Nanoparticle complexes and therapeutic uses therefor

Abstract

Orally administrable nanoparticle complexes of biological molecules and biopolymers are described herein, and include formulations for the oral administration of peptides, proteins, nucleic acids, and antibodies.

Description

[0001] 1392-17709

[0002] NANOPARTICLE COMPLEXES AND THERAPEUTIC USES THEREFOR

[0003] TECHNICAL FIELD

[0004] The invention described herein pertains to nanoparticle complexes of biological molecules and biopolymers.

[0005] BACKGROUND

[0006] Biological molecules, both endogenous and exogenous to the host animal, hold a great promise as potential therapies for a wide variety of medical conditions. However, many biological molecules, such as, for example, peptides, proteins, antibodies, and nucleic acids are not administered orally due to exceedingly low bioavailability. Orally administered biological molecules are also prone to degradation in the upper gastrointestinal (GI) tract due to low pH and enzymatic activity. Notable examples are found in the GLP-1 agonist family of peptide drugs, such as Mounjaro® (tirzepatide) and Ozempic® (semaglutide) for diabetes, and Zepbound® and Wegovy® for obesity. Those drugs show oral bioavailability of «0.1%, and are therefore, cannot be administered orally, and are only administered by injection. Similarly, insulin for treating diabetes, and antibody drugs such as Humira® (adalimumab) and Stelara® (usetkinumab) for treating arthritis and other auto-immune disorders are also only administered by injection.

[0007] Rybelsus® is an oral formulation of semaglutide and sodium N-(8-[2-hydroxylbenzoyl] aminojcaprylate (SNAC), but still suffers from low bioavailability of only about 0.4-0.8% and has to be administered daily, and in a fasted state. In addition, Rybelsus® is released in the stomach and is directly absorbed through the stomach epithelial layer. That passive absorption results in several adverse side effects, including nausea, stomach (abdominal) pain, diarrhea, decreased appetite, vomiting, and constipation.

[0008] It has been reported that in addition to problems associated with the uptake in the stomach, the pharmacokinetic (PK) profile of Rybelsus® also contributes to adverse side effects. Injected Mounjaro® and Ozempic® have a relatively flat PK profile, with a relatively low Cmax and / or relatively late Tmax, and consistent AUC over 96 hours. That PK profile allows for those GLP-1 agonists to be used in escalating dose profiles that slowly load the patient with drug over 6-10 weeks. In contrast, Rybelsus® exhibits an early Tmax, high Cmax, and rapidly changing AUC over 96 hours, which does not effectively mirror the profile needed for GLP-1 agonists.

[0009] Oral administration, especially when compared to parenteral administration such as injection, is a desired route of administration for many reasons, not the least of which include consistent and accurate dosing, higher patient compliance, and increased patient convenience. 1392-17709

[0010] For example, it has been reported that 42% of patients fail to maintain diabetes treatment due to injection concerns using an injectable GLP1. Discontinuing treatment often results in GLP1 withdrawal causing patients to regain 30-50% of lost weight and leads to a 33% higher likelihood of adverse cardiovascular events.

[0011] New compositions and formulations are needed to provide for oral administration of biological molecules that survive degradation in the upper GI as well as exhibit bioavailability in the host animal of at least 1%, and greater. Higher bioavailability allows for the administration of lower doses having comparable therapeutic effectiveness, and fewer adverse side effects.

[0012] SUMMARY OF THE INVENTION

[0013] It has been unexpectedly discovered herein that nanoparticle complexation of biological molecules with biopolymers provides a generalized approach to adapting biomolecules for oral administration.

[0014] In one illustrative embodiment of the invention, compounds and compositions are described herein that include one or more biomolecules and one or more biopolymers, where the one or more biomolecules and the one or more biopolymers form nanoparticle complexes.

[0015] In another illustrative embodiment, pharmaceutical compositions are described herein that include nanoparticle complexes formed from one or more biomolecules and the one or more biopolymers.

[0016] In another embodiment, unit doses and unit dosage forms are described herein that include a therapeutically effective amount of one or more nanoparticle complexes formed from one or more biomolecules and the one or more biopolymers for use in treating a host animal. It is to be understood that the compositions may include other components and / or ingredients, including, but not limited to, other therapeutically active compounds, and / or one or more carriers, vehicles, diluents, adjuvants, excipients, and the like, and combinations thereof.

[0017] In another embodiment, methods for treating host animals with a medical condition are also described herein, where the methods include administering one or more of the compounds and / or compositions and / or unit doses described herein to a host animal. In another embodiment, the compounds and compositions are used in the manufacture of a medicament for treating the host animal.

[0018] It is to be understood herein that the compounds, compositions, unit doses, and methods described herein may be used alone or in combination with other compounds, including those compounds that may be therapeutically effective by the same or different modes of action. In addition, it is to be understood herein that the compounds, compositions, unit doses, and 1392-17709 methods described herein may be used in combination with other compounds that are administered to treat other symptoms of the medical condition.

[0019] In another embodiment, processes are described herein for preparing the compounds and compositions. In another embodiment, compounds and compositions are described herein that are prepared by the foregoing processes.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 shows illustrative transmission electron micrographs (TEMs) of a plurality of tirzepatide (TZP)-pUDCA nanoparticle complexes. A suspension of the nanoparticles were absorbed onto a carbon coated TEM grid followed by phosphotungstic acid negative stain contrast agent. The nanoparticles were prepared according to Example 3 from tirzepatide and 6 kd pUDCA.

[0022] FIG. 2 shows illustrative transmission electron micrographs (TEMs) of a plurality of semaglutide (SEM)-pUDCA nanoparticle complexes. A suspension of the nanoparticles were absorbed onto a carbon coated TEM grid followed by phosphotungstic acid negative stain contrast agent. The nanoparticles were prepared according to Example 3 from semaglutide and 6 kd pUDCA.

[0023] FIG. 3 shows an illustrative transmission electron micrograph (TEM) of a plurality of semaglutide (SEM)-pUDCA complexes. A suspension of the nanoparticles were absorbed onto a carbon coated TEM grid followed by phosphotungstic acid negative stain contrast agent. The nanoparticles were prepared according to Example 3 from semaglutide and 6 kd pUDCA in 75:25 DMF / DMSO in the absence of water, and show the highly spherical morphology7of the NCs described herein.

[0024] FIG. 4 shows an illustrative transmission electron micrograph (TEM) of a plurality of semaglutide (SEM)-pUDCA complexes (composition Example 3A). A suspension of the nanoparticles were absorbed onto a carbon coated TEM grid followed by phosphotungstic acid negative stain contrast agent. The nanoparticles were prepared according to Example 3 from semaglutide and 6 kd pUDCA in 90: 10 v / v DMF / CH2CI2 and H2O (WFI), and show the highly spherical morphology of the NCs described herein.

[0025] FIG. 5 shows the activity profile of (c) recombinant human insulin released from an illustrative nanoparticle complex prepared according to (Process Example 4) from recombinant human insulin and 3 kd pUDCA, and compared to control samples of (a) commercially available human insulin and (b) commercially available recombinant human insulin using an IR-B phosphorylation activation assay.

[0026] FIG. 6 shows the PK profile of (c) recombinant human insulin released from an 1392-17709 illustrative nanoparticle complex prepared according to (Process Example 4) from semaglutide and 6 kd pUDCA, and compared to Rybelsus® as a positive control in pigs.

[0027] DETAILED DESCRIPTION

[0028] Several illustrative embodiments of the invention are described by the following delineated clauses:

[0029] A nanoparticle complex comprising one or more active pharmaceutical ingredients and a carrier, where at least one of the one or more active pharmaceutical ingredients is a biomolecule or an analog or derivative thereof, and the carrier comprises a plurality of biomonomers or biopolymers, or a combination thereof.

[0030] A orally administrable nanoparticle complex comprising one or more active pharmaceutical ingredients and a carrier, where at least one of the one or more active pharmaceutical ingredients is a biomolecule or an analog or derivative thereof, and the carrier comprises a plurality of biomonomers or biopolymers, or a combination thereof, and where the nanoparticle complex is characterized by an average diameter of about 400 nm, about 350 nm, about 300 nm, about 250 nm, about 200 nm, about 150 nm, about 100 nm, or about 50 nm or less.

[0031] The nanoparticle complex of any preceding clause wherein the biological molecule comprises a peptide, a protein, a nucleic acid, both a peptide and a nucleic acid, or an antibody.

[0032] The nanoparticle complex of any preceding clause wherein the biological molecule is a protein or peptide.

[0033] The nanoparticle complex of any preceding clause wherein the biological molecule is a glucagon-like peptide- 1 (GLP-1) or an analog or derivative thereof.

[0034] The nanoparticle complex of any preceding clause wherein the biological molecule is a glucagon-like peptide- 1 (GLP-1) receptor agonist.

[0035] The nanoparticle complex of any preceding clause wherein the biological molecule is a glucose-dependent insulinotropic polypeptide (GIP) or an analog or derivative thereof.

[0036] The nanoparticle complex of any preceding clause wherein the biological molecule is a glucose-dependent insulinotropic polypeptide (GIP) receptor agonist.

[0037] The nanoparticle complex of any preceding clause wherein the biological molecule is a dual GLP-l / GIP receptor agonist.

[0038] The nanoparticle complex of any preceding clause wherein the biological molecule is a triple GLP-1 / GIP / GR receptor agonist. 1392-17709

[0039] The nanoparticle complex of any preceding clause wherein the biological molecule is a glucocorticoid receptor (GR) or an analog or derivative thereof.

[0040] The nanoparticle complex of any preceding clause wherein the biological molecule is a glucocorticoid receptor (GR) receptor agonist.

[0041] The nanoparticle complex of any preceding clause wherein the biological molecule is selected from the group consisting of albiglutide, dulaglutide, exenatide, liraglutide, lixisenatide, semaglutide, and tirzepatide, and analogs and derivatives thereof, and any combinations of the foregoing.

[0042] The nanoparticle complex of any preceding clause wherein the biological molecule is a nucleic acid.

[0043] The nanoparticle complex of any preceding clause wherein the biological molecule is an antibody or antibody mimetic.

[0044] The nanoparticle complex of any preceding clause wherein the biological molecule is adalimumab or ofatumumab.

[0045] The nanoparticle complex of any preceding clause wherein the biological molecule is hydrophilic.

[0046] The composition of any preceding clause wherein the loading of biological molecule in the plurality of nanoparticles is about 10%, about 20%, about 30%, about 35%, about 40%, or about 50% or greater.

[0047] The nanoparticle complex of any preceding clause wherein the biomonomer or biopolymer is a substrate for or is capable of binding to an intestinal receptor or intestinal transporter.

[0048] The nanoparticle complex of any preceding clause wherein the biomonomer or biopolymer is a substrate for or is capable of binding to a bile acid receptor or a cholesterol receptor.

[0049] The nanoparticle complex of any preceding clause wherein the biomonomer or biopolymer is capable of increasing passive transport of the biomolecule across or through the intestine.

[0050] The nanoparticle complex of any preceding clause wherein the biomonomer or biopolymer is selected from the group consisting of poly-arginine; bile acids, poly bile acids, including primary, secondary, tertiary, conjugated, and / or synthetic poly bile acids; PEGylated polymers; lipidated polymers, including reduced P -glycoprotein (P-gp) efflux lipidated polymers; lipids, ePLL, PLA, PGA, and PLGA, and combinations thereof. 1392-17709

[0051] The nanoparticle complex of the preceding clause wherein the polymer comprises one or more bile acids, or analogs or derivatives thereof, a polymer of one or more bile acids, or analogs or derivatives thereof, or a combination of the foregoing.

[0052] The nanoparticle complex of the preceding clause wherein the bile acid or analog or derivative thereof comprises a cholesterol or analog or derivative thereof.

[0053] The nanoparticle complex of any preceding clause wherein the bile acid or analogs or derivatives thereof comprises a cholic acid or analog or derivative thereof.

[0054] The nanoparticle complex of any preceding clause wherein the polymer comprises one or more polymerized ursodeoxycholic acids (pUDCAs).

[0055] The nanoparticle complex of any preceding clause wherein the polymer comprises one or more polymerized deoxy cholic acids (pDCAs).

[0056] The nanoparticle complex of any preceding clause wherein the polymer comprises one or more polymerized polymerized glycocholic acids (pGCAs).

[0057] The nanoparticle complex of any preceding clause wherein the polymer comprises one or more polymerized lactic acids (PLAs), polymerized glycolic acids (PGAs), co-polymerized lactic and glycolic acids (PLGAs), or a combination thereof.

[0058] The nanoparticle complex of any preceding clause wherein the polymer comprises one or more polymerized lysines (ePLLs).

[0059] The nanoparticle complex of any preceding clause wherein the biomonomers or biopolymers are amphiphilic.

[0060] The nanoparticle complex of any preceding clause wherein the biomonomer is not sodium N-(8-[2-hydroxylbenzoyl] amino)caprylate (SNAC).

[0061] The nanoparticle complex of any preceding clause wherein the biopolymer is not a polymer of methacry lic acid, trimethylsilyl methacrylate, methyl methacry late, tert-buty l methacrylate, or cyclohexylmethacrylate.

[0062] The composition of any preceding clause wherein the weight average molecular weight of the biopolymer is less than about 100,000, less than about 90,000, less than about 80,000, less than about 70,000, or less than about 60,000.

[0063] The composition of any preceding clause wherein the weight average molecular weight of the biopolymer is about 30,000 or less, about 20,000 or less, about 15,000 or less, about 12,000 or less, about 11,000 or less, about 10,000 or less, about 9,000 or less, about 8,000 or less, about 7,000 or less, about 6,000 or less, about 5,000 or less, about 4,000 or less, about 3,000 or less, about 2,000 or less, or about 1,000 or less.

[0064] The composition of any preceding clause wherein the weight average molecular weight of the biopolymer is about 6,000. 1392-17709

[0065] The composition of any preceding clause wherein the weight average molecular weight of the biopolymer is in the range from about 300 to about 30,000

[0066] The composition of any preceding clause wherein the weight average molecular weight of the biopolymer is in the range from about 300 to about 20.000

[0067] The composition of any preceding clause wherein the ratio of the biopolymer to the biological molecule is in the range from about 10:1 to about 1 : 1, from about 8:1 to about 1: 1, from about 6 : 1 to about 1 : 1, from about 4 : 1 to about 1 : 1 , or from about 2 : 1 to about 1 : 1 by weight.

[0068] The composition of any preceding clause wherein the ratio of the biopolymer to the biological molecule is about 1 : 1 by weight.

[0069] The composition of any preceding clause wherein the mass concentration of the biopolymer is in the range from about 0. 1 to about 10 mg / mL.

[0070] The nanoparticle complex of any preceding clause further comprising one or more permeation agents.

[0071] The nanoparticle complex of any preceding clause wherein the permeation agent comprises Sodium N Caprylate, sodium N-(8-[2-hydroxylbenzoyl] amino)caprylate (SNAC) (SNAC)*, sodium octanoate (C8). sodium decanoate (CIO)*, sodium lauryl sulphate, sodium taurodihydrofusidate, palmitoycamitine, sodium glycodeoxy cholate, bile acids, poly bile acids, fatty acids; cholesterol; chitosan; salicylates; chelators; glycerides; poly-cations; 5-CNAC; cellpenetrating peptides such as oligo-arginine, or a combination thereof.

[0072] The nanoparticle complex of any preceding clause wherein the permeation agent comprises sodium N-(8-[2-hydroxylbenzoyl] amino)caprylate (SNAC).

[0073] The nanoparticle complex of any preceding clause wherein the permeation agent comprises sodium decanoate (CIO).

[0074] The nanoparticle complex of any preceding clause further comprising a cryoprotectant.

[0075] The nanoparticle complex of the preceding clause wherein the cryoprotectant comprises one or more polyols.

[0076] The nanoparticle complex of any preceding clause wherein the cry oprotectant comprises one or more sugar alcohols.

[0077] The nanoparticle complex of any preceding clause wherein the cryoprotectant comprises one or more monosaccharides, polysaccharides, or a combination thereof.

[0078] The nanoparticle complex of any preceding clause wherein the cry oprotectant is selected from the group consisting of mannitol, sucrose, trehalose, and combinations thereof. 1392-17709

[0079] The nanoparticle complex of any preceding clause wherein the biological and the cryoprotectant have a ratio in the range from about 1 : 1 to about 1:50, from about 1 : 1 to about 1 : 10. or from about 1 : 3 to about 1:9.

[0080] The nanoparticle complex of any preceding clause further comprising one or more reconstitution agents.

[0081] The nanoparticle complex of any preceding clause further comprising one or more reconstitution agents selected from the group consisting of glycols, polyols, saccharides, and polysaccharides, and combinations thereof.

[0082] The nanoparticle complex of any preceding clause wherein the reconstitution agents are selected from the group consisting of mannitol, glycerol, ery thritol, threitol, arabitol, xylitol, ribitol, trehalose, sucrose, sorbitol, galactitol, fucitol, iditol. inositol, volemitol, isomalt, maltitol, lactitol. ethylene glycol, propylene glycol, glycerol, cyclodextrin, tert-butyl alcohol, DMSO, organic acids, such as succinic acid and oxalic acid, phosphates, such as tripolyphosphate (STPP), hexametaphosphate (HMP), sodium triphosphates (STP), tetrasodium pyrophosphate (TSPP) and pyrophosphate (SPP), monosaccharides, disaccharides, oligosaccharides and polysaccharides, lactose, Mannan oligosaccharides, xylo-oligosaccharides. Konjac oligoglucomannan, trehalose, alginate oligosaccharides, Curdlan, short-clustered maltodextrin, carboxymethyl chito-oligosaccharides, Raffinose, stachyose, fructooligosaccharide, maltose oligosaccharide, cottonseed oligosaccharide, kappa-carrageenan (KC), carrageenan oligosaccharides (CGO), chitosan oligosaccharides (COS), oligogalactose, isomaltooligosaccharides, soybean oligosaccharides, dextrans, amino acids, such as proline and glycine, anti-freeze proteins derived from fish, insects, plants, and micro-organisms, natural deep eutectic solvents prepared from mixtures of two or more solid or liquid components, such as sugars, organic acids, polyols, and choline derivatives, and any combination of the foregoing.

[0083] The nanoparticle complex of any preceding clause wherein the reconstitution agents are selected from the group consisting of mannitol, trehalose, propylene glycol, glycerol, cyclodextrin, and any combination of the foregoing.

[0084] The nanoparticle complex of any preceding clause further comprising one or more surfactants.

[0085] The nanoparticle complex of any preceding clause wherein the surfactants are selected from the group consisting of bile acids, poly bile acids, cationic polymers, PVA, SDS, Tween, Poloxamer 188, F127, PEG, BAs, CTAB (Cetyltrimethylammonium bromide), Didodecyldimethy lammonium bromide (DDAB), PVAm (poly vinylamine), cationic, anionic, and neutral lipids, an other lipids, such as glycolipids, lipopeptides. phospholipids, rhamnolipid, and sophorolipid, oleyl amidopropyl betaine (OAPB), 3-|N,N- 1392-17709 dimethyltetradecylammonio] propane- 1 -sulfonate (SB3-14), PVA, PEG, SDS, glyceryl monostearate (GMS), Span60, gly cerol, hydroxypropyl methylcellulose (HPMC), carboxy methyl cellulose (CMC), hydroxypropyl cellulose (HPC), lipopeptide, fatty acids, polysaccharide, Alasan, Emulsan. Liposan, Surfactin. Fengycin, Iturin-PEGylated hpid ALC- 0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide), cationic lipid ALC-0315 ((4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2-hexyldecanoate), and any combination of the foregoing.

[0086] The nanoparticle complex of any preceding clause wherein the surfactants are selected from the group consisting of Tween, Didodecyldimethylammonium bromide (DDAB), PVAm (polyvinylamine), cationic lipids, and any combination of the foregoing.

[0087] The nanoparticle complex of any preceding clause further comprising a stabilizer.

[0088] The nanoparticle complex of the preceding clause wherein the stabilizer is selected from the group consisting of PVA, PEG, glycerol, bile salts, HPMC, HPC, citric acid, proteolytic enzyme inhibitors, chelating agent (e.g., ORMD-0801: insulin formulation using trypsin inhibitor and chelating agent), PVAm (polyvinylamine), cationic, anionic, and neutral lipids, an other lipids, such as glycolipids, lipopeptides. phospholipids, rhamnolipid, and sophorolipid,, oleyl amidopropyl betaine (OAPB), 3-[N,N- dimethyltetradecylammonio] propane- 1 -sulfonate (SB3-14), PVA, PEG, SDS, glyceryl monostearate (GMS), Span60, Tween, Poloxamer 188, F127, glycerol, hydroxypropyl methylcellulose (HPMC), carboxy methyl cellulose (CMC), hydroxypropyl cellulose (HPC), lipopeptide, fatty acids, polysaccharide. Alasan, Emulsan, Liposan, Surfactin, Fengycin, Iturin- PEGylated lipid ALC-0159 (2-[(poly ethylene glycol)-2000]-N,N-ditetradecylacetamide), the cationic hpid ALC-0315 ((4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2- hexyldecanoate), and combinations thereof

[0089] The nanoparticle complex of the preceding clause wherein the stabilizer is selected from the group consisting of PVAm (polyvinylamine), cationic, anionic, and neutral lipids, an other lipids, such as glycolipids, lipopeptides, phospholipids, rhamnolipid, and sophorolipid, and combinations thereof

[0090] The nanoparticle complex of any preceding clause wherein the nanoparticle complex comprises a core and one of or more coatings, and where the core substantially comprises the biological molecule, and the coating substantially comprises the biomonomer or biopolymer, and where the coating covers at least a portion of the core.

[0091] The nanoparticle complex of any preceding clause further comprising one or more additional coatings / layers. 1392-17709

[0092] In another embodiment, the layer is tunable to a predetermined thickness to achieve desired physicochemical changes such as size or charge, amount / dose of substance in layer such as API, permeation enhancer, surfactant, etc., kinetic modification such as decreasing burst release by coating a layer of dense API with a layer of empty polymer matrix or polymer matrix plus permeation enhancer, surfactant, etc. Without being bound by theory, it is believed herein that surface thickness is primarily a function of the concentration of the coating compound added, but is also influenced by the molecular weight of the coating compound.

[0093] The nanoparticle complex of any preceding clause wherein at least one layer is an enteric coating.

[0094] The nanoparticle complex of any preceding clause wherein the enteric coating comprises a polymer of methacrylic acid, a polymer of methyl methacrylate, a copolymer of methacrylic acid and methyl methacrylate, cellulose acetate phthalate, HPMCP 55 (hydroxypropyl methylcellulose),HPMCAS (hydroxypropyl methylcellulose succinate), pectin, or a combination thereof.

[0095] Illustrative “enteric coating polymers’’ are substantially insoluble and / or substantially stable under acidic conditions exhibiting a pH of less than about 5 and which are substantially soluble or can decompose under conditions exhibiting a pH of about 5 or more. Illustrative examples of such enteric polymers include carboxymethylethylcellulose, cellulose acetate phthalate, cellulose acetate succinate, methylcellulose phthalate, hydroxymethylethylcellulose phthalate, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, polyvinyl alcohol phthalate, polyvinyl butyrate phthalate, polyvinyl acetal phthalate, a copolymer of vinyl acetate / maleic anhydride, a copolymer of vinylbutylether / maleic anhydride, a copolymer of styrene / maleic acid monoester, a copolymer of methyl acrylate / methacrylic acid, a copolymer of styrenc / acrylic acid, a copolymer of methyl acrylate / methaciylic acid / octyl acrylate, a copolymer of methacrylic acid / methyl methacrylate and mixtures thereof.

[0096] The nanoparticle complex of any preceding clause wherein the enteric coating comprises a polymer or copolymer of methacry lic acid, methyl methacry late, or a combination thereof.

[0097] The nanoparticle complex of any preceding clause wherein the enteric coating comprises a Eudragit polymer.

[0098] The nanoparticle complex of any preceding clause wherein at least one layer is a permeation agent.

[0099] The nanoparticle complex of any preceding clause wherein at least one layer is a cryoprotectant. 1392-17709

[0100] The nanoparticle complex of any preceding clause wherein at least one layer is a reconstitution agent.

[0101] The nanoparticle complex of any preceding clause wherein at least one layer is a surfactant.

[0102] A composition comprising a plurality of nanoparticles according to any one of the preceding clauses.

[0103] The composition of any preceding clause wherein the plurality of nanoparticles has an average particle size (D-average) in the range from about 1 nm to about 900 nm, from about 1 nm to about 800 nm, from about 1 nm to about 700 nm, from about 1 nm to about 600 nm, from about 1 nm to about 500 nm, from about 1 nm to about 400 nm, from about 1 nm to about 350 nm, from about 1 nm to about 300 nm, from about 1 nm to about 250 nm, from about 1 nm to about 225 nm. from about 1 nm to about 200 nm. from about 1 nm to about 175 nm, from about 1 nm to about 150 nm, from about 1 nm to about 125 nm, from about 1 nm to about 100 nm, or in the range from about 1 nm to about 75 nm.

[0104] The composition of any preceding clause wherein the plurality of nanoparticles has an average particle size (D-average) in the range from about 5 nm to about 900 nm, from about 5 nm to about 800 nm, from about 5 nm to about 700 nm, from about 5 nm to about 600 nm, from about 5 nm to about 500 nm, from about 5 nm to about 400 nm, from about 5 nm to about 350 nm, from about 5 nm to about 300 nm, from about 5 nm to about 250 nm, from about 5 nm to about 225 nm, from about 5 nm to about 200 nm. from about 5 nm to about 175 nm. from about 5 nm to about 150 nm. from about 5 nm to about 125 nm. from about 5 nm to about 100 nm, or in the range from about 5 nm to about 75 nm.

[0105] The composition of any preceding clause wherein the plurality of nanoparticles has an average particle size (D-average) in the range from about 10 nm to about 900 nm, from about 10 nm to about 800 nm, from about 10 nm to about 700 nm, from about 10 nm to about 600 nm, from about 10 nm to about 500 nm, from about 10 nm to about 400 nm. from about 10 nm to about 350 nm, from about 10 nm to about 300 nm, from about 10 nm to about 250 nm, from about 10 nm to about 225 nm, from about 10 nm to about 200 nm, from about 10 nm to about 175 nm, from about 10 nm to about 150 nm, from about 10 nm to about 125 nm, from about 10 nm to about 100 nm. or in the range from about 10 nm to about 75 nm.

[0106] The composition of any preceding clause wherein the plurality of nanoparticles has a D90 or Dv90 of about 900 nm, about 500 nm, about 250 nm, about 200 nm, about 150 nm, about 100 nm, about 90 nm, about 80 nm, about 70 nm, about 60 nm, or about 50 nm or less. 1392-17709

[0107] The composition of any preceding clause wherein the plurality of nanoparticles has a D90 in the range from about 10 nm to about 900 nm, or in the range from about 50 nm to about 500 nm.

[0108] The composition of any preceding clause wherein the poly dispersity index (PDI) of the plurality of nanoparticles is about 0.4, about 0.3, about 0.25, about 0.22, about 0.20 about 0.19, about 0.18, about 0.17, about 0.16, about 0.15, about 0.14, about 0.13, about 0.12 about 0. 11, about 0. 10, about 0.09, or about 0.08 or less.

[0109] In another embodiment, the particle size ranges described herein refer to the particles before isolation, such as by lyophilization. In another embodiment, the particle size ranges described herein refer to the particles before isolation, such as by lyophilization. In another embodiment, the particle size ranges described herein refer to uncoated particles either before or after isolation, such as by lyophilization. In another embodiment, the particle size ranges described herein refer to coated particles either before or after isolation, such as by lyophilization.

[0110] It is to be understood that in some embodiments that include a cryoprotectant and / or lyoprotectant, and / or a surfactant, the particles may temporarily aggregate. It is to be further understood that such temporary aggregates are considered to fall within the particle size ranges described herein because the individual particles in such temporary aggregates fall within the particle size ranges described herein.

[0111] It is to be understood that in some embodiments that include a cryoprotectant and / or lyoprotectant, and / or a surfactant, the particles may temporarily take on an excessive coating. It is to be further understood that such temporary excessive coated particles fall within the particle size ranges described herein because the parent particles within such temporary excessive coatings fall within the particle size ranges described herein.

[0112] In another embodiment, the PDI ranges described herein refer to the particles before isolation, such as by lyophilization. In another embodiment, the PDI ranges described herein refer to the particles before isolation, such as by lyophilization. In another embodiment, the PDI ranges described herein refer to uncoated particles either before or after isolation, such as by lyophilization. In another embodiment, the PDI ranges described herein refer to coated particles either before or after isolation, such as by lyophilization.

[0113] It is to be understood that in some embodiments that include a cryoprotectant and / or lyoprotectant, and / or a surfactant, the particles may temporarily aggregate. It is to be further understood that such temporary aggregates are considered to fall within the PDI ranges described herein because the individual particles in such temporary aggregates fall within the particle size ranges descnbed herein. 1392-17709

[0114] It is to be understood that in some embodiments that include a cryoprotectant and / or lyoprotectant, and / or a surfactant, the particles may temporarily take on an excessive coating. It is to be further understood that such temporary excessive coated particles fall within the PDI ranges described herein because the parent particles within such temporary excessive coatings fall within the particle size ranges described herein.

[0115] The composition of any preceding clause wherein the plurality7of nanoparticles is characterized by a charge zeta in the range from about -50 mV to about +50 mV, or in the range from about -40 mV to about +40 mV. or in the range from about -35 mV to about +35 mV, or in the range from about -30 mV to about +30 mV, or in the range from about -25 mV to about +25 mV, or in the range from about -20 mV to about +20 mV, or in the range from about -40 mV to about OmV, or in the range from about 0 mV to about +25 mV.

[0116] In another embodiment, the charge zeta is tunable by the addition of one or more surfactants and / or stabilizers, such as but not limited to PVAm

[0117] Without being bound by theory it is believed herein that the charge zeta contributes to and or regulated clearance of the biomolecule after administration to the host animal. In addition, it is believed herein that extreme values of zeta, such as values greater than +70 mV or less than about -70 mV may be cytotoxic in certain instances with specific biomolecules and / or specific biomonomers or biopolymers.

[0118] The composition of any preceding clause wherein the plurality7of nanoparticles is substantially free of nanoparticles of the polymer, nanoparticles of the biological, or both.

[0119] The composition of any preceding clause wherein the plurality of nanoparticles is substantially free of organic solvent.

[0120] The composition of any preceding clause wherein the plurality of nanoparticles comprises less than about 3%, about 2.5%, about 2%, about 1.5%, or about 1% by weight each of one or more Class 3 solvents.

[0121] The composition of any preceding clause wherein the plurality of nanoparticles comprises less than about 0.25%, about 0.20%, or about 0.15% each of one or more Class 2 solvents by yveight.

[0122] The composition of the preceding clause wherein the solvent is DMSO. THF, EtOH, tert-butyl alcohol, dimethylformamide, dichloromethane, or a combination thereof.

[0123] The composition of the preceding clause wherein the solvent is EtOH.

[0124] The composition of the preceding clause yvherein the solvent is dichloromethane. Illustrative Class 2 solvents are listed in the FDA Q3C — Tables and List Guidance for Industry. Revision 3, June 2017. and include, but are not limited to, dichloromethane, 1,2- dimethoxy ethane, N,N-dimethylacetamide, N,N-dimethylformamide, 1,4-di oxane, 2- 1392-17709 ethoxyethanol, ethyleneglycol, formamide, hexane, methanol, 2-methoxyethanol, methylbutyl ketone, methylcyclohexane, methylisobutylkctonc2. N-methylpyrrolidone, nitromethane, pyridine, sulfolane, tetrahydrofuran, tetralin, toluene, 1.1.2-tri chloroethene, and xylene.

[0125] Illustrative Class 3 solvents are listed in the FDA Q3C — Tables and List Guidance for Industry, Revision 3, June 2017, and include, but are not limited to, acetic acid, heptane, acetone, isobutyl acetate, anisole, isopropyl acetate, 1 -butanol, methyl acetate, 2-butanol, 3- methyl-1 -butanol, butyl acetate, methylethyl ketone, tert-butylmethyl ether, 2-methyl-l- propanol, dimethyl sulfoxide, pentane, ethanol, 1-pentanoL ethyl acetate. 1-propanol, ethyl ether, 2-propanol, ethyl formate, propyl acetate, formic acid, and triethylamine. performance

[0126] The composition of any preceding clause wherein the plurality of nanoparticles are characterized by preferential absorption / uptake in the lower GI tract compared to the upper GI tract or stomach, where the preference is about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 95%, or about 98% or greater.

[0127] The composition of any preceding clause wherein the plurality of nanoparticles are characterized by a total release of about 50%, about 60%, about 70%, about 80%, about 90%. or about 95% or more.

[0128] The composition of any preceding clause wherein the plurality of nanoparticles are characterized by a burst release of about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, or about 10% or less.

[0129] The composition of any preceding clause wherein the plurality of nanoparticles are characterized by a burst release of about 35%, about 30%, about 25%, about 20%, about 15%, or about 10% or less.

[0130] A unit dose comprising a therapeutically effective amount, in single or divided form, of the composition of any preceding clause.

[0131] A unit dose comprising a therapeutically effective amount of the composition or pharmaceutical composition of any preceding clause for use in treating a medical condition or disease.

[0132] The unit dose of any preceding clause further comprising one or more carriers, diluents, or excipients, or any combination of the foregoing.

[0133] The unit dose of any preceding clause configured for oral administration to a host animal, the unit dose comprising a therapeutically effective amount of a composition of any of the preceding clauses.

[0134] The unit dose of any preceding clause in the form of a tablet or a capsule. 1392-17709

[0135] The unit dose of the preceding clause wherein the tablet or the capsule includes an enteric coating.

[0136] The unit dose of any preceding clause in divided form.

[0137] It is to be understood that unit doses described herein may be in single or divided form, and may correspond to a daily dosage amount, or adjusted to a periodic amount that is shorter, including for multiple daily doses, or longer, including weekly or monthly doses. It is to be understood that the compositions may include other components and / or ingredients, including, but not limited to, other therapeutically active compounds, and / or one or more carriers, vehicles, diluents, adjuvants, excipients, and the like, and combinations thereof.

[0138] In vivo performance

[0139] The unit dose of any preceding clause wherein the in vivo Tmax is about 12 h, about 24 h, about 36 h, or about 48 h or later in beagle dogs.

[0140] The unit dose of any preceding clause wherein the in vivo Tmax is about 12 h, about 24 h, about 36 h, or about 48 h or later in pigs.

[0141] The unit dose of any preceding clause capable of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% bioavailability in beagle dogs.

[0142] The unit dose of any preceding clause capable of at least about 1%. 2%, 3%, 4%. 5%, 6%, 7%, 8%, 9%, or 10% bioavailability in pigs.

[0143] A method for treating a medical condition in a host animal, the method comprising administering the unit dose of any preceding clause or a therapeutic amount of the composition of pharmaceutical composition of any of the preceding clauses.

[0144] Use of a composition of any of the preceding clauses in the manufacture of a medicament for treating a medical condition in a host animal.

[0145] A pharmaceutical composition for use in the treatment of a medical condition in a host animal, where the pharmaceutical composition comprises the composition of any of the preceding clauses.

[0146] The pharmaceutical composition of the preceding clause further comprising one or more carriers, diluents, or excipients, or any combination of the foregoing.

[0147] The use or pharmaceutical composition of any preceding clause wherein the medical condition is diabetes.

[0148] The use or pharmaceutical composition of any preceding clause wherein the medical condition is obesity.

[0149] The use or pharmaceutical composition of any preceding clause wherein the medical condition is cardiovascular disease in overweight or obese host animals. 1392-17709

[0150] The use or pharmaceutical composition of any preceding clause wherein the medical condition is sleep apnea.

[0151] The use or pharmaceutical composition of any preceding clause wherein the medical condition is addiction.

[0152] A process for preparing a nanoparticle complex according to any preceding clause, the process comprising contacting (a) a first mixture comprising the biopolymer and an organic solvent with (b) a second mixture comprising the biological molecule and water to prepare a third mixture, and optionally with (c) a third mixture comprising a surfactant, a stabilizer, or a combination thereof; optionally adding a cry oprotectant to the third mixture; lyophilizing the third mixture, including the optionally added cryoprotectant to obtain the nanoparticle complex.

[0153] A process for preparing a coated nanoparticle complex according to any preceding clause, the process comprising contacting (a) a first mixture comprising the biopolymer and an organic solvent with (b) a second mixture comprising the biological molecule and water to prepare a third mixture, and optionally with (c) a third mixture comprising a surfactant, a stabilizer, or a combination thereof; stirring the third mixture for a predetermined period of time to form an intermediate nanoparticle complex core; contacting the third mixture with a fourth mixture comprising a coating reagent and an organic solvent to prepare a fifth mixture; stirring to fifth mixture for a predetermined period of time to form a coated nanoparticle complex core; optionally adding a cryoprotectant to the fifth mixture; lyophilizing the fifth mixture, including the optionally added cryoprotectant to obtain the coated nanoparticle complex.

[0154] The process of the preceding clause including an organic phase volume and an aqueous phase volume, wherein the ratio of the organic phase volume to the aqueous phase volume is in the range from about 10: 1 to about 1 : 10, or from about 3 : 1 to about 1 : 6.

[0155] The process of the preceding clause including an organic phase volume and an aqueous phase volume, wherein the ratio of the organic phase volume to the aqueous phase volume is about 3: 1, about 1: 1, about 1:2, about 1:4, or about 1:6. 1392-17709

[0156] The process of the preceding clause including an organic phase volume and an aqueous phase volume, wherein the ratio of the organic phase volume to the aqueous phase volume is about 1 :2.

[0157] The process of the preceding clause wherein the organic phase volume comprises an alcohol, amide, ether, sulfoxide, or combination thereof.

[0158] The process of any preceding clause wherein the organic phase volume comprises an DMSO, THF, or a combination thereof.

[0159] The process of any preceding clause wherein the organic phase volume comprises an alcohol or amide.

[0160] The process of any preceding clause wherein the organic phase volume comprises tert-butyl alcohol, dimethylformamide, or a combination thereof.

[0161] The process of any preceding clause wherein the weight ratio of the biopolymer to the biological molecule is in the range from about 1 : 1 to about 8: 1.

[0162] The process of the preceding clause wherein the weight ratio of the biopolymer to the biological molecule is about 1: 1, about 3:1, about 5: 1, or about 10:1.

[0163] The process of the preceding clause wherein the weight ratio of the biopolymer to the biological molecule is about 3: 1.

[0164] The process of any preceding clause wherein the total concentration of the biopolymer and the biological molecule is in the range from about 1:4 to about 5: 1 mg / mL.

[0165] The process of the preceding clause wherein the total concentration of the biopolymer and the biological molecule is about 1 : 4, about 1 :2. about 1: 1, about 3 : 1. or about 5: 1 mg / mL.

[0166] The process of the preceding clause wherein the total concentration of the biopolymer and the biological molecule is about 1: 1 mg / mL.

[0167] The process of any preceding clause wherein the organic phase volume is added to the aqueous phase volume.

[0168] The process of any preceding clause wherein purifying includes dialysis.

[0169] The process of any preceding clause wherein purifying includes tangential flow fdtration.

[0170] The process of any preceding clause wherein the cryoprotectant is sucrose.

[0171] The process of any preceding clause wherein the weight ratio of the cryoprotectant to the sum of the biopolymer and the biological molecule is in the range from about 3 : 1 to about 9:1. 1392-17709

[0172] The process of the preceding clause wherein the weight ratio of the cryoprotectant to the sum of the biopolymer and the biological molecule is about 3: 1, about 6: 1, or about 9: 1.

[0173] The process of the preceding clause wherein the weight ratio of the cryoprotectant to the sum of the biopolymer and the biological molecule is about 9: 1.

[0174] It has been unexpectedly discovered that nanocomplexes of high molecular weight biomolecules and low to moderate molecular weight biopolymers rapidly form upon mixing. Without being bound by theory, it is believed herein that the use of low to moderate molecular weight biopolymers provides small average particle sizes, lower PDIs, highly monodispersed nanoparticles, and high spherical morphology, thus avoiding the need for purifications, including size-exclusion purification reported by others.

[0175] In addition, contrary to reports by others, high biomolecule / biopolymer ratios are lead more readily to self-assembly of nanoparticle complexes, and higher API loading and loading encapsulation.

[0176] As used herein, the term “biomolecule” or “biological molecule” generally refers to a molecule produced by a living organism, or a molecule consumed by a living organism, that is important or essential to one or more biological processes in the organism. Such biomolecules and biological molecules may be endogenous, produced in the host animal, or exogenous and consumed by the host animal. Illustrative biomolecules are proteins and peptides, including synthetic peptides and analogs and derivatives of peptides. It is to be understood that biomolecules or biological molecules described herein are not necessarily naturally occurring and instead, may also be synthetically derived.

[0177] As used herein, the term “peptide” generally refers to plurality of covalently bond amino acids. Illustratively, the plurality may be as few as about 10 amino acids. In other embodiments, the plurality is at least about 15, at least about 20, at least about 25, at least about 30. at least about 35. at least about 40. at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 amino acids.

[0178] Illustratively, the plurality may have a combined molecular weight of as little as about 1 kd. In other embodiments, the plurality has a combined molecular weight of at least about 1.5, at least about 2, at least about 2.5. at least about 3, at least about 3.5, at least about 4. at least about 4.5, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10 kd.

[0179] As used herein, the term “amino acid” refers generally to beta, gamma, and longer amino acids, and including cyclic groups, that have both an amino group and an acid group 1392-17709 from each of which a radical can be formed. Illustrative acyclic amino acids include the formula:

[0180] -N(R)-(CR'R")q-C(O)- where R is hydrogen, alkyl, acyl, or a suitable nitrogen protecting group. R' and R" are hydrogen or a substituent, each of which is independently selected in each occurrence, and q is an integer such as 1, 2, 3, 4, or 5. Illustratively, R' and / or R" independently correspond to, but are not limited to, hydrogen or the side chains present on naturally occurring amino acids, such as methyl, benzyl, hydroxymethyl, thiomethyl, carboxyl, carboxylmethyl, guanidinopropyl, and the like, and derivatives and protected derivatives thereof. The above described formula includes all stereoisomeric variations. For example, the amino acid may be selected from asparagine, aspartic acid, cysteine, glutamic acid, lysine, glutamine, arginine, serine, ornithine, threonine, and the like.

[0181] Illustrative biomolecules include, but are not limited to, peptides, proteins, antibodies, antibody mimetics, nucleic acids, and the like.

[0182] Illustrative peptides may be naturally occurring or synthetic, and include, but are not limited to, linear, cyclic, lipopeptides, glycopeptides, lipogly copeptides, and the like.

[0183] Illustrative proteins may be naturally occurring or synthetic, and include, but are not limited to, enzymes, protein toxins, bispecific fusion proteins, hormones, cytokines, growth factors, and the like.

[0184] As used herein, the term “glucagon-like peptide-1” or “GLP-1” generally refers to the 30 or 31 amino acid cleavage product of proglucagon, including GLP-1 (7-36) amide and GLP- 1 (7-37), as well as analogs and derivatives of GLP-1, such as but not limited to albiglutide, dulaglutide, exenatide, liraglutide, lixisenatide, semaglutide, tirzepatide, and the like. It is also to be understood that in each of the foregoing, any corresponding pharmaceutically acceptable salt is also included in the illustrative embodiments described herein.

[0185] As used herein, the term “glucose-dependent insulinotropic polypeptide” or “GIP” generally refers to the 42 amino acid cleavage product of the 153 amino acid proprotein encoded by the GIP gene, as well as analogs and derivatives of GIP. It is also to be understood that in each of the foregoing, any corresponding pharmaceutically acceptable salt is also included in the illustrative embodiments described herein.

[0186] As used herein, the term “nucleic acid” or “oligonucleotide” generally refers to plurality of covalently bond ribonucleic and / or deoxyribonucleic acids, and / or analogs and derivatives of the foregoing. Illustratively, the plurality may be as few as about 10 nucleic acids or analogs and derivatives thereof. In other embodiments, the plurality is at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at 1392-17709 least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 nucleic acids or analogs and derivatives thereof. It is understood that the foregoing ranges to single stranded oligonucleotides, and the corresponding double stranded oligonucleotides are double in number.

[0187] Illustrative nucleic acids and oligonucleotides may be naturally occurring or synthetic, and include, but are not limited to, antisense nucleotides, small interfering RNAs, microRNAs, messenger RNAs, aptamers, gene therapies, and the like.

[0188] Illustrative antibodies may be naturally occurring or synthetic, and include, but are not limited to, monoclonal antibodies, including monospecific and bispecific, antibody-drug conjugates, and the like.

[0189] Illustrative antibody mimetics may be naturally occurring or synthetic, and include, but are not limited to, antibody-derived-Fab, antibody-derived-scFv, antibody-derived-nanobodies, and the like; small protein scaffolds, including adnectins, anticalins, DARPins, knottins, affibodies, Kunitz domains, affimers, nanofitins®, avimers, centyrins, affilin® molecules, ADAPTs, cell-penetrating alphabodies, ADDobodies (attrimers), fynomers, OBody, repebodies, and the like.

[0190] Without being bound by theory, it is believed herein that the nanoparticle complexed described herein arise through a self-assembly mechanism where an energetically favorable interaction occurs between the biopolymer and the biomolecule. It has been unexpectedly observed that at higher ratios of biomolecule to biomonomer and / or biopolymer there is a lower concentration of unbound biomolecule, leading to very high encapsulation efficiency. In addition, it has been unexpectedly observed that at higher ratios of biomolecule to biomonomer and / or biopolymer there is a lower concentration of biomolecule on the surface of the nanoparticle, leading to very' high encapsulation efficiency.

[0191] Without being bound by theory, it is believed herein that NCs prepared with bile acid based biopolymers may demonstrate improved bioavailability due to the in vivo active transport mechanisms available for those biopolymers. For example, bile acid recycling in the lower gastrointestinal tract via the intestinal lamina propia is 90% or greater. In addition, and without being bound by theory, it is believed herein that that NCs prepared with bile acid based biopolymers may demonstrate improved efficacy due to the localization of those biopolymers to the pancreas.

[0192] In another embodiment, the NCs described herein optionally include additional active pharmaceutical ingredients. Illustrative additional active pharmaceutical ingredients include, but are not limited to, lipids, carbohydrates, secondary metabolites, and the like. 1392-17709

[0193] Illustrative lipids may be naturally occurring or synthetic, and include, but are not limited to, hormones, steroids, prostaglandins, fatty acid-based lipid medications, lipid injectable emulsions for nutrition supplementation, lipid-based drug delivery systems, including liposomes, niosomes, transferosomes. solid lipid nanoparticles, nanostructured lipid carriers, and the like.

[0194] Illustrative carbohydrates may be naturally occurring or synthetic, and include, but are not limited to, anticoagulants, antibiotics, vaccines, antidiabetic drugs, and the like.

[0195] Illustrative secondary metabolites may be naturally occurring or synthetic, and include, but are not limited to, phytochemicals, statins, antibiotics, and the like.

[0196] The compounds described herein may contain one or more chiral centers, or may otherwise be capable of existing as multiple stereoisomers. It is to be understood that in one embodiment, the invention described herein is not limited to any particular stereochemical requirement, and that the compounds, and compositions, methods, uses, and medicaments that include them may be optically pure, or may be any of a variety of stereoisomeric mixtures, including racemic and other mixtures of enantiomers, other mixtures of diastereomers, and the like. It is also to be understood that such mixtures of stereoisomers may include a single stereochemical configuration at one or more chiral centers, while including mixtures of stereochemical configuration at one or more other chiral centers.

[0197] Similarly, the compounds described herein may include geometric centers, such as cis, trans, E, and Z double bonds, or spatial arrangements, such as cis, trans, syn, and anti, relative configurations on a ring. It is to be understood that in another embodiment, the invention described herein is not limited to any particular geometric isomer requirement, and that the compounds, and compositions, methods, uses, and medicaments that include them may be pure, or may be any of a variety of geometric isomer mixtures. It is also to be understood that such mixtures of geometric isomers may include a single configuration at one or more double bonds, while including mixtures of geometry at one or more other double bonds.

[0198] The compounds described herein can be used for both human clinical medicine and veterinary applications. Thus, the host animal treated with the compounds described herein can be human or, in the case of veterinary applications, can be domestic animals, companion animals, laboratory animals, agricultural animals, livestock, or wild animals, including wild animals in captivity, such as, but not limited to, rodents (mice, rats, hamsters, and the like), rabbits, monkeys, chimpanzees, dogs, cats, cows, horses, pigs, sheep, goats, bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins, and whales.

[0199] It is to be understood that in every instance disclosed herein, the recitation of a range of integers for any variable describes the recited range, every individual member in the range, and 1392-17709 every possible subrange for that variable. For example, the recitation that n is an integer from 0 to 8, describes that range, the individual and selectable values of 0, 1, 2, 3, 4, 5, 6, 7, and 8, such as n is 0, or n is 1, or n is 2, etc. In addition, the recitation that n is an integer from 0 to 8 also describes each and every subrange, each of which may for the basis of a further embodiment, such as n is an integer from 1 to 8, from 1 to 7, from 1 to 6, from 2 to 8, from 2 to 7, from 1 to 3, from 2 to 4, etc.

[0200] It is also to be understood that unless otherwise indicated the recitation of a numerical value necessarily reflects the relative precision of the numerical value. For example, the recitation of a number with a specified precision based on significant figures necessarily includes a range of values that would match that number after appropriate rounding. For example, the recitation of the number 1 with a single significant figure is understood to properly refer to a range of values from 0.5 to 1.4. Similarly, the recitation of the number 1.0 with two significant figures is understood to properly refer to a range of values from 0.95 to 1.04. The relative precision of the numerical value can be further indicated by modifying with the term “abouf ’ to indicate that the modified number has low er precision.

[0201] As used herein, the term “about'’ when used with numerical values or limits generally means that the number is approximate and that, as recited, it is understood to include a range of values. For example, a real number that is recited with a single significant figure, would by definition include a so-called rounding range; the number about 5 would at the very least include the range 4.5-5.4, as each of those values rounds to 5. The same is to be understood for real numbers expressed with additional significant figures, where the corresponding rounding range applies to the last significant figure. Integers are to be understood to at least include the values ±1 for single-digit numbers, ±10 for two-digit numbers, etc. Depending upon the context and the variable recited, the term “about” is also interpreted to contemplate a range based on a percentage of the recited number, such as about 5 construed to include 5 ±10% or 5 ±20%. Notwithstanding the foregoing, it is understood that the range of values, unless otherwise indicated, should not be interpreted to include a negative range for a positively recited number, and vice-versa. In addition, depending up on the context, the recited number, unless otherwise indicated, should not be interpreted to include a value of zero when used in conjunction with an added component.

[0202] As used herein, the term “composition” generally refers to any product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combinations of the specified ingredients in the specified amounts. It is appreciated that certain functional groups, such as the hydroxy, amino, and like groups form complexes and / or coordination compounds with water and / or various solvents, in the various 1392-17709 physical forms of the compounds. It is to be understood that the compositions described herein may be prepared from isolated compounds described herein or from salts, solutions, hydrates, solvates, and other forms of the compounds described herein. It is also to be understood that the compositions may be prepared from various amorphous, non-amorphous, partially crystalline, crystalline, and / or other morphological forms of the compounds described herein. It is also to be understood that the compositions may be prepared from various hydrates and / or solvates of the compounds described herein. In addition, it is to be understood that the compositions may be prepared from various co-crystals of the compounds described herein. Accordingly, such pharmaceutical compositions that recite compounds described herein are to be understood to include each of, or any combination of, the various morphological forms and / or solvate or hydrate forms of the compounds described herein.

[0203] Illustratively, compositions may include one or more carriers, diluents, and / or excipients. The compounds described herein, or compositions containing them, may be formulated in a therapeutically effective amount in any conventional dosage forms appropriate for the methods described herein. The compounds described herein, or compositions containing them, including such formulations, may be administered by a wide variety of conventional routes for the methods described herein, and in a wide variety’ of dosage formats, utilizing known procedures (see generally, Remington: The Science and Practice of Pharmacy, (21sted., 2006)).

[0204] As used herein, the term “host animaf’ or “subject” or “patient” generally refers to mammals, including humans, companion animals, and livestock animals. A host animal in need may be a patient who has or is suffering from a disease described herein.

[0205] As used herein, the term “inhibiting” generally includes its generally accepted meaning which includes prohibiting, preventing, restraining, slowing, stopping, and / or reversing progression, severity of the disease and / or any resultant symptom of the disease. As such, the methods described herein include both clinical therapeutic and / or prophylactic administration, as appropriate.

[0206] The term “therapeutically effective amount” as used herein, refers to that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated. In one aspect, the therapeutically effective amount is that which may treat or alleviate the disease or symptoms of the disease at a reasonable benefit / risk ratio applicable to any medical treatment. However, it is to be understood that the total daily usage of the compounds and compositions described herein may be decided by the attending physician 1392-17709 within the scope of sound medical judgment. The specific therapeutically-effective dose level for any particular patient will depend upon a variety of factors, including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, gender and diet of the patient: the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidentally with the specific compound employed; and like factors well known to the researcher, veterinarian, medical doctor or other clinician of ordinary skill.

[0207] It is also appreciated that the therapeutically effective amount, whether referring to monotherapy or combination therapy, is advantageously selected with reference to any toxicity, or other undesirable side effect, that might occur during administration of one or more of the compounds described herein. Further, it is appreciated that the co-therapies described herein may allow for the administration of lower doses of compounds that show such toxicity, or other undesirable side effect, where those lower doses are below thresholds of toxicity or lower in the therapeutic window than would otherwise be administered in the absence of a cotherapy.

[0208] In addition to the illustrative dosages and dosing protocols described herein, it is to be understood that an effective amount of any one or a mixture of the compounds described herein can be readily determined by the attending diagnostician or physician by the use of known techniques and / or by observing results obtained under analogous circumstances. In determining the effective amount or dose, a number of factors are considered by the attending diagnostician or physician, including, but not limited to the species of mammal, including human, its size, age, and general health, the specific disease or disorder involved, the degree of or involvement or the severity of the disease or disorder, the response of the individual patient, the particular compound administered, the mode of administration, the bioavailability characteristics of the preparation administered, the dose regimen selected, the use of concomitant medication, and other relevant circumstances.

[0209] The dosage of each compound of the claimed combinations may depend on several factors, including: the administration method, the condition to be treated, the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the person to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular patient may affect the dosage used.

[0210] It is to be understood that in the methods described herein, the individual components of any co-administration. or combination can be administered by any suitable means, contemporaneously, simultaneously, sequentially, separately or in a single pharmaceutical 1392-17709 formulation. Where the co-administered compounds or compositions are administered in separate dosage forms, the number of dosages administered per day for each compound may be the same or different. The compounds or compositions may be administered via the same or different routes of administration. The compounds or compositions may be administered according to simultaneous or alternating regimens, at the same or different times during the course of the therapy, concurrently in divided or single forms.

[0211] The term “administering” as used herein includes all means of introducing the compounds and compositions described herein to the host animal, including, but are not limited to, oral (po), intravenous (iv), intramuscular (im), subcutaneous (sc), transdermal, inhalation, buccal, ocular, sublingual, vaginal, rectal, and the like. The compounds and compositions described herein may be administered in unit dosage forms and / or formulations containing conventional nontoxic pharmaceutically-acceptable carriers, adjuvants, and / or vehicles.

[0212] As used herein, the term “carrier” generally refers to any ingredient other than the active components in a formulation. The choice of carrier will to a large extent depend on factors such as the particular mode of administration, the effect of the carrier on solubility' and stability', and the nature of the dosage form.

[0213] Illustrative formats for oral administration include tablets, capsules, gelcaps, enteric capsules, elixirs, syrups, solutions, suspensions, and the like.

[0214] Illustrative routes for parenteral administration include intravenous, intraarterial, intraperitoneal, epidurial, intraurethral, intrastemal, intramuscular and subcutaneous, as well as any other art recognized route of parenteral administration.

[0215] In making the pharmaceutical compositions of the compounds described herein, a therapeutically effective amount of one or more compounds in any of the various forms described herein may be mixed with one or more excipients, diluted by one or more excipients, or enclosed within such a carrier which can be in the form of a capsule, sachet, paper, or other container. Excipients may serve as a diluent, and can be solid, semi-solid, or liquid materials, which act as a vehicle, carrier or medium for the active ingredient. The compositions may contain anywhere from about 0.1% to about 99.9% active ingredients, depending upon the selected dose and dosage form.

[0216] In another embodiment, the unit dose is a solid dosage forms for oral administration. Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers; granulating and disintegrating agents; binding agents; and lubricating agents, glidants, and antiadhesives. Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like. 1392-17709

[0217] The tablets may be uncoated or they may be coated by known techniques, optionally to delay disintegration and absorption in the gastrointestinal tract and thereby providing a sustained action over a longer period. The coating may be adapted to release the active drug substance in a predetermined pattern (e.g., in order to achieve a controlled release formulation) or it may be adapted not to release the active drug substance until after passage of the stomach (enteric coating). The coating may be a sugar coating, a film coating, or an enteric coating. Furthermore, a time delay material may be employed.

[0218] The solid tablet compositions may include a coating adapted to protect the composition from unwanted chemical changes, such as chemical degradation prior to the release of the active drug substance.

[0219] The effective use of the compounds, compositions, and methods described herein for treating or ameliorating one or more medical conditions, or effects of a medical condition, using one or more compounds described herein may be based upon animal models, such as murine, canine, porcine, and non-human primate animal models of disease.

[0220] In addition, it is to be understood that therapeutically effect doses administered in animal models may be used to calculate corresponding therapeutically effect doses for administration to other host animals, including humans. Illustrative corresponding doses may be calculated using the “Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers” published by FDA, and found at https: / / www.fda.gov / media / 72309 / download. and which is incorporated herein in its entirety’ by reference.

[0221] In each of the foregoing and each of the following embodiments, and each of the claims, unless otherwise indicated, it is also to be understood that the transitional phrase “consisting essentially of’ means that the scope of the corresponding composition, unit dose, method or use is understood to encompass the specified compounds or recited steps, and those that do not materially affect the basic and novel characteristics of the invention described herein. For example, a method described herein that consists essentially of a single compound, or genus of compounds, is understood to represent a monotherapy for the recited disease. Though the monotherapy may include co-administration of one or more carriers, vehicles, diluents, adjuvants, excipients, and the like, and combinations thereof, and / or include co-administration of one or more additional active pharmaceutical ingredients, those latter additional active pharmaceutical ingredients are to be understood to be for treating diseases and / or symptoms distinct from treating the underlying conditions described herein, such as the treatment of the medical condition itself.

[0222] The following examples further illustrate specific embodiments of the invention; 1392-17709 however, the following illustrative examples should not be interpreted in any way to limit the invention. Unless otherwise indicated, all starting compounds, reagents, and solvents used in the following examples are available from commercial suppliers.

[0223] EXAMPLES

[0224] PROCESS EXAMPLE 1. General Preparation of Nanoparticle Complex (NC) via Benchtop Sonication (BTS). A solution of the biopolymer is prepared in an organic solvent. A solution of the biological molecule is prepared in the same or a different organic solvent. The solutions of the biological molecule and the biopolymer are mixed, and added to a 0-10% weight / volume PVA solution in buffered deionized water. The resulting mixture is sonicated for a predetermined length of time to form nanoparticle complexes. The progress of the formation of the nanoparticle complexes is optionally monitored. The sonicated mixture is added to a 0.2% PVA solution in deionized water, and the resulting mixture is centrifuged, redispersed into deionized water, optionally treated with a cryoprotectant and / or lyoprotectant, and lyophilized to obtain the NCs as a solid.

[0225] PROCESS EXAMPLE 2. General Preparation of Nanoparticle Complex (NC) via Microfluidics (MF). A solution of the biopolymer is prepared in an organic solvent. A solution of the biological molecule is prepared in the same or a different organic solvent. The solutions of the biological molecule and the biopolymer are mixed, and processed in a microfluidics apparatus using deionized water or water for injection as the aqueous phase. The resulting eluent is purified by tangential flow filtration TFF against deionized water or water for injection. A cryoprotectant, and / or lyoprotectant, is optionally added to the resulting solution, and the final solution is lyophilized to obtain the NCs as a solid.

[0226] PROCESS EXAMPLE 3. General Preparation of Nanoparticle Complex (NC). A solution of the biopolymer is prepared in an organic solvent. An aqueous solution of the biological molecule is prepared. The aqueous solution of the biological molecule is added to the solution of the biopolymer is the organic solvent, and the mixture is stirred for a predetermined length of time, generally 30-60 minutes, to form nanoparticle complexes. The progress of the formation of the nanoparticle complexes is optionally monitored.

[0227] The solution containing the nanoparticle complexes is optionally purified by dialysis against deionized water. The dialyzed solution is centrifuged, redispersed in an aqueous solution, and a cryoprotectant and / or lyoprotectant is optionally added to the resulting solution. The final solution is lyophilized.

[0228] The solution containing the nanoparticle complexes is optionally purified by TFF against deionized water. A cryoprotectant and / or lyoprotectant is optionally added to the 1392-17709 resulting solution. The final solution is lyophilized to obtain the NCs as a solid.

[0229] PROCESS EXAMPLE 4. General Preparation of Nanoparticle Complex (NC). Using a dropwise addition method, the required amounts of biomolecule and biopolymer, such as pUDCA or ePLL, are weighed. The biopolymer is dissolved in water or a water-miscible organic solvent, such as EtOH, DMSO, DMF, or a mixture thereof, such as 80:20 v / v EtOH / DMSO, and the like. Additional organic solvents such as CH2CI2 may included. The biomolecule is dissolved or suspended in H2O, such as water for injection (WFI) or deionized water (DI), mixed well, and transferred to a reaction vessel, such as a beaker with magnetic stirring. The biopolymer solution is added slowly dropwise to the aqueous API solution, and stirred for 30-60 minutes. Nanocomplexes described herein self-assemble. A cryoprotectant and / or lyoprotectant is optionally added, and the mixture is frozen, then lyophilized to obtain the NCs as a dried powder.

[0230] PROCESS EXAMPLE 5. General Preparation of Biomonomer or Biopolymer Coated Nanoparticle Complex (core-shell NC). Using a dropwise addition method, the required amounts of biomolecule and biopolymer, such as pUDCA or ePLL, are weighed. The biopolymer is dissolved in water or a water-miscible organic solvent, such as EtOH, DMSO, DMF, or a mixture thereof, such as 80:20 v / v EtOH / DMSO, and the like. Additional organic solvents such as CH2CI2 may included. The biomolecule is dissolved or suspended in H2O, such as water for inj ection (WFI) or deionized water (DI), mixed well, and transferred to a reaction vessel, such as a beaker with magnetic stirring. The biopolymer solution is added slowly dropwise to the aqueous API solution, and stirred for 30-60 minutes. In the case of ePLL biopolymers, the addition can be rapid in a single aliquot. Nanocomplexes described herein self-assemble.

[0231] The biomonomer or biopolymer, or mixture thereof, for coating, such as PLGA, pUDCA, pDCA. or pGCA, is dissolved in water or a water-miscible organic solvent, such as EtOH, DMSO. DMF, or a mixture thereof, such as 80:20 v / v EtOH / DMSO, and the like. Additional organic solvents such as CH2CI2 may included. The biomonomer or biopolymer, or mixture thereof, for coating is added quickly in one aliquot to the stirring NC core reaction mixture, and stirring is continued for 1 h.

[0232] A cryoprotectant and / or lyoprotectant is optionally added, and the mixture is frozen, then lyophilized to obtain the biomonomer or biopolymer coated NCs as a dried powder.

[0233] PROCESS EXAMPLE 6. General Preparation of Enteric Coated Nanoparticle Complex (core-enteric shell NC). Using a dropwise addition method, the required amounts of biomolecule and biopolymer, such as pUDCA or ePLL, are weighed. The biopolymer is dissolved in water or a water-miscible organic solvent, such as EtOH, DMSO, DMF, or a 1392-17709 mixture thereof, such as 80:20 v / v EtOH / DMSO, and the like. Additional organic solvents such as CH2CI2 may included. The biomolecule is dissolved or suspended in H2O, such as water for injection (WFI) or deionized water (DI), mixed well, and transferred to a reaction vessel, such as a beaker with magnetic stirring. The biopolymer solution is added slowly dropwise to the aqueous API solution, and stirred for 30-60 minutes. In the case of ePLL biopolymers, the addition can be rapid in a single aliquot. Nanocomplexes described herein self-assemble.

[0234] The enteric polymer for coating is dissolved in water or a water-miscible organic solvent, such as isopropyl alcohol. The solution of the enteric coating polymer is added quickly in one aliquot to the stirring NC core reaction mixture, and stirring is continued for about 15 minutes.

[0235] A cryoprotectant and / or lyoprotectant is optionally added, and the mixture is frozen, then lyophilized to obtain the enteric coated NCs as a dried powder.

[0236] COMPOSITION EXAMPLES. GLP-l / bile acid nanocomplexes. The following example is prepared from tirzepatide (TZP) and pUDCA using Process Example 3.

[0237] (a)(concentration, solvent);(b)(concentration, solvent);(c)cryoprotectant / biological molecule weight ratio;(d)particle size after lyophilization;(e)PDI=polydispersity index;(f)DIW=deionized water;(g)calculated quantitative yield.

[0238] COMPOSITION EXAMPLES. GLP-l / mixed bile acid nanocomplexes. The following example is prepared from tirzepatide (TZP) and pUDCA using Process Example 4. 1392-17709

[0239] COMPOSITION EXAMPLES. GLP-l / bile acid nanocomplexes. The following example is prepared from semaglutide (SEM) and pUDCA using Process Example 3.

[0240] (a)(concentration, solvent);(b)(concentration, solvent);(c)cryoprotectant / biological molecule weight ratio;(d)particle size after lyophilization;(e)PDI=polydispersity index;(f)DIW=deionized water;(g)calculated quantitative yield.

[0241] COMPOSITION EXAMPLE. GLP-l / bile acid nanocomplexes. The following examples are prepared from semaglutide (SEM) and pUDCA using Process Example 4. 1392-17709

[0242] COMPOSITION EXAMPLE. GLP-l / bile acid nanocomplexes. The following examples are prepared from semaglutide (SEM) and 6 kd pUDCA using Process Example 4. COMPOSITION EXAMPLE. GLP-l / polylysine nanocomplexes. The following example is prepared from semaglutide (SEM) and ePLL using Process Example 4.

[0243] (a) Biopolymer solvent / Biomolecule solvent.

[0244] COMPOSITION EXAMPLE. GLP-l / PLGA nanocomplexes. The following examples are prepared from 1.5 mg / mL semaglutide (SEM) in DI, and a PLGA in 95:5 EtOH / DCM using Process Example 4. 1392-17709

[0245] (a) Biopolymer solvent / Biomolecule solvent; (b) Loading concentration of biomolecule in the nanoparticle complex; (c) Release

[0246] COMPOSITION EXAMPLE. Insulin / bile acid nanocomplexes. The following example is prepared from 1 mg / mL recombinant human insulin (R-HI) in WFI, and 2 mg / mL 3 kd pUDCA + 10% UDCA in (90: 10 EtOH / DCM) using Process Example 4.

[0247] (a) Biopolymer solvent / Biomolecule solvent.

[0248] COMPOSITION EXAMPLE. Monoclonal antibody / bile acid nanocomplexes. The following examples are prepared from 0.53 mg / mL ofatumumab and 2. 15 mg / mL pUDCA in 92:8 EtOH / DCM using Process Example 4.

[0249] (a) 4: 1 w / w ratio of Cry oprotectant to nanoparticle complex.

[0250] COMPOSITION EXAMPLE. Monoclonal antibody / bile acid nanocomplexes. The following example is prepared from Humira® (adalimumab) and 4 kd pUDCA using Process Example 4.

[0251] COMPOSITION EXAMPLES. Nanocomplexes with tunable particle size. The following uncoated NCs were prepared from semaglutide and pUDCA using Process Example 4. 1392-17709

[0252] It is observed that the size of the NCs is tunable and dependent upon the relative concentration of the biopolymer and the molecular weight of the biopolymer. It is also observed that particle is maintained when the relative concentration of the biopolymer is increased, and the molecular weight of the biopolymer is decreased. It is also observed that particle is maintained when the relative concentration of the biopolymer is decreased, and the molecular weight of the biopolymer is increased.

[0253] COMPOSITION EXAMPLES. Nanocomplexes with tunable particle size. The following uncoated NCs were prepared from semaglutide using Process Example 4.

[0254] (a) Biopolymer solvent / biomolecule solvent.

[0255] It is observed that the size of the NCs is tunable and dependent upon the relative concentration of the biopolymer. Higher relative concentrations of the pUDCA biopolymer included in Examples 12C, 12D, and 12E provide NCs with higher particle diameter. The tunable NCs are produced from a variety of biomonomers and / or biopolymers, and a variety of molecular weights. Both smaller and larger D-average particle size compositions are characterized by a low PDI. It is also observed that the size of the NCs is tunable and dependent the molecular 1392-17709 weight of the biopolymer, where increasing molecular weight provides increased particle sizes. It is also observed that particle is maintained when the relative concentration of the biopolymer is increased, and the molecular weight of the biopolymer is decreased. It is also observed that particle is maintained when the relative concentration of the biopolymer is decreased, and the molecular weight of the biopolymer is increased.

[0256] COMPOSITION EXAMPLES. Nanocomplexes with tunable charge zeta. The following uncoated NCs were prepared from semaglutide and 6 kd pUDCA, at a ratio of 40 / 100 using Process Example 4. In addition, the nanocomplexes were processed with a SIF wash, and

[0257] 2 pm filter.

[0258] (a) 1:2 Biomolecule / sucrose ratio.

[0259] COMPOSITION EXAMPLES. Biopolymer coated nanocomplexes. The biopolymer coated NCs were prepared from 0.5 mg / ML semaglutide in DI. 0.2 mg / mL 2.8 kd ePLL in DI. and a biopolymer coating in DMF using Process Example 5 and a 1 :4 core / coating ratio.

[0260] (a) 23% glycolic acid (GA) and 77% lactic acid (LA).

[0261] COMPOSITION EXAMPLES. Biopolymer coated nanocomplexes with tunable particle size. The biopolymer coated NCs were prepared using Process Example 5. 1392-17709

[0262] (a) Biopolymer solvent / Biomolecule solvent; (b) 23% glycolic acid (GA) and 77% lactic acid (LA).

[0263] It is observed that the size of the NCs is tunable and dependent upon the relative concentration of the cryoprotectant. Higher relative concentrations of the cryoprotectant included in Examples 15B and 15C provide NCs with higher particle diameter. The tunable NCs are produced from a variety of cryoprotectants, a variety of biomonomers and / or biopolymersand a variety of molecular weights. Both smaller and larger D-average particle size compositions are characterized by a low PDI. Alternative coating biopolymers include PLGA62, 62% glycolic acid (GA) and 38% lactic acid (LA), and the like.

[0264] COMPOSITION EXAMPLES. Enteric coated nanocomplexes. The enteric coated NCs were prepared from semaglutide / 11 kd pUDCA core NCs, and Eudragit FS-30D using Process

[0265] Example 6.

[0266] METHOD EXAMPLE. Particle Size and PDI. Particle size disclosed herein is 1392-17709 generally the average particle size (D-average) of a entire sample, and generally determined by dynamic light scattering (DLS) unless otherwise indicated. The corresponding PDI is also determined by DLS , unless otherwise indicated.

[0267] METHOD EXAMPLE. High-performance liquid chromatography (HPLC). HPLC standards of the biomolecules are analyzed in triplicate. Test articles of the biomolecule are used in a sham process variation that excludes the biopolymer. The resulting test articles of the biomolecule are analyzed by HPLC and the chromatograms are compared to the standard HPLC chromatograms to determine the presence of any differences, including the appearance of degradation products as evidenced by, for example, retention time, peak shape, and the appearance of additional peaks.

[0268] Following sample preparation using Process Examples 3 and 4, no significant differences in the HPLC chromatograms were observed for GLP-1 proteins, monoclonal antibodies, and endogenous proteins, such as insulin.

[0269] In contrast, sample preparation of either insulin or a series of monoclonal antibodies using an industry -standard T-mixing formulation process resulted in 50% degradation of the insulin and monoclonal antibodies.

[0270] METHOD EXAMPLE. In vitro Percent Bioavailability. NCs described here are weighed and dissolved in a solvent that solubilizes the biopolymer away from the NC, such as dimethylformamide. Sonication and vortexing are used to further break apart the NC and release the encapsulated biomolecule API. PBS is added to solubilize the biomolecule API and the solution is filtered. HPLC is used with API standards to determine the concentration which is used to calculate the mass of API (loading) in pg per mg of NC.

[0271] The NC is weighed and dissolved in simulated intestinal fluid (SIF) at pH 6.8. A small amount is optionally removed for pre-SIF particle size evaluation using DLS before incubation in an incubator shaker at 100 rpm and 37°C. The SIF solution is then incubated at 37°C. After 1 hour of incubation, another small amount is optionally removed for post-SIF particle size evaluation using DLS. The incubated SIF solution is filtered. HPLC is used with API standards to determine the concentration which is used to calculate the mass of API (released) as an estimate of API that leave the in vivo system without being absorbed. 1 hour corresponds to the gastric emptying time. The % bioavailability is obtained by dividing the difference (loading-released) by (loading).

[0272] METHOD EXAMPLE. Insulin Bioactivity, IR-B Phosphorylation. To determine receptor ty rosine phosphory lation, CHO-IRB cells are plated in 96-well plates, grown for 48 h and serum starved for 2 h. The cells are then stimulated with (a) Human Insulin (HI from Sanofi), (b) recombinant Human Insulin (Sigma 91077C), and (c) Nanoparticle complexes 1392-17709 described herein formed from Human Insulin and a biopolymer (Example 4).

[0273] Activation is assessed through an in-Cell Western blot by stimulation of CHO cells overexpressing the human insulin receptor isoform B(CHO-IRB). Once a test article binds and activates the receptor, a primary phosphotyrosine antibody is used to detect insulin receptor signaling.

[0274] Each test article is serially diluted 3-fold from a top concentration of 900 nM to generate the concentration range down to 50 pM. Cells are treated for 20 minutes and fixed immediately. After fixation, the cells are permeabilized, blocked, incubated overnight with Anti-pTyr 4G10 (Millipore) antibody and subsequently treated with secondary antimouse-IgG- 800-CW antibody for 1 h. The results are normalized by the quantification of DNA with DRAQ DNA dye and presented as percentage phosphorylated.

[0275] Each test article is tested on three separate plates, with triplicate technical replicates per plate. Human Insulin is used as a control and compared to recombinant Human Insulin and nanoparticle complexes described herein.

[0276] The calculated ECso values and standard errors for each test article are as follows:

[0277] As shown in FIG. 5 . the normalized phosphorylation for each test article is nearly identical. The calculated ECso values and FIG. 5 demonstrate that the biomolecules forming the nanoparticle complexes described herein retain 100% of their biological activity compared to control samples.

[0278] METHOD EXAMPLE. Semaglutide Pharmacokinetics (PK). Test animals, such a pigs. dogs, or rats were fasted overnight. Test articles containing the biomolecule-biopolymer nanocomplexes are administered in capsule form by oral gavage tube and compared to control samples or comparator samples doses according to the product label. To confirm dose administration, the gavage tube is cleared using water or degassed non-caffeinated soda.

[0279] At various times post administration, blood is collected via a jugular catheter into K2 EDTA blood collection tubes. Blood samples are inverted multiple times to prevent clotting and cooled at 4°C. Blood samples are immediately centrifuged at 8000 rpm for 8 minutes for plasma separation. Following centrifugation, replicate >75 pL aliquots of each plasma sample are placed in multiple tubes and stored at -80°C until used for testing.

[0280] The biomolecule is extracted from each plasma sample through protein precipitation. Briefly, the solids are centrifuged down, and the supernatant is extracted and adjusted to the 1392-17709 appropriate composition for conventional LC / MS injection by adding and diluting the sample with appropriate solutions, as necessary. The identity of the biomolecule obtained from the plasma samples is confirmed by the parent-daughter fragmentation pattern, and corresponding peak ratios compared to control standards of the biomolecule. A spiked plasma calibration line is also used where predetermined amounts of the control biomolecule is added to the plasma sample prior to LC / MS and used for quantification of biomolecule in each sample as a function of time.

[0281] The quantified blood samples are averaged for each time point, and plotted to obtain a fitted curve showing the Cmax, Tmax, and AUC for each of the various time intervals.

[0282] A representative sample of nanoparticles prepared from semaglutide and 6 kd pUDCA was prepared according to Example 4, and administered to pigs (n=4). As a positive control, the Rybelsus SNAC formulation was administered to pigs (n=8) at the same absolute API dosage for comparison. The compared PK profiles for Example are shown in FIG. 6.

[0283] It is observed that the NCs described herein demonstrate a greatly improved PK profile. At equivalent administered doses, the NCs described herein provide 2-fold higher exposure of the therapeutic molecule to the host animal as evidenced by the AUCo-96h. In addition, the NCs described herein show a 3-fold delayed Tmax and a 50% lower Cmax compared to the Rybelsus SNAC formulation. It has been reported that a fast onset to Tmax generally results in a higher number of adverse events due to the early achieving of the Cmax and the rapid increase in exposure to the biomolecule. The NCs described herein provide a more gradual concentration gradient of exposure to the subject animal leading to a lower number of adverse events. The nanoparticle complexes described herein exhibit a superior 50% increase in bioavailability.

[0284] The PK limitations of Rybelsus® cannot be overcome by increasing the administered dose because Rybelsus® is dose-limited at current approved levels. It has been reported that higher doses result in an increase in adverse side effects, and an increase in their severity. In contrast, because the nanoparticle complexes described herein are absorbed in the lower GI tract, increases in dosing for higher therapeutic exposure is possible.

[0285] Each of the publications and / or websites cited herein is incorporated herein by reference.

Claims

1392-17709WHAT IS CLAIMED IS:1 . A nanoparticle complex comprising one or more active pharmaceutical ingredients and a carrier, where at least one of the one or more active pharmaceutical ingredients is a biomolecule or an analog or derivative thereof, and the carrier comprises a plurality of biomonomers or biopolymers, or a combination thereof.

2. A orally administrable nanoparticle complex comprising one or more active pharmaceutical ingredients and a carrier, where at least one of the one or more active pharmaceutical ingredients is a biomolecule or an analog or derivative thereof, and the carrier comprises a plurality of biomonomers or biopolymers, or a combination thereof, and where the nanoparticle complex is characterized by an average diameter of about 400 nm, about 350 nm. about 300 nm, about 250 nm, about 200 nm, about 150 nm, about 100 nm, or about 50 nm or less.

3. The nanoparticle complex of claim 1 or claim 2 wherein the biological molecule comprises a peptide, a protein, a nucleic acid, both a peptide and a nucleic acid, or an antibody.

4. The nanoparticle complex of claim 1 or claim 2 wherein the biological molecule is a protein or peptide.

5. The nanoparticle complex of claim 1 or claim 2 wherein the biological molecule is a glucagon-like peptide-1 (GLP-1) or an analog or derivative thereof, or a GLP receptor agonist.

6. The nanoparticle complex of claim 1 or claim 2 wherein the biological molecule is a glucose-dependent insul inotropic polypeptide (GIP) or an analog or derivative thereof, or a GIP receptor agonist.

7. The nanoparticle complex of claim 1 or claim 2 wherein the biological molecule is a dual GLP-1 / GIP receptor agonist.

8. The nanoparticle complex of claim 1 or claim 2 wherein the biological molecule is a triple GLP-l / GIP / GR receptor agonist.

9. The nanoparticle complex of claim 1 or claim 2 wherein the biological molecule is a glucocorticoid receptor (GR) or an analog or derivative thereof, or aGR receptor agonist.

10. The nanoparticle complex of claim 1 or claim 2 wherein the biological molecule is selected from the group consisting of albiglutide, dulaglutide, exenatide, liraglutide, lixisenatide, semaglutide, and tirzepatide. and analogs and denvatives thereof, and any combinations of the foregoing.1392-1770911. The nanoparticle complex of any one of claims 1-11 wherein the biological molecule is a nucleic acid.

12. The nanoparticle complex of any one of claims 1-11 wherein the biological molecule is an antibody or antibody mimetic.

13. The nanoparticle complex of any one of claims 1-11 wherein the biological molecule is adalimumab or ofatumumab.

14. The nanoparticle complex of any one of claims 1-11 wherein the biomonomer or biopolymer is selected from the group consisting of poly-arginine; bile acids, poly bile acids, including primary, secondary, tertiary, conjugated, and / or synthetic poly bile acids; PEGylated polymers; lipidated polymers, including reduced P-gly coprotein (P-gp) efflux lipidated polymers; lipids, ePLL, PLA, PGA, and PLGA, and combinations thereof.

15. The nanoparticle complex of any one of claims 1-11 wherein the polymer comprises one or more bile acids, or analogs or derivatives thereof, a polymer of one or more bile acids, or analogs or derivatives thereof, or a combination of the foregoing.

16. The nanoparticle complex of any one of claims 1-11 wherein the bile acid or analog or derivative thereof comprises a cholesterol or analog or derivative thereof.

17. The nanoparticle complex of any one of claims 1-11 wherein the bile acid or analogs or derivatives thereof comprises a cholic acid or analog or derivative thereof.

18. The nanoparticle complex of any one of claims 1-11 wherein the polymer comprises one or more polymerized ursodeoxycholic acids (pUDCAs).

19. The nanoparticle complex of any one of claims 1-11 wherein the polymer comprises one or more polymerized deoxy cholic acids (pDCAs).

20. The nanoparticle complex of any one of claims 1-11 wherein the polymer comprises one or more polymerized polymerized glycocholic acids (pGCAs).

21. The nanoparticle complex of any one of claims 1-11 wherein the polymer comprises one or more polymerized lactic acids (PLAs), polymerized glycolic acids (PGAs), co-polymerized lactic and glycolic acids (PLGAs), or a combination thereof.

22. The nanoparticle complex of any one of claims 1-11 wherein the polymer comprises one or more polymerized lysines (ePLLs).

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