Cyclic peptides and uses thereof

Abstract

Described herein are cyclic polypeptides comprising the sequence cycloSQKCHEA or cycloCHEASAAQK. Also described herein are a composition comprising the polypeptide, as well as a method of modulating HSP70 activity, and a method of treating, ameliorating, and / or preventing diseases and disorders using the polypeptide.

Description

Attorney Docket No. 046528-7146W01(01103)CYCLIC PEPTIDES AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONSThe present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 724,688, filed November 25, 2024, and U.S. Provisional Patent Application No. 63 / 760,951, filed February 20, 2025, both of which are incorporated herein by reference in their entireties.SEQUENCE LISTINGThe XML file named "046528-7146W01(01103)_Sequence Listing.xml" created on November 12, 2025, comprising 5,293 Kbytes, is hereby incorporated by reference in its entirety.BACKGROUNDToxic protein accumulations or condensates are implicated in various diseases, especially neurodegenerative diseases. In addition, they were understood as features of relatively rare blood diseases, classified generally as amyloidosis. Methods of promoting the removal of such toxic protein accumulations remain lacking, and existing methods are most specific to specific protein types with, for example, antibodies.Heat shock proteins, such as HSP70, promote the proper folding of proteins and the removal of misfolded proteins, which forms condensates. It has been proposed that the activation of HSP70 is a viable strategy of removing both intracellular and intercellular toxic protein accumulations, and a potential therapy for human aggregate diseases. However, HSP70 activators are not widely available.There is a need for compounds that can remove toxic protein accumulations, and compounds for activating HSP70. The present invention addresses this need.SUMMARYIn some aspects, the present invention is directed to the following non-limiting embodiments:156618988.2Attorney Docket No. 046528-7146W01(01103)PolypeptideIn some aspects, the present invention is directed to a polypeptide.In some embodiments, the polypeptide comprises the sequence set forth in SEQ ID NO: 1 (cycloSQKCHEA) or SEQ ID NO:2 (cycloCHEASAAQK).In some embodiments, the polypeptide consists of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.CompositionIn some aspects, the present invention is directed to a composition.In some embodiments, the composition comprises the polypeptide herein.In some embodiments, the composition further comprises a carrier, such as a pharmaceutically acceptable carrier.Method of activating HSP70In some aspects, the present invention is directed to a method of activating HSP70, such as in a cell or in a subject.In some embodiments, the method comprises contacting the cell with or introducing into the subject the polypeptide, or the composition herein.In some embodiments, the method reduces oxidative stress, increase free thiol level, and / or reduces protein aggregation, optionally amyloidogenesis.In some embodiments, the activation of HSP70 in the subject reduces a protein aggregation, optionally amyloidogenesis, in a blood circulation of the subject.In some embodiments, the cell is a primary cell, a cell of a cell line, or a cell in a subject, optionally a human subject.In some embodiments, the cell is a mammalian cell, a human cell, or a cell derived therefrom.In some embodiments, the subject is a mammalian subject, optionally a human subject.Method of treating, ameliorating, and / or preventing a disease or disorderIn some aspects, the present invention is directed to a method of treating, ameliorating, and / or preventing a disease or disorder in a subject in need thereof.256618988.2Attorney Docket No. 046528-7146W01(01103)In some embodiments, the method comprises administering to the subject an effective amount of the polypeptide or the composition herein.In some embodiments, the disease or disorder is caused or aggravated by a toxic protein aggregate, an oxidative stress, an insufficient HSP70 activity, a hyperglycemic condition.In some embodiments, the disease or disorder is diabetes, a complication of diabetes, hyperglycemia, a neurodegenerative disease, a cardiovascular disease, an inflammatory condition, a protein aggregate disorder, and / or an autoimmune disease.In some embodiments, the diabetes is type I diabetes or type II diabetes.In some embodiments, the complication of diabetes is kidney damage, eye damage, foot damage, a skin condition, a mouth condition, accelerated aging, or cognitive decline caused by diabetes.In some embodiments, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, or amyotrophic lateral sclerosis (ALS).In some embodiments, the cardiovascular disease is myocardial infarction, ischemia or reperfusion, heart failure, atherosclerosis, hypertension, or arrhythmia.In some embodiments, the autoimmune disease is multiple sclerosis (MS), rheumatoid arthritis (RA).In some embodiments, the subject is a mammal, optionally a human.Method of increasing longevityIn some aspects, the present invention is directed to a method of increasing longevity of a subject.In some embodiments, the method comprises administering to the subject an effective amount of the polypeptide or the composition herein.In some embodiments, the subject is a mammal.In some embodiments, the mammal is a non-human mammal or a human.BRIEF DESCRIPTION OF THE DRAWINGSThe following detailed description of exemplary embodiments will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating, nonlimiting embodiments are shown in the drawings. It should be understood, however, that the356618988.2Attorney Docket No. 046528-7146W01(01103) instant specification is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.Fig. 1 A illustrates certain aspects of the structure and composition of cSKE7, in accordance with some embodiments. Fig. IB illustrates certain aspects of the structure and composition of cSKE9, in accordance with some embodiments. The sequences shown in Figs. 1A and IB are cycloSQKCHEA (SEQ ID NO: 1) and cycloCHEASAAQK (SEQ ID NO:2), respectively.Fig. 2A: Solubility predictions of cSKE7 and CHEC-7 peptides based on amino acid composition using the online software from Cambridge University (https: / / www- cohsoftware.ch.cam.ac.uk / index.php / camsolintrinsic). Fig. 2B: The differences were tested directly with different aqueous mixtures of 2-propanol (shown are 4 examples of solubility in mg quantities of peptide in 200pl of 50% 2-propanol / water mixture).Fig. 3 demonstrates that the increased fructosamine levels in plasma treated with methylglyoxal (MG) for 8 days at 37°C, in accordance with some embodiments. Values were normalized to average of control samples (-MG). MG increased fructosamine both with and without pretreatment with bioactive concentrations of cSKE7 (p=.OOOl for all MG comparisons with no MG except OpM cSKE7 where p=.0002. MG treated samples, whether peptide treated or not, were not statistically different (n.s.).Fig. 4: plasma HSP70 immunostaining with different concentrations of cSKE7 immobilized on nitrocellulose discs, in accordance with some embodiments. The plasma was incubated with and without methylglyoxal for 8 days at 37°C.Fig. 5A: Thioflavin-t (Tht) fluorescence of smears from samples of the different plasma pools showing the range of amyloid accumulations after 8-day exposure to methylglyoxal (ImM) and after subsequent treatment with POE vehicle, 1 or lOpM cSKE7 for 24hrs. The two panels on the right from the 70yo pool were also treated with HSP70 inhibitor pifithrin-p (200pM) or DMSO vehicle for 1 hr, prior to peptide treatment. Large areas of the smears were sampled for the micrographs (bar = 100pm). Fig. 5B: A subset of samples from the 30yo and 70yo pools was processed for the Tht solution assay and normalized to average value for vehicle-treated samples. The HSP inhibitor pifithrin-p abrogated the loss of THt fluorescence at all concentrations. DMSO controls were not statistically different than the original measurements. Significance values are from unpaired t-test with Welch’s correction.456618988.2Attorney Docket No. 046528-7146W01(01103)Fig. 6: Free thiols were detected with 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) and normalized to the average of vehicle treated samples. Significant elevations (fewer oxidized thiols) were detected at 1 and lOpM peptide concentrations, representing combined samples from the 70yo and 30yo plasma pools. Included in these experiments for comparison are measurements from plasma samples incubated for 8 days without MG. A subset of these samples was pretreated with pifithrin-p or DMSO. The HSP70 inhibitor eliminated the increase. Values from companion samples pretreated with DMSO were not statistically different and were included with the initial samples. Note that the apparent loss of free thiols with the inhibitor at IpM peptide was statistically greater than with the initial vehicle treated samples suggesting additional inhibition of endogenous HSP70 activity after MG treatment. Significance levels via unpaired t-test with Welch’s correction.Figs. 7A-7B: Paired data from the 70 and 30 year old pools after cSKE7 treatments of 1 and lOpM and normalized to average of vehicle treated samples (see text for details). Fig. 7C: A representative subset of all MG-treated samples from the 30 yo pool was pretreated with pifithrin-p or DMSO suggested that the inhibitor eliminates both peptide mediated rescue effect of activity as well as endogenous surviving GAPDH activity. A “floor effect”, encountered with the present assay (too many values at or near the detection limit), precluded a similar analysis of inhibitor effects in the 70yo pool. Significance levels via paired t-test for Figs. 7A-7B, and unpaired test for Fig. 7C.DETAILED DESCRIPTIONThe following disclosure provides many different embodiments, or examples, for implementing different features of the provided subject matter. Specific examples of components and arrangements are described below to simplify the present disclosure. These are, of course, merely examples and are not intended to be limiting. For example, the formation of a first feature over or on a second feature in the description that follows may include embodiments in which the first and second features are formed in direct contact, and may also include embodiments in which additional features may be formed between the first and second features, such that the first and second features may not be in direct contact. In addition, the present disclosure may repeat reference numerals and / or letters in the various examples. This repetition556618988.2Attorney Docket No. 046528-7146W01(01103) is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and / or configurations discussed.The present study generated a cyclic peptide, which has the sequence cycloSQKCHEA (SEQ ID NO: 1) (referred to herein as cSKE7). The present study confirmed that the new cSKE7 peptide has significantly better solubility than the CHEC-7 (CHEASQC, SEQ ID NO:4) peptide. Furthermore, unlike the CHEC-7 peptide which has decreased activity at higher concentrations, the activity of the cSKE7 peptide displayed good linearity in relation to peptide concentrations.The present study further generated the cSKE9 peptide having the sequence cycloCHEASAAQK (SEQ ID NO:2), based on the CHEC-9 peptide (CHEASAAQC, SEQ ID NO:3). Since the strategies behind cSKE7 and cSKE9 are similar: e.g., circularization plus replacing the cysteine (C) residue with a lysine residue (K), it is expected that the cSKE9 peptide has the desirable features of cSKE7, e.g., better solubility and linear concentration-activity relationship, as well.Accordingly, in some aspects, the present invention is directed to a cyclic peptide, as well as compositions comprising the same.The present study shows that the cyclic peptide cSKE7 herein has antioxidative effect, was able to increase the free thiols, and was able to reverse toxic protein aggregations, such as reversing the amyloidogenesis in the blood plasma caused by methylglyoxal (MG) exposure. Furthermore, the present study shows that the peptide herein achieves these effects through HSP70, as the inhibition of HSP70 abolishes such effects. Since cSKE9 and cSKE7 peptides are derived from the same protein and have virtually the same binding partners, cSKE9 has similar effects, as well.Accordingly, in some aspects, the present invention is directed to a method of activating HSP70, such as with the cSKE7 or cSKE9 peptide herein.Since oxidative stress and toxic protein aggregations are known to cause an array of diseases and disorders, such as diabetes, complications of diabetes, hyperglycemia, neurodegenerative diseases, cardiovascular diseases, inflammatory conditions, and an autoimmune disease, it is expected that the peptide herein would be effective in treating, ameliorating, and / or preventing such diseases or disorders.In some aspects, the present invention is directed to a method of treating, ameliorating, and / or preventing diseases or disorders, such as with the cSKE7 or cSKE9 peptide herein.656618988.2Attorney Docket No. 046528-7146W01(01103)HSP70 is a molecular chaperone that maintains cellular health by preventing and clearing protein aggregates, and its levels decline with age. Increased HSP70 levels in lab animals has been shown to increase lifespan. As such, it is expected that the activation of HSP70 by the polypeptides or compositions herein is able to increase the longevity of subjects.Accordingly, in some aspects, the present invention is directed to a method of increasing longevity of a subject.DefinitionsAs used herein, each of the following terms has the meaning associated with it in this section. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Generally, the nomenclature used herein and the laboratory procedures in animal pharmacology, pharmaceutical science, peptide chemistry, and organic chemistry are those well-known and commonly employed in the art. It should be understood that the order of steps or order for performing certain actions is immaterial, so long as the present teachings remain operable. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.In the application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components and can be selected from a group consisting of two or more of the recited elements or components.In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.756618988.2Attorney Docket No. 046528-7146W01(01103)In this document, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. The statement "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B.""About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, in certain embodiments ±5%, in certain embodiments ±1%, in certain embodiments ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.Polypeptide and CompositionIn some aspects, the present invention is directed to a polypeptide.In some aspects, the polypeptide is a cyclic polypeptide.In some embodiments, the polypeptide comprises the sequence set forth in SEQ ID NO: 1 (cycloSQKCHEA) or SEQ ID NO:2 (cycloCHEASAAQK).In some embodiments, the polypeptide consists of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.In some aspects, the present invention is directed to a composition.In some embodiments, the composition comprises the polypeptide herein; and a pharmaceutically acceptable carrier.Method of Activating HSP70In some aspects, the present invention is directed to a method of activating HSP70.In some embodiments, the HSP70 is inside a cell.In some embodiments, the HSP70 is extracellular, such as in the blood circulation of a subject.In some embodiments, the HSP70 is in a subject.In some embodiments, the method comprises contacting the cell containing HSP70 with the polypeptide or composition herein.In some embodiments, the method reduces oxidative stress, increases free thiol level, and / or reduces protein aggregation, such as amyloidogenesis, in the cell.856618988.2Attorney Docket No. 046528-7146W01(01103)In some embodiments, the cell is a primary cell, a cell of a cell line, or a cell in a subject, such as a human subject.In some embodiments, the cell is a mammalian cell, a human cell, or a cell derived therefrom.In some embodiments, the method comprises introducing into the subject the polypeptide or composition herein.In some embodiments, the method reduces oxidative stress, increases free thiol level, and / or reduces protein aggregation, such as amyloidogenesis, in the subject.In some embodiments, the activation of HSP70 in the subject reduces a protein aggregation, such as amyloidogenesis, in a blood circulation of the subject.In some embodiments, the subject is a mammalian subject, such as a human subject.Method of treating, Ameliorating, and / or Preventing Disease or DisorderIn some aspects, the present invention is directed to a method of treating, ameliorating, and / or preventing a disease or disorder in a subject in need thereof.In some embodiments, the method comprises administering to the subject an effective amount of the polypeptide or composition herein.In some embodiments, the disease or disorder is caused or aggravated by a toxic protein aggregate, an oxidative stress, an insufficient HSP70 activity, or a hyperglycemic condition.In some embodiments, the disease or disorder is diabetes, a complication of diabetes, hyperglycemia, a neurodegenerative disease, a cardiovascular disease, an inflammatory condition, a protein aggregate disorder, and / or an autoimmune disease.In some embodiments, the diabetes is type I diabetes or type II diabetes.In some embodiments, the complication of diabetes is kidney damage, eye damage, foot damage, a skin condition, a mouth condition, accelerated aging, or cognitive decline caused by diabetes.In some embodiments, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, or amyotrophic lateral sclerosis (ALS).In some embodiments, the cardiovascular disease is myocardial infarction, ischemia or reperfusion, heart failure, atherosclerosis, hypertension, or arrhythmia.956618988.2Attorney Docket No. 046528-7146W01(01103)In some embodiments, the autoimmune disease is multiple sclerosis (MS), rheumatoid arthritis (RA).In some embodiments, the subject is a mammal, such as a human.Combination TherapiesIn some embodiments, the method of treating, ameliorating, and / or preventing the disease / disorders herein includes administering to the subject the effective amount of at least one compound and / or composition contemplated within the disclosure.In some embodiments, the subject is further administered at least one additional agent that treats, ameliorates, and / or prevents a disease and / or disorder contemplated herein. In other embodiments, the compound and the at least one additional agent are co-administered to the subject. In yet other embodiments, the compound and the at least one additional agent are coformulated.The compounds contemplated within the disclosure are intended to be useful in combination with one or more additional compounds. These additional compounds may comprise at least one additional agent that treats one or more diseases or disorders contemplated herein.A synergistic effect may be calculated, for example, using suitable methods such as, for example, the Sigmoid-Emax equation (Holford & Scheiner, 1981, Clin. Pharmacokinet. 6:429- 453), the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul. 22:27-55). Each equation referred to above may be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively.Methods of Increasing LongevityIn some embodiments, the present invention is directed to a method of increasing longevity of a subject.In some aspects, the method comprises administering to the subject an effective amount of the polypeptide herein.1056618988.2Attorney Docket No. 046528-7146W01(01103)In some embodiments, the subject is a mammal.In some embodiments, the subject is a non-human mammal, optionally a dog, a cat, a horse, a sheep, a goat, a pig, a cattle, a camel, a llama, an alpaca, a mouse, a rat, a rabbit, or a non-human primate.In some embodiments, the subject is a human.Administration / Dosage / FornuilationsThe regimen of administration may affect what constitutes an effective amount. The therapeutic formulations contemplated within the disclosure may be administered to the subject either prior to or after the onset of a disease and / or disorder contemplated herein. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations contemplated within the disclosure may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.Administration of the compositions contemplated within the disclosure to a patient, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a disease and / or disorder contemplated herein in the patient. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound contemplated within the disclosure to treat a disease and / or disorder contemplated herein in the patient. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound contemplated within the disclosure is from about 1 and 5,000 mg / kg of body weight / per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.Actual dosage levels of the active ingredients in the pharmaceutical compositions contemplated within the disclosure may be varied so as to obtain an amount of the active1156618988.2Attorney Docket No. 046528-7146W01(01103) ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.In particular, the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts.A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds contemplated within the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms contemplated within the disclosure are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such a therapeutic compound for the treatment of a disease and / or disorder contemplated herein.In certain embodiments, the compositions of the disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the disclosure comprise a therapeutically effective amount of a compound of the disclosure and a pharmaceutically acceptable carrier.The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of1256618988.2Attorney Docket No. 046528-7146W01(01103) microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.In certain embodiments, the compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more. In another embodiment, the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the disclosure varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physical taking all other factors about the patient into account.Compounds of the disclosure for administration may be in the range of from about 1 pg to about 10,000 mg, about 20 pg to about 9,500 mg, about 40 pg to about 9,000 mg, about 75 pg to about 8,500 mg, about 150 pg to about 7,500 mg, about 200 pg to about 7,000 mg, about 3050 pg to about 6,000 mg, about 500 pg to about 5,000 mg, about 750 pg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.In some embodiments, the dose of a compound of the disclosure is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound of the disclosure used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as1356618988.2Attorney Docket No. 046528-7146W01(01103) described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.In certain embodiments, the present disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of the disease / disorders in a patient.Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for intracranially, intrathecal , oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and / or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.Routes of administration of any of the compositions of the disclosure include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical. The compounds for use in the disclosure may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized1456618988.2Attorney Docket No. 046528-7146W01(01103) formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein.Oral AdministrationFor oral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps. The compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate. The tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.For oral administration, the compounds of the disclosure may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropylmethylcellulose); fdlers (e g., cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrates (e.g., sodium starch glycollate); or wetting agents (e.g., sodium lauryl sulphate). If desired, the tablets may be coated using suitable methods and coating materials such as OPADRY™ fdm coating systems available from Colorcon, West Point, Pa. (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY™ White, 32K18400). Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions. The liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxy benzoates or sorbic acid).1556618988.2Attorney Docket No. 046528-7146W01(01103)The present disclosure also includes a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds of the disclosure, and a further layer providing for the immediate release of another medication. Using a wax / pH-sensitive polymer mix, a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release.Parenteral AdministrationFor parenteral administration, the compounds of the disclosure may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and / or continuous infusion. Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and / or dispersing agents may be used.Additional Administration FormsAdditional dosage forms of this disclosure include dosage forms as described in U.S.Patents Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms of this disclosure also include dosage forms as described in U.S. Patent Applications Nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Additional dosage forms of this disclosure also include dosage forms as described in PCT Applications Nos. WO 03 / 35041; WO 03 / 35040; WO 03 / 35029; WO 03 / 35177; WO 03 / 35039; WO 02 / 96404; WO 02 / 32416; WO 01 / 97783; WO 01 / 56544; WO 01 / 32217; WO 98 / 55107; WO 98 / 11879; WO 97 / 47285; WO 93 / 18755; and WO 90 / 11757.Controlled Release Formulations and Drug Delivery SystemsIn certain embodiments, the formulations of the present disclosure may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended1656618988.2Attorney Docket No. 046528-7146W01(01103) time period. The period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds. As such, the compounds for use the method of the disclosure may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.In certain embodiments of the disclosure, the compounds of the disclosure are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.DosingThe therapeutically effective amount or dose of a compound of the present disclosure depends on the age, sex and weight of the patient, the current medical condition of the patient1756618988.2Attorney Docket No. 046528-7146W01(01103) and the progression of the disease / disorders in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors.A suitable dose of a compound of the present disclosure may be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day. The dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses.It is understood that the amount of compound dosed per day may be administered, in nonlimiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.In the case wherein the patient's status does improve, upon the doctor's discretion the administration of the modulator of the disclosure is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (z.e., a "drug holiday"). The length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.Once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced, as a function of the patient's condition, to a level at which the improved disease is retained. In certain embodiments, patients require intermittent treatment on a long-term basis upon any recurrence of symptoms and / or infection.The compounds for use in the method of the disclosure may be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosage1856618988.2Attorney Docket No. 046528-7146W01(01103) for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LDso (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50. Capsid assembly modulators exhibiting high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human. The dosage of such capsid assembly modulators lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.Those skilled in the art recognizes, or is able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this disclosure and covered by the claims appended hereto. For example, it should be understood, that modifications in assay and / or reaction conditions, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application.

[0001] It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present disclosure. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application.ExamplesThe instant specification further describes in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless so specified. Thus, the instant specification should in no way1956618988.2Attorney Docket No. 046528-7146W01(01103) be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.Example 1: Redesigned anti-inflammatory peptide stimulates HSP70-mediated repair of human plasma stressed with glucose metabolite.A seven amino acid cyclic peptide has been applied to human blood plasma treated with glucose metabolite Methylglyoxal (MG). In the hyperglycemic condition, elevated MG levels in the blood lead directly to diabetic complications and accelerated aging, including cognitive decline. These pathologies are attributed to oxidant stress and amyloidogenesis, the latter involving toxic accumulations of blood and tissue proteins. The applied peptide, cycloSKE7 or cSKE7, was redesigned from cell survival -promoting and anti-inflammatory fragments of human / primate “orphan” protein DSEP / Dermcidin and incubated at low micromolar concentrations with the MG-stressed plasma for 24 hours. The modified structure of the new compound offers several practical advantages over predecessors including cyclic stability and a marked increase in aqueous solubility. Most importantly, the peptide dispersed thioflavin-t- stained amyloid aggregates and reduced oxidant stress as measured by plasma levels of free thiols and the rescue of glyceraldehyde 3-phosphate-dehydrogenase (GAPDH) activity. Since these n-terminal fragments of DSEP / Dermcidin have been shown to bind and influence the activity of heat shock protein 70 (HSP70), HSP70 inhibitor pifithrin-p was added to the plasma prior to peptide treatment. The inhibitor disrupted amyloid dispersion and both peptide-induced and, in some cases, normally-occurring antioxidant effects, suggesting these reparative activities are HSP70 dependent. The results are discussed in terms of their potential relevance to new therapies for the complications of metabolic disease and other disorders of aging including lifespan limitation.Example 2:Attention to protein aggregate diseases has increased dramatically over the last several decades due to their involvement in age-related neurodegenerative disorders. Toxic protein accumulations were essentially rediscovered for the brain, since for most of the last century they were understood as features of relatively rare blood diseases, classified generally as amyloidosis.2056618988.2Attorney Docket No. 046528-7146W01(01103)While the causes of amyloidosis, or other instances aggregate pathology, are diverse, one generalized approach to these disorders has remained therapeutically elusive. That approach consists of enhancing endogenous mechanisms of protein quality control, an important part of the balanced process of proteostasis. For this the prominent role of Heat Shock Proteins, especially HSP70, has been emphasized although attempts at regulation of this protein have so far not been successfully translated to a workable therapy for human aggregate diseases. HSP70 also exists in the circulation where its role is less well understood but may have functions both similar and distinct from its intracellular versions.Targeting toxic protein aggregation and associated oxidative stress has been considered also for metabolic diseases like diabetes and its complications. This is especially the case as these pertain to vascular disorders, but also pancreatic and kidney disease as well as peripheral neuropathies. It is now well-known that chronic elevations in blood glucose and its metabolites lead to toxic protein aggregation. The aggregates form from blood proteins and accumulate in blood vessel walls, and in the parenchyma of organs tissues like pancreas, kidney and peripheral nerves establishing a direct link between hyperglycemia and diabetic complications, including age-related cognitive decline. The proteins aggregate in a hyperglycemic environment, at least in part, because elevations in glucose’s principal metabolite, Methyl glyoxal (MG), stimulates the nonenzymatic formation of Advanced Glycation End Products (AGEs); the activities of AGEs are diverse, but a primary effect of their excess is to block redox balance elevating oxidative stress through a complex chemistry that also promotes disruption of protein structure via direct glycation of protein amines as well as by thiol oxidation and crosslinking. These changes create hydrophobic patches in the proteins that stick together, often in an amyloid configuration, disrupting interactions with binding partners and protein function. Thus, the destructive cycle of persistent hyperglycemia is perpetuated, further highlighting the role that a failure of protein quality control could play in the pathogenesis of diabetes and its complications.It is not surprising therefore that HSP70 has been proposed as a therapeutic target for diabetes and its many complications, as well as other conditions generally associated with inflammatory and oxidant stressors, especially as these relate to aging. Peptide fragments near the n-terminus of an endogenous human protein known as Dermcidin or DSEP. The fragments included Y-P30, and two shorter peptides, CHEC-9 (CHEASAAQC, SEQ ID NO:3), and CHEC- 7 (CHEASQC, SEQ ID NO:4), all with cell survival-promoting and anti-inflammatory2156618988.2Attorney Docket No. 046528-7146W01(01103) properties, in vitro and in vivo. The shorter peptides were cyclized by adding a terminal cysteine and verifying cross linking prior to use by the loss of both DTNB absorbance and of 2 protons by mass spectroscopy. Subsequently, and most importantly for the present study, a DSEP / Dermcidin fragment that included the CHEC-9 sequence was shown independently to bind HSP70 in detailed peptide mapping studies. Both CHEC-9 and CHEC-7 were also shown to bind and influence HSP70 activity, including the dissolution of amyloid forming aggregates, and for CHEC-7, this was demonstrated for oxidant-stressed human plasma ex vivo. The present study is a proof-of-concept report designed to extend these observations to a hyperglycemic model using Methylglyoxal stressed human plasma, a compound that is also implicated in cognitive decline. The present study tested the effectiveness of a peptide like CHEC-7 but in this case substituting the native lysine at the C-terminal end and then cyclizing the new peptide end- to-end giving cycloSQKCHEA (SEQ ID NO: 1) (Fig. 1). It is called cSKE7. This strategy was considered a necessary step for further therapeutic development of these peptide fragments because the nonnative disulfide bond, susceptible to reduction under various uncontrolled conditions, was replaced with the native lysine. End-to-end cyclization was expected to provide a more robust and stable therapeutic. In addition, and perhaps most important, we found limitations of the solubility of CHEC-7 in scaling up formulation efforts for potential human applications. While this problem may eventually be overcome as more sophisticate peptide delivery methods are developed, Savjani, et al. (and many others more recently) have noted that “Low aqueous solubility is the major problem encountered with formulation development of new chemical entities as well as generic development.” Substitution of the lysine for the second cysteine dramatically increases aqueous solubility, both theoretically and practically, mainly by increasing the polarity of the peptide (Results, Fig. 2).The data show that cSKE7 inhibits and reverses MG toxicity in human blood plasma, including amyloidogenesis and elements of oxidative stress as measured by glyceraldehyde 3- phosphate-dehydrogenase (GAPDH) activity and thiol redox status, all in an HSP-dependent manner. The present study used well-tested assays of the entirety of blood plasma, rather than focusing on specific plasma proteins, to document the reparative properties of this peptide. The present study also used human-derived plasma noting that the peptide is a re-designed fragment of a particularly human / primate protein (“orphan”) that is not present in other mammals. The goal was to encourage further work with this peptide that might lead to a new treatment strategy2256618988.2Attorney Docket No. 046528-7146W01(01103) for the complications of persistent hyperglycemia and other systemic metabolic disorders. The present study also considers the possibility of a novel approach to life extension therapy where aged proteostasis mechanisms become overwhelmed and simply wear out.Example 3: MethodsPeptide cSKE7 was synthesized, cyclized and purified. Purity was >98% by LC / MS. Endotoxin levels were =5-0.01 EU / g.Handling of Blood PlasmaPlasma from individuals of known age and both genders was prepared in ACD anticoagulant by BioIVT (Westbury, NY) and frozen within one hour of collection. BioIVT provides viral-negative normal human blood products collected from consented donors under IRB-approved protocols at facilities located in the United States and Europe. The plasma was from both genders (3-4 individuals per group) and pooled according to age groups with 3-4 individuals per group :“70 yo”, mean age = 72.6+ 2.1, n=3; “70 yo -G2”, mean age = 72.3+0.6; “30 yo”, mean age = 32+ 3.5, n=3; and “Mixed” mean age = 37.7+ 19, n=4;. The purpose of these pools was not to systematically compare the response of plasma samples from different aged individuals to MG treatment. The present study aimed simply to include samples collected from different groups to test the general applicability of the chosen hyperglycemic model. The present study included samples collected from different groups to test the general applicability of the chosen hyperglycemic model for producing pathological changes in plasma and then test the effect of the peptide on this modified plasma chemistry. Differences between groups were minimal, except as noted in Results, and therefore combined accordingly. The plasma samples from the 70 yo-G2 group were used primarily to document MG effects of plasma fructosamine levels and plasma HSP70 binding to immobilized cSKE7 (Figs. 3 & 4).In initial experiments, plasma from each pool was treated with different concentrations of methylglyoxal (MG) and compared daily to parallel samples without MG treatment using plasma smears and thioflavin-t fluorescent staining to detect amyloidogenesis (see below). All age groups showed significant amyloid accumulations without peptide treatment at 8 days when incubated with ImM MG (Fig. 5). Subsequently, plasma samples were handled as described in2356618988.2Attorney Docket No. 046528-7146W01(01103)Cunningham et al. (Rejuvenation Res. 2018;21 (6):527-34. Epub 20180530) in order to control for variations in baseline chemistry that might result from ex vivo procedures. Briefly, aliquots from individual pools were treated with methylglyoxal, ATP levels were restored at lOOpM, and only then was the aliquot divided into different peptide or vehicle control groups for side-by-side assay analysis. In some cases (as noted below) the plasma was also pretreated with HSP70 inhibitor (200pM pifithrin-p or DMSO vehicle) for 1 hr at 37°C prior to group assignment. The specific targeting of this inhibitor to plasma HSP70 was confirmed in a previous study demonstrating identical effects on plasma HSP70 activity using a HSP70 antibody.Solubility determinationThe present study applied the Chemistry of Health software, University of Cambridge, which assigns a solubility value for each residue taking into account its charge, polarity and capability to interact with water molecules, as well as these properties of surrounding of amino acids (Pietro Somanni, University of Cambridge, personal communication). To test software predictions directly, we used different water / 2-propanol mixtures in a common aqueous two- phase system that allows determination of aqueous solubility following adjustments of solution polarity.FructosamineFructosamine, an AGE intermediary, was measured after incubation of plasma with or without MG for 8 days at 37°C. The present study applied the method of Johnson et al. (Clinica Chimica Acta. 1983; 127(l):87-95) with minor modifications. This method clearly distinguishes diabetic patients from healthy controls and correlates well with fasting glucose. Briefly, 20pl plasma samples were reacted with nitroblue tetrazolium (Gold Bio) in carbonate buffer (pH 10.1) at 37°C with 5 minutes agitation. The samples were read after 20 additional minutes at 550nm with the Tecan Infinite 200 PRO reader. MG-exposed and non MG-exposed samples were run side by side and compared (see data analysis). Additional sets of cSKE7 treated samples were measured and compared to these initial baseline values to determine if peptide treatment interfered with fructosamine formation (see Fig. 3).Binding of MG stressed plasma HSP70 to immobilized cSKE72456618988.2Attorney Docket No. 046528-7146W01(01103)Plasma samples were incubated at 37°C with or without ImM MG. The plasma was diluted to 10% in 4mM phosphate buffer and reacted overnight at 4°C with nitrocellulose dots to which 4pl of different concentrations of cSKE7 had been added and dried. The dots were blocked with Intercept Blocking Buffer (Licor) and incubated overnight (4°C) with HSP70 Rabbit pAb (A12948, AbClonal). Secondary antibodies and all additional reagents were from Licor and the blots viewed and captured on the Odyssey M Imaging System (Licor).AmyloidogenesisAfter 8 days of MG exposure, the samples of MG treated plasma were centrifuged at 20,000 X g to sediment amyloid fibrils and other aggregates. Supernatants were removed and stored at -80° for later assays of free thiols and GAPDH enzyme activity. Fifteen microliters of 4mM Phosphate buffer (pH 7.5) were added to the tube and the precipitate (not always visible) was disrupted with a tissue homogenizer, subsequent vigorous scraping and vortexing, followed by addition of 7.5 pl of ImM thioflavin-t (THt). After further vortexing, a smear of the plasma sample was prepared on poly-L-lysine coated gelatinized slides, examined and photographed with a Leitz Fluorescent microscope. For the THt solution assay, a subset of samples representing all age groups and cSKE7 concentrations, as well as those pretreated with the HSP70 inhibitor, was further diluted 1 :25, because of the different concentration requirements of the solution assay. The samples were read with the Tecan reader at 450 / 5 lOnm (ex / em).Determination of Free ThiolsThe measurements were conducted in lOOpl of 50mM Tris (pH 7.4) to which was added lOpl plasma, and finally 20pl lOmM DTNB (5,5 ' -dithio-bis-(2-nitrobenzoic acid, Cayman Chemical) in ,4M Tris pH 8.2. The progress of the absorbance at 412nm was measured via the kinetic software in the Tecan reader to determine the time to maximum absorbance (20 minutes) which was used in all subsequent assays.Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) assayThe present study used a standard method as described for serum and plasma samples measuring the conversion of NAD+to NADH at 340nm absorbance. Ten microliters of plasma were mixed with 40pl 3X sample buffer and the reaction was initiated by addition of lOOpl of a2556618988.2Attorney Docket No. 046528-7146W01(01103)1 ,5X mix of NAD+and glycerol 3-phosphate (G3P). Final concentration of buffer reagents was triethanolamine 50mM, EDTA ImM in 50mM Tris (pH 8.9), and of reactants G3P 3mM and NAD+ImM. The maximum velocities reached during the initial 5 minutes of the reaction was recorded for each peptide and control pair and taken as a proportional estimate of the concentration of active enzyme in each sample. The procedure was repeated 3 times for each sample pair with systematic variation of the position of the samples on the plate. The average of the measurements was normalized and analyzed as described below.Data AnalysisData sets from different plasma pools and different assay procedures were obtained after side-by-side processing in all experiments to avoid variations introduced by ex vivo handling of blood plasma (see above). The data points from in each data set and for a particular assay session were normalized to average vehicle / contol value for that session and expressed as percentage change from this average value according to a standard normalization formula.% Value Reported= ((100*(Value Measuredvehicie / Controi or Experimental / Average Vehicle / Control Measurement))- 100)).This procedure also reflects the variability of all measurements around an average baseline control value. The resulting data was compared statistically by unpaired t-tests with Weich’s correction for unequal variances, when necessary, except for the GAPDH measurements (as also noted in“Results”). GAPDH measurements from individual side by side comparisons were paired because of session and age variability. Statistical analysis was via Prism software (GraphPad).Example 4:SolubilityReferring to Fig. 2, these experiments were intended to test the aqueous solubility the CSKE7 peptide compared to predecessor peptide CHEC-7. The Cambridge University software suggested a marked solubility difference between the redesigned peptide and CHEC-7. This was2656618988.2Attorney Docket No. 046528-7146W01(01103) tested directly and confirmed by adjustments of solvent polarity with various aqueous mixtures of 2-propanol.FructosamineFructosamine represents early-stage glycation by glucose and methylglyoxal (MG) and is an intermediate in the formation of advanced glycation end products. The present study compared samples incubated with or without MG for 8 days and found that fructosamine formation was increased by over 50% (Fig. 3). Pretreatment of the samples with bioactive concentrations of cSKE7 was ineffective at reducing fructosamine levels, either following MG treatment (Fig. 3) or that which occurs spontaneously without MG treatment (data not shown)Binding of MG stressed plasma HSP70 to immobilized cSKE7Plasma stressed for 8 days with MG had increased levels of HSP70 and / or increased HSP70 affinity for cSKE7 as shown in Fig. 4. The results are consistent with previous reports of HSP70 affinity for peptide fragments near the n-terminus of the DSEP / Dermcidin protein.AmyloidogenesisMethylglyoxal treatment results in a range of amyloid fibrillation and globulation and consistent inhibition by cSKE7 addition at both 1 and lOpM. Shown are thioflavin-t (THt) stained smears of the resuspended precipitates of treated plasma and the typical appearance of fields from the different experimental conditions. The difference in amyloid staining between experimental groups and at both peptide concentrations was obvious upon inspection in all age groups. Large areas of fibers and globules were absent after peptide treatment. However, these accumulations were apparent once again following pretreatment 200pM pifithrin-p (shown for the 70 yo pool) compared to DMSO vehicle where the dissolution of amyloid accumulations was still observed (Fig. 1 A, far-right panels). An equivalent subset of samples from the 30yo pool and the 70 yo pools including after vehicle and 1 and lOpM peptide treatment were selected for the THt solution assay (Fig. IB). Samples pretreated with the HSP70 inhibitor were not different than vehicle controls without inhibitor. The solution assay supported the fluorescent staining observations and revealed also a significant effect at 0.1 pM cSKE7, a result that was not consistently obvious upon examination amyloid staining of the plasma smears. The solution2756618988.2Attorney Docket No. 046528-7146W01(01103) assay may be sensitive to prefibrillar amyloid forms, even though these have a less robust interaction with Tht, and disruption of these was realized at even the lowest concentration of the peptide tested in these experiments.Free ThiolsThe present study investigated free thiol levels in MG-treated plasma because of the close relationship of thiol oxidation to the redox status in the circulation and the reported influence of HSP70. Samples included all age groups and revealed a significant increase in free thiols (nonoxidized) with peptide treatment at both 1 and lOpM cSKE7 (Fig. 6). The increase in free thiols was eliminated in a subset of samples pretreated with pifithrin-p suggesting the effect is HSP70 dependent. There was a trend for even lower values with the inhibitor than that recorded initially with vehicle treatment (reaching significance at IpM) suggesting inhibition of residual endogenous HSP70 activity in the MG-treated samples (see also “GAPDH” below).Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH)GAPDH enzyme activity was examined because of its sensitivity to both MG levels and redox status and therefore the possible relationship of enzyme activity to plasma HSP70 activity (see Discussion). These measurements were by far the most variable and required not only that individual cSKE7-treated samples be run side by side with individual vehicle-treated samples, but also these samples be paired for statistical analysis. The present study also analyzed pairs of samples from the 70 yo pool separately from those of the 30 yo pool because of near significant differences in baseline (vehicle-treated) responses to MG treatment between the two pools after 8 days (30yo pool, 1140.03 + 46.7 vs 70 yo pool, 880.44 116.7 mAU / min / ml at 340nm, p=0.073). No firm conclusion was possible with this very limited sample but the result may be consistent with the decline in GAPDH activity reported in aging mammalian systems. Despite the variability in these measurements, both pools showed significant elevations in GAPDH activity with cSKE7 treatment after pairing and combining data from 1 and lOpM peptide treatments (Figs. 7A & 7B). Inhibition of HSP70 activity had a profound effect on GAPDH activity in MG treated samples, whether peptide treated or not. This is shown for a subset of samples from the 30 yo pool where peptide and vehicle samples were also pretreated with pifithrin-p or DMSO (Fig. 7C). This result would be expected if plasma GAPDH activity is supported generally (i.e.,2856618988.2Attorney Docket No. 046528-7146W01(01103) without peptide treatment) by plasma HSP70 under conditions of hyperglycemic stress (see also “Free Thiols” above).Example 5:Methylglyoxal (MG) model. One of the goals of the present study was to model the well-documented cumulative effect of elevated MG levels on plasma protein amyloidogenesis and oxidative stress (and related metabolic disease states) and to test the redesigned peptide, cSKE7, for its reparative properties under these conditions. Elevated levels of fructosamine after MG treatment is consistent with an accelerated process of non-enzymatic protein glycation and the resulting toxic changes documented in this study. MG is a highly reactive dicarbonyl compound, making it a much more potent glycating agent than glucose. The fact that cSKE7 treatment of MG-exposed samples had no effect on this “fructation” suggests that the reparative activities found in plasma with the peptide are realized after the hyperglycemic modifications of blood proteins have occurred as would be expected if HSP70 quality control processes were involved.The selection of a ImM MG concentration and of an 8 day incubation was also based on the consistent and marked appearance of amyloid globular and fibrous aggregates without peptide treatment. This is also the level measured in the plasma of mice fed MG to model metabolic and vascular dysfunction. Studies of MG effects on amyloidogenesis of identified plasma proteins generally apply higher concentrations, while sporadic measures of MG are lower in diabetic patients but significantly higher than in healthy subjects. MG, fructosamine, and fasting glucose levels all correlate with HbAlc, the routine clinical measure of glycemic control. Importantly, persistent elevations of any of these is associated with heightened risk for diabetic complications. Therefore, it is suggested that the present model is sufficiently robust to demonstrate cSKE7 influence on amyloidogensis as well as the involvement of HSP70 in this effect. The use of human plasma for these experiments may also be important since, as noted above, the parent DSEP / Dermcidin is restricted to humans and primates, i.e., an “orphan” protein, not present in other mammals.Plasma thiol and redox status. The loss of free thiols in blood plasma, although not a specific biomarker per se, nevertheless indicates pro-oxidant changes that accompany or contribute to several disease states. Intracellular HSP70 is antioxidant and promotes thiol2956618988.2Attorney Docket No. 046528-7146WOI(01103) reduction. The simple explanation for the increase in free thiols after peptide treatment therefore is the reduction of oxidant conditions due to the action of HSP70. This has been demonstrated in cells where there is a direct effect of HSP70 on the glutathione redox balance and suggested by several studies in patient serum where HSP70 levels are negatively correlated with stress enzymes in metabolic disorders. Altered levels of thiols in plasma have been linked with specific pathological conditions including increased risk for cardiovascular and cerebrovascular diseases. In addition, the pro-oxidant effects of MG in the hyperglycemic condition also are strongly- linked to the progression of age-related and chronic metabolic diseases.Glyceraldehyde Phosphate Dehydrogenase (GAPDH), MG, and HSP70. GAPDH is an ancient and multifunctional protein with properties that go well beyond intracellular glycolysis. Plasma and serum GAPDH are less well understood but the circulating enzyme is described as an enzymatically active multimer consisting of several high-molecular-weight subunits besides the ~37 kDa monomer. In the present study, GAPDH activity was examined because of its role in redox balance and the relationship between MG levels and inhibition of the enzyme. It is unclear how the partial rescue of GAPDH activity occurs in plasma after cSKE7 treatment, as well as the demonstrated relationship to HSP70 activity, but several possibilities are evident. First, HSP70 might simply reduce or dissociate GAPDH aggregates as one of its target proteins such that there is an increase in the relative concentration of an active multimeric form. A GAPDH gel shift after treatment of oxidized human plasma with the cSKE7 predecessor peptide, CHEC-7, which also regulates plasma HSP70 activity, has also been shown. In fact, the pro-oxidant conditions present in the current model can induce GAPDH to form inactive amyloid like structures, an effect attributed to oxidation the enzyme’s active site cystine under such conditions. For example, oxidative stress produced by hyperglycemic MG levels occurs via a decrease of the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG), which can further induce the reversible S-glutathionylation of GAPDH thus inactivating the enzyme. Finally, intracellular GAPDH’ s interaction with multiple protein partners may allow cells to utilize the enzyme as a cleaning agent that sequesters and flushes out misfolded molecules arising because of cellular stress, and, in a hypoxic cellular environment, HSP70 targets and disaggregates GAPDH to assist in this activity. As noted however, most of the reports reviewed above relate to the activity of intracellular GAPDH so it remains to be determined if similar processes occur in the circulation. Nonetheless, the human pathologies associated with the3056618988.2Attorney Docket No. 046528-7146W01(01103) oxidant-induced dysregulation of GAPDH highlight the importance of the redox regulation of the enzyme, especially in metabolic and neurodegenerative disorders, and, as reviewed above, there are several possible mechanisms by which CSKE7 and HSP70 can intervene to protect enzyme activity.Potential for HSP70 Therapy: While the cell survival (anti-aptotic) and antiinflammatory properties of HSP70 are generally appreciated, therapeutic strategies for targeting HSP70 activity, in theory, seem to be disease dependent: Inhibition for neoplastic disease if the focus is on its cell survival and anti-tumor suppression properties, but enhancement for inflammatory and protein aggregate disorders where the emphasis is targeting systemic pathologies.It is just reasonable to suggest that distinct, or more likely repurposed intracellular HSP70s, some perhaps housed in extracellular microvesicles and in a systemic environment. Here they are recruited to combat systemic oxidant stressors and amyloidogenesis of blood proteins. In support of this suggestion are the systemically administered recombinant human HSP70 on protection against muscle denervation including in the SOD model of Amyotrophic Lateral Sclerosis. HSP70 administration in models of CNS vascular injury and clinical manifestations of sepsis in an LPS-treated rat model has been tested. The cSKE7 predecessor CHEC-7, applied orally to LPS injected rats or to an autoimmune model (Experimental Autoimmune Myloencephalitis) reduced inflammation and promoted recovery of motor function. However, one difficulty with direct application of HSP70 in clinical models, a problem that is likely to reappear in human applications, may be the limited bioavailability demonstrated in pharmacokinetic studies of the protein. Several investigators therefore have opted for indirect stimulation of HSP70 via stressors such as hyperthermia, or alternatively, have intensified the search for chemical stimulants of HSP70 activity. The CHEC peptides, including the current modification cSKE7, are among the latter category and so far, the only compounds with demonstrated reparative activities in stressed human blood plasma, including for cSKE7 in the current model of hyperglycemic stress. Present results from this model may be significant because promising clinical outcomes in diabetic monkeys and humans have been obtained with indirect stimulation of HSP70 via hyperthermia.Lifespan extension. The link between glycative stress and life expectancy has been suggested for decades, as has the role of HSP70 and an intact system of proteostasis as an3156618988.2Attorney Docket No. 046528-7146W01(01103) important factor in establishing this relationship. Not only do the protein handling functions in cells and in the blood get overburdened (regardless of glycative status), hyperglycemia and accompanying inflammation and oxidative stress contribute significantly to this burden. Further study is required to determine if cycloSKE7 will provide some relief for the accumulated aggregate toxicity in aging paradigms, whether under metabolically compromised conditions or simply as the expected age-related decline in the detoxification functions of proteostasis.Example 6: cSKE9. The experiments detailed above are repeated with cSKE9. Comparable solubility and activity are observed.Enumerated EmbodimentsIn some aspects, the present invention is directed to the following non-limiting embodiments:Embodiment 1 : A polypeptide comprising the sequence set forth in SEQ ID NO:1 (cycloSQKCHEA) or SEQ ID NO:2 (cycloCHEASAAQK).Embodiment 2: The polypeptide of Embodiment 1, wherein the polypeptide consists of the sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2.Embodiment 3: A composition, comprising: the polypeptide of any one of Embodiments 1-2; and a pharmaceutically acceptable carrier.Embodiment 4: A method of activating HSP70 in a cell or in a subject, the method comprising: contacting the cell with or introducing into the subject the polypeptide of any one of Embodiments 1-2, or the composition of Embodiment 3.Embodiment 5: The method of Embodiment 4, which reduces oxidative stress, increase free thiol level, and / or reduces protein aggregation, optionally amyloidogenesis.Embodiment 6: The method of any one of Embodiments 4-5, wherein the activation of HSP70 in the subject reduces a protein aggregation, optionally amyloidogenesis, in a blood circulation of the subject.Embodiment 7: The method of any one of Embodiments 4-6, wherein at least one of the following applies:3256618988.2Attorney Docket No. 046528-7146W01(01103)(a) the cell is a primary cell, a cell of a cell line, or a cell in a subject, optionally a human subject,(b) the cell is a mammalian cell, a human cell, or a cell derived therefrom,(c) the subject is a mammalian subject, optionally a human subject.Embodiment 8: A method of treating, ameliorating, and / or preventing a disease or disorder in a subject in need thereof, the method comprising: administering to the subject an effective amount of the polypeptide of any one of Embodiments 1-2, or the composition of Embodiment 3, wherein the disease or disorder is caused or aggravated by a toxic protein aggregate, an oxidative stress, an insufficient HSP70 activity, a hyperglycemic condition.Embodiment 9: The method of Embodiment 8, wherein the disease or disorder is diabetes, a complication of diabetes, hyperglycemia, a neurodegenerative disease, a cardiovascular disease, an inflammatory condition, a protein aggregate disorder, and / or an autoimmune disease.Embodiment 10: The method of Embodiment 9, wherein at least one of the following applies:(a) the diabetes is type I diabetes or type II diabetes,(b) the complication of diabetes is kidney damage, eye damage, foot damage, a skin condition, a mouth condition, accelerated aging, or cognitive decline caused by diabetes,(c) the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, or amyotrophic lateral sclerosis (ALS),(d) the cardiovascular disease is myocardial infarction, ischemia or reperfusion, heart failure, atherosclerosis, hypertension, or arrhythmia,(e) the autoimmune disease is multiple sclerosis (MS), rheumatoid arthritis (RA).Embodiment 11 : The method of any one of Embodiments 8-10, wherein the subject is a mammal, optionally a human.Embodiment 12: A method of increasing longevity of a subject, the method comprising: administering to the subject an effective amount of the polypeptide of any one of Embodiments 1-2, or the composition of Embodiment 3.Embodiment 13: The method of Embodiment 12, wherein the subject is a mammal.3356618988.2Attorney Docket No. 046528-7146W01(01103)Embodiment 14: The method of any one of Embodiments 12-13, wherein the mammal is a non-human mammal or a human.The foregoing outlines features of several embodiments so that those skilled in the art may better understand the aspects of the present disclosure. Those skilled in the art should appreciate that they may readily use the present disclosure as a basis for designing or modifying other processes and structures for carrying out the same purposes and / or achieving the same advantages of the embodiments introduced herein. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure.3456618988.2

Claims

Attorney Docket No. 046528-7146W01(01103)CLAIMSWhat is claimed is:

1. A polypeptide comprising the sequence set forth in SEQ ID NO: 1 (cycloSQKCHEA) or SEQ ID NO:2 (cycloCHEASAAQK).

2. The polypeptide of claim 1, wherein the polypeptide consists of the sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2.

3. A composition, comprising: the polypeptide of any one of claims 1 -2; and a pharmaceutically acceptable carrier.

4. A method of activating HSP70 in a cell or in a subject, the method comprising: contacting the cell with or introducing into the subject the polypeptide of any one of claims 1-2, or the composition of claim 3.

5. The method of claim 4, which reduces oxidative stress, increase free thiol level, and / or reduces protein aggregation, optionally amyloidogenesis.

6. The method of any one of claims 4-5, wherein the activation of HSP70 in the subject reduces a protein aggregation, optionally amyloidogenesis, in a blood circulation of the subject.

7. The method of any one of claims 4-6, wherein at least one of the following applies:(a) the cell is a primary cell, a cell of a cell line, or a cell in a subject, optionally a human subject,(b) the cell is a mammalian cell, a human cell, or a cell derived therefrom,(c) the subject is a mammalian subject, optionally a human subject.

8. A method of treating, ameliorating, and / or preventing a disease or disorder in a subject in need thereof, the method comprising:3556618988.2Attorney Docket No. 046528-7146W01(01103) administering to the subject an effective amount of the polypeptide of any one of claims 1-2, or the composition of claim 3, wherein the disease or disorder is caused or aggravated by a toxic protein aggregate, an oxidative stress, an insufficient HSP70 activity, a hyperglycemic condition.

9. The method of claim 8, wherein the disease or disorder is diabetes, a complication of diabetes, hyperglycemia, a neurodegenerative disease, a cardiovascular disease, an inflammatory condition, a protein aggregate disorder, and / or an autoimmune disease.

10. The method of claim 9, wherein at least one of the following applies:(a) the diabetes is type I diabetes or type II diabetes,(b) the complication of diabetes is kidney damage, eye damage, foot damage, a skin condition, a mouth condition, accelerated aging, or cognitive decline caused by diabetes,(c) the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, or amyotrophic lateral sclerosis (ALS),(d) the cardiovascular disease is myocardial infarction, ischemia or reperfusion, heart failure, atherosclerosis, hypertension, or arrhythmia,(e) the autoimmune disease is multiple sclerosis (MS), rheumatoid arthritis (RA).

11. The method of any one of claims 8-10, wherein the subject is a mammal, optionally a human.

12. A method of increasing longevity of a subject, the method comprising: administering to the subject an effective amount of the polypeptide of any one of claims 1-2, or the composition of claim 3.

13. The method of claim 12, wherein the subject is a mammal.

14. The method of any one of claims 12-13, wherein the mammal is a non-human mammal or a human.3656618988.2

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