patients who have shown a positive response to therapeutics targeting one molecular pathway, can be treated with a rotational combinatorial therapy containing therapeutics that target that same pathway. Information from patients who were not previously responsive, or did not show amelioration of symptoms, after treatment with a monotherapy targeting one molecular pathway, can be used to determine the therapeutics for inclusion in a combination therapy herein. For example, if a patient was not previously responsive to a particular pharmaceutical, a plurality of pharmaceuticals targeting the molecular pathway can be included in a combination therapy. In other examples, if a patient was not previously responsive to a particular pharmaceutical, the pharmaceutical can be excluded from the combination therapy. In other examples, if a patient was not previously responsive to a particular pharmaceutical, the pharmaceutical can be included in the combination therapy with other therapeutics predicted to act synergistically with the pharmaceutical.

[0423] Treatments that target different pathways associated with satiation, metabolism, hunger and weight gain or loss can be included in a rotational combinatorial therapy herein for treating overweight, obesity or for weight loss, or for treating comorbidities of overweight or obesity, such as diabetes (e.g., diabetes mellitus type 2), cardiovascular disease (z.e., cardiovascular disease leading to heart attack or stroke), high blood pressure, high blood cholesterol, high triglyceride levels, persisting neurodegenerative disorders, metabolic syndrome, obstructive sleep apnea, cancer, depression, and non-alcoholic fatty liver disease. For example, therapeutics that mimic the physiological effects following gastric bypass can include in a rotational combinatorial regimen as provided herein.

[0424] 4) Identify combinations that include at least 2 therapeutics known to activate different molecular and / or cellular pathways and design a regimen for administration of the combinations

[0425] The combination can include therapeutics that target the same pathways or that target different pathways. Therapeutics that can provide complementary benefits (z.e., modulate similar or the same molecular pathways to compound the effects), or therapeutics that act through different molecular pathways to effect positive benefits through a variety of pathways can each be included in the rotational combinatorial therapy described herein. The therapeutics in the combinations generally target different pathways. Selecting therapeutics that target different pathways is generally to prevent redundancy in the pathways that the therapeutics target, such as, for example, where redundancy does not increase the therapeutic benefit over the benefit of a single therapeutic, or when redundancy can increase the likelihood or severity of adverse side effects. Selecting therapeutics that target different pathways also overcomes the decreased therapeutic effect that occurs after desensitization of a particular molecular pathway after prolonged treatment. For example, if a therapeutic in a first combination targets a pathway that is desensitized after use, the therapeutic in the second combination targets a second pathway, which is not desensitized, and, thus, not downregulated; a third combination can contain the first therapeutic, if the desensitization no longer exists, or can contain a third therapeutic that targets a third pathway. The rotation of therapeutics that target different pathways overcomes the reduced therapeutic effect after desensitization of a pathway.

[0426] The therapeutics for use in the rotational combinatorial therapy described herein can be therapeutics that are complementary in that one effects treatment of secondary effects (z.e., inflammation) and the other effects treatment of the primary cause of the disorder. For example, one therapeutic in the combination can inhibit production of proinflammatory cytokines that are a downstream effect of the primary disorder, such as, cancer and obesity. In other examples, the combination can include therapies that treat or ameliorate the symptoms of concomitant disease states, such as a treatment (z.e., medication) for hypertension included in a combination for treatment of the primary condition of obesity.

[0427] Therapeutics that can provide complementary benefits by effecting treatment of different disease phases can be selected for inclusion in the rotational combinatorial therapy described herein. For example, in multi-phasic diseases states, one therapeutic or combination of therapeutics can effect treatment or amelioration of effects of an acute phase of the disease and the other therapeutic(s) can effect treatment or amelioration of effects of a chronic phase or state of the disease.

[0428] In some combinations that include therapeutics that target the same pathways, the overlap (redundancy) is, for example, to ensure the efficacy of the combination, to ensure activation or inhibition of a particular pathway. Therapeutics with overlapping targets also can be included in separate combinations in the therapeutic regimen, such as, for example, combinations that are administered at different points in the therapeutic regimen, and generally with a cycle of the regimen separating administration of the redundant therapeutics.

[0429] 5) Create a regimen to administer multiple rounds of treatment, with combinations of therapeutics

[0430] The combinatorial rotational therapy described herein includes a regimen for administering the plurality of combinations multiple times, such that there are at least two rounds, and generally at least three, four, five or more rounds of treatment, with different combinations. Various factors can influence the timing for administering the plurality of combinations in the rotational combinatorial therapy provided herein including, but not limited to: the particular therapeutics in the combinations; the particular disease to be treated; the predicted and / or known efficacy of the therapeutics or therapies, alone or in combination with other therapeutics and / or therapies; the time for the therapeutics to demonstrate therapeutic activity (e.g., onset of action); the recommended time for therapeutic administration; the maximum recommended time for therapeutic administration; medication dosages; the characteristics of a patient to be treated, including, but not limited to, gender, weight, age, overall health, comorbidities, and other characteristics; and other particulars of the therapeutic(s), patient and disease, disorder, or condition.

[0431] In examples where the rotational combinatorial therapy is for treating a rapidly progressing disease, disorder, or condition, the first combination in the rotational combinatorial therapy can include a therapeutic or therapeutics with a high (z.e., the highest) probability of activity and / or efficacy for treating the disease, disorder, or condition. For treating a disease, disorder, or condition that is less rapidly progressing, where an extended time period for administering a second- or third-line combination therapy can be considered, lower dosages or a less aggressive first combination of therapeutics can be administered. The therapeutics for inclusion in the first line therapy (i.e., therapeutics in the first cycle of the rotation) can depend on the disease, disorder, or condition, to be treated, the disease state, state, the rate of progression of the disease, disorder, or condition, the severity of the disease, disease, disorder, or conditions. As detailed above and below, the skilled physician can modify the therapeutics for inclusion in the rotational combinatorial therapy described herein based on the available therapeutics, and, for example, the disease to be treated, the disease state, the rate of disease progression, the severity of the disease, the particular patient to be treated, and other factors. The rotational aspect of the combination therapy provided herein provides for changing of doses and replacement of ineffective or less effective therapeutics with others that have increase efficacy, for example, based on the patient response.

[0432] The time for the drug or therapeutic to exhibit therapeutic activity also can be considered when selecting a drug or other therapeutic for treatment in a rotational treatment regimen provided herein. In one example, a drug or therapeutic used in the rotational combination therapy herein should be active prior to rotation and administration of the next therapeutic combination. For example, if a medication takes one week to show therapeutic activity, the combinations will not be rotated at less than one week. For example, if a medication takes one week to show therapeutic activity, the combinations can be rotated after at least one month, two months, three months or more, in order to show therapeutic activity of the initial combination.

[0433] As detailed herein, the timing for rotation of the therapeutic combinations can be determined. Various factors can be considered to identify indicators for a switch from one therapy (z.e., treatment or drug or therapeutic or combination thereof) to another, and the timing for a switch. In some examples, the following factors can influence the decision to switch medications: one or more adverse side effects; change in clinical status (e.g., improvement or worsening of the disorder or condition to be treated); poor drug activity despite administration of a therapeutically effective amount of drug; concomitant pharmacotherapy (i.e., drug-drug interactions); need for a different route of administration (e.g., oral vs. injection), such as, for convenience or for improved adherence to the administration regimen or for drug uptake and / or activity; subject responsiveness to the drug (i.e., poor responsiveness), such as because of receptor down-regulation or due to sex or age or race; and others. Thus, the timing for switching combinations will depend on many factors, including the particular therapeutics in each combination. The information provided herein can be used to develop guidelines for a rotation schedule. In examples, combinations are administered in rotation, for predetermined periods, such as for at least 1 month, 2 months, 3 months or 4 months or 5 months or 6 months or 9 months or 1 year or 15 months or 18 months or 2 years or 27 months or 30 months or 33 months or 4 years or more, and in some examples rotated at least every 3 months. As downregulation can occur after at or about 3 months or less, the combinations can be rotated every three months.

[0434] After an administration regimen, such as a rotational regimen described herein, has been implemented in a particular patient, the regimen can be modified to improve the therapeutic effects. The patient’s clinical status can be used to modify the treatment regimen or schedule or drugs administered. For example, if a patient develops drug sensitivities, the regimen can be modified to switch to a new therapeutic under an expedited schedule. In other examples, the rotational schedule can be extended if previously unrealized benefits of the therapeutic are achieved with a particular medication.

[0435] As detailed herein, the dosage of the individual therapeutics in the combinations for rotation can be determined. Various factors can be considered to identify the dosage for each therapeutic (ie., drug). The examples and disclosure provided herein can be used to develop guidelines for a rotation schedule, and identify the medications for inclusion in the regimen / schedule. The dose of an individual therapeutic can vary depending on the other therapeutics in the combination, and can vary depending on the doses of the therapeutics in the other combinations in the rotation. The regimen can include guidelines for dose adjustment based on the action of the previously administered medications, relative potency, and other facts that can influence efficacy of the therapeutic(s).

[0436] The dose range for each of the individual therapeutics in the combinations provided herein can be adjusted by monitoring the subjects (z.e., patients), such as monitoring plasms or monitoring symptoms or monitoring adverse side effects after administration of the combination(s). The dose administered can be such that the subject will maintain a plasma level to effect amelioration of symptoms of the disease, disorder, or condition for which treatment is administered. The dose also can be a dosage that will effect amelioration of symptoms of the disease, disorder, or condition for which treatment is administered where the subject does not exhibit adverse side effects, or does not exhibit adverse side effects at a severity that will effect cessation of the treatment.

[0437] F. METHODS OF TREATMENT AND USES

[0438] The combinatorial and rotational combinatorial therapy provided herein can be used in methods of therapy for treating chronic conditions, such as, for example, overweight and obesity. In other examples, the combinatorial and rotational combinatorial therapy described herein can be used in methods of therapy for treating chronic conditions such as diabetes (e.g., diabetes mellitus type 2), cardiovascular disease (i.e., cardiovascular disease leading to heart attack or stroke), high blood pressure, high blood cholesterol, high triglyceride levels, persisting neurodegenerative disorders, such as Parkinson’s disease, metabolic syndrome, obstructive sleep apnea, cancer, osteoarthritis, depression, and non-alcoholic fatty liver disease.

[0439] In the methods, a combination therapy is administered in a rotational regimen to a subject having a chronic condition. As described herein, the first combination of therapeutics, in a combination regimen with a second (and third or more) combination(s) far exceeds the effects of a monotherapy or combination therapy that is not rotated, for treating a chronic condition, such as, for example, obesity. For example, the effects can be synergistic. For example, extent and level of weight loss observed following the combination therapy provided herein achieves results that have heretofore not been achieved with existing non-surgical weight loss therapies, including increased fat loss and muscle gain that surpasses existing treatment regimens.

[0440] 1. Therapeutic Uses of the Combinatorial Therapy

[0441] Combinatorial and pulsed-dose pharmacology have been used for a variety of diseases, disorders, and conditions, such as pain control, birth control and treatment of cancer, COPD, Parkinson’s disease, Alzheimer’s disease, and bacterial infections. These previous uses do not contemplate administering a combination of at least two, generally, at least three, different therapeutics / treatments (i.e., therapeutics having a different target or different mode of action) according to a schedule in which different therapeutics are rotated, and / or the combinations selected are not selected to target different pathways. Treatment strategies to deal with complex medical issues such as cancer, pain control and pulmonary issues also are described herein to integrate more than one medication (combinatorial pharmacology) administered in a rotational regimen, to improve clinical outcomes. The methods and regimens provided herein are designed to target different pathways involved in disease, disorder, or condition, and to rotate combinations of treatments.

[0442] In accord with methods herein, rotational combinatorial therapy for treating complex medical disorders include first identifying known treatments / therapies for each disease state or pathways, and mechanisms of action or targets associated with the disease. Then, treatments / therapies that activate different pathways, have different mechanism of actions or targets, and / or that are compatible with a rotational therapy can be identified. Using previous treatments and pathways / mechanisms / targets associated with the disease state, combinations that include at least 2 therapeutics known to activate different molecular and / or cellular pathways can be selected and a regimen to administer multiple rounds of treatment, with combinations of therapeutics, can be designed. As set forth herein, the methods for selecting therapeutics for inclusion in a rotational combinatorial regimen, and the timing for administration of the therapeutic combinations can be prepared. In examples, the combinations can be administered in rotational regimens for treating, for example, any of the diseases, disorders and / or conditions set forth below. a. Combination Therapies in Cancer

[0443] Several cancer therapeutics are used in combination but without a rotational component to treat tumors in patients. For example, ABVD (Adriamycin, Bleomycin Vinblastine Dacarbazine) can be used as the initial chemotherapy treatment for newly diagnosed Hodgkin lymphoma. ABVD has been the most effective and least toxic chemotherapy regimen available for treating early-stage Hodgkin Lymphoma. One cycle of ABVD chemotherapy is typically given over 4 weeks in two doses, where the first dose of the drug combination is administered on day 1 and the second dose is administered on day 15, where all four of the chemotherapy drugs (Adriamycin, Bleomycin Vinblastine Dacarbazine) are administered intravenously.

[0444] Another combination therapy for cancer treatment is known by the acronym CHOP, which includes a combination of cyclophosphamide, doxorubicin hydrochloride (hydroxydaunorubicin), vincristine sulfate (Oncovin), and prednisone for the treatment of non-Hodgkin lymphoma. Cyclophosphamide, methotrexate, and fluorouracil (5FU), also known as CMF, also are used in combination for treatment of breast cancer. Other chemotherapy regimens used in the clinical setting include the Stanford V protocol, which generally includes a two to three month treatment period with a chemotherapy regimen (doxorubicin, vinblastine, mechlorethamine, vincristine, bleomycin, etoposide, and prednisone) followed by radiation treatment; and the BEACOPP regimen, which includes administering a combination of bleomycin, etoposide, doxorubicin hydrochloride (Adriamycin), cyclophosphamide, vincristine (Oncovin), and procarbazine for treatment of Hodgkin lymphoma. The BEACOPP treatment generally includes four to eight 14- or 21 -day cycles with no drugs given on days 15-21. A course of BEACOPP therapy also can be combined with ABVD. An exemplary BEACOPP therapy protocol is set forth in Table 7, below:

[0445] Table 7: BEACOPP regimen

[0446] Previous combination therapies for treatment of cancer, including those described above, thus exist. These combination therapies, however, lack a rotational component of rotational combinatorial therapies and regimens provided herein. Any of the previously described combinations of cancer therapeutics can be modified in accord with the rotational combinatorial therapy described herein. For example, to prevent adverse side effects associated with administration of multiple medications on day one (z.e., 5 medications on day one of the BEACOPP protocol), the protocols can be modified to administer fewer medications in combination, and rotate the combinations of medications, such as, but not limited to, chemotherapeutics. b. Combination Therapies in Pain Management Some individuals with chronic pain undergoing long- term treatment with painkillers, such as opioids, can develop decreased responsiveness to the opioids despite dose titration (Knotkova et al., J. Pain Symptom Manage. 38(3): 426-39 (2009)). Opioid rotation is a strategy implemented to increase efficacy of the combination of opiates; one opiate is initially administered and when efficacy begins to wane, the first opiate is replaced by another to optimize clinical outcomes and lessen side effects. This drug “exchange” substitutes one opioid for another where both medications work through a similar pathway, both targeting the mu receptor; the medications are “exchanged” rather than rotated.

[0447] With pain treatment, opioids are substituted when efficiency begins to wane, not at predetermined intervals, and based on a “shared decision-making approach” between the treating physician and the patient (Fine el al.. J Pain Symptom Manage (2009) 38(3): 418-425). There is no fixed period at which opioids are exchanged, the exchange is based on clinical efficacy which may or may not correlate with the biological process of receptor downregulation.

[0448] In contrast, rotational combinatorial therapies described herein exchanges a group of medications for a second group of medications, where the second group targets different pathways or targets than the first group. With opioid rotation, the rotated opioids are the same, and generally is one drug, not a combination of drugs, exchanged for a second drug. Also, in embodiments herein the rotational therapy described herein includes a regularly defined period for the rotation to occur, including regularly defined intervals, to avoid receptor downregulation and to target different receptors. c. Combination Therapies in Oral Contraception

[0449] Treatment with oral contraceptives employs both combinatorial and pulse- dosed pharmacology treatment strategies; treatment with oral contraceptives is not rotational, the different medications are not rotated. Oral contraceptives come in a variety of formulations; some formulations contain both estrogen analogs and progestin, and some contain only progestin; some pills are monophasic, delivering the same dose of hormones each day, and others are multiphasic, where the doses vary each day. Doses of the component hormones also varies among commercial oral contraceptive products. The monophasic pills employ a strategy that targets the same, one pathway for birth control each month, with a pulse of hormones, a “pulse-based strategy.”

[0450] Multiphasic birth control pills have both and estrogen analogs and progestin components with a fixed increasing dose that is given for set periods of time for a set period (z.e., biphasic dosing includes tablets of one strength for 7 to 10 days, then tablets of a second strength for the next 11 to 14 days; triphasic dosing includes tablets of one strength for 5 to 7 days, then tablets of a second strength for the next 5 to 9 days, and then tablets of a third strength for the next 5 to 10 days). The birth control pills target the same estrogen and progesterone receptors.

[0451] Thus, birth control pills can contain a combination of two medications and multiphasic birth control pills can increase in dosage over the month. There is a continuous activation of the same receptors (ie., one or both of estrogen and progesterone receptors) throughout the month, and activation of these same receptors during the next month. Activation of the same set of receptors differs from the rotational combinational therapy described herein because treatment with birth control pills does not contain a rotational element where different combinations of therapeutics (z.e., medications) are administered and rotated to prevent decreased efficacy due to downregulation of receptor activity; each therapeutic in the rotational combinatorial therapy described herein has a different target or mode of action, unlike birth control pills which target the same receptors each month.

[0452] Present combination therapies used for treatment of cancer or for pain management or for contraceptive purposes differ from the rotational combinatorial therapy described herein, and can be modified in accord with the rotational combinatorial regimen described herein. For example, efficacy of chemotherapeutic combinations can be improved by applying the rotational combinatorial regimen described herein; chemotherapeutic combinations can be rotated in accord with a specified schedule to target different receptors or pathways to prevent receptor downregulation or decreased efficacy or increased toxicity, such as adverse side effects, which occurs after prolonged continuous use of the single combination. d. Combination Therapies to Treat Pathogens

[0453] Regular use of antibiotics, such as treatment with a monotherapy or combination therapy, eventually leads to antibiotic resistance in the targeted microorganism (z.e., bacterium). Combination therapies are strategies for treatment of bacterial infections to overcome or prevent drug resistance, broaden the antimicrobial spectrum, improve the efficacy, treat multi-drug resistant bacteria, and lower the dose of the individual drugs to reduce the side effects. Combination therapy with multiple antibiotics have been described (Drusano et al. (2014) PLos ONE 9 7):el01311; U.S. Patent Publication No. 2021 / 0236589).

[0454] US Patent Publication No. 2021 / 0236589 describes selecting combinations of three antibiotics and rotating among the three different-member combinations to eliminate the possibility of developing resistance.

[0455] The combinations of such antibiotics can be administered in a rotational combinatorial regimen to ameliorate side effects from the particular antibiotics and to avoid development of antibiotic resistance. e. Combination Therapies to Treat Alzheimer’s Disease

[0456] There is no cure for the progressive neurodegeneration and resulting phenotypes that occur in patients with Alzheimer’s disease (AD). A wide spectrum of approaches exist for treatment of AD, with the majority focusing on targeting the Ap peptide to slow disease progression (Galimberti el al. , Ther Adv Neurol Disord 4:203- 216 (2011)). Medications and management strategies can improve symptoms, albeit temporarily. Aducanumab (sold under the trade name Aduhelm™) is an intravenous infusion therapy that targets beta-amyloid, and is approved to treat AD. Other medications, such as cholinesterase inhibitors, such as, but not limited to, medications sold under the trademarks Aricept®, Exelon®, and Razadyne®, glutamate regulators, such as, but not limited to the medication sold under the name Namenda®), and combinations of a cholinesterase inhibitor and a glutamate regulator (sold under the trade name Namzaric®). Non-cognitive symptoms, such as behavioral and psychological symptoms, can be treated by orexin receptor agonists (such as the medication sold under the trademark Belsomra®). All of the medications have adverse side effects, including one or more of nausea, vomiting, headache, constipation, confusion and dizziness, impaired alertness and motor coordination, worsening of depression or suicidal thinking, complex sleep behaviors, sleep paralysis, and compromised respiratory function. A combinatorial pharmacological approach has been described to attenuate the microglial activation and chronic inflammation characteristic of AD (McLarnon (2019) Current Alzheimer Research 16: 1007-1017). The anti-inflammatory combination therapy described by McLarnon does not contemplate rotation of the combinations, and includes administration of a cocktail of compounds to modulate inflammatory pathways activated by microglia in response to the proinflammatory AD brain microenvironment. McLarnon does not consider combination therapy for treatment of aspects of AD pathology that are not related to the chronic inflammation, such as pathology that is related to amyloid deposition or other factors, such as amyloid beta activation or altered cell signaling.

[0457] Additional medications for inclusion in the rotational combinatorial therapy for treatment of Alzheimer’s are set forth in Table 8, below:

[0458] Table 8: Exemplary Medications for Alzheimer’s Treatment

[0459] Additionally, these rotational combinatorial treatments for Alzheimer’s disease can include the weight loss regimens or weight loss drugs. For example, glucagon-like peptide 1 (GLP-1) receptor agonists, including those described herein, exhibit neurotrophic and neuroprotective effects in amyloid-P (AP) toxicity models of Alzheimer’s disease (AD). Hence these drugs can be included in a rotational combinatorial regimen for Alzheimer’s disease. f. Combination Therapies to Treat Hypertension

[0460] Combination therapies are not generally used to treat hypertension because of concerns about lowering blood pressure to unsafe levels and increased risk of adverse side effects. Generally, if medications are used in combination to treat hypertension, a first medication is administered and a second can be added to the treatment if the first medication is ineffective and / or there are no significant adverse side effects. There are combination therapies have previously been shown to be superior to monotherapy for treatment of hypertension. A combination of losartan at 50-100 mg with hydrochlorothiazide at 12.5 -25 mg lowered systolic blood pressure significantly (p<0.001) more than either drug alone (MacDonald et al.. J of the American Heart Association DOI: 10.1161 / JAHA.117.006986 (2017)).

[0461] Combinations of medications for the treatment of hypertension generally include two antihypertensive agents with different mechanisms of action that demonstrate enhanced efficacy compared to either medication alone. The medications can be used at lower dosages than treatment with individual medications, to reduce the risk or severity of adverse side effects. Combinations of drugs used for treatment of hypertension are set forth in Table 9, below:

[0462] Table 9: Exemplary Medications for Hypertension Treatment

[0463] Adapted from Skolnik et al., Am Fam Physician (2000) 61(10):3049-3056

[0464] There are combination therapies that have been employed for treatment of hypertension, including those described above, but not rotational of combinations. The medications can be administered initially as a combination of two drugs, or therapy can begin with one medication and one or more medications is added to the monotherapy. Any of the previously described combinations of medications for treatment of hypertension or medications known to treat the molecular and cellular pathways associated with hypertension can be modified in accord with the rotational combinatorial therapy and regimens as described herein. For example, to prevent adverse side effects associated with administration of multiple medications at once or to target alternative molecular pathways, the protocols can be modified to administer fewer medications in combination, and rotate the combinations of medications (ie., ACE inhibitors and / or diuretics). g. Combination Therapies to Treat Parkinson’s Disease

[0465] Parkinson’s Disease (PD) is a progressive neurodegenerative disorder characterized by altered body movements, including tremor, stiffness, slowed movement (bradykinesia), loss of automatic or autonomic movements, and speech changes. PD is not curable; a number of medical treatments are used in the management or improvement of PD symptoms, including levodopa alone or in combination with carbidopa, dopamine agonists, MAO B inhibitors (z.e., selegiline (sold under the trademark Zelapar®), rasagiline (sold under the trademark Azilect®) and safinamide (sold under the trademark Xadago®), Catechol O-methyltransferase (COMT) inhibitors. Entacapone (sold under the trademark Comtan®) and opicapone (sold under the trademark Ongentys®), Anticholinergics, such as, but not limited to benztropine (sold under the trademark Cogentin®), trihexyphenidyl, and amantadine.

[0466] Any of the previously described combinations of medications for treatment of PD can be administered in accord with the rotational combinatorial therapy described herein. In other examples, molecular and cellular pathways associated with PD progression and / or symptoms of PD can be targeted with therapeutics for inclusion in the rotational combinatorial therapy herein. For example, therapeutics that target dopaminergic pathways or neurons or neurotransmitters or receptors can be included in the rotational combination therapy. To prevent adverse side effects associated with administration of multiple therapeutics at once or to target alternative molecular pathways, the protocols can be adjusted to administer fewer medications in combination, and rotate the combinations of therapeutics. h. Combination Therapies to Treat Chronic Obstructive Pulmonary Disease (COPD)

[0467] Chronic obstructive pulmonary disease (COPD) is a type of progressive lung disease characterized by shortness of breath and cough. The two classic COPD phenotypes are emphysema and chronic bronchitis. COPD is not curable; a number of medical treatments are used in the management of stable COPD and exacerbations, including bronchodilators, corticosteroids, inhibitors of the enzyme phosphodiesterase-4, and antibiotics. Bronchodilators for treatment of COPD include short-acting beta? agonists [SABAs], long-acting beta? agonists [LABAs], shortacting muscarinic antagonists [SAMAs], and long-acting muscarinic antagonists [LAMAs],

[0468] Previous studies have shown that long-term treatment with a combination inhaler containing inhaled corticosteroids and long-acting beta? antagonist is more effective than either agent alone in improving COPD symptoms and in reducing exacerbation frequency. If a dual therapy is not effective, triple therapy with a combination of ICS, LABA and LAMA is indicated. Different, complementary pharmacological mechanisms of action of the three medications improves clinical benefits. For example, a triple fixed-dose combination of extra fine beclomethasone dipropionate (100 pg / puff), formoterol fumarate (6 pg / puff), and glycopyrronium bromide (12.5 pg / puff) administered via a hydrofluoroalkane pressurized metered dose inhaler is superior to fixed ICS / LABA combined therapy and also superior to the LAMA tiotropium for improving lung function and exacerbation prevention in COPD patients at risk of exacerbation.

[0469] Thus, there previously are combination therapies used for treatment of diseases, disorders, and conditions, including those described above. Ultimately, this combination will not avoid the problems associated with desensitization to long-term treatment. These combination therapies can be improved by rotating combinations of the drugs. Any of the previously described combination therapies, where there are different targets for therapeutic intervention or can be modified by rotating combinations of therapeutics or treatments to produce a rotational combinatorial therapy regimen described herein. For example, to prevent receptor downregulation upon continuous administration or adverse side effects associated with administration of multiple medications on one day, the protocols can be modified to administer fewer medications in combination, and rotate the combinations of medications. i. Combination Therapies to Treat Obesity-Associated Diseases and Conditions

[0470] Rotational combinatorial therapy, such as rotational combinatorial pharmacology provided herein can be used to treat diseases, disorders and conditions that are comorbid with overweight and obesity, including, but not limited to, metabolic syndrome, obstructive sleep apnea, non-alcoholic fatty liver disease, diabetes (e.g., diabetes mellitus type 2), cardiovascular disease (e.g., heart attack, stroke), elevated blood pressure, elevated blood cholesterol, and elevated triglyceride levels, and others.

[0471] Combinatorial therapy and rotational combinatorial therapy, such as rotational combinatorial pharmacology provided herein can be used to treat cardiovascular disease. Rotational combinatorial therapy can be used in the treatment of cardiovascular diseases including ischemia reperfusion injury resulting from stroke, myocardial infarction, cardiopulmonary bypass, coronary artery bypass graft, angioplasty, or hemodialysis. Rotational combinatorial therapy also can be used in the treatment of the inflammatory response associated with cardiopulmonary bypass that can contribute to tissue injury. For example, a combination of therapeutics can be administered in accord with a regimen described herein prior to, or in order to prevent cardiovascular disease or an adverse cardiac event. In other examples, a combination(s) of therapeutics can be administered in accord with a regimen described herein subsequent to a cardiac event to prevent further injury from the cardiac event or to aid in recovery from the adverse cardiac event (e.g., heart attack or stroke). In one example, a combination(s) of therapeutics can be administered to a subject in accord with a regimen described herein in order to ameliorate the symptoms of cardiovascular disease or the secondary damage or effects resulting from cardiovascular disease. Amelioration of symptoms can be assessed by methods described herein or those known to the skilled physician. j. Combination Therapies to Treat Overweight and Obesity

[0472] There is a lack of therapeutics for obese patients that also focus on polypharmacy for diabetes, metabolic syndrome, and other associated diseases, disorders, and conditions. The approach to weight loss and muscle development described herein is shown to produce results using pharmacological approaches that are at least as effective as bariatric surgery, but without the risks of surgery. This is described in the following sections and detailed in the working examples.

[0473] G. COMBINATORIAL AND ROTATIONAL COMBINATORIAL THERAPY FOR WEIGHT LOSS

[0474] As described in the next sections, obesity is an exemplary condition for which combinatorial therapies, and rotational combinatorial therapies can be designed. Fat can be stored subcutaneously or as viscerally subcutaneous and visceral adipose tissue. Subcutaneous fat lies underneath the skin and is visible, and is the fat that changes after body composition improves (i.e., fat decrease) with cardiovascular resistance training. Visceral adipose tissue cannot always be seen directly and distributes around the organs, making it more dangerous to health and is more strongly associated with metabolic syndrome and diabetes compared to subcutaneous fat.

[0475] Visceral fat actively contributes to health because it produces cytokines and immunoregulatory hormones. Excess of cytokines and immunoregulatory hormones are seen with obesity, causing inflammation and increased risk of cardiovascular disease, immune-dysregulation and a negative effect on cells sensitivity to insulin further contributing to diabetes.

[0476] Obesity can induce a series of chronic metabolic diseases, such as diabetes, dyslipidemia, hypertension and nonalcoholic fatty liver disease.

[0477] 1. Limitations of Existing Treatments for Weight Loss

[0478] Given the limitations of lifestyle interventions and bariatric surgery, pharmacotherapeutic approaches for the treatment of obesity are important options. Development of anti-obesity medications has been slow and ineffective. During the past 20 years, several anti-obesity drugs have been discovered, marketed, and subsequently withdrawn from the market; despite showing efficacy during initial stages of treatment, therapeutics for obesity have been accompanied by adverse sideeffects following long-term use.

[0479] With the exception of the GLP1R agonist, semaglutide, and the combination medication tirzepatide, which activates the GLP-1 and GIP receptors, the average percent body weight reduction for registered drug treatments varies, but they are in the single-digit range (Bray et al.. Lancet 387: 1947-1956 (2016)). An improved antiobesity treatment(s) is needed to correct excess weight while reducing risk for cardiovascular-associated adverse effects and psychological adverse effects. Likewise, an improved treatment for overweight or obesity can decrease risk for obesity-related comorbidities including metabolic syndrome, obstructive sleep apnea, non-alcoholic fatty liver disease, diabetes (e.g., diabetes mellitus type 2), cardiovascular disease (e.g., heart attack, stroke), cancer, elevated blood pressure, elevated blood cholesterol, and elevated triglyceride levels, and others.

[0480] Significant and harmful side effects are associated with weight loss therapies. Several limitations on surgery exist, including high cost, and the potential for intraabdominal abscess formation, thrombosis, dehydration, and type 1 diabetic ketoacidosis. As noted above, pharmaceutical weight loss treatments are not as effective as surgical interventions (ie., generally do not show high percent weight loss) and increase the risk of adverse side effects, including nausea, vomiting, headache, constipation, confusion and dizziness, impaired alertness and motor coordination, worsening of depression or suicidal thinking, complex sleep behaviors, sleep paralysis, and compromised respiratory function.

[0481] The robust escalations in obesity and associated health complications constitute major driving forces for the discovery of targets and for the development of safe and effective weight loss therapeutics. The combinatorial and rotational therapy provided herein is different from previous approaches for obesity and other disease processes because they are designed with combinatorial and rotational pharmacology (CRP) in mind.

[0482] 2. Obesity and the Challenges of Treatment

[0483] Treatment for obesity can be challenging because of the multitude of causes. A primary treatment for obesity includes dieting and physical exercise. The combination of dieting and exercising, however, rarely produces sustained weight loss, generally resulting from slow weight regain over time.

[0484] Altered eating (e.g., dieting) has been recommended as a behavioral change to increase weight loss. CDC recommends a variety of lifestyle interventions for weight loss, including: calorie restriction; time restricted feeding, where meals are consumed within a limited time window (ie., 6-8 hours) during the day; altemate-day fasting, where food consumption is unrestricted every other day and minimal or no calories are consumed on the other days; “5:2” eating patterns, where meals are unrestricted for 5 days each week, followed by 2 days of restricted calorie intake; and periodic fasting, where calorie intake is restricted for several days in a row (ie., 5 days) once per month, and food consumption is unrestricted on the other days.

[0485] Increased exercise is recommended as a behavioral change to increase and sustain weight loss. The American College of Sports Medicine (ACSM) and CDC Guidelines propose increasing exercise to augment weight loss. For example, the Guidelines indicate that healthy adults aged 18-65 years should participate in moderate intensity aerobic physical activity a minimum of 30 min on five days per week, or vigorous intensity aerobic activity for a minimum of 20 minutes on three days per week.

[0486] Behavioral changes targeting lifestyle changes has been studied extensively, with weight loss typically ranging from 3-10% of body weight with 12 months of intensive treatment in research settings. Nonetheless, most of that weight lost is regained within 12 months of initiating a program. Lifestyle modifications can be used alone, or as an adjunct to medical or surgical treatments for overweight and obesity, and for long-term treatment and management. (Jin (2018) JAMA 320(17): 1210). There are some metabolic and hormonal counterforces that reduce or reverse weight loss; these include an increase in appetite, reduction in energy expenditure, reduced insulin sensitivity that favors growth of adipocyte size and numbers. For example, weight loss is accompanied by persistent endocrine adaptations that cause an increase in appetite and decrease satiety, and there can be a physiological downregulation of pathways associated with weight loss and metabolism, thereby resisting continued weight loss and conspiring against long-term weight maintenance. This can lead to more robust weight regains as the weight lost progresses further from the “weight thermostat” set by years of overeating and all the offsetting mechanisms being activated.

[0487] Lifestyle and behavioral modifications are inadequate to provide long lasting weight loss and significant metabolic changes. Pharmacotherapy is necessary in many cases to assist with the metabolic derangements that occur in obese patients but results, as noted below, are very limited. A variety of weight loss treatments have been developed; exemplary therapeutics and treatments are summarized in the following sections.

[0488] 3. Pharmacological Treatments

[0489] Obesity is a chronic degenerative disease that can stem from the rewiring of biological mechanisms that hinder weight loss and promote weight gain. Although lifestyle and behavioral interventions provide moderate efficacy, these strategies are limited by complications in adherence. They can be augmented by surgical intervention and / or pharmacological approaches (Muller TD. et al. Nat Rev Drug Discov 21(3):201-223 (2021)). Bariatric surgery generally is considered the most effective approach to weight loss, but it is expensive and limited to single patients, lacking the reach necessary to treat obesity at a global scale. Pharmacological agents can offer such treatment.

[0490] The historical development of anti-obesity medications is challenging because of the limited understanding of the molecular mechanisms that control appetite and adverse effects. Historical anti-obesity drug classes include mitochondrial uncouplers, amphetamines, thyroid hormones, drug cocktails, neuromodulators, lipase inhibitors, cannabinoid receptor antagonists, and gastrointestinal-derived peptides chemically optimized for pharmaceutical use (Muller TD. et al. Nat Rev Drug Discov 21(3):201- 223 (2021)). Therapeutic approaches regulate the function of pathways related to energy balance and systemic energy sensing. Mechanisms of action can involve limiting fat absorption or food intake, increase satiety, or facilitate energy expenditure.

[0491] Medical standard of care for weight loss focuses on pharmacological monotherapies and occasionally dual therapies. FDA-approved monotherapy options include phentermine (sold under the trademark Adipex-P®), orlistat (sold under the trademark Xenical®), lorcaserin (sold under the trademark Belviq®), liraglutide (sold under the trademark Saxenda®), phentermine-topiramate (sold under the trademark Qysmia®), naltrexone-bupropion (sold under the trademark Contrave®) and semaglutide (sold under the trademark Wegovy®) medications. These medications, and others, can target components of the central nervous system or peripherally, such as in the gastrointestinal system, including, but not limited to the stomach, small intestine and colon, and pancreas (see e.g., FIG. 2).

[0492] As described and exemplified herein, monotherapies have limited efficacy, in part due to the metabolic redundancies and recruitment of alternate and counter- regulatory pathways, and desensitization. Obesity is a disease with multiple etiologies; as described herein can be treated with a multi -targeted approach. As exemplified and described herein, a multi-target approach provides greater benefit than any single medication alone. A multi-targeted approach includes a combination therapy or a rotational combinatorial therapy.

[0493] A number of medications have been administered for long-term use for weight loss; these medications, include, but are not limited to, phentermine-topiramate, orlistat, lorcaserin, naltrexone-bupropion, and liraglutide. Treatment with these medications as monotherapies results in weight loss after one year that ranges from 3.0 to 6.7 kg (6.6-14.8 lbs) compared to placebo (Heymsfield et al., (2017) The New England Journal of Medicine 376 (3):254- 266). Information on how these drugs affect longer-term complications of obesity, such as cardiovascular disease or death is sparse. Obesity drugs do not target weight loss per se, but on appetite suppression and / or are label expansions of other marketed drugs. As a result, treated subjects have significant adverse side effects, and the medications have relatively poor efficacy. For example, semaglutide (sold under the trademark Wegovy®) medication is a therapy designed for treatment of type-2 diabetes. An effect of the drug was weight loss and it has been rebranded for weight loss treatment.

[0494] The following are exemplary weight loss drugs that can be combined as described herein for combination therapy and also in a rotational combinatorial therapy regimen for treating overweight, obesity, for effecting weight loss and for treating secondary complications of each of the preceding conditions. The combinatorial therapy can include a combination of therapeutics known to effect weight loss and / or pharmaceuticals previously used as weight loss monotherapies and dual therapies. Medications that previously were used for non-weight loss indications that exhibit weight loss as a secondary effect also can be included in the combination therapies herein. The medications listed herein, such as, for example, below can be included in the combinations herein for treating overweight, obesity and / or for weight loss. Pathways associated with satiation, metabolism, hunger and weight gain or loss can be identified and used to formulate targets and / or therapeutics for the combination therapy, such as therapeutics listed below. Combinatorial regimens employing combinations, such as three or more of the medications below can be included in the combinations for weight loss, and / or treatment of overweight or obesity. These drugs can be part of a rotational combinatorial regimen as well, in which two or more of the drugs are combined and administered for a predetermined time, and then are rotated with another drug or combination of drug for a predetermined time. a. Amphetamines (e.g., phentermine-topiramate)

[0495] Amphetamines stimulate norepinephrine release that can result in increased blood pressure, heart rate, and cardiac excitability via binding to vasculature and heart adrenergic receptors. These drugs belong to the class of drugs known as sympathomimetic amines. Amphetamines used as anti-obesity medication include, but are not limited to Methamphetamine (Desoxy ephedrine), Phenmetrazine (sold under the trademark Preludin®), Phendimetrazine, Phentermine, benzphetamine and Diethylpropion. Phentermine (sold under the trademark Adipex-P®) and Diethylpropion (sold under the trademark Tenuate®) were generally designed to retain anorectic activity, but with reduced effects on the cardiovascular and brain reward system (Colman, Ann., Intern. Med. 143, 380-385 (2005)). Several clinical studies report the absence of major adverse effects of phentermine or diethylpropion on blood pressure and heart rate; nonetheless, their use is contraindicated in patients with hypertension or elevated risk for cardiovascular disease (Muller etal., Nat Rev DrugDiscov 21 : 201-223 (2022)). Patients also quickly develop a tolerance to these medications.

[0496] Phentermine is a weight loss medication approved for short term use, such as less than a month (Hendricks et al., Obesity 17: 1730-35 (2009)). Patients can rapidly develop a tolerance to the medication. Amphetamines can be used in combination with other medications for treatment of overweight and obesity. For example, phentermine is used in combination with topiramate (combination sold under the brand name Qsymia®), which can be used for a longer period of time than phentermine alone. Previous studies show that the combination therapy results in an average of 5-10% weight loss, where weight loss corresponds to dosage. These can be administered in combination with liposomes provided herein, or incorporated into the liposomes. b. Lipase inhibitors (e.g., Orlistat)

[0497] Lipases are a class of digestive enzymes produced in the pancreas, mouth, and stomach to metabolize fat and to facilitate nutrient uptake. Orlistat (sold under the trade name Xenical®) is a lipase inhibitor that reduces the uptake of dietary fat in the gastrointestinal tract. The resulting fat malabsorption after treatment with lipases facilitates a negative energy state leading to a placebo-normalized weight loss in the range of 2.5%. Orlistat has shown beneficial effects on blood glucose, non-alcoholic fatty liver disease, and blood pressure (Muller et al. (2018) Pharmacol. Rev. 70:712- 746; Khera et al. (2018) Gastroenterology 154'.1309-1319. e7). The most common adverse events after orlistat treatment are of a gastrointestinal or digestive nature. c. Serotonergic agonists - Neuromodulators (e.g., lorcaserin)

[0498] Serotonergic agonists act as anti-obesity medication by suppressing appetite through the activation of serotonin receptors. Clinical serotonergic agonists include Fenfluramine, Dexfenfluramine, Sibutramine, Lorcaserin (sold under the trademark Belviq®), and Tesofensine. Cardiovascular safety concerns accompany Fenfluramine, Dexfenfluramine, and Sibutramine (Muller et al. (2018) Pharmacol. Rev.70 712- 746). The FDA requested withdrawal of Lorcaserin due to clinical trials showing an increased occurrence of cancer (Muller et al., Nat Rev Drug Discov 21 : 201-223 (2022)). Tesofensine, an inhibitor of norepinephrine, serotonin, and dopamine reuptake that was initially advanced for treatment of Alzheimer disease. A phase II study, it was reported to dose-dependently decrease body weight by 4.4-10.4% (Wharton et al. (2015) J. Curr. Cardiol. Rep. 17:35,' Astrup A. et al. (2008) Lancet 372:1906-1913). d. Bupropion / Naltrexone (such as the product sold under the trademark Contrave®) Naltrexone, an opioid antagonist, blocks the inhibitory effects of opioid receptors activated by P-endorphin released in the hypothalamus, which stimulates feeding. Naltrexone reduces food intake in combination with bupropion, reuptake inhibitor of dopamine and norepinephrine. Naltrexone does not cause weight loss in monotherapy; patients treated with Naltrexone 32 mg plus Bupropion showed a body weight reduction of 6.1% after 56 weeks of treatment (Greenway et al. Lancet 376, 595-605 (2010)). No increased adverse cardiovascular events were noted in an analysis of a cardiovascular outcome trial after bupropion / naltrexone treatment (Nissen et al., JAMA 315:990-1004 (2016)). Another study showed that addition of a modified diet and increased exercise to the bupropion / naltrexone combination lost an average of 9.3% of their baseline body weight. e. Glucagon-like peptide-1 receptor (GLP1R) agonists

[0499] Glucagon-like peptide-1 (GLP1; SEQ ID NO: 1) acts at the pancreas to enhance the expression and secretion of insulin and to inhibit the release or glucagon. GLP1 and glucose-dependent insulinotropic polypeptide (GIP; SEQ ID NO: 10) are primary incretin hormones secreted from the intestine on ingestion of glucose or nutrients to stimulate insulin secretion from pancreatic P cells. Although the specific mechanism of action is multifactorial, with gut, brain, and systemic improvements in insulin sensitivity, GLPR1 antagonism can lead to decreased body weight via the inhibition of food intake (Muller et al. (2018) Pharmacol. Rev. 70:712-746).

[0500] Chronic treatment with GLP-1 receptor agonists causes weight loss in diabetic humans. Liraglutide (sold under the trademark Saxenda®), is a once-daily injectable GLPR1 agonist, and was the first GLP1R agonist approved for treatment of obesity. Liraglutide is short-acting, so it is administered daily. After one year of treatment, there was a reported mean decrease of 8% body weight in subjects treated with liraglutide compared to 2.6% in subjects treated with vehicle controls (Pi-Sunyer et al. (2015) N. Engl. J. Med. 373: 11-22). In 2020, treatment with 3 mg liraglutide was approved for weight management in adolescents with obesity.

[0501] GLP-1 increases insulin metabolism and plays a role in appetite and digestion. It is among the incretins, which are hormones released by the small intestine into the bloodstream following a meal to help lower blood sugar by triggering insulin and blocking other sugar sources. Medications that are GLP-1 agonists are referred to as incretin mimetics since they “mimic” the incretin hormone effects.

[0502] Semaglutide, which is similar to and acts like a GLP-1 hormone, is used as an anti-diabetic medication. Semaglutide also slows down the rate at which food leaves the stomach (called gastric emptying). These actions cause a feeling of fullness, lowering appetite and resulting in weight loss. Semaglutide (sold under the brand name Wegovy®) is an injectable GLPR1 agonist FDA-approved in 2021 for chronic weight management in adults with obesity (URL: fda.gov / news-events / press- announcements / fda-approves-new-drug-treatment-chronic-weight-management-first- 2014). Wegovy® weight loss medication is approved for treatment of adults with a body mass index (BMI) greater than or equal to 30mg / kg2alone or 27 mg / kg2with at least one weight-related comorbidity (e.g., high blood pressure, high cholesterol). Semaglutide administered at a dose of 2.4 mg lowers mean body weight to -12-15% after 68 weeks of treatment (relative to -2.4% in placebo controls) with once-weekly treatment (Wilding et al., N. Engl. J. Med. 384(11): 989-1002 (2021)). A longer mechanism of action compared to other weight loss medications allows semaglutide to be administered weekly; semaglutide is the first once-weekly GLP-1 agonist approved for weight loss. Daily dosing of 2.4 mg achieved >15% weight loss in half of the study participants, whereas one third experienced more than a 20% reduction (O’Neil et al., Lancet 392:637-649 (2018)). Semaglutide is well-tolerated although the adverse effects (nausea, diarrhea, vomiting and constipation) typified by GLP1- related remain. The high level of side effects can limit a patient’s ability to maintain or reach a target level of the drug. In such cases, a provider can extend the period to reach the target dose, or may have to discontinue treatment if the patient is unable to tolerate the target dose, due to the adverse side effects.

[0503] Several peptide and small-molecule GLP1R agonists are in clinical development, including formulations designed for oral administration (Muller et al., Nat Rev Drug Discov 21 : 201-223 (2022)). GLP1R agonists in clinical development include Efpeglenatide (s a long-acting GLP-1RA that is a single amino acid-modified exendin conjugated to a fragment crystallizable region of human immunoglobulin 4 via a 3.4-kDa mini-poly ethylene glycol linker), semaglutide sold under the trademark Rybelsus®, Danuglipron (formerly PF-06882961), GLPR-NPA, and lotiglipron (formerly PF-07081532). Several GLP-1 receptor agonists approved to improve glycemic control and lower blood sugar in adults with type 2 diabetes exhibit a secondary effect of increased weight loss. For example, type 2 diabetes patients taking >1500 mg / day metformin in combination with the GLP-1 receptor agonist sold under the tradename dulaglutide, sold under the tradename Trulicity®, at 1.5 mg, 3.0 mg, or 4.5 mg once weekly lost an average of 6.6, 8.4 and 1.1 pounds, respectively, after 36 weeks on the medication. f. Mitochondrial uncouplers

[0504] An alternative approach to decreasing food intake and / or absorption is to increase the metabolic efficiency by which food is converted into useful energy. Highly thermogenic tissues such as brown adipose tissue show high expression of Uncoupling Protein 1 (UCP1, SEQ ID NO:41) that uncouples oxygen consumption from ATP synthesis. Pharmacological upregulation of UCP1 activity can be induced by catecholamines or small molecule uncouplers. Small molecule uncouplers for antiobesity application include 2,4-dinitrophenol (DNP) and BAM15 ((2- fhiorophenyl){ 6-[(2-fluorophenyl)amino]( 1 ,2, 5 -oxadiazolo [3 ,4e] pyrazin-5-yl) } amine). DNP is the first small molecule uncoupler tested clinically, but has limited utility owing to toxicity. Controlled-release oral formulation of DNP are under investigation to achieve an enhanced therapeutic index (Muller el al.. Nat Rev Drug Discov 21 : 201-223 (2022)). BAM15 is an orally administered therapeutic that reverses diet-induced obesity and insulin resistance in mice (Alexopoulos et al., Nat. Commun. 11(1): 2397 (2020)). g. Thyroid hormones

[0505] Thyroid hormone can decrease body weight and body fat by stimulating energy expenditure (Muller et al., Pharmacol. Rev. 70:712-746 (2018)). Thyroid hormone also can improve hepatic lipid metabolism and decrease low-density lipoprotein (LDL) cholesterol via enhanced reverse cholesterol transport and clearance of LDL via the liver (Baxter et al., Nat Rev Drug Discov 8:308-320 (2009)). The biologically active form of thyroid hormone is tri-iodothyronine (T3), which promotes its pharmacology through two specific nuclear thyroid receptor (TR) isoforms, TRa and TRp. Administration of T3 increases metabolic rate in a variety of species, including mice, rats, and humans (Muller et al., Pharmacol. Rev. 70, 712-746 (2018)). The molecular mechanism underlying T3 modulation of metabolic rate includes uncoupling of oxidative phosphorylation from mitochondrial ATP synthesis in skeletal muscle and other peripheral tissues, regulation of lipogenesis, activation of Na+ / K+ ATPase, enhanced mitochondrial biogenesis, and stimulation of futile cycling (Muller el al.. Pharmacol. Rev. 70:712-746 (2018)). Excess thyroid hormone also leads to muscle and bone catabolism, as well as several cardiovascular adverse effects including cardiac arrhythmia, tachycardia, and heart failure, severely limiting its use as a weight loss therapeutic. h. Drug cocktails

[0506] Metabolic redundancies and recruitment of alternate and counter-regulatory pathways can limit the efficacy of monotherapies. Clarkotabs, developed in 1941, were among the first commercially distributed combination diet pills; the combination sought to harness the anorectic effect of amphetamines with the thermogenic effect of thyroid hormone, with Aloin and Atropine sulfate to counteract potential adverse cardiovascular effects (Muller et aL, Pharmacol. Rev. 70:712-746 (2018)). Later combinations included cocktails of weight-reducing substances, including d- amphetamine or related analogs (e.g., Diethylpropion, Fenfluramine, Sibutramin, or Fenproporex), thyroid hormones, diuretics, laxatives, Chlorthalidon, Ephedrine, and / or Phenolphthalein. Substances such as Digitalis, Belladonna, benzodiazepines, barbiturates, corticosteroids, cardiac glycosides, beta-blocker, and potassium were common additives used to counteract or mask adverse cardiovascular effects of the drug cocktail (Cohen PA. et al. , Am J Public Health 102: 1676-1686 (2012)).

[0507] The combination of the amphetamine phentermine and topiramate was approved by the FDA in 2012. Topiramate is a sulfamate-substituted monosaccharide derived from D-fructose, commonly used to treat epilepsy and migraine. Although the mechanism of how the combination improves systemic metabolism is unknown, placebo-normalized weight loss ranges from 5.9%-9.6% (Muller et al., Pharmacol. Rev. 70:712-746 (2018)).

[0508] The weight loss combination of the medications naltrexone and bupropion combines two medications approved for indications other than weight loss. Bupropion, a dopamine-norepinephrine reuptake inhibitor is approved to treat depression and aid in smoking cessation, and naltrexone, an opioid receptor antagonist is approved to treat alcohol and opioid dependence. The naltrexonebupropion combination is marketed under the trade name Contrave® and is FDA approved for chronic weight management. i. Cannabinoid receptor antagonists

[0509] Rimonabant, an endocannabinoid 1 receptor (CB1) antagonist, acts by modulating neurons in both homeostatic and hedonic feeding circuits, leading to placebo- subtracted weight loss of ~2.6-6.3 kg (Muller etal., Pharmacol. Rev. 70: 712-746 (2018); Pi-Sunyer et al. JAMA 295:761-775 (2006)). Rimonabant was discontinued in 2009 due to serious adverse psychiatric effects (Sam et al., J Obes 2011 :432607 (2011)). j. GIPR agonists and GIPR / GLP1R combination agonists

[0510] Glucose-dependent insulinotropic polypeptide (GIP; SEQ ID NO: 10) is a hormone involved in blood sugar control. GIP receptor (GIPR) agonists have been shown to decrease food intake, increase energy expenditure, decrease body weight and to improve glucose handling in preclinical studies (Mroz et al., Mol. Metab. 20:51-62 (2019); Zhang et al., Cell Metab. 33:833-844.e5 (2021)). GIP is known to be upregulated following Rou-en-Y gastric bypass.

[0511] Body weight loss associated with GLP-1 agonist treatment is enhanced when GLP-1 and GIP are co-administered (Matthias Tschbp oral presentation at ADA (American Diabetes Association), 2011; Tschbp et al., Diabetes 66: 1766-1769 (2017)). GIPR / GLP1R dual agonists and co-admini strati on of GLP-1R agonists and GIPR agonists have demonstrated metabolic benefits and reduced body weight in mice when compared to GLP1R agonists (Coskun et al., Mol. Metab. 18: 3-14 (2018); Finan et al., Sci. TranslMed. 5, 209ral51 (2013)). Thus, in addition to improving blood glucose control, GIP can also enhance GLP-1 -mediated body weight loss. GIP / GLP1 dual agonists include Tirzepatide, GIP / GLP peptide I, GIP / GLP peptide II, and NN9709.

[0512] Tirzepatide is a combination medication that activates the GLP-1 and GIP receptors; tirzepatide contains GLP-R and GIPR agonists). Tirzepatide (available under the trademark Mounjaro®) is a dual glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptor agonist. The drug is manufactured by Eli Lilly & Co. and was approved in May 2022. Tirzepatide works similarly to the other drugs in the GLP-1 receptor agonist family, but it has additional effects that appears to give it a slight edge. It has a dual-action design, mimicking the action of two incretin hormones involved in blood glucose control: Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). This is the first-in-class medicine to act on both of these receptors.

[0513] Tirzepatide is a 39 amino acid peptide GLP-1 receptor agonist based on the GIP sequence. It contains 2 noncoded amino acids in positions 2 and 13, a C-terminal amide, and a lysine residue at position 20 that is attached to a 1, 20 eicosanoic acid via a linker. Studies of tirzepatide were conducted through two placebo-controlled trials (SURP ASS-1 and -5), three trials in combination with metformin, sulfonylureas and / or SGLT2 inhibitors (SURPASS-2, -3, -4), and two additional trials conducted in Japan. A phase III trial of tirzepatide in patients with type 2 diabetes of excess weight revealed a body weight reduction of >15% relative to 9% in patients treated with Img semaglutide (Frias etal., N. Engl. J. Med. 385, 503-515 (2021); URL: fda.gov / news- events / press-announcements / fda-approves-novel-dual-targeted-treatment-type-2- diabetes; published May 13, 2022).

[0514] A separate study looking at efficacy of tirzepatide (5 mg, 10 mg, 15mg) was conducted via the SURMONT-1 clinical trial, where participants received once a week GLP-1 receptor agonist. Patients receiving the GLP-1 receptor agonist lost up to 22.5% (52 pounds or 24 kg) of their body weight. The study enrolled 2,539 participants across the US, Argentina, Brazil, China, India, Japan, Mexico, Russia, and Taiwan and was the first phase 3 global registration trial evaluating the efficacy and safety of tirzepatide in adults with obesity, or overweight with at least one comorbidity, who do not have diabetes. Study participants were assessed for mean percent change in body weight at week 72 and the percentage of patients achieving >5% weight loss from baseline to week 72 as primary end points. Secondary end points included the percentage of patients achieving greater than or at 10 and 20% weight loss from baseline to week 72. The study results are set forth in the table below:

[0515] The study results show that participants taking tirzepatide achieved average weight reductions of 16.0% (35 lb. or 16 kg on 5 mg), 21.4% (49 lb. or 22 kg on 10 mg) and 22.5% (52 lb. or 24 kg on 15 mg), compared to placebo (2.4%, 5 lb. or 2 kg). Additionally, 89% (5 mg) and 96% (10 mg and 15 mg) of people taking tirzepatide achieved at least 5% body weight reductions compared to 28% of those taking placebo. Tirzepatide met both co-primary endpoints of superior mean percent change in body weight from baseline and greater percentage of participants achieving body weight reductions of at least 5% compared to placebo. The study also achieved all key secondary endpoints at 72 weeks. There was no data on actual fat loss or muscle mass loss or retention. Safety and adverse reactions also were assessed; the investigators found that 18%, 17%, 9%, 7% and 5% of trial participants experienced nausea, diarrhea, vomiting, constipation, and abdominal pain, respectively.

[0516] Tirzepatide (sold under the trademark Mounjaro®) was approved by the US Food and Drug Administration as a once-weekly subcutaneous injection to treat adults with type 2 diabetes, to improve blood sugar levels. As detailed above, and herein, Tirzepatide showed significantly better long-term blood sugar control (i.e., A1C) and weight loss compared to semaglutide.

[0517] Glucagon and glucagon agonists have been shown to promote satiety and to increase energy expenditure when used in combination in both rodents and human (Kleinert et al., Int J Mol Set. 20(21): 5407 (2019)). A triagonist peptide that act as an agonist of the glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon receptors was previously described to effect weight loss in animal models (Finan et al., Nat Med 21(l):27-36 (2015)). k. GLPIR / glucagon dual agonists

[0518] GLP-1 receptor (GLP1R) agonists in combination with glucagon (GcgR) agonists participate in several mechanisms of body weight reduction, including appetite suppression, thermogenesis and lipolysis, while minimizing the risk of hyperglycemia (Muller et al., Nat Rev Drug Discov 21 (3):201 -223 (2021)). Combination strategies of these agonists have included Cotadutide (MEDI0382), BI 456906, Efinopegdutide (LAPSGLP / GCG), and oxyntomodulin (OXM; SEQ ID NO:4). Several GIP / GLP1 / glucagon tri-agonists also have been developed for weight loss, including HM15211 (LAPSTriple Agonist), GGG tri-agonist, LY3298176 and NN9423.

[0519] 1. Summary

[0520] With few exceptions, the field of pharmaceutically assisted weight loss has focused on monotherapy and the most effective pharmaceuticals include versions of a GLP-1 agonist. Although approved for use in human, several therapeutics that show efficacy are not widely used because of adverse side effects or toxicity associated with administration of the medications at the therapeutic or FDA approved dosages. For example, patients taking Rimonabant experience adverse psychiatric effects, patients taking thyroid hormones and amphetamines experience adverse cardiac effects, patients taking lorcaserin show increased occurrence of cancer, and patients taking orlistat experience gastrointestinal issues.

[0521] A combination of therapeutics administered in accord with the rotational protocol / regimen described herein incorporates pharmacological principles known to occur after a gastric bypass, as well as overcoming tolerance and / or desensitization to medications due to receptor downregulation / desensitization to medications. The combination therapy can be administered at a lower dosage than the monotherapeutic dosage to decrease side effects associated with administration of the monotherapy. The combination therapy is rotated at intervals (ie., monthly, bi-monthly, or trimonthly) wherein the patient is not administered an individual therapeutic for an extended period; the finite time period for administration of the medication decreases the time for developing adverse side effects (z.e., severe adverse effects) or downregulation of the pathways activated by the medications.

[0522] The rotational combinatorial therapy described herein can include a combination of a plurality of different known therapeutics and / or treatments. For example, the combination for treatment of obesity or for weight loss can include more than one previously characterized weight loss treatment. In some examples, the rotational combinatorial therapy can include one or more FDA-approved monotherapy options, such as, for example, phentermine (sold under the trademark Adipex-P®), orlistat (sold under the trademark Xenical®), lorcaserin (sold under the trademark Belviq®), liraglutide (sold under the trademark Saxenda®), phenterminetopiramate (sold under the trademark Qysmia®), naltrexone-bupropion (sold under the trademark Contrave ®), tirzepatide (sold under the trademark Mounjaro®), and semaglutide (sold under the trademark Wegovy®) medications. The rotational combinatorial therapy provided herein also can be used for treatment of any condition with a previously characterized mechanism of action or pathway, where a combination of therapeutics can be developed for treating the disease or disorder or condition. For example, a treatment of obesity or overweight, for which a plurality of different known molecular targets for treatment or multiple known molecular or cellular pathways are known to be involved in disease progression; a combinatorial therapy including one or more of amphetamines, lipase inhibitors, serotonergic agonists, opioid agonists, Glucagon-like peptide-1 receptor (GLP1R) agonists, mitochondrial uncouplers, thyroid hormones, diuretics, dopamine-norepinephrine reuptake inhibitors, cannabinoid receptor antagonists, GIP agonists, GIPR agonists, and GLPIR / glucagon dual agonists can be administered in a rotational combinatorial therapy to target multiple pathways that are associated with a disease or disorder, but where a pharmacotherapy has not yet been developed.

[0523] 4. Surgical Treatments

[0524] The most effective treatment for obesity has been bariatric surgery (Kissler et al., (2013) Semin Nephrol. 33(l):75-89). Bariatric procedures include, for example, laparoscopic adjustable gastric banding, Roux-en-Y gastric bypass, vertical-sleeve gastrectomy, and biliopancreatic diversion. Surgery for severe obesity is associated with long-term weight loss, improvement in obesity -related conditions, and decreased overall mortality. A resolution of co-morbidities, such as diabetes, hypertension, fatty liver, urinary distress, and hyperthyroidism, also have been shown to occur following bariatric surgery (Susmallian et al., Medicine 98(3): el3824 (2019)).

[0525] Weight loss of between 12% and 35% (depending on the type of procedure performed) is expected at 1-2 years after surgery. The average weight loss in clinical practice after 2 years for adjustable gastric banding is about 20%, weight loss after Roux-en-Y gastric bypass is about 30%, and weight loss after biliopancreatic diversion or duodenal switch is about 35% (Cummings et al., (2004) J Clin Endocrinol Metab 59:2608-2615). Complications occur in about 17% of cases and reoperation is needed in 7% of cases (Chang etal., JAMA Surgery (Meta-analysis, Review) 149 (3): 275- 87 (2014); doi: 10.1001 / jamasurg.2013.3654). Weight loss after bariatric surgery results, not only from the bypass and other physical changes, but also from factors other than the physical changes to the gastrointestinal tract and calorie red...

Claims

What is Claimed:

1. A delivery vehicle, comprising a combination of therapeutic peptides, wherein: the peptides are linked to the surface directly or indirectly via a linker or are part of the surface of the delivery vehicle or are in the delivery vehicle; the surface of the delivery vehicle optionally is modified for linkage of the peptides; the combination of peptides comprises at least three different peptides; at least two of the peptides target different pathways and / or have different activities; and the peptides target pathways involved in obesity and / or diabetes, or have activity for treating obesity and / or diabetes or other obesity comorbidity.

2. The delivery vehicle of claim 1, further comprising a small molecule drug for treatment of obesity or an associated comorbidity.

3. A composition, comprising a mixture of delivery vehicles, wherein: each delivery displays at least one therapeutic peptide on the surface and / or contains the at least one peptide; and the composition comprises delivery vehicles that are selected so that the composition comprises at least three different peptides.

4. The composition of claim 3 that is formulated for injection.

5. The delivery vehicle or composition of any of claims 1-4, wherein the delivery vehicle selected from among a liposome, lipid nanoparticle, exosome, and extracellular vesicle.

6. The delivery vehicle or composition of any of claims 1-5, wherein the peptides comprise fat loss and muscle enhancement peptides, wherein muscle enhancement polypeptides reduce or eliminate loss of muscle mass or increase muscle mass.

7. The delivery vehicle or composition of any of claims 1-6, wherein two of the peptides are fat loss peptides, and one is a muscle enhancement polypeptide.

8. The delivery vehicle or composition of any of claims 1-7, wherein the polypeptides for fat loss (PeptideFL) are selected from among:PeptideFLl=GLP-l,PeptideFL 2=Adiponectin,PeptideFL3=Leptin,PeptideFL4= Oxyntomodulin,PeptideFL5=PYY,PeptideFL6= Amylin,PeptideFL7=Pancreatic peptide,PeptideFL8=Enterostatin / GIP (Gastroinhibitory Polypeptide),PeptideFL9= Glicentin,PeptideFL 10 = Glucagon,PeptideFL 11=GRPP,PeptideFL 12=HGH 176-191,Peptide FL13= CCK,PeptideFL 14= Neurotensin,PeptideFL15= Secretin,PeptideFL 16= IP1, andPeptideFL17= MPGF (major proglucagon fragment).

9. The delivery vehicle or composition of any of claims 1-8, wherein the peptides for muscle enhancement (PeptideME) are selected from among:PeptideMEl=Sermorelin;PeptideME2=Tesamorelin; andPeptideME2=IGFl or human growth hormone to induce IGF1.

10. The delivery vehicle or composition of any of claims 1-9, wherein: a) the polypeptides for fat loss are selected from among:PeptideFL 1=GLP-1,PeptideFL 2=Adiponectin,PeptideFL3=Leptin,PeptideFL4= Oxyntomodulin,PeptideFL5=PYY,PeptideFL6= Amylin,PeptideFL7=Pancreatic peptide,PeptideFL8=Enterostatin / GIP (Gastroinhibitory Polypeptide),PeptideFL9= Glicentin,PeptideFLIO = Glucagon,PeptideFLl 1=GRPP,PeptideFLl 2=HGH 176-191,Peptide FL13= CCK,PeptideFLl 4= Neurotensin,PeptideFLl 5= Secretin,PeptideFLl 6= IP1, andPeptideFLl 7= MPGF (major proglucagon fragment); and b) the peptides for muscle enhancement are selected from among:PeptideMEl=Sermorelin;PeptideME2=Tesamorelin; andPeptideME2=IGFl or human growth hormone to induce IGF1.

11. The delivery vehicle or composition of any of claims 1-10, wherein the peptides comprise GLP-1, Oxyntomodulin, enterostatin / GIP (Gastroinhibitory Peptide); and Sermorelin.

12. The delivery vehicle or composition of any of claims 1-11, wherein the delivery vehicle is a liposome.

13. The delivery vehicle or composition of claim 12, wherein the liposome is a large multilamellar vesicle (LMV).

14. The delivery vehicle or composition of any of claims 1-13, wherein: the delivery vehicle comprises a liposome; and the liposome comprises phospholipids.

15. The delivery vehicle or composition of claim 14, wherein the phospholipids are selected from one or more of phosphatidyl choline (PC), phosphatidyl ethanol amine (PE), and phosphatidyl serine (PS), and phosphatidic acid (PA).

16. The delivery vehicle or composition of any of claims 1-15, wherein: the delivery vehicle is a liposome; and the phospholipid is from a natural source.

17. The delivery vehicle or composition of any of claims 1-16, wherein: the delivery vehicle comprises a liposome; the liposome comprises cholesterol; andthe amount of cholesterol is sufficient to increase the permeability of the liposome compared to the liposome that does not contain the cholesterol.

18. The delivery vehicle or composition of any of claims 1-17, wherein: the delivery vehicle is a liposome; the liposome comprises cholesterol; and the molar percentage of cholesterol in the liposome is less than 60%, 50%, 40%, 30%, 20%, 10%, or less.

19. The delivery vehicle or composition of any of claims 1-18, wherein: the delivery vehicle is a liposome; and the liposome comprises lipids modified with a reactive group for coupling with a peptide or peptide modified with a reactive group.

20. The delivery vehicle or composition of any of claims 1-19, wherein: the delivery vehicle is a liposome; the liposome comprises lipids modified with a reactive group for coupling with a peptide or peptide modified with a reactive group; and the reactive group for the coupling reaction is selected from among amino, thiol, maleimide, bromo- or iodoacetyl, pyridyl di thio, carboxylic, hydrazide, p- nitrophenyl carbonate, azide, and alkyne.

21. The delivery vehicle or composition of any of claims 1-20, wherein: the delivery vehicle is a liposome; the liposome comprises lipids modified with a reactive group for coupling with a peptide or peptide modified with a reactive group; the reactive group is an amino group that forms an amide bond with an activated carboxylic ester, or is a thiol group that binds with maleimide, bromo- or iodo acetyl, pyridyldithio groups, or is a hydrazide that bind with carbonyl groups, or is p-nitrophenyl carbonate that reacts with amines forming an amide bond, or comprise azide and alkyne group that bind with each other in the presence of a copper ion catalyst, to attach the protein to the liposome.

22. The delivery vehicle or composition of claim 21, wherein the peptides are linked to the liposomes via the bonds formed by reaction of the reactive groups.

23. The delivery vehicle or composition of any of claims 1-22, wherein: the delivery vehicle is a liposome; andthe peptide and liposome are linked via an amide / peptide bond or linker, a thioester bond or linker, a disulfide bond or linker, a hydrazone bond or linker, a carbamate bond or linker, and a 1,2, 3 -triazole linker.

24. The delivery vehicle or composition of any of claims 1-20, wherein: the delivery vehicle is a liposome; wherein the peptide and / or liposome is / are PEGylated for linking the peptide to the liposome.

25. The delivery vehicle or composition of any of claims 1-20, wherein: the delivery vehicle is a liposome; and the liposome and / or peptide is PEGylated for linkage of the peptide to the liposome.

26. The delivery vehicle or composition of any of claims 1-25, wherein: the delivery vehicle is a liposome; the linkage of the peptide to the liposome comprises a spacer.

27. The delivery vehicle or composition of claim 26, wherein the spacer comprises polyethylene glycol (PEG) and / or and an oligonucleotides bound to the liposome and to the peptide linked to a complementary oligonucleotide.

28. The delivery vehicle or composition of any of claims 1-26, wherein: the delivery vehicle is a liposome; the liposome comprises streptavidin bound to biotin-linked peptide or the streptavidin is bound to the liposome and to biotin-linked peptide.

29. The delivery vehicle or composition of claim 28, wherein: the liposomes are coated with a monolayer of streptavidin and linked to peptides functionalized with biotin-PEG-NHS to form liposomes that display the peptides upon mixing these peptide derivatives with streptavidin liposomes.

30. The delivery vehicle or composition claim 28, wherein: the linkage of the peptide to the liposome comprises a PEG spacer; one end of the spacer is attached biotin, and the other comprises a reactive group, such as an NHS active ester, that easily forms an amide bond with the PEG.

31. A combination of the delivery vehicle or composition of any of claims 1-30 that comprises one or more small molecule drug(s), wherein:the small molecule drug enhances weight loss or treats a comorbidity associated with obesity; and the small molecule is formulated in or with the delivery vehicle or is for administrations separately from the delivery vehicle.

32. The combination of claim 31, wherein the small molecule drug is / are selected from among one or more of: Phentermine, Topiramate, Metformin, Empagliflozin, Dapagliflozin, Bexagliflozin, Ertugliflozin, Linagliptin, Canagliflozin, NS-2330, Liothyronine, Diethylpropion, Zonisamide, Albuterol, Clenbuterol, Levothyroxine, Naltrexone, Orlistat, Testosterone Cypionate, and Testosterone Enanthate.

33. The combination of any of claims 30-32, wherein the delivery vehicle and small molecule drugs are for administration together in a single composition, sequentially, or intermittently.

34. A container, comprising the delivery vehicle or composition of any of claims 1-30.

35. The container of claim 34 that comprises a pen or a syringe for administering the delivery vehicle.

36. The container of claim 34 or claim 35 that is an autoinjector.

37. The container of any of claims 34-36 that is a multicompartment container, wherein one compartment contains the delivery vehicles or a mixture of different delivery vehicles, which is / are lyophilized; and the other contains a pharmaceutically acceptable vehicle for mixing with the delivery vehicles to produce a composition for injection.

38. A pharmaceutical composition, comprising the composition or delivery vehicle of any of claims 1-30.

39. The delivery vehicle, composition, container, or pharmaceutical composition of any of claims 1-38 for use for treating obesity.

40. A method of treatment of obesity or diabetes, comprising administering a delivery vehicle or composition of any of claims 1-30 and 38.

41. A method of treating a disease, disorder, or condition, comprising administering and rotating combinations of drugs, wherein: drugs include peptides and small molecule drugs;the combinations of drugs comprise delivery vehicles or compositions of any of claims 1-30; the disease, disorder, or condition is obesity and / or diabetes; at least two combinations are rotated for each cycle of treatment; and treatment comprises at least two cycles.

42. A regimen for treating a disease, disorder, or condition, comprising combinations of peptides for use in a rotational combinatorial regimen, wherein: the disease, disorder, or condition is obesity or diabetes, a chronic disease, disorder, or condition that requires treatment for at least 6 months; each combination comprises at least two different peptides that target different pathways or intervention targets; and the combinations of peptides comprise a delivery vehicle or composition of any of claims 1-33.

43. The regimen of claim 42, further comprising a small molecule drug for weight loss and / or for treating a comorbidity associated with obesity.

44. The regimen of claim 43, wherein the small molecule drug is selected from one or more of Phentermine, Topiramate, Metformin, Empagliflozin, Dapagliflozin, Bexagliflozin, Ertugliflozin, Linagliptin, Canagliflozin, NS-2330, Liothyronine, Diethylpropion, Zonisamide, Albuterol, Clenbuterol, Levothyroxine, Naltrexone, Orlistat, Testosterone Cypionate, and Testosterone Enanthate, and a statin.

45. The regimen of claim 44, wherein the small molecule drug is incorporated in or on the delivery vehicle and / or the small molecule drug is coadministration, simultaneously, sequentially, or intermittently with the delivery vehicle(s).

46. The method of claim 41 or regimen of any of claims 42-45, comprising a plurality of cycles of administration of different combinations of peptides and optionally the small molecules, wherein: each combination is administered at least once a cycle; a cycle comprises administration of each combination at least once; a cycle comprises at least two different combinations; a cycle can be repeated a plurality of times; andat least one of the combinations comprises at least two different drugs that target different targets or pathways.

47. The method or regimen of any of claims 41-46, wherein each cycle comprises one delivery vehicle that comprises at least 3 different peptides.

48. The method or regimen of any of claims 41-46, wherein each cycle comprises a mixture of delivery vehicles; each delivery vehicle comprises at least peptide, whereby at least three different peptides are administered in each cycle; and the different delivery vehicles are administered together or serially.

49. The method or regimen of any of claims 41-48, wherein the different vehicles are administered within 24 hours of each other.

50. A method for treating obesity, comprising administering a delivery vehicle of any of claims 1-30, wherein each peptide targets a different pathway or different target for intervention for treatment of obesity.

51. The method or regimen of any of claims 41-50, wherein the combination of peptides mimic effects of gastric bypass.

52. The method or regimen of any of claims 41-51, wherein the combination of peptides on the delivery vehicle comprises at least three selected from among: a peptide that inhibits gastric emptying selected from among one or more of GLP1, Amylin, and Pancreatic Polypeptide Therapeutic; a peptide drug that enhances satiety comprising one or more drugs selected from among glucagon-like peptide- 1 (GLP-1), peptide YY (PYY), amylin, enterostatin / gastric inhibitory peptide (GIP), cholecystokinin (CCK), and glicentin; a peptide that increases insulin release and / or sensitivity comprising one or both of GLP1 and adiponectin; and a peptide that modulates energy expenditure comprising leptin, oxyntomodulin, and glicentin.

53. The delivery vehicle, composition, method, or regimen of any of claims 1-51, wherein the vehicle comprises or displays combinations of peptides selected from among combinations of drugs for fat loss + a drug for muscle enhancement:a) GLPl / GIPl / Oxyntomodulin + Sermorelin or Tesamorelin or IGF1; b) GLPl / GIPl / Amylin + Sermorelin or Tesamorelin or IGF1; c) GLP1 / GIP1 / Glucagon + Sermorelin or Tesamorelin or IGF1; d) GLP1 / GIP1 / CCK+ Sermorelin or Tesamorelin or IGF1; e) GLP1 / GIP1 / PYY + Sermorelin or Tesamorelin or IGF1; and f) GLP1 / GIP1 / Leptin + Sermorelin or Tesamorelin or IGF1.

54. The delivery vehicle, composition, method, or regimen of claim 53, wherein the amino acid sequence of each of the peptides for each of a)-f) are as set forth set forth in the following SEQ IDs, or are variants thereof that have at least 90% or at least 95% sequence identity thereto and retain activity for effecting fat loss or muscle enhancement: a) SEQ ID NOs: 1, 10, 4 or 47 + SEQ ID NOs: 5, 8, 44; b) SEQ ID NOs: 1, 10, 7 + SEQ ID NOs: 5, 8, 44; c) SEQ ID NOs: 1, 10, 27 + SEQ ID NOs: 5, 8, 44; d) SEQ ID NOs: 1, 10, 11 + SEQ ID NOs: 5, 8, 44; e) SEQ ID NOs: 1, 10, 6 or 37 + SEQ ID NOs: 5, 8, 44; and f) SEQ ID NOs: 1, 10, 3 + SEQ ID NOs: 5, 8, 44.

55. The delivery vehicle, composition, method, or regimen of any of claims 1-52 that includes a delivery vehicle that comprises a peptide that results in muscle enhancement and / or a drug or polypeptide that inhibits the myostatin pathway.

56. The delivery vehicle, composition, method, or regimen, wherein the delivery vehicle comprises one or more of sermorelin, tesamorelin and / or growth hormone, and testosterone.

57. The method or regimen of any of claims 41-56, further comprising administering a delivery vehicle that comprises a peptide that promotes intestinal smooth muscle relaxation, such as vasoactive intestinal peptide (VIP).

58. The method or regimen of any of claims 41-57 wherein: the peptides are selected from among:GLP-1, Adiponectin, leptin, oxyntomodulin, peptide tyrosine-tyrosine (PYY), amylin, pancreatic peptide, enterostatin / gastric inhibitory polypeptide (GIP), cholecystokinin (CCK), vasoactive intestinal peptide (VIP), glicentin, human growth hormone or an active portion thereof or an analog of human growth hormone or anactive portion thereof, ephedrine, caffeine, aspirin (EC A), oxyntomodulin, neuropeptide Y (NPY), antimicrobial peptide 2 (LEAP2), vaccine CYT009-GhrQb, the peptide-binding compound Nox-Bl 1, and the ghrelin analog AZP-531 (SEQ ID NO: 15); and / or the peptides are a GLP-1 agonist, an appetite suppressant, a thyroid hormone, a carbonic anhydrase inhibitor, an alpha-glucosidase inhibitor, a dipeptidyl peptidase- R (DPP-4) inhibitor, a sodium-glucose co-transporter 2 (SGLT2) inhibitor, a muscle enhancer, drugs that modulate energy expenditure, a GLP-1 agonist, peptides that increase gastric inhibitory polypeptide (GIP), drugs that modulate GIP2, and mitochondrial uncouplers; and the peptides are combined by displaying a plurality on each delivery vehicle or by mixing delivery vehicles that display different peptides.

59. The method or regimen of any of claims 41-58, wherein the peptides and drugs are selected from among: dulaglutide, bydureon, semaglutide, exenatide, liraglutide, phentermine, liothyronine, topiramate (carbonic anhydrase inhibitor), acarbose (alpha-glucosidase inhibitor), sitagliptin (dipeptidyl peptidase-4 (DPP -4) inhibitor), canagliflozin (sodium-glucose co-transporter 2 (SGLT2) inhibitor), dapagliflozin ( SGLT2 inhibitor), sermorelin, mirabegron (beta-3 adrenergic agonist), and amylin.

60. The method or regimen of any of claims 41-59, wherein the peptides and drugs are selected from among: a GLP-1 agonist, phentermine, thyroid hormone, carbonic anhydrase inhibitor, carbonic anhydrase inhibitor, alpha-glucosidase inhibitor, DPP-4 inhibitor, SGL2 inhibitor, muscle enhancer, and an appetite suppressant.

61. The delivery vehicle, composition, method, use, or regimen of any of claims 1-58, further comprising a mitochondrial uncoupler, wherein the mitochondrial uncoupler is provided linked to a delivery vehicle or mixed in the composition or administered as a free molecule not bound to a delivery vehicle.

62. The delivery vehicle, composition, method, use, or regimen of claim 59, wherein the mitochondrial uncoupler is selected from among uncoupling protein 1 (UCP1), a catecholamine, and a small molecule uncoupler, such as 2,4-dinitrophenol(DNP) and BAM15 (N5,N6-bis(2-Fluorophenyl)[l,2,5]oxadiazolo[3,4-b]pyrazine- 5,6-diamine).

63. The delivery vehicle or composition of any of claims 1-33 for use for treating obesity and / or diabetes.

64. A method of preparing a PEGylated peptide, comprising preparing a PEGylated lysine and adding it to peptide during solid phase synthesis.

65. The method of claim 64, wherein the PEGylated lysine is prepared by reacting the epsilon-amino group of the lysine with a carboxyl terminated PEG moiety via an amide group to produce a PEGylated lysine.

66. The method of claim 65, further comprising adding the PEGylated lysine to a peptide during solid phase synthesis of the peptide.

67. The method of any of claims 64-66, wherein the PEG moiety comprises a functional group for conjugation with a reactive group, such as one on a liposome.

68. The method of claim 67, wherein the functional group is acetylene that is reacted with azide in the liposome (click chemistry).

69. A method of conjugating a polypeptide to a liposome, comprising adding a lysine to a polypeptide during solid phase synthesis by reacting the epsilon-amino group of lysine with a carboxyl terminated PEG moiety via an amide group to produce a peptide comprising a lysine comprising the PEG moiety, which comprises a functional group for conjugation with the liposome; and conjugating the PEGylated peptide to the liposome.

70. The method of claim 69, wherein the functional group is acetylene that is reacted with azide in the liposome (click chemistry).

71. The method of any of claims 64-70, wherein the peptides comprise a is a GLP-1 agonist and a muscle enhancer peptide.

72. The method of claim 71, wherein the peptide is selected from among:GLP-1 pathway agonist, adiponectin, leptin, oxyntomodulin, PYY (peptide YY), amylin, pancreatic peptide, enterostatin / gastric inhibitory polypeptide (GIP), glicentin, glucagon, GRPP (glicentin-related pancreatic polypeptide), HGH (human growth hormone), CCK (cholecystokinin), neurotensin, secretin, IIP (myo-inositol 1- phosphate), and MPGF (major proglucagon fragment); and / orthe peptides are sermorelin, tesamorelin, and IGF1 (or HGH).

73. The method of claim 69, comprising preparing at least three pegylated polypeptides and linking the peptides to one liposome or each to a different liposome, or two to one liposome and the third on a separate liposome.

74. The method of any of claims 64-73, wherein the peptides linked to the liposome or to different liposomes comprise a polypeptide for fat loss, and a polypeptide for muscle enhancement or a polypeptide that inhibits the myostatin pathway.

75. The method of claim 74, wherein: a) the polypeptides for fat loss (FL) are selected from among:PeptideFL 1=GLP-1,PeptideFL 2=Adiponectin,PeptideFL3=Leptin,PeptideFL4= Oxyntomodulin,PeptideFL5=PYY,PeptideFL6= Amylin,PeptideFL7=Pancreatic peptide,PeptideFL8=Enterostatin / GIP (Gastroinhibitory Polypeptide),PeptideFL9= Glicentin,PeptideFL 10 = Glucagon,PeptideFL 11=GRPP,PeptideFL 12=HGH 176-191,Peptide FL13= CCK,PeptideFL 14= Neurotensin,PeptideFL15= Secretin,PeptideFL 16= IP1, andPeptideFL 17= MPGF (major proglucagon fragment); and b) the peptides for muscle enhancement (ME) are selected from among:PeptideMEl=Sermorelin;PeptideME2=Tesamorelin; andPeptideME2=IGFl (or human growth hormone to induce IGF1).

76. A liposome, comprising one or more of a fat loss peptide and a muscle enhancement peptide, wherein the liposome is produced by a method of any of claims 69-75.

77. The delivery vehicle, composition, use, method, regimen, or liposome of any of claims 1-64 and 76, further comprising a small molecule drug, wherein the drug is for weight loss and / or a co-morbidity associated.

78. The delivery vehicle, liposome, use, method, or liposome of claim 77, wherein the co-morbidity is metabolic syndrome.

79. The delivery vehicle, composition, use, method, regimen, or liposome of claim 77 or claim 78, wherein the co-morbidity is diabetes, hypertension, dyslipidemia, and heart disease.

80. The delivery vehicle, composition, method, regimen, or liposome, wherein the small molecule drug is Phentermine, Topiramate, Metformin, Empagliflozin, Dapagliflozin, Bexagliflozin, Ertugliflozin, Linagliptin, Canagliflozin, NS-2330, Liothyronine, Diethylpropion, Zonisamide, Albuterol, Clenbuterol, Levothyroxine, Naltrexone, Orlistat, Testosterone Cypionate, and Testosterone Enanthate, and a statin.

81. The delivery vehicle, composition, use, method, regimen, or liposome of any of claims 1-64, and 76-80, wherein the delivery vehicle comprises phentermine or wherein phentermine is combined with a composition comprising the delivery vehicle or liposome for combination therapy.

82. The delivery vehicle, composition, use method, regimen, or liposome of any of claims 1-81, wherein the delivery vehicle is an extracellular vesicle or an LPN, or an exosome, or a liposome.

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