Compositions and methods for stimulating islet viability and enhancing insulin secretion

Abstract

FIELD: biotechnology.SUBSTANCE: provided is a method for restoring glucose-stimulated insulin secretion, comprising contacting pancreatic islet cells with an effective amount of a composition comprising a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide or pharmaceutically acceptable salt thereof comprises an amino acid sequence comprising any of SEQ ID NO: 1-44, 49-60 or 62, or any of SEQ ID NO: 45-58 with a Q to N substitution.EFFECT: enhancement of insulin secretion.20 cl, 2 tbl, 9 dwg, 7 ex

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATION

[0002] The subject matter described herein claims priority to U.S. Provisional Patent Application Serial No. 62 / 817,790, filed March 13, 2019, the entire contents of which are incorporated herein by reference.

[0003] STATE INTEREST

[0004] This invention was made with government support under Grants No. EY 024327 and EY 026171 awarded by the National Institutes of Health (USA). The government has certain rights in this invention.

[0005] STATE OF THE ART

[0006] At least 8.5% of the world's population suffers from diabetes, and the prevalence of the disease is increasing (Tatum et al., 2017). Types 1 and 2 diabetes generated global drug sales totaling $31 billion in 2016, with an expected increase to $45 billion by 2021. Most of these are injectable forms of insulin or are designed to increase incretin levels to counteract elevated blood levels or inhibit sodium-glucose cotransporter 2, available in oral tablets or injectables. Global sales in the equally large market for devices such as insulin pumps and continuous glucose meters are projected to exceed $35 billion by 2024.Although both approaches can be beneficial, diabetics still suffer from a significantly increased risk of stroke, heart attack, kidney failure, limb amputation, and blindness associated with diabetic peripheral neuropathy, nephropathy, and blood vessel damage.

[0007] Attempts to reverse the underlying biological deficits experienced by diabetics continue, but with limited success. For example, regeneration of diabetic islets could be revolutionary, but research in this area is in its early stages and does not address neural innervation. For example, molecules that can modulate beta cell replication have been identified, but they have yet to be successfully implemented in clinical settings. Such molecules include certain DYRK1A kinase inhibitors, which facilitate calcineurin-dependent dephosphorylation of NFAT, a prerequisite for NFAT translocation to the nucleus to act as a transcription factor for beta cell replication (Wang et al., 2015a). Furthermore, PIAA and amlexanox molecules have been found in zebrafish, which suppress TBK1 / IKKs to activate mTOR and thus beta cell replication (Xu et al., 2018).Genetic restoration of mTOR in an Akita dog model of neonatal diabetes also activates beta cell replication (Riahi et al., 2018).

[0008] SUMMARY OF THE INVENTION

[0009] The Summary of the Invention section lists several embodiments of the described subject matter and, in many cases, lists variants and combinations of proposed embodiments of the described subject matter. The Summary of the Invention section is merely an example of the numerous and varied embodiments. The mention of one or more illustrative features of a given embodiment is also illustrative. Such an embodiment can generally exist with or without the mentioned feature(s); likewise, the mentioned features may relate to other embodiments of the described subject matter, regardless of whether they are listed in the Summary of the Invention or not. To avoid unnecessary repetition, the Summary of the Invention section does not list or suggest all possible combinations of such features.

[0010] In some embodiments, the disclosed subject matter relates to methods for restoring glucose-stimulated insulin secretion. In some embodiments, the disclosed methods comprise contacting pancreatic islets in vitro, ex vivo, and / or in vivo with an effective amount of a composition comprising a peptide and / or a pharmaceutically acceptable salt thereof, and / or a biologically active fragment, analog, or derivative thereof, wherein said peptide, pharmaceutically acceptable salt thereof, and / or a biologically active fragment, analog, or derivative thereof comprises, consists essentially of, or consists of an amino acid sequence comprising, consisting essentially of, or consisting of any of SEQ ID NOs: 1-60, or any combination thereof.

[0011] In some embodiments, the described subject matter also relates to methods for restoring the viability and / or cellular proliferation of transplanted or endogenous pancreatic islets.In some embodiments, the disclosed methods include contacting pancreatic islets before, simultaneously with, and / or after transplantation or without transplantation to diabetics with an effective amount of a composition comprising a peptide and / or a pharmaceutically acceptable salt thereof, and / or a biologically active fragment, analog, or derivative thereof, wherein said peptide, its pharmaceutically acceptable salt, and / or its biologically active fragment, analog, or derivative comprises, consists essentially of, or consists of an amino acid sequence comprising, consisting essentially of, or consisting of any of SEQ ID NOs: 1-60, or any combination thereof, wherein the viability and / or proliferation of the transplanted pancreatic islets or endogenous islets is restored relative to the level of islet cells not contacted with an effective amount of said composition.

[0012] In some embodiments, the described subject matter also relates to methods for preventing and / or suppressing rejection of transplanted pancreatic islets or preventing further degeneration of endogenous pancreatic islets.In some embodiments, the disclosed methods include contacting isolated pancreatic islets before, simultaneously with, and / or after transplantation, or contacting endogenous diabetic islets with an effective amount of a composition comprising a peptide and / or a pharmaceutically acceptable salt thereof, and / or a biologically active fragment, analog, or derivative thereof, wherein said peptide, pharmaceutically acceptable salt thereof, and / or a biologically active fragment, analog, or derivative thereof comprises, consists essentially of, or consists of an amino acid sequence comprising, consisting essentially of, or consisting of any of SEQ ID NOs: 1-60, or any combination thereof, wherein the rejection of the transplanted islet cells is prevented and / or suppressed compared to a similar parameter of islet cells not contacted with an effective amount of said composition.

[0013] In some embodiments, the described subject matter also relates to methods of transplanting pancreatic islets.In some embodiments, the disclosed methods comprise transplanting pancreatic islets into a transplant recipient, wherein the islets have been contacted before, simultaneously and / or after the transplantation step with an effective amount of a composition comprising a peptide and / or a pharmaceutically acceptable salt thereof and / or a biologically active fragment, analog or derivative thereof, wherein said peptide, its pharmaceutically acceptable salt and / or its biologically active fragment, analog or derivative comprises, consists essentially of or consists of an amino acid sequence comprising, consisting essentially of or consisting of any of SEQ ID NOs: 1-60 or any combination thereof, wherein the rejection of the transplanted pancreatic islet cells is prevented and / or suppressed compared to a similar parameter of pancreatic islet cells not contacted with an effective amount of said composition.

[0014] In some embodiments, the disclosed subject matter relates to methods for restoring the healthy status of the nerves innervating the pancreatic islets. In some embodiments, the disclosed methods comprise contacting the nerves of the pancreatic islets in vitro, ex vivo and / or in vivo with an effective amount of a composition comprising a peptide and / or a pharmaceutically acceptable salt thereof and / or a biologically active fragment, analog or derivative thereof, wherein said peptide, pharmaceutically acceptable salt thereof and / or a biologically active fragment, analog or derivative thereof comprises, consists essentially of, or consists of an amino acid sequence comprising, consisting essentially of, or consisting of any of SEQ ID NOs: 1-60 or any combination thereof.

[0015] In some embodiments, the described subject matter also relates to methods for treating a symptom of a pathological condition, disorder, or disease associated with an impaired insulin response to glucose in a subject, wherein said pathological condition, disorder, or disease is not necessarily type 1 or type 2 diabetes.In some embodiments, the disclosed methods comprise administering to a subject an effective amount of a composition comprising a peptide and / or a pharmaceutically acceptable salt thereof and / or a biologically active fragment, analog or derivative thereof, wherein said peptide, pharmaceutically acceptable salt thereof and / or a biologically active fragment, analog or derivative thereof comprises, consists essentially of, or consists of an amino acid sequence comprising, consisting essentially of, or consisting of any of SEQ ID NOs: 1-60 or any combination thereof, wherein the rejection of transplanted pancreatic islets is prevented and / or suppressed compared to a similar parameter of islets not brought into contact with an effective amount of said composition.

[0016] In some embodiments of the described methods, the proposed composition is formulated for administration to a subject, optionally a human, via intravenous, intramuscular, oral, intranasal, and / or transdermal delivery. In some embodiments, the composition is formulated as nanoparticles, nanovesicles, microparticles, microvesicles, liposomes, packaged in PEG, lyophilized into pill form, or any combination thereof.

[0017] In some embodiments of the described methods, the provided peptide, its pharmaceutically acceptable salt and / or its biologically active fragment, analog or derivative comprises at least one modification selected from the group consisting of N- and / or C-terminal amidation, N- and / or C-terminal acylation, N- and / or C-terminal acetylation, attachment of N- and / or C-terminal cysteine, PEGylation and combinations thereof. In some embodiments, PEGylation comprises attachment of a PEG group to the N-terminal cysteine, the C-terminal cysteine, or both. In some embodiments, the PEG group has a molecular weight of from about 1 kilodalton (kDa) to about 40 kDa. In some embodiments, the N-terminal amidation, the C-terminal amidation, or both include a substituted amide and / or the N-terminal acylation, the C-terminal acylation, or both include a substituted acyl group.

[0018] In some embodiments of the described methods, the proposed composition does not contain any type of enzymatic, chemical or biochemical molecules capable of destroying the said peptide at its ends, which is a sequential degradation of the peptide, its pharmaceutically acceptable salt and / or its biologically active fragment, analog or derivative at its end in the absence of N- and / or C-terminal amidation, N- and / or C-terminal acylation, N- and / or C-terminal acetylation, addition of N- and / or C-terminal cysteine, pegylation or a combination thereof.

[0019] In some embodiments of the described methods, the proposed composition is stabilized against any type of enzymatic, chemical or biochemical degradation of the peptide at its ends, which is a sequential degradation of the peptide, its pharmaceutically acceptable salt and / or its biologically active fragment, analog or derivative at its end in the absence of N- and / or C-terminal amidation, N- and / or C-terminal acylation, N- and / or C-terminal acetylation, addition of N- and / or C-terminal cysteine, pegylation or a combination thereof.

[0020] In some embodiments of the described methods, the proposed composition is stabilized against any type of enzymatic, chemical or biochemical degradation of the peptide in the side chains of the amino acids included in its composition, wherein said peptide, its pharmaceutically acceptable salt and / or its biologically active fragment, analog or derivative is stabilized in a helical structure by N- and / or C-terminal amidation, N- and / or C-terminal acylation, N- and / or C-terminal acetylation, addition of N- and / or C-terminal cysteine, pegylation or a combination thereof.

[0021] In some embodiments of the described methods, the proposed composition further comprises a pharmaceutically acceptable carrier, an excipient, a diluent, tonicity agents, viscosity-increasing agents and / or encapsulating agents, and wherein said composition is further formulated for administration to a subject in need thereof via a systemic, oral or transdermal delivery method, wherein the subject in need thereof is not necessarily a human.

[0022] In some embodiments of the described methods, the proposed peptide, its pharmaceutically acceptable salt and / or its biologically active fragment, analog or derivative is present in the composition at a concentration of 1.0 nM to 100 μM.

[0023] In some embodiments of the described methods, the proposed composition further comprises one or more stabilizing agents, wherein the one or more stabilizing agents stabilize the peptide, its pharmaceutically acceptable salt, its biologically active fragment, its analog and / or degradation derivative and / or stabilize the peptide, its pharmaceutically acceptable salt, its biologically active fragment, its analog and / or derivative in a certain conformation to increase its chemical stability. In some embodiments, the stabilizing agent includes tyloxapol.

[0024] In some embodiments of the disclosed methods, the subject has a disease, disorder, or pathological condition associated with an abnormal response to glucose. In some embodiments, the disease, disorder, or pathological condition associated with an abnormal response to glucose is type 1 or type 2 diabetes. In some embodiments, the disclosed methods further comprise administering to the subject one or more additional antidiabetic therapies. In some embodiments, the one or more additional antidiabetic therapies are selected from the group consisting of immune therapy, optionally an immune therapy comprising administration of IgM; administration of a calcineurin inhibitor, optionally tacrolimus; administration of a glucagon-like peptide-1 (GLP-1) analog, optionally exendin-4; and any combination thereof.

[0025] In some embodiments of the described methods, the proposed composition is formulated for use in humans, and / or the pancreatic islet cell is a human islet cell, and / or the pancreatic islet cell is in the subject's body.

[0026] In some embodiments, the described subject matter also relates to compositions for use in restoring pancreatic islet viability and / or cell proliferation in vitro, ex vivo, and / or in vivo. In some embodiments, the provided composition comprises, consists essentially of, or consists of a peptide and / or a pharmaceutically acceptable salt thereof, and / or a biologically active fragment, analog, or derivative thereof, wherein said peptide, pharmaceutically acceptable salt thereof, and / or a biologically active fragment, analog, or derivative thereof comprises, consists essentially of, or consists of an amino acid sequence comprising, consisting essentially of, or consisting of any of SEQ ID NOs: 1-60, or any combination thereof.

[0027] In some embodiments, the described subject matter also relates to compositions for use in restoring pancreatic islet viability and / or cellular proliferation in vitro, ex vivo and / or in vivo; and / or in restoring glucose-stimulated insulin secretion; and / or in restoring the viability and / or cellular proliferation of transplanted islets; and / or in preventing and / or suppressing rejection of transplanted islets; and / or in islet transplantation; and / or in treating a symptom of a condition, disorder or disease associated with an impaired insulin response to glucose in a subject.In some embodiments, the proposed composition comprises, consists essentially of, or consists of a peptide and / or a pharmaceutically acceptable salt thereof, and / or a biologically active fragment, analog, or derivative thereof, wherein said peptide, pharmaceutically acceptable salt thereof, and / or a biologically active fragment, analog, or derivative thereof comprises, consists essentially of, or consists of an amino acid sequence comprising, consisting essentially of, or consisting of any of SEQ ID NOs: 1-60, or any combination thereof.

[0028] In some embodiments, the described subject matter also relates to compositions for the preparation of a medicament for restoring pancreatic islet viability and / or cellular proliferation in vitro, ex vivo and / or in vivo; and / or for restoring glucose-stimulated insulin secretion; and / or for restoring viability and / or cellular proliferation of transplanted pancreatic islets; and / or for preventing and / or suppressing rejection of transplanted islets; and / or for islet transplantation; and / or for treating a symptom of a pathological condition, disorder or disease associated with an impaired insulin response to glucose in a subject.In some embodiments, the proposed composition comprises, consists essentially of, or consists of a peptide and / or a pharmaceutically acceptable salt thereof, and / or a biologically active fragment, analog, or derivative thereof, wherein said peptide, pharmaceutically acceptable salt thereof, and / or a biologically active fragment, analog, or derivative thereof comprises, consists essentially of, or consists of an amino acid sequence comprising, consisting essentially of, or consisting of any of SEQ ID NOs: 1-60, or any combination thereof.

[0029] In some embodiments of the described subject matter, the proposed compositions are formulated for administration to a subject, optionally a human, by intravenous, intramuscular, oral, intranasal and / or transdermal delivery.

[0030] In some embodiments of the described subject matter, the proposed compositions are formulated in the form of nanoparticles, nanovesicles, microparticles, microvesicles, liposomes, packaged in PEG, lyophilized in the form of pills, or any combination thereof.

[0031] In some embodiments of the described subject matter, the subject peptide, its pharmaceutically acceptable salt, and / or its biologically active fragment, analog, or derivative comprises at least one modification selected from the group consisting of N- and / or C-terminal amidation, N- and / or C-terminal acylation, N- and / or C-terminal acetylation, attachment of an N- and / or C-terminal cysteine, PEGylation, and combinations thereof. In some embodiments, PEGylation comprises attachment of a PEG group to an N-terminal cysteine, a C-terminal cysteine, or both. In some embodiments, the PEG group has a molecular weight of from about 1 kilodalton (kDa) to about 40 kDa. In some embodiments, the N-terminal amidation, the C-terminal amidation, or both include a substituted amide and / or the N-terminal acylation, the C-terminal acylation, or both include a substituted acyl group.

[0032] In some embodiments of the described subject matter of the invention, the proposed compositions do not contain any type of enzymatic, chemical or biochemical molecules capable of destroying the said peptide at its ends, which is a sequential degradation of the peptide, its pharmaceutically acceptable salt and / or its biologically active fragment, analog or derivative at its end in the absence of N- and / or C-terminal amidation, N- and / or C-terminal acylation, N- and / or C-terminal acetylation or a combination thereof.

[0033] In some embodiments of the described subject matter, the proposed compositions further comprise a pharmaceutically acceptable carrier, excipient, diluent, or encapsulating agent, and wherein said composition is further formulated for administration to a subject in need thereof via a systemic, oral, or transdermal delivery method, wherein the subject in need thereof is not necessarily a human.

[0034] In some embodiments of the described subject matter, the proposed peptide, its pharmaceutically acceptable salt and / or its biologically active fragment, analog or derivative is present in the composition at a concentration of 1.0 nM to 100 μM.

[0035] In some embodiments of the described subject matter, the proposed composition further comprises one or more pharmaceutically acceptable carriers, diluents, excipients, tonicity agents, and / or viscosity-enhancing agents. In some embodiments, the proposed composition further comprises one or more stabilizing agents, wherein the one or more stabilizing agents stabilize the peptide, its pharmaceutically acceptable salt, its biologically active fragment, analog, and / or degradation derivative and / or stabilize the peptide, its pharmaceutically acceptable salt, its biologically active fragment, its analog, and / or derivative in a certain conformation to increase its chemical stability. In some embodiments, the stabilizing agent comprises tyloxapol.

[0036] In some embodiments of the described subject matter, the proposed compositions are formulated for use in humans, and / or the pancreatic islet cell is a human islet cell, and / or the pancreatic islet cell is located in the subject's body.

[0037] Accordingly, the object of the described subject matter of the invention is to provide compositions and methods for restoring pancreatic islet viability and / or cellular proliferation in vitro, ex vivo and / or in vivo; and / or for restoring glucose-stimulated insulin secretion; and / or for restoring viability and / or cellular proliferation of transplanted pancreatic islets; and / or for preventing and / or suppressing rejection of transplanted islets; and / or for islet transplantation; and / or for treating a symptom of a pathological condition, disorder or disease associated with an impaired insulin response of a subject to glucose. The described subject matter of the invention provides a complete or partial solution to these and other problems.Furthermore, the objects of the described subject matter, as well as other objectives and advantages thereof, will become apparent to those skilled in the art upon examination of the following description, drawings, and examples. Furthermore, various aspects and embodiments of the described subject matter are described in more detail below.

[0038] BRIEF DESCRIPTION OF DRAWINGS

[0039] Fig. 1A and 1B are bar graphs of isolated mouse pancreatic islets showing that 4 μM N-94 peptide, but not 4 μM control peptide C-95, promoted mouse islet viability (Fig. 1A; the left bar of each pair represents N-94 peptide; the right bar of each pair represents C-95 peptide) and glucose-dependent insulin secretion (Fig. 1B; the left bar of each triplicate represents baseline; the middle bar of each triplicate represents N-94 peptide; the right bar of each triplicate represents C-95 peptide) in vitro. The latter treatment was performed 24 hours after islet isolation. Low: 2.8 mM glucose; High: 28 mM glucose.

[0040] Figure 2A depicts the structures of some exemplary embodiments of peptides according to the described subject matter of the invention, including lacritin (SEQ ID NO: 61) and several derivatives. The sequences of these peptides are presented in Table 2.

[0041] Fig. 2B is a bar graph showing the results of glucose-dependent insulin secretion analysis 24 hours after treatment with 1 μM of the listed exemplary peptides. One hundred human islets were left untreated (white, leftmost columns) or treated overnight with 1 μM peptide C-95 (second columns from left), peptide N-94 (third columns from left), peptide N-94 / C-6 (SEQ ID NO: 62; fourth columns from left), peptide N-104 (SEQ ID NO: 49; fifth columns from left), peptide Tearp3 (SEQ ID NO: 21; second columns from right), or peptide Tearp3 / C-6 (SEQ ID NO: 33; black, rightmost columns) for 24 h, after which glucose-dependent insulin secretion was determined. N-104 was the most active, followed by Tearp3 / C-6. NS: not significant.

[0042] Fig. 3 is a photomicrograph of isolated mouse pancreatic islets. The inset shows a higher magnification image.

[0043] Fig. 4 is a graph of blood glucose levels for pancreatic islets treated with the indicated peptides and transplanted into diabetic mice, which were observed for 60 days. A graph of blood glucose levels after minimal transplantation of 75 C57B1 / 6 islets treated with the N-94 peptide, treated with the C-95 peptide, or treated with saline under the renal capsule of C57BL / 6 mice in which diabetes was induced before transplantation by a single injection of streptozocin (220 mg / kg; two mice per group) is shown. Pretreatment was carried out for 24 hours. Blood was collected from the tail vein daily for glucose analysis. Each line represents the changing blood glucose level of one diabetic mouse.Six mice received islets treated with the N-94 peptide (black), six others received islets treated with the C-95 peptide (blue), and four mice received islets treated with saline (orange). The N-94 peptide consistently returned mice to normoglycemia after transplantation, with glucose levels <200 ng / dL for 9 days post-transplantation, with efficacy continuing beyond 40 days post-transplantation. With the C-95 peptide, glucose levels on day 12 post-transplantation were <250 mg / dL. Mean blood glucose levels measured between days 22 and 43 were 142.9 ± 17.6 mg / dL for the N-94 group compared with 275 ± 34.8 mg / dL for the C-95 peptide group. This figure shows that pancreatic islets pre-treated with the N-94 peptide transplanted into diabetic mice provided rapid restoration of normoglycemia.

[0044] Figure 5 is a graph of the experimental results, showing that human islets treated with 4 μM Tearpep3 / N-104 exhibited superior viability twenty days after isolation compared with 4 μM Tearpep3 / C-6 and N-94 lacritin. The synthetic lacritin peptide C-95 (4 μM), derived from the inactive N-terminus of lacritin, was used as a negative control.

[0045] Fig. 6 is a graph of blood glucose levels in NOD mice administered three doses of 200 μg IgM on days 1, 3, and 5, starting from the initial onset of hyperglycemia (i.e., baseline value (BG) 180-340 mg / dL). Diabetes was reversed in 70% of NOD mice throughout the observation period.

[0046] Fig. 7 is a graph showing that IgM therapy suppresses the occurrence of T1D. 80% of saline-treated control NOD mice developed diabetes by 18-20 weeks of age. Mice treated with IgM twice weekly (~50 μg in 100 μL PBS) showed significant protection against T1D when treatment was started early (at 5 weeks of age) (p<0.0001). [BSA resulted in 70% T1D incidence, whereas IgG administration resulted in a 50% reduction in incidence (n=10 / group) at 25 weeks of age]. Treatment cessation resulted in diabetes in only 9 of 33 mice 22 weeks after cessation, indicating the development of resistance.

[0047] Fig. 8 is a graph showing that treatment of NOD mice with IgM reversed the diagnosed disease in combination with islet transplantation.

[0048] Figures 9A-9C show the results of experiments demonstrating that the N-94 / C-6 peptide according to the described subject matter of the invention restored the viability of human sensory neurons exposed to inflammatory cytokines. Figure 9A is a series of immunofluorescence micrographs for the analysis of sensory neuron markers in cells at day 0, day 10, and day 21. Figure 9B is a series of bar graphs showing the results of the analysis of mRNA levels of the TRPM8, TUJ1, and PMCA genes at day 0, day 10, and day 18, which were analyzed by quantitative real-time PCR. Fig. 9C is a bar graph of the percentage viability of sensory neuron cells at day 21 incubated with IFN-γ (1000 U / ml)+TNF-α (100 ng / ml) with or without 1 mM N-94 / C-6 peptide or negative control C-95 peptide for 72 hours in a 96-well plate.The obtained data are presented as columns of mean ± standard deviation (SD) from pools consisting of 3 independent experiments, with a value **p<0.001.

[0049] BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0050] SEQ ID NO: 1-60 and 62 are amino acid sequences of exemplary peptides of the described subject matter.

[0051] SEQ ID NO: 61 is the amino acid sequence of the human lacritin gene product.

[0052] SEQ ID NO: 63 is the amino acid sequence of exendin-4.

[0053] DETAILED DESCRIPTION OF THE INVENTION

[0054] I. General Provisions

[0055] Currently, there are no approved treatments available to address the causes, drivers, or underlying pathology of diabetes, including, in particular, type 1 diabetes (T1D). The only treatments currently approved for T1D focus on insulin replacement therapy, blood glucose control, insulin action, and organ transplantation. T1D is described as a progressive and deficient state of insulin caused by the immune-mediated loss of functional insulin-producing beta cells. During this process, various factors (genetic, environmental, and immune), individually or in combination, induce beta cell stress, leading to loss of beta cell function and cell death. Both these factors lead to insulin deficiency and lifelong dependence on insulin replacement therapy.

[0056] Therapies that directly modify beta cell biology may halt the loss of function and number of insulin-producing cells that occurs in T1D and increase their numbers if they are lost. Recent studies have suggested that impaired beta cell function is an early sign of disease pathogenesis, with decreased beta cell mass more closely associated with clinical manifestations. Beta cells are not solely passive victims in the development of T1D; pathological beta cell stress occurs very early in the development of T1D and plays a role in the loss of beta cell function and mass in T1D, possibly by triggering or amplifying a beta cell-specific autoimmune response. Research is ongoing to elucidate the dynamic nature of the beta cell population.In the healthy pancreas, this population is heterogeneous in both function and phenotype: for example, changes in insulin secretion levels and endoplasmic reticulum stress are observed.

[0057] Beta cell survival therapy can delay and halt the progression of T1D at all stages of the disease: maintaining insulin independence for individuals without beta cell loss, preventing insulin dependence for those experiencing asymptomatic beta cell loss, and maintaining residual beta cell mass in those already insulin dependent. Beta cell survival therapy is also being used to protect restored or replaced beta cell mass to achieve insulin independence. Beta cell survival therapy nourishes and protects cell number and function when exposed to stressors unique to T1D.

[0058] Recent clinical studies have confirmed the validity of approaches based on enhancing beta cell survival. Both Gleevec and verapamil have been shown to delay or slow the loss of insulin production in adults at stage 3 of the disease, improve glucose control, and reduce the incidence of diabetic ketoacidosis.

[0059] Loss of beta cell function occurs early in the T1D pathology process and precedes the loss of beta cell mass. Furthermore, dysfunctional beta cells can be found in many individuals with long-standing T1D. It has recently been described that abnormal hormonal processing, cellular senescence, and other indicators of decreased beta cell function occur in the prodromal period of T1D. Strategies to improve beta cell function may be incorporated into approaches aimed at increasing beta cell mass. Functional and dysfunctional beta cells can be detected before diagnosis and decades after the initial diagnosis of T1D, indicating the need for therapies aimed at increasing residual mass and function at all stages of the disease. Strategies to increase beta cell mass can be applied to proliferation, differentiation from other cell types, and / or new growth.

[0060] Therapy targeting beta cell survival and beta cell regeneration, in some embodiments in combination with appropriate immunotherapy, can modify the course of T1D to prevent, halt, and in some embodiments, eliminate T1D. Therapy targeting beta cell restoration can also provide a non-invasive treatment option for individuals living with T1D by providing therapeutic agents that increase the number and function of the subject's remaining beta cells.

[0061] There are two basic strategies for correcting beta cell deficiency in T1D: (1) implanting insulin-producing cells into the individual (beta cell replacement); and (2) treating the individual with agents that increase the number and function of insulin-producing cells in their body (beta cell reconstitution). Beta cell replacement strategies have various advantages but also disadvantages. On the other hand, reconstitution therapy can provide improved glucose control in individuals at stage 3 of the disease and ultimately insulin independence. Such therapies are designed to replenish beta cell mass and / or improve the function of the remaining cells in individuals at stage 2 of the disease to prevent the development of insulin dependence. Both beta cell reconstitution and replacement strategies can be combined with strategies to combat autoimmune or transplant-directed anti-beta cell responses.

[0062] Trials of beta-cell-targeted therapies (and other drugs) in T1D rely on a single clinical trial design and a single clinical outcome: maintenance of fasting insulin secretion stimulation at 1 year post-intervention. This makes testing potential therapeutics expensive and time-consuming and highlights the need for more efficient clinical development of treatments. Recent advances in platform study designs, combination regimens, specific patient subpopulations (e.g., testing agents in the most clinically relevant patient population (stage 2 versus stage 3)), and innovative therapeutic data address this need.Similarly, the use of continuous glucose monitoring devices in T1D studies may provide valuable and early assessment of drug efficacy for those agents that, by maintaining or increasing beta cell mass, lead to improved glucose control.

[0063] As described herein, the synthetic peptide N-94 restores islet beta cell function, reversing the disease in streptozocin-induced diabetic mice, consistent with a mechanism that preserves lacritin signaling in islets. This mechanism involves NFAT and mTOR. Like harmine and INDY, lacritin accelerates NFAT nuclear translocation, and like PIAA and amlexanox, it activates mTOR, promoting both HSG / HeLa cell proliferation (Wang et al., 2006) and eye healing in NOD mice (Wang et al., 2015b). This occurs through the C-terminal domain of N-94. The C-terminal N-94 of lacritin is also responsible for restoring the health of inflamed eyes in two different models of autoimmune dry eye disease by: (1) rapidly stimulating autophagy to scavenge inflammation-damaged proteins and organelles, and (2) restoring oxidative phosphorylation (Wang et al., 2013). This occurs primarily through FOX01 and FOX03 signaling.The fact that N-94-like peptides are constant components of plasma suggests that islets are constantly floating in them and, due to dry eye (Karnati et al., 2013), they may be selectively deficient in diabetes.

[0064] Transplantation of N-94-treated islets requires surgical intervention. In some embodiments, regeneration of endogenous islets is achieved by systemic [self-administered] treatment. F Definitions

[0065] In the description and claims of the described subject matter of the invention, the following terminology is used in accordance with the definitions given below.

[0066] Singular terms are used in this description to denote one or more (i.e., at least one) grammatical object of the description. For example, "element" means one element or more than one element.

[0067] The term "about" in this context means approximately, in the region of, roughly, or around. When used in conjunction with a numerical range, the term "about" modifies the stated range by extending the boundaries above and below the stated numerical values. For example, in some embodiments, the term "about" is used in this context to modify a numerical value above and below the stated value by a difference of 10%. Thus, about 50% means a range of 45%-55%. Numerical ranges specified herein by endpoints include all integer and fractional values ​​included in the range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It should also be understood that all integer and fractional values ​​are intended to be modified by the term "about".

[0068] As used herein, the term "biological specimen" refers to a sample isolated from a subject (e.g., a biopsy, blood, serum, etc.) or isolated from a cell or tissue obtained from a subject (e.g., RNA and / or DNA, and / or a protein or polypeptide isolated therefrom). Biological specimens may be any biological tissue or fluid, or cells from any organism, as well as cells grown in vitro, such as cell lines and tissue culture cells. A sample is often a "clinical specimen," which is a sample obtained from a subject (i.e., a subject undergoing a diagnostic procedure and / or treatment).Typical clinical specimens include, but are not limited to, cerebrospinal fluid, serum, plasma, blood, saliva, skin, muscle, olfactory tissue, tears, synovial fluid, nail tissue, hair, stool, urine, specific tissue or cell types and combinations thereof, tissue biopsy specimens, or fine-needle aspiration biopsy specimens, and cells from them. Biological specimens may also include tissue sections, such as frozen sections or formalin-embedded sections, taken for histological purposes.

[0069] As used herein, the term "comprising," which is synonymous with the terms "including," "having," or "characterized by," is inclusive or open-ended and does not exclude additional, unlisted elements and / or method steps. "Comprising" is a special term used in the formulation of claims that means that the named elements are present, but other elements may be added and still form a composition or method within the scope of the described subject matter. By way of example, but not limitation, a pharmaceutical composition comprising a particular active agent and a pharmaceutically acceptable carrier may also contain other components, including, but not limited to, other active agents, other carriers and excipients, as well as any other molecule that may be appropriate for inclusion in the pharmaceutical composition without any limitation.

[0070] As used herein, the term "consisting of" excludes any element, step, or ingredient not specifically recited in a claim. When the term "consists of" appears in the preamble of a claim, rather than immediately following the preamble, it limits only the element recited in that claim; other elements are not excluded from the claim as a whole. By way of example, but not limitation, a pharmaceutical composition comprising an active agent and a pharmaceutically acceptable carrier contains no components other than the specified active agent and the specified pharmaceutically acceptable carrier. It should be understood that any molecule whose content is below a reasonable detection limit is considered absent.

[0071] In this context, the expression "consisting essentially of" limits the scope of a claim to specific materials or steps, as well as those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter. By way of example, but not limitation, a pharmaceutical composition consisting essentially of an active agent and a pharmaceutically acceptable carrier contains the active agent and the pharmaceutically acceptable carrier, and may also contain any additional elements that may be present, but which do not materially affect the biological functions of the composition in vitro or in vivo.

[0072] With respect to the terms "comprising," "consisting essentially of," and "consisting of," when one of these three terms is used in this context, the described and claimed subject matter includes the use of any of the other two terms. For example, "comprising" is a transitional term that is broader than the terms "consisting essentially of" and "consisting of," and therefore the term "comprising" explicitly includes both "consisting essentially of" and "consisting of." Likewise, the transitional expression "consisting essentially of" is broader than "consisting of," and therefore the expression "consisting essentially of" explicitly includes "consisting of."

[0073] In this context, the term "biologically active fragments" or "bioactive fragment" of polypeptides includes natural or synthetic portions of a full-length protein that are capable of specifically binding to their natural ligand or of performing the function of the protein.

[0074] As used herein, the term "lacritin polypeptide" and similar terms are defined as any peptide comprising the amino acid sequence of SEQ ID NO: 1 and / or any biologically active fragment, homologue or derivative thereof. As used herein, the term "biologically active fragments" or "bioactive fragment" of a lacritin polypeptide includes natural or synthetic portions of the amino acid sequence MKFTTLLFLAAVAGALVYAEEDASSDSTGADPAQEAGTSKPNEEISGPAEPASPRETTTAQETSAAAVQGTAKVTSSRQELNPLKSIVEKSILLTEQALAKAGKGMHGGVPGGKQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 61). Lacritin fragments (SEQ ID NO: 61) include, for example: KQFIENGSEFAQKLLKKFS (SEQ ID NO: 62; 'N-94 / C-6'; Wang et al., 2006) and KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 1; 'N-94'; see Zhang et al., 2013).

[0075] As used herein, an "acylated" amino acid is an amino acid containing an acyl group that is not native to the natural amino acid, regardless of the method of production. Examples of methods for producing acylated amino acids and acylated peptides are known in the art and include acylation of an amino acid prior to incorporation into a peptide or synthesis of a peptide followed by chemical acylation of the resulting peptide. In some embodiments, the acyl group causes the resulting peptide to have one or more of (i) a prolonged half-life in the bloodstream, (ii) a delayed onset of action, (iii) an increased duration of action, and (iv) improved resistance to proteases.

[0076] As used herein, an "alkylated" amino acid is an amino acid containing an alkyl group that is not native to the natural amino acid, regardless of the method of production. Examples of methods for producing alkylated amino acids and alkylated peptides are known in the art and include alkylation of an amino acid prior to incorporation into a peptide or peptide synthesis followed by chemical alkylation of the resulting peptide. Without wishing to be bound by any particular theory, it is believed that alkylation of peptides achieves similar, if not the same, effects as acylation of peptides, such as prolonged circulating half-life, delayed onset of action, increased duration of action, and improved resistance to proteases.

[0077] In this context, the term "improved survival" refers to a reduction in the amount or rate of cell death in a cell population (e.g., a population of islet cells). Improved survival may result solely from preventing cell death (e.g., cell death due to apoptosis) or from reducing the rate of cell death. Reduced cell death may also result from indirect effects, such as initiating proliferation of some cells, an indirect effect that effectively replenishes at least some or all cells in the population as they die. Improved cell survival may also be achieved through a combination of initiating proliferation and reducing cell death or the rate of cell death. "Improved survival" and "improved survival" are used interchangeably with "enhanced survival" in this context.

[0078] The term "identity" in this context refers to the similarity of two or more sequences. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the result by 100 to obtain a percentage. Thus, two exact copies of the same sequence have 100% identity, while two sequences that contain deletions, additions, or substitutions of amino acids / nucleic acids relative to each other have a lower degree of identity. Those skilled in the art are aware that several computer programs exist for determining sequence identity, such as BLAST (Basic Local Alignment Search Tool, Altschul et al., 1993).

[0079] In this context, “modification” of an amino acid refers to the substitution of an amino acid or the derivatization of an amino acid by adding and / or removing chemical groups to / from an amino acid and includes the substitution of any of the 20 amino acids commonly found in human proteins, as well as atypical or non-naturally occurring amino acids. Commercial suppliers of atypical amino acids include Sigma-Aldrich (Milwaukee, WI, USA), ChemPep Inc. (Miami, FL, USA), and Genzyme Pharmaceuticals (Cambridge, MA, USA). Atypical amino acids may be purchased from commercial suppliers, synthesized from scratch, or chemically modified or derivatized from naturally occurring amino acids.

[0080] In this context, “substitution” of an amino acid refers to the replacement of one amino acid residue with another amino acid residue.

[0081] In this context, the term "conservative amino acid substitution" is defined as substitutions in one of the following five groups:

[0082] • Small aliphatic non-polar or slightly polar residues: Ala, Ser, Thr, Pro, Gly;

[0083] • Polar, negatively charged residues and their amides: Asp, Asn, Glu, Gin, cysteine ​​acid and homocysteine ​​acid;

[0084] • Polar, positively charged residues: His, Arg, Lys; ornithine (Orn);

[0085] • Large aliphatic non-polar residues: Met, Leu, Ile, Val, Cys, norleucine (Nle), homocysteine;

[0086] • Large aromatic residues: Phe, Tyr, Trp, acetylphenylalanine.

[0087] "Peptidomimetic" refers to a chemical compound having a structure that differs from the general structure of an existing peptide, but which functions in the same manner as the existing peptide, for example, by mimicking the biological activity of said peptide. Peptidomimetics typically contain naturally occurring amino acids and / or unnatural amino acids, but may also contain modifications in the peptide backbone. For example, a peptidomimetic may contain a sequence of naturally occurring amino acids with the insertion or substitution of a non-peptide moiety, such as a retro-inverso fragment, or with the insertion of non-peptide linkages, such as an azapeptide linkage (CO replaced by NH) or a pseudopeptide linkage (e.g., NH replaced by CH2), or an ester linkage (e.g., depsipeptides in which one or more amide (--CONHR--) linkages are replaced by ester (COOR) linkages). Alternatively, a peptidomimetic may not contain any naturally occurring amino acids.

[0088] As used herein, the term "amino acid" includes any molecule containing both an amine and a carboxyl functional group, wherein the amine group and the carboxylate group are attached to the same carbon atom (the alpha carbon atom). The alpha carbon atom may optionally have one or two additional organic substituents. As used herein, a description of an amino acid without specifying its stereochemistry is intended to include either the L or D form of the amino acid or a racemic mixture. However, when an amino acid is described by a three-letter code and contains a numeric superscript, the D form of the amino acid is designated by including a lowercase letter d before the three-letter code and the numeric superscript (e.g., dLys1), while a designation without a lowercase letter d (e.g., Lys1) is intended to indicate the native L form of the amino acid.In this nomenclature, the inclusion of a numerical superscript denotes the position of the amino acid in the peptide sequence, numbered sequentially from the N-terminus. The term "amino acid" in this context includes both natural and synthetic amino acids, as well as D- and L-amino acids. "Standard amino acid" refers to any of the twenty L-amino acids commonly found in natural peptides.

[0089] The term "subject" as used herein refers to a member of any invertebrate or vertebrate animal species. Accordingly, the term "subject" includes any member of the kingdom Animalia, including, but not limited to, the phylum Chordata (i.e., members of the classes Osteichythyes (bony fish), Amphibia (amphibians), Reptilia (reptiles), Aves (birds), and Mammalia (mammals)), and all classes and families within them. In some embodiments, the subject is a human.

[0090] It should be noted that all genes, gene names, gene products, and other products described herein correspond to orthologs or other similar products of any biological species to which the compositions and methods described herein are applicable. Thus, these terms include, but are not limited to, genes and gene products of humans and mice. It should be understood that when describing a gene or gene product of a particular biological species, such description is illustrative only and should not be construed as limiting unless the context in which it appears clearly indicates otherwise.Thus, for example, any genes specifically mentioned herein and genes for which various illustrative gene products are described in the GENBANK® biological sequence database by accession numbers include homologous genes and gene variants, as well as gene products from humans and other animals, including, but not limited to, other mammals.

[0091] The methods according to the described subject matter of the invention are particularly suitable for warm-blooded vertebrates. Thus, the described subject matter of the invention relates to mammals and birds. More particularly, the isolation, processing and use of stem cells of mammals, such as humans and other primates, as well as mammals that are important due to the threat of their extinction (such as Siberian tigers), economic importance (animals raised on farms for human consumption) and / or social importance (animals kept as pets or in zoos) for humans, for example, non-human carnivores (such as cats and dogs), swine (pigs, boars and wild boars), ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison and camels), rodents (such as mice, rats and rabbits), marsupials and horses.The application of the described methods and compositions to birds, including endangered species kept in zoos, as well as poultry, and more specifically domesticated birds, such as turkeys, chickens, ducks, geese, guinea fowl, etc., is also proposed, as they are also of economic importance to humans. Thus, the isolation, processing, and use of stem cells from farm animals is also envisaged, including, but not limited to, domesticated pigs (pigs and boars), ruminants, horses, poultry, etc.

[0092] As used herein, the term "substantially" refers to a condition wherein, in some embodiments, no more than 50%, in some embodiments no more than 40%, in some embodiments no more than 30%, in some embodiments no more than 25%, in some embodiments no more than 20%, in some embodiments no more than 15%, in some embodiments no more than 10%, in some embodiments no more than 9%, in some embodiments no more than 8%, in some embodiments no more than 7%, in some embodiments no more than 6%, in some embodiments no more than 5%, in some embodiments no more than 4%, in some embodiments no more than 3%, in some embodiments no more than 2%, in some embodiments no more than 1%, and in some embodiments no more than 0% of the components in a population of objects do not have the characteristic.

[0093] The terms "additional therapeutically active compound" or "additional therapeutic agent" in the context of the described subject matter refer to the use or administration of a compound for additional therapeutic use in the treatment of a specific injury, disease, or disorder. Such a compound may, for example, include a compound used to treat unrelated diseases or disorders, or a disease or disorder that does not respond to the primary treatment of the injury, disease, or disorder being treated. Diseases and disorders treated with an additional therapeutically active agent include, for example, hypertension and diabetes. Additional compounds can also be used to treat symptoms associated with the injury, disease, or disorder, including, but not limited to, pain and inflammation.

[0094] The term "adult" in this context refers to any subject that is not an embryo or immature.

[0095] As used herein, an "agonist" is a composition of matter that, when administered to a mammal, such as a human, enhances or prolongs the biological activity associated with the level or presence of a target compound or molecule of interest in the subject.

[0096] A disease or disorder is "alleviated" if the severity of a symptom of the disease, condition, or disorder is reduced, or if the frequency of occurrence of the symptom in the subject is reduced, or if both are present.

[0097] In this context, amino acids are described by their full name, their corresponding three-letter code, or their corresponding one-letter code, as shown in Table 1:

[0098]

[0099]

[0100] The term "amino acid" in this context includes both natural and synthetic amino acids, as well as D- and L-amino acids. "Standard amino acid" means any of the twenty standard L-amino acids commonly found in natural peptides. "Non-standard amino acid residue" means any amino acid other than the standard amino acids, regardless of whether it is produced synthetically or from a natural source. In this context, "synthetic amino acid" also includes chemically modified amino acids, including, but not limited to, salts, amino acid derivatives (such as amides), and substitutions. The amino acids contained in the peptides according to the described subject matter of the invention, and in particular at the carboxy-terminus or at the amino-terminus, can be modified by methylation, amidation, acetylation or substitution with other chemical groups that can change the half-life of the peptide from the bloodstream without impairing its activity.In addition, the peptides according to the described subject matter of the invention may or may not contain a disulfide bond.

[0101] The term "amino acid" is used interchangeably with "amino acid residue" and can refer to a free amino acid or an amino acid residue of a peptide. The context in which the term is used will determine whether it refers to a free amino acid or a peptide residue.

[0102] Amino acids can be divided into seven groups based on the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxyl (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused with an amino group.

[0103] The term "basic" or "positively charged" amino acid, as used herein, refers to amino acids in which the R groups have a net positive charge at pH 7.0, and includes, but is not limited to, the standard amino acids lysine, arginine, and histidine.

[0104] As used herein, an "analog" of a chemical compound is a compound that is, for example, structurally similar to another compound, but is not necessarily an isomer of that compound (e.g., 5-fluorouracil is an analog of thymine). With respect to an amino acid, an "analog" of an amino acid in some embodiments may be another amino acid that is structurally similar to the amino acid, or a compound other than the amino acid that is structurally similar to the amino acid. A large number of known analogs of the 20 amino acids commonly found in proteins ("standard" amino acids) are known.Thus, with respect to a peptide, the term "analog" refers to peptides that differ from a reference peptide (in some embodiments, a naturally occurring peptide) in the identity or location of one or more amino acid residues (e.g., by deletion, substitution, and / or insertion), and yet have some or all of the same properties as the reference peptide, provided that they have the desired characteristics of the reference peptide (e.g., the biological activity of a lacritin peptide or derivative thereof according to the described subject matter).

[0105] An "antagonist" is a composition of matter that, when administered to a mammal, such as a human, inhibits the biological activity associated with the level or presence of a compound or molecule of interest in the subject.

[0106] The term "antibody" as used herein refers to an immunoglobulin molecule that can specifically or selectively bind to a particular epitope of an antigen. Antibodies may be intact immunoglobulins obtained from natural sources or from recombinant sources, and may be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies according to the described subject matter may exist in a variety of forms. The term "antibody" refers to polyclonal and monoclonal antibodies and derivatives thereof (including chimeric, synthesized, humanized, and human antibodies), including a whole immunoglobulin or antibody, or any functional fragment of an immunoglobulin molecule that binds to a target antigen, and / or combinations thereof. Examples of such functional particles include whole antibody molecules, antibody fragments such as F v , single-stranded Fv , complementarity determining regions (CDRs), V L (light chain variable region), V H (heavy chain variable region), Fab, F(ab')2, and any combinations of these or any other functional portions of the immunoglobulin peptide capable of binding to the target antigen.

[0107] Antigens exist, for example, in the form of intact immunoglobulins or in the form of a variety of well-characterized fragments obtained by cleavage by various peptidases. For example, pepsin cleaves the antibody under the disulfide bonds in the hinge region to form the Fab dimer F(ab')2, which itself is a light chain linked to V H -C H1disulfide bond. F(ab')2 can be reduced under mild conditions to break the disulfide bond in the hinge region, converting the F(ab')2 dimer to a Fab1 monomer. The Fab1 monomer is essentially a Fab with a portion of the hinge region. Although various antibody fragments have been described in terms of cleavage of an intact antibody, those skilled in the art will appreciate that such fragments can be synthesized chemically or recombinantly from scratch. Thus, the term "antibody" in this context also includes antibody fragments that are either produced by modifying whole antibodies or synthesized from scratch using recombinant DNA methodologies.

[0108] "Antibody heavy chain" in this context refers to the larger of the two types of polypeptide chains present in all intact antibody molecules.

[0109] "Antibody light chain" in this context refers to the smaller of the two types of polypeptide chains present in all intact antibody molecules.

[0110] The term "single-chain antibody" refers to an antibody in which the genetic information encoding the functional fragments of the antibody is located in a single contiguous DNA sequence. A detailed description of single-chain antibodies is provided in Bird et al., 1988; Huston et al., 1993.

[0111] The term "humanized" refers to an antibody whose constant regions are at least about 80% or more homologous to human immunoglobulin. In addition, some amino acid residues of a non-human, such as a mouse, variable region may be modified so that they contain amino acid residues of human origin. Humanized antibodies are called "reshaped" antibodies. Alteration of the complementarity determining regions (CDRs) is a method for producing humanized antibodies. See, for example, U.S. Patent Nos. 4,816,567; 5,482,856; 6,479,284; 6,677,436; 7,060,808; 7,906,625; 8,398,980; 8,436,150; 8,796,439; and 10,253,111; and U.S. Patent Application Publications Nos. 2003 / 0017534, 2018 / 0298087, 2018 / 0312588, 2018 / 0346564, and 2019 / 0151448, the entire contents of each of which are incorporated by reference.

[0112] The term "synthetic antibody" in this context means an antibody that is produced using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage, as described herein. This term should also be understood as meaning an antibody that is produced by synthesizing a DNA molecule encoding an antibody, and wherein said DNA molecule expresses an antibody protein or an amino acid sequence defining the antibody, wherein the DNA or amino acid sequence is produced using technology for producing synthetic DNA or amino acid sequences that is available and known in the art.

[0113] The term "antigen" in this context is defined as a molecule that elicits an immune response. Such an immune response may involve either the production of an antibody, the activation of specific immunologically competent cells, or both. An antigen may be derived from organisms, protein / antigen subunits, dead or inactivated whole cells, or lysates.

[0114] The term "antimicrobial agents" as used herein refers to any natural, synthetic, or semi-synthetic compound or composition, or mixture thereof, that is safe for use in humans or animals in the practice of the methods of the described subject matter of the invention and that is effective in killing or significantly inhibiting the growth of microbes. "Antimicrobial" as used herein includes antibacterial, antifungal, and antiviral agents.

[0115] As used herein, the term "antisense oligonucleotide" or antisense nucleic acid means a nucleic acid polymer, at least a portion of which is complementary to a nucleic acid present in a normal cell or a diseased cell. "Antisense" refers in particular to a nucleic acid sequence of the non-coding strand of a double-stranded DNA molecule encoding a protein, or to a sequence that is substantially homologous to the non-coding strand. In this context, the antisense sequence is complementary to the sequence of a double-stranded DNA molecule encoding a protein. It is not necessary for the antisense sequence to be complementary only to the coding portion of the coding strand of the DNA molecule.The antisense sequence may be complementary to regulatory sequences identified in the coding strand of a DNA molecule encoding a protein, and said regulatory sequences regulate the expression of the coding sequences. Antisense oligonucleotides according to the described subject matter include, but are not limited to, phosphate thioate oligonucleotides and other oligonucleotide modifications.

[0116] The term "autologous" in this context refers to something that occurs naturally and is normal for a particular tissue type or structure in the body. In transplantation, it refers to a graft in which the donor and recipient areas are from the same individual, or to blood that the donor previously donated and then received back, usually during surgery.

[0117] The term "basic medium" in this context refers to the minimum essential medium type, such as Dulbecco's Modified Eagle's Medium, Ham's F12 Medium, Eagle's Medium, RPMI, AR8, etc., to which other ingredients may be added. This term does not exclude media that have been prepared or intended for a specific use but which, after modification, can be used for other cell types, etc.

[0118] The term "biocompatible" in this context refers to a material that does not cause a significant adverse response in the host.

[0119] The term "biodegradable" in this context means capable of being biologically degraded. A biodegradable material differs from a non-biodegradable material in that a biodegradable material can be biodegraded into components that can be either removed from the biological system and / or chemically incorporated into the biological system.

[0120] The term "biological specimen" in this context refers to specimens obtained from a living organism, including skin, hair, tissue, blood, plasma, cells, sweat and urine.

[0121] The term "bioresorbable" in this context refers to the ability of a material to be resorbed in vivo. "Complete" resorption means that no significant extracellular fragments remain. The resorption process involves the removal of the original implant materials by bodily fluids, enzymes, or cells. For example, resorbed calcium carbonate can be redeposited as bone mineral, reused in the body in other ways, or excreted. The term "substantially bioresorbable" in this context means that at least 80% of the total mass of the implanted material is resorbed within one year.

[0122] The expressions "cell culture medium", "culture medium" (in each case, the plural form of "media") and "medium composition" refer to a nutrient solution for cell culture and can be used interchangeably.

[0123] "Conditioned medium" is the medium obtained by culturing a first population of cells or tissue in the medium and then harvesting the medium. The conditioned medium (along with anything secreted by the cells into that medium) can then be used in any desired manner, such as to treat a disease or disorder in a subject or to support the growth or differentiation of a second population of cells.

[0124] A "control" cell, tissue, sample, or subject is a cell, tissue, sample, or subject of the same type as the experimental cell, tissue, sample, or subject. A control may be studied, for example, at exactly the same time or nearly the same time as the experimental cell, tissue, sample, or subject. A control may also be studied, for example, at a time different from the time the experimental cell, tissue, sample, or subject is studied, and the results of studying the control may be recorded so that the recorded results can be compared with the results obtained when studying the experimental cell, tissue, sample, or subject. A control may also be obtained from a different source or a similar source than the experimental group or experimental subject, in which case the experimental sample is obtained from a subject suspected or confirmed to have the disease or disorder being tested.

[0125] An "experimental" cell, tissue, specimen, or subject is one on which research or treatment is performed.

[0126] A "pathologically indicative" cell, tissue, or sample is one that, when present, indicates that the animal in which such cell, tissue, or sample is located (or from which the tissue was obtained) suffers from a disease or disorder. For example, the presence of one or more mammary gland cells in an animal's lung tissue indicates that the animal suffers from metastatic mammary cancer.

[0127] A tissue "normally contains" a cell if one or more such cells are present in that tissue in an animal not suffering from a disease or disorder.

[0128] "Compound" as used herein refers to any type of substance or agent that is generally considered to be a drug, or a candidate for use as a drug, or combinations and mixtures of any of the foregoing, as well as polypeptides and antibodies according to the described subject matter.

[0129] "Cytokine" in this context refers to intercellular signaling molecules, the most well-known of which are involved in the regulation of mammalian somatic cells. Several families of cytokines have been described, with both growth-promoting and growth-inhibiting effects, including, for example, interleukins, interferons, and transforming growth factors. Many other cytokines are familiar to those skilled in the art. The sources, characteristics, targets, and effector actions of these cytokines are described.

[0130] "Chemokine" in this context refers to an intercellular signaling molecule involved in the chemotaxis of white blood cells such as T cells.

[0131] The term "delivery carrier" refers to any type of device or material that can be used to deliver cells in vivo or that can be added to a composition containing cells to be administered to an animal. Delivery carriers include, but are not limited to, implantable devices, cell aggregates, matrix materials, gels, etc.

[0132] As used herein, a "derivative" of a compound refers to a chemical compound that can be prepared from another compound of similar structure in one or more steps, such as by replacing H with an alkyl, acyl, or amino group. In the context of a peptide or polypeptide sequence, a "derivative" is a peptide or polypeptide that has one or more modifications in its amino acid sequence such that it differs in at least one respect from a reference sequence (e.g., a naturally occurring peptide or polypeptide) either in terms of the amino acid sequence (e.g., due to one or more insertions, deletions, and / or amino acid substitutions) or in terms of some of its modifications.Illustrative non-limiting modifications include N- and / or C-terminal additions of one or more amino acids, in some embodiments functional amino acids (e.g., cysteine), N- and / or C-terminal amidation, N- and / or C-terminal acylation, N- and / or C-terminal acetylation, and N- and / or C-terminal pegylation.

[0133] In some embodiments, a peptide derivative according to the described subject matter is a cross-linked peptide. Cross-linked peptides are stabilized peptides that use one or more intramolecular cross-linking agents to hold the peptide in the desired configuration, for example, using disulfide bonds, amide bonds and / or carbon-carbon bonds to link amino acid side chains. The cross-linking agents connect at least two amino acids of the peptide. The cross-linking agents can contain at least 5, 6, 7, 8, 9, 10, 11, or 12 consecutive carbon-carbon bonds. The cross-linking agents can contain at least 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms.

[0134] The use of the term "detection" and its grammatical variants refers to the identification of particles without quantification, while the use of the term "determination" or "measurement" and their grammatical variants refers to the identification of particles with quantification. The terms "detection" and "identification" are used interchangeably in this context.

[0135] As used herein, a "detectable marker" or "reporter molecule" is an atom or molecule that enables the specific detection of a compound containing the said marker in the presence of the same compounds without the marker. Detectable markers or reporter molecules include, for example, radioactive isotopes, antigenic determinants, enzymes, hybridizable nucleic acids, chromophores, fluorophores, chemiluminescent molecules, electrochemically detectable molecules, and molecules that provide a change in fluorescence polarization or a change in light scattering.

[0136] "Disease" is a health condition of an animal in which the animal cannot maintain homeostasis, and if the disease condition is not improved, the animal's health continues to deteriorate.

[0137] In contrast, a "disorder" in a mammal is a health condition in which the animal can maintain homeostasis, but in which the animal's health is less favorable than in the absence of the disorder. If left untreated, a disorder does not necessarily cause further deterioration in the animal's health.

[0138] In this context, "effective amount" means an amount sufficient to provide the intended effect. "Therapeutically effective amount" means an amount effective for treating or preventing a disease or disorder.

[0139] The term "epitope" in this context is defined as small chemical groups on an antigen molecule that can elicit antibody production and interact with it. An antigen may have one or more epitopes. Most antigens have multiple epitopes; that is, they are polyvalent. Typically, an epitope is approximately five amino acids or sugars in size. Those skilled in the art will appreciate that, as a rule, the overall three-dimensional structure, rather than the specific linear sequence of the molecule, is the primary criterion for antigen specificity.

[0140] A "fragment" or "segment" is a portion of an amino acid sequence containing at least one amino acid, or a portion of a nucleic acid sequence containing at least one nucleotide. The terms "fragment" and "segment" are used interchangeably in this context.

[0141] In this context, the term "fragment" in relation to a protein or peptide can generally mean at least about 3-15 amino acids in length, at least about 15-25 amino acids in length, at least about 25-30 amino acids in length, at least about 75-100 amino acids in length, and more than 100 amino acids in length.

[0142] As used herein, the term "fragment" when used in relation to a nucleic acid can typically be at least about 20 nucleotides in length, at least about 50 nucleotides, more typically from about 50 to about 100 nucleotides, in some embodiments from at least about 100 to about 200 nucleotides, in some embodiments from at least about 200 nucleotides to about 300 nucleotides, in other embodiments from at least about 300 to about 350, in some embodiments from at least about 350 nucleotides to about 500 nucleotides, in some embodiments from at least about 500 to about 600, in some embodiments from at least about 600 nucleotides to about 620 nucleotides, in some embodiments from at least about 620 to about 650,and in some embodiments, the nucleic acid fragment is greater than about 650 nucleotides in length.

[0143] In this context, a "functional" molecule is a molecule in a form in which it exhibits the property or activity by which it is characterized.

[0144] In this context, a "functional biological molecule" is a biological molecule in a form that exhibits the property it is characterized by. For example, a functional enzyme is an enzyme that exhibits the characteristic catalytic activity that characterizes that enzyme.

[0145] The term "ingredient" refers to any compound of chemical or biological origin that can be used in a cell culture medium to maintain or enhance the proliferation, survival, or differentiation of cells. The terms "component," "nutrient," "additive," and "ingredient" may be used interchangeably and refer to such compounds. Typical, non-limiting ingredients used in cell culture medium include amino acids, salts, metals, sugars, lipids, nucleic acids, hormones, vitamins, fatty acids, proteins, and the like. Other ingredients that enhance or support ex vivo cell growth can be selected by one skilled in the art according to specific needs.

[0146] The term "inhibit" as used herein refers to the ability of a compound, agent, or method to reduce or delay the described function, level, activity, rate, etc., in the context of which the term "inhibit" is used. In some embodiments, the inhibition is at least 10%, in some embodiments at least 25%, in some embodiments at least 50%, and in some embodiments the function is inhibited by at least 75%. The term "inhibit" is used interchangeably with the terms "reduce" and "block".

[0147] The term "inhibitor" in this context refers to any compound or agent that, when administered, results in inhibition of the process or function in question, including, but not limited to, differentiation and activity. Inhibition can be judged by a decrease in the activity or function in question.

[0148] As used herein, "injection or administration" includes administration of a compound or composition according to the described subject matter by any of the methods and approaches, including, but not limited to, topical, oral, buccal, intravenous, intratumoral, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, vaginal, ophthalmic, pulmonary or rectal routes.

[0149] In this context, "damage" usually refers to a defeat, negative action, or injury; it is usually used to refer to damage caused to the body by external forces.

[0150] As used herein, "instructional material" includes a publication, recording, diagram, or any other means of expression that can be used to convey information about the practical value of the composition of the described subject matter in a kit for providing alleviation of various diseases or disorders specified herein. Optionally or alternatively, the instructional material may describe one or more methods for alleviating diseases or disorders in a mammalian cell or tissue. The instructional material from a kit according to the described subject matter may be, for example, attached to a container containing an identified compound according to the described subject matter, or may be supplied together with the container containing the identified compound. Alternatively, the instructional material may be supplied separately from the container, allowing for shared use by the recipient of the instructional material.

[0151] The terms "selection" and "selection" are used interchangeably in this context.

[0152] The terms "isolate," "isolated," "isolation," and grammatical variations thereof, when used in relation to cells, refer to a single cell in question or to a population of cells in question, at least partially separated from other cell types or other cellular material with which it is found in the original culture or tissue. A sample is "substantially pure" if it is free of cells or other cellular material other than the cells in question, in some embodiments at least 60%, in some embodiments at least 75%, in some embodiments at least 90%, and in some cases, in some embodiments, at least 99%.

[0153] "Isolated nucleic acid" refers to a segment or fragment of nucleic acid that is separated from the sequences that naturally flank it, such as a fragment of DNA separated from the sequences that are normally adjacent to that fragment, such as the sequences adjacent to that fragment in the genome in which it naturally occurs. The term also refers to nucleic acids that have been substantially purified from other components that normally accompany nucleic acid, such as RNA or DNA, or from proteins that naturally accompany them in cells.Thus, this term includes, for example, recombinant DNA that is inserted into a vector, an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or that exists as a separate molecule (e.g., as cDNA or genomic DNA, or a cDNA fragment obtained by PCR or restriction enzyme digestion), independent of other sequences. It also includes recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence.

[0154] Unless otherwise specified, "a nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

[0155] As used herein, a "ligand" is a compound that specifically binds to a target compound. A ligand (e.g., an antibody) "specifically binds to" or "is specifically immunoreactive with" a compound if the ligand participates in a binding reaction that detects the presence of the compound in a sample of heterogeneous compounds. Thus, under the conditions of the intended assay (e.g., an immunoassay), the ligand binds preferentially to a particular compound and does not bind significantly to other compounds present in the sample. For example, an antibody specifically binds under the conditions of an immunoassay to an antigen bearing the epitope against which the antibody was raised. Various immunoassay formats can be used to select antibodies that are specifically immunoreactive with a particular antigen.For example, enzyme-linked immunosorbent assays are commonly used to select monoclonal antibodies specifically immunoreactive with an antigen. A description of immunoassay formats and conditions that can be used to determine specific immunoreactivity is presented in Harlow & Lane, 1988.

[0156] A “receptor” is a compound that binds specifically or selectively to a ligand.

[0157] In this context, the term "bond" refers to the connection between two groups. Such connection may be covalent or non-covalent, including, but not limited to, ionic bonds, hydrogen bonding, and hydrophobic / hydrophilic interactions.

[0158] As used herein, the term "linker" refers to a molecule, or a divalent group derived therefrom, that joins two other molecules covalently or non-covalently, such as through ionic or hydrogen bonds, or van der Waals interactions.

[0159] The term "expression level measurement" or "expression level determination" in this context refers to any measurement method or analysis that can be used to correlate the results of the analysis with the expression level of the gene or protein in question. Such analyses include the measurement of mRNA levels, protein levels, etc., and can be performed using assays such as Northern and Western blotting, binding assays, immunoblotting, etc. The expression level may include the rate of expression and can be measured in terms of the actual amount of mRNA or protein present. Such assays involve processes or systems for storing and processing information, facilitating the quantitative determination of levels, signals, etc., and for digitizing the information for use at the comparative level.

[0160] MicroRNAs are typically approximately 16-25 nucleotides in length. In some embodiments, microRNAs are RNA molecules 22 nucleotides or less in length. Such molecules have been found to be significantly involved in the pathology of certain types of cancer. Although microRNA molecules are typically stable when bound to serum and its components after treatment with EDTA, the incorporation of locked nucleic acids (LNAs) into microRNAs via PCR further enhances miRNA stability. LNAs are a class of nucleic acid analogs in which the ribose ring is "locked" via a methylene bridge linking the 2'-O atom and the 4'-C atom of the ribose ring, resulting in increased affinity of the molecule for other molecules.MicroRNAs (miRNAs) are a type of small, noncoding, single-stranded regulatory RNA that interact with the 3'-untranslated region (3'-UTR) of target mRNA molecules through partial sequence homology. They participate in regulatory networks as regulatory elements that direct comprehensive gene expression. Bioinformatic analysis has predicted that a single miRNA can regulate hundreds of target genes, facilitating the combined and fine-tuned regulation of numerous genetic pathways.

[0161] The term "modulate" in this context refers to a change in the level of an activity, function, or process. The term "modulate" includes both inhibition and stimulation of an activity, function, or process. The term "modulate" is used in this context interchangeably with the term "regulate."

[0162] The term "nucleic acid" generally refers to large polynucleotides. By "nucleic acid" is meant any nucleic acid, regardless of whether it consists of deoxyribonucleotides or ribonucleotides, and regardless of whether it consists of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethyl ester, acetamidate, carbamate, thioester, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate, thiophosphate, methylphosphonate, dithiophosphate, bridged thiophosphate or sulfone linkages, and combinations of these linkages. The term "nucleic acid" also includes, in particular, nucleic acids consisting of bases other than the five bases found in biological objects (adenine, guanine, thymine, cytosine, and uracil).

[0163] As used herein, the term "nucleic acid" includes RNA, as well as single-stranded and double-stranded DNA and cDNA. Furthermore, the terms "nucleic acid," "DNA," "RNA," and similar terms also include nucleic acid analogs, i.e., analogs having a backbone other than the phosphodiester backbone. For example, so-called "peptide nucleic acids," which are known in the art and contain peptide bonds in the backbone instead of phosphodiester bonds, are considered to be within the scope of the described subject matter.By "nucleic acid" is meant any nucleic acid, whether composed of deoxyribonucleotides or ribonucleotides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethyl ester, acetamidate, carbamate, thioester, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate, thiophosphate, methylphosphonate, dithiophosphate, bridged thiophosphate or sulfone linkages, and combinations of these linkages. The term "nucleic acid" also includes, in particular, nucleic acids consisting of bases other than the five bases found in biological objects (adenine, guanine, thymine, cytosine, and uracil).In this context, the conventional notation for describing polynucleotide sequences is used: the left end of a single-stranded polynucleotide sequence is the 5' end; the left direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The 5'-3' direction of nucleotide addition to nascent RNA transcripts is referred to as the direction of transcription. The DNA strand that has the same sequence as the mRNA is referred to as the "coding strand"; sequences in the DNA strand that are located in the 5' direction relative to the DNA origin are referred to as "upstream sequences"; sequences in the DNA strand that are located in the 3' direction relative to the DNA origin are referred to as "downstream sequences".

[0164] The term "nucleic acid construct" as used herein includes DNA and RNA sequences encoding a specific desired gene or gene fragment, whether produced by genomic or synthetic means.

[0165] Unless otherwise specified, "a nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

[0166] The term "oligonucleotide" generally refers to short polynucleotides, typically no more than approximately 50 nucleotides in length. It should be understood that if a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it also includes an RNA sequence (i.e., A, U, G, C), in which "U" replaces "T."

[0167] When two polynucleotides are described as "operably linked," it is meant that a single-stranded or double-stranded nucleic acid fragment comprises two polynucleotides arranged within the nucleic acid fragment in such a way that at least one of the two polynucleotides can exert a physiological effect characteristic of the other polynucleotide. For example, a promoter operably linked to the coding region of a gene can promote transcription of the coding region.

[0168] As used herein, "parenteral administration" of a pharmaceutical composition includes any method of administration characterized by physically creating an opening in the tissue of a subject and introducing the pharmaceutical composition through the opening in the tissue. Thus, parenteral administration includes, but is not limited to, administering the pharmaceutical composition by injection of the composition, by introducing the composition through a surgical incision, by introducing the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is intended to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular injection, intratumor administration, and renal dialysis infusion technology.

[0169] "Permeation enhancement" and "permeation enhancers" in this context refer to processes and added materials that result in improved skin penetration of a pharmacologically active agent with poor skin penetration, i.e., increasing the rate at which the drug penetrates the skin and enters the bloodstream. "Permeation enhancer" is used interchangeably with "penetration enhancer."

[0170] The term "pharmaceutical composition" means a composition containing at least one active ingredient, wherein said composition is suitable for testing for a specific effective result in mammals (for example, without limitation, in humans). Those skilled in the art are familiar with and understand technologies suitable for determining whether an active ingredient provides the desired effective result based on the needs of the specialist.

[0171] As used herein, the term "pharmaceutically acceptable carrier" means a chemical composition with which the corresponding compound or derivative can be combined and which, after such combination, can be used to administer the corresponding compound to a subject.

[0172] As used herein, the term "physiologically acceptable" ester or salt means an ester or salt form of the active ingredient that is compatible with any other ingredients of the pharmaceutical composition and that is not deleterious to the subject to whom the composition is administered.

[0173] As used herein, the term "pharmaceutically acceptable salt" refers to salts of compounds that retain the biological activity of the parent compound and that are not biologically or otherwise undesirable. Many of the compounds described herein can form acid and / or base salts due to the presence of amino and / or carboxyl groups or similar groups.

[0174] As used herein, the term "hydrophilic moiety" refers to any compound that is readily soluble in water or readily absorbs water and that is tolerated by mammals in vivo without toxic effects (i.e., are biocompatible). Examples of hydrophilic moieties include polyethylene glycol (PEG), polylactic acid, polyglycolic acid, polylactic-polyglycolic acid copolymer, polyvinyl alcohol, polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxyethyl methacrylate, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide and cellulose derivatives such as hydroxymethylcellulose or hydroxyethylcellulose and copolymers thereof, as well as natural polymers including, for example, albumin, heparin and dextran.

[0175] "Many" means at least two.

[0176] "Polynucleotide" means a single strand or parallel and counter-parallel strands of nucleic acid. Thus, a polynucleotide may be a single-stranded or double-stranded nucleic acid.

[0177] "Polypeptide" refers to a polymer consisting of amino acid residues, related naturally occurring structural variants and their synthetic analogues not occurring in nature, linked by peptide bonds, related naturally occurring structural variants and their synthetic analogues not occurring in nature.

[0178] "Synthetic peptides or polypeptides" means a peptide or polypeptide not found in nature. Synthetic peptides or polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. Various solid-phase methods for peptide synthesis are known to those skilled in the art.

[0179] The term "prevent" in this context means to prevent something from occurring or to take precautions against something that may or is likely to occur. In the context of medicine, "prevention" typically refers to actions taken to reduce the likelihood of acquiring a disease or pathological condition.

[0180] "Primer" refers to a polynucleotide that can specifically hybridize with a given polynucleotide template and provide the initiation site for the synthesis of a complementary polynucleotide. Such synthesis occurs upon exposure of the polynucleotide primer to conditions that initiate synthesis, i.e., in the presence of nucleotides, a complementary polynucleotide template, and a polymerization agent such as DNA polymerase. The primer is typically single-stranded, but may also be double-stranded. Primers are typically deoxyribonucleic acids, but a variety of synthetic and natural primers are suitable for many applications. A primer is complementary to the template to which it is intended to hybridize to provide the initiation site for synthesis, but it does not necessarily reflect the exact sequence of the template. In this case, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions.Primers may be labeled, for example with chromogenic, radioactive or fluorescent moieties, and may be used as fragments suitable for detection.

[0181] "Prophylactic" treatment is treatment given to a subject who does not show signs of a disease or injury, or who only shows early signs of a disease or injury, with the aim of reducing the risk of developing a pathology associated with the disease or injury.

[0182] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence that is necessary for the expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, such a sequence may be a core promoter sequence, while in other cases, such a sequence may also contain an enhancer sequence and other regulatory elements necessary for the expression of the gene product. A promoter / regulatory sequence may be, for example, a sequence that expresses the gene product in a tissue-specific manner.

[0183] A "constitutive" promoter is one that consistently drives the expression of the gene to which it is functionally linked in a cell. For example, promoters that drive the expression of cellular housekeeping genes are considered constitutive promoters.

[0184] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the production of the gene product in a living cell substantially only if an inducer corresponding to the promoter is present in the cell.

[0185] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the production of the gene product in a living cell substantially only if the cell is a cell of the tissue type that corresponds to the promoter.

[0186] As used herein, a "protecting group" with respect to a terminal amino group refers to a terminal amino group of a peptide, wherein said terminal amino group is linked to any of a variety of terminal amino group protecting groups conventionally used for peptide synthesis. Such protecting groups include, for example, acyl protecting groups such as formyl, acetyl, benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl; aromatic urethane protecting groups such as benzyloxycarbonyl; and aliphatic urethane protecting groups such as tert-butoxycarbonyl or adamantyloxycarbonyl. Suitable protecting groups are described in Gross & Mienhofer, 1981.

[0187] As used herein, a "protecting group" with respect to a terminal carboxyl group refers to a terminal carboxyl group of a peptide, wherein said terminal carboxyl group is linked to any of a variety of protecting groups for a terminal carboxyl group. Such protecting groups include, for example, tert-butyl, benzyl, and other suitable groups linked to the terminal carboxyl group by an ester or ether linkage.

[0188] The term "protein" typically refers to large polypeptides. In this context, the standard notation for describing polypeptide sequences is used: the left end of the polypeptide sequence is the amino terminus; the right end of the polypeptide sequence is the carboxyl terminus.

[0189] The term "protein regulatory pathway" in this context refers to both the upstream regulatory pathway that regulates a protein and the downstream events that regulate that protein. Such regulation includes, but is not limited to, transcription, translation, levels, activity, post-translational modification, and function of the protein in question, as well as the subsequent events that the protein regulates.

[0190] The terms "protein pathway" and "protein regulatory pathway" are used interchangeably in this context.

[0191] In this context, the term "purified" and similar terms refer to an increase in the content of a molecule or compound relative to other components typically found with that molecule or compound in nature. The term "purified" does not necessarily mean achieving complete purity of a particular molecule during the specified process. A "highly pure" compound in this context refers to a compound with a purity greater than 90%.

[0192] "Recombinant polynucleotide" refers to a polynucleotide having sequences that are not naturally related to each other. The amplified or assembled recombinant polynucleotide can be incorporated into a suitable vector, and the vector can be used to transform a suitable host cell.

[0193] The recombinant polynucleotide can also perform a non-coding function (e.g., promoter, replication origin, ribosome binding site, etc.).

[0194] A host cell that contains a recombinant polynucleotide is referred to as a "recombinant host cell." A gene expressed in a recombinant host cell, wherein said gene contains a recombinant polynucleotide, produces a "recombinant polypeptide."

[0195] A "recombinant polypeptide" is a polypeptide that is produced by expressing a recombinant polynucleotide.

[0196] The term "regulate" refers to the stimulation or suppression of the function or activity in question.

[0197] In this context, the term "regulatory elements" is used interchangeably with "regulatory sequences" and refers to promoters, enhancers, and other expression regulating elements, or any combination of such elements.

[0198] A “reversibly implantable” device is a device that can be implanted (e.g. surgically or by insertion into an animal’s natural orifice) into the body of an animal and then removed without significant harm to the animal.

[0199] "Sample" in this context, in some embodiments, refers to a biological sample obtained from a subject, including, but not limited to, normal tissue samples, diseased tissue samples, biopsy samples, blood, saliva, feces, semen, tears, and urine. A sample may also be any other source of material obtained from a subject that contains cells, tissues, or fluid of interest. A sample may also be obtained from a cell or tissue culture.

[0200] “Significant detectable amount” is the amount of contamination that is visible in the submitted data and that would need to be addressed / explained during forensic evidence analysis.

[0201] The term "signal sequence" refers to a polynucleotide sequence that encodes a peptide that determines the polypeptide's pathway within a cell, i.e., it directs the cellular processing of the polypeptide within the cell, including, but not limited to, the final release of the polypeptide from the cell. A signal sequence is a sequence of amino acids, typically, but not exclusively, found at the amino terminus of a polypeptide, that directs polypeptide synthesis to the endoplasmic reticulum. In some cases, the signal peptide is proteolytically removed from the polypeptide and is therefore absent from the mature protein.

[0202] "Small interfering RNA (siRNA)" means, among other things, isolated dsRNA molecules consisting of a sense and antisense strand. In some embodiments, they are more than 10 nucleotides in length. siRNA also refers to an individual transcript that contains both the sense and the complementary antisense sequence of a target gene, such as a hairpin. siRNA further includes any form of dsRNA (products of proteolytic cleavage of larger dsRNA, in particular, purified RNA, substantially pure RNA, synthetic RNA, recombinantly produced RNA), as well as altered RNA that differs from natural RNA by the addition, deletion, substitution, and / or change of one or more nucleotides.

[0203] In this context, the term “secondary antibody” refers to an antibody that binds to the constant region of another antibody (primary antibody).

[0204] In this context, the term "single-chain variable fragment (scFv)" refers to a single-chain fragment of an antibody consisting of heavy and light chains linked by a peptide linker. In some cases, scFv are expressed on the surface of an engineered cell to select specific scFv that bind to the antigen in question.

[0205] The terms "solid support," "surface," and "substrate" are used interchangeably and refer to a structural element of any size, wherein said structural element or substrate has a surface suitable for immobilizing a molecular structure or modifying said structure, and said substrate is made of a material such as, but not limited to, metal, metal films, glass, fused silica, synthetic polymers, and membranes.

[0206] The term "specifically binds" in this context means a molecule that recognizes and binds to a specified molecule but does not substantially recognize or bind to other molecules in a sample, or means binding between two or more molecules, such as as part of a cellular regulatory process, in which said molecules do not substantially recognize or bind to other molecules in a sample.

[0207] The term "standard" in this context refers to something used for comparison. For example, it may be a known standard agent or compound that is administered and used to compare results when an experimental compound is administered, or it may be a standard parameter or function that is measured to obtain a reference value when measuring the effect of an agent or compound on the specified parameter or function. "Standard" may also refer to an "internal standard," such as an agent or compound that is added in a known amount to a sample and is suitable for determining parameters such as purification or recovery during sample processing or during its purification or extraction, prior to measuring the marker of interest.Internal standards are often, but not always, limited to a purified marker of interest that has a label, such as a radioactive isotope, allowing it to be distinguished from the endogenous substance in the sample.

[0208] The term "stimulate" as used herein means initiating or increasing the level of an activity or function to a higher value than a reference value. The stimulation may be achieved by direct or indirect mechanisms. In some embodiments, the activity or function is stimulated by at least 10% than a reference value, in some embodiments by at least 25%, and in some embodiments by at least 50%. The term "stimulant" as used herein refers to any composition, compound, or agent whose use results in stimulation of the process or function of interest.

[0209] The "subject" of diagnosis or treatment is an animal, including humans. It also includes pets and farm animals.

[0210] In this context, "subject in need thereof" is a patient, animal, mammal or human who will benefit from the method or compositions of the described subject matter of the invention.

[0211] The term "substantially pure" describes a compound, molecule, or the like, separated from the components that naturally accompany it. Typically, a compound is substantially pure if at least 10%, more preferably in some embodiments at least 20%, more preferably in some embodiments at least 50%, more preferably in some embodiments at least 60%, more preferably in some embodiments at least 75%, more preferably in some embodiments at least 90%, and most preferably in some embodiments at least 99% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the compound in question.Purity can be measured by any suitable method, such as, but not limited to, column chromatography, gel electrophoresis, or HPLC analysis for polypeptides. A compound, such as a protein, is also considered substantially purified if it is substantially free of naturally occurring components or if it is separated from naturally occurring impurities.

[0212] A "surface active agent" or "surface active substance" is a substance that has the ability to reduce the surface tension of materials and allow penetration into and through the materials.

[0213] The term "symptom" in this context refers to any abnormality or deviation in structure, function, or sensation experienced by a patient, indicating disease. In contrast, a "sign" is objective evidence of disease. For example, a nosebleed is a sign. It is obvious to the patient, physician, nurse, and other observers.

[0214] "Therapeutic" treatment is a treatment that is carried out on a subject showing signs of pathology with the aim of alleviating or eliminating said signs.

[0215] A “therapeutically effective amount” of a compound is that amount of the compound that is sufficient to provide a beneficial effect to a subject to whom the compound is administered.

[0216] “Tissue” means (1) a group of similar cells that perform a specific function together; (2) a part of an organism consisting of a collection of cells that have the same structure and function; or (3) a group of cells that are similarly characterized in their structure and function, such as muscle or nerve tissue.

[0217] The term "topical administration" in this context refers to application to a surface, such as the skin. This term is used interchangeably with "application to the skin" in the case of the skin. "Topical administration" refers to "direct administration."

[0218] "Transdermal delivery" means delivery by passage of a drug through the skin or muscle tissue and into the bloodstream. Transdermal also refers to the skin as a portal for administration of drugs or compounds through topical application of the drug or compound. "Transdermal" is used interchangeably with "transdermal."

[0219] The term "transfection" is used interchangeably with "gene transfer," "transformation," and "transduction" and refers to the intracellular introduction of a polynucleotide. "Transfection efficiency" refers to the relative amount of transgene taken up by transfected cells. In practice, transfection efficiency is assessed by the amount of reporter gene product expressed after transfection.

[0220] As used herein, the term "transgene" means an exogenous nucleic acid sequence comprising a nucleic acid that encodes a promoter / regulatory sequence operably linked to a nucleic acid that encodes an amino acid sequence, wherein the exogenous nucleic acid is encoded by a transgenic mammal.

[0221] As used herein, the term "treatment" may include the prevention of a specific injury, disease, disorder, or pathological condition, or the alleviation of symptoms associated with a specific injury, disease, disorder, or pathological condition, and / or the prevention or elimination of said symptoms. "Prophylactic" treatment is treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs of a disease, with the aim of reducing the risk of developing a pathology associated with the disease. "Treatment" is used herein interchangeably with the term "therapy."

[0222] A "vector" is a composition of matter that contains an isolated nucleic acid and that can be used to deliver the isolated nucleic acid into the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides linked to ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or virus. This term should also be understood as including compounds other than a plasmid and virus that facilitate the transfer or delivery of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, recombinant viral vectors, and the like.Examples of non-viral vectors include, but are not limited to, liposomes, polyamine DNA derivatives, etc.

[0223] "Expression vector" refers to a vector containing a recombinant polynucleotide that contains expression control sequences operably linked to the nucleotide sequence to be expressed. The expression vector contains a sufficient number of cis-acting elements for expression; other expression elements may be provided by the host cell or in an in vitro expression system. Expression vectors include all expression vectors known in the art, such as cosmids, plasmids (e.g., non-enveloped or liposome-enclosed), and viruses containing the recombinant polynucleotide.

[0224] The term "peptide" includes a sequence of 3 or more amino acids, wherein said amino acids are natural or synthetic (not naturally occurring) amino acids. Peptidomimetics include peptides having one or more of the following modifications:

[0225] 1. Peptides in which one or more peptidyl bonds --C(O)NR-- are replaced by a non-peptidyl bond such as a --CH2-carbamate bond (--CH2OC(O)NR--), a phosphonate bond, a --CH2-sulfonamide bond (--CH2-S(O)2NR--), a urea bond (-NHC(O)NH--), a --CH2--(secondary amine) bond, or an alkylated peptidyl bond (--C(O)NR--), wherein R is C1-C4 alkyl;

[0226] 2. peptides in which the N-terminus is derivatized to a -NRR1 group, to a --NRC(O)R group, to a --NRC(O)OR group, to a --NRS(O)2R group, to a --NHC(O)NHR group, wherein R and R1 are hydrogen or C1-C4 alkyl, provided that R and R are not simultaneously hydrogen;

[0227] 3. peptides in which the C-terminus is derivatized to --C(O)R2, where R2 is selected from the group consisting of C1-C4 alkoxy, and --NR3R4, where R3 and R4 are independently selected from the group consisting of hydrogen and C1-C4 alkyl.

[0228] Synthetic or unnatural amino acids refer to amino acids that are not naturally occurring in vivo, but which can nevertheless be incorporated into the peptide structures described herein. The resulting "synthetic peptide" contains amino acids other than the 20 naturally occurring, genetically encoded amino acids at one, two, or more positions of the peptide. For example, naphthylalanine can be replaced with tryptophan to facilitate synthesis. Other synthetic amino acids that can be substituted in peptides include L-hydroxypropyl, L-3,4-dihydroxyphenylalanyl, alpha-amino acids such as L-alpha-hydroxylysyl and D-alpha-methylalanyl, L-alpha-methylalanyl, beta-amino acids, and isoquinolyl. D-amino acids and unnatural synthetic amino acids can also be incorporated into peptides. Other derivatives involve replacing the natural side chains of the 20 genetically encoded amino acids (or any L or D amino acid) with other side chains.

[0229] The term "fusion polypeptide" or "fusion protein" refers to a chimeric protein comprising a reference protein (e.g., a protein or peptide according to the described subject matter) linked at the N- and / or C-terminus to one or more heterologous sequences (e.g., a polypeptide that is not lacritin). Polypeptide molecules are said to have an "amino-terminus" (N-terminus) and a "carboxy-terminus" (C-terminus) because the peptide bonds occur between the backbone amino group of the first amino acid residue and the backbone carboxyl group of the second amino acid residue. The terms "N-terminal" and "C-terminal" with respect to polypeptide sequences refer to regions of the polypeptides comprising portions of the N-terminal and C-terminal regions of the polypeptide, respectively. A sequence that contains a portion of the N-terminal region of a polypeptide contains amino acids predominantly from the N-terminal half of the polypeptide chain, but is not limited to such sequences.For example, the N-terminal sequence may comprise an internal portion of the polypeptide sequence containing bases from both the N-terminal and C-terminal halves of the polypeptide. The same applies to the C-terminal regions. The N-terminal and C-terminal regions may, but do not necessarily, contain the amino acid that defines the final N-terminus and C-terminus of the polypeptide, respectively.

[0230] Fusion proteins can be produced by any recombinant methods or solid-phase chemical peptide synthesis methods. Such methods have been known in the art since the early 1960s (Merrifield, 1963; see also Stewart et al., 1984) and have recently been used in laboratory kits for the design and synthesis of peptides available commercially (Cambridge Research Biochemicals). In addition, numerous peptide synthesis systems based on the FMOC strategy are available. For example, assembly of a polypeptide or fragment can be performed on a solid support using the Applied Biosystems, Inc. 431A automated peptide synthesizer. Such equipment provides easy access to the peptides of the present invention by direct synthesis or by synthesis of a series of fragments that can be linked by other known technologies.

[0231] Modification and Production of Peptides. The production of peptides is described above and in the Examples section. It should, of course, be understood that proteins or peptides according to the described subject matter may contain amino acid residues that are modified without impairing their activity. For example, the termini can be derivatized to contain blocking groups, i.e., chemical substituents suitable for protecting and / or stabilizing the N- and C-termini from "undesirable degradation," and this term includes any type of enzymatic, chemical, or biochemical degradation of a compound at its termini that can impair the function of the compound, i.e., sequential degradation of the compound at its termini.

[0232] Blocking groups include protecting groups commonly used in peptide chemistry that do not adversely affect the in vivo activity of the peptide. For example, suitable N-terminal blocking groups can be introduced by alkylation or acylation of the N-terminus. Examples of N-terminal blocking groups include C1-C5 branched or unbranched alkyl groups, acyl groups such as formyl and acetyl groups, and their substituted forms such as the acetamidomethyl (Acm) group. Deaminated amino acid analogs are also suitable N-terminal blocking groups and can either be linked to the N-terminus of the peptide or used in place of the N-terminal residue. Suitable C-terminal blocking groups, which may or may not include the carboxyl group of the C-terminus, include esters, ketones, or amides.Ester- or ketone-forming alkyl groups, particularly lower alkyl groups such as methyl, ethyl, and propyl, as well as amide-forming amino groups such as primary amines (-NH2) and mono- and dialkylamino groups such as methylamino, ethylamino, dimethylamino, diethylamino, methylethylamino, and the like, are examples of C-terminal capping groups. Decarboxylated amino acid analogs such as agmatine are also suitable C-terminal capping groups and can either be linked to the C-terminal residue of the peptide or used in place of them. Furthermore, it should be understood that all free amino groups and carboxyl groups at the ends of the peptide can be removed to form its deaminated and decarboxylated forms without impairing the activity of the peptide.

[0233] Acid addition salts of the described subject matter are also provided as functional equivalents. Thus, a peptide according to the described subject matter, treated with an inorganic acid such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, etc., or an organic acid such as acetic, propionic, glycolic, pyruvic, oxalic, malic, malonic, succinic, maleic, fumaric, tartaric, citric, benzoic, cinnamic, mandelic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicylic, etc., to form a water-soluble salt of the peptide, is suitable for use according to the described subject matter.

[0234] The described subject matter also provides protein analogs. Analogs may differ from naturally occurring proteins or polypeptides by conservative differences in amino acid sequence or by modifications that do not affect the sequence, or both. For example, conservative amino acid substitutions may be made that, although they alter the primary sequence of the protein or peptide, typically do not alter its function. In this regard, peptide function is typically unaffected by 10 or more conservative amino acid substitutions.

[0235] Modifications (which generally do not alter the primary sequence) include in vivo or in vitro chemical derivatization of polypeptides, such as acetylation or carboxylation. Also included are glycosylation modifications, such as modifications achieved by altering the glycosylation pattern of a polypeptide during its synthesis and processing or at further processing steps; for example, by treating the polypeptide with enzymes that affect glycosylation, such as mammalian glycosylation or deglycosylation enzymes. Also included are sequences that contain phosphorylated amino acid residues, such as phosphotyrosine, phosphoserine, or phosphothreonine.

[0236] Also included are polypeptides modified using conventional molecular biology techniques to improve their resistance to proteolytic degradation or to optimize solubility properties, or to enhance their suitability as a therapeutic agent. Analogs of such polypeptides include those containing residues other than natural L-amino acids, such as D-amino acids, or non-naturally occurring or non-standard synthetic amino acids. The peptides according to the described subject matter are not limited to the products of any particular illustrative process specified herein.

[0237] The described subject matter of the invention includes the use of beta-alanine (also referred to as β-alanine, β-Ala, bA and βpA, having the structure:

[0238]

[0239] This document presents sequences that use the designation "βA", but in the sequence listing appended hereto, "Xaa" is used instead of "βA", and the reference in the text of the sequence listing indicates that Xaa is beta-alanine.

[0240] It should be understood, of course, that polypeptides, their derivatives, or fragments may contain amino acid residues that are modified without impairing their activity. For example, the termini may be derivatized to contain capping groups, i.e., chemical substituents suitable for protecting and / or stabilizing the N- and C-termini from "undesirable degradation," and this term includes any type of enzymatic, chemical, or biochemical degradation of the compound at its termini that may impair the function of the compound, i.e., sequential degradation of the compound at its termini.

[0241] Other modifications that do not adversely affect activity may also be introduced, including, but not limited to, the substitution of one or more amino acids in the natural L-isomeric form for amino acids in the D-isomeric form. Thus, the peptide may contain one or more D-amino acid residues or may contain amino acids all of which are in the D-form. Retro-inverso forms of the peptides according to the described subject matter are also provided, for example, inverted peptides in which all amino acids are replaced by HaD-amino acid forms.

[0242] The substantially pure protein obtained as described herein can be purified by known protein purification methods that utilize immunological, enzymatic, or other assays to monitor purification at each step of the process. Protein purification methods are well known in the art and are described, for example, in Deutscher et al., 1990.

[0243] As noted above, modifications or optimizations of peptide ligands according to the described subject matter are within the scope of the present application. Modified or optimized peptides are included within the definition of a peptide-binding ligand. In particular, an identified peptide sequence may be modified to optimize its potency, pharmacokinetic properties, stability, and / or other biological, physical, and chemical properties.

[0244] Amino Acid Substitutions. In some embodiments, the described methods and compositions may include producing polypeptides with one or more substituted amino acid residues.

[0245] In various embodiments, the structural, physical and / or therapeutic characteristics of peptide sequences can be optimized by substituting one or more amino acid residues.

[0246] Other modifications that do not adversely affect activity may also be introduced, including, but not limited to, the substitution of one or more amino acids in the natural L-isomeric form with amino acids in the D-isomeric form. Thus, the peptide may contain one or more D-amino acid residues or may contain amino acids all of which are in the D-form. Retro-inverso forms of the peptides according to the described subject matter are also provided, for example, inverted peptides in which all amino acids are replaced with D-amino acid forms.

[0247] Those skilled in the art understand that, in general, amino acid substitutions typically involve replacing an amino acid with another amino acid with relatively similar properties (i.e., conservative amino acid substitutions). The properties of various amino acids and the impact of amino acid substitutions on protein structure and function have been the subject of extensive research and understanding in the art.

[0248] For example, the following isosteric and / or conservative amino acid substitutions can be made in the original polypeptide sequence with the expectation that the resulting polypeptides will have the same or improved property profile as described above:

[0249] Substitution of alkyl-substituted hydrophobic amino acids: including alanine, leucine, isoleucine, valine, norleucine, S-2-aminobutyric acid, S-cyclohexylalanine or other simple alpha-amino acids substituted with an aliphatic side chain containing C1-10 carbon atoms, including substitutions with branched, cyclic and unbranched alkyl, alkenyl or alkynyl.

[0250] Replacement of hydrophobic amino acids substituted with an aromatic group: including phenylalanine, tryptophan, tyrosine, biphenylalanine, 1-naphthylalanine, 2-naphthylalanine, 2-benzothienylalanine, 3-benzothienylalanine, histidine, amino, alkylamino, dialkylamino, aza, halogenated (fluorine, chlorine, bromine or iodine) or alkoxy-substituted forms of the above aromatic amino acids, illustrative examples of which are: 2-, 3- or 4-aminophenylalanine, 2-, 3- or 4-chlorophenylalanine, 2-, 3- or 4-methylphenylalanine, 2-, 3- or 4-methoxyphenylalanine, 5-amino-, 5-chloro-, 5-methyl- or 5-methoxytryptophan, 2'-, 3'- or 4'-amino-, 2'-, 3'- or 4'-chloro-, 2,3- or 4-biphenylalanine, 2',3'- or 4'-methyl-2,3- or 4-biphenylalanine and 2- or 3-pyridylalanine.

[0251] Substitution of amino acids containing basic functional groups: including arginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid, homoarginine, alkyl-, alkenyl or aryl-substituted (from C1-C10 branched, linear or cyclic) derivatives of the above-mentioned amino acids, regardless of whether the substituent is located at heteroatoms (such as the alpha nitrogen atom or the distal nitrogen atom or atoms) or at the alpha carbon atom, for example, at the pro-R-position. Compounds serving as illustrative examples include: N-upsilon-isopropyl lysine, 3-(4-tetrahydropyridyl)glycine, 3-(4-tetrahydropyridyl)alanine, N,N-gamma,gamma'-diethylhomoarginine. Also included are compounds such as alpha-methylarginine, alpha-methyl-2,3-diaminopropionic acid, alpha-methylhistidine, alpha-methylornithine, in which the alkyl group occupies the pro-R-position of the alpha carbon atom.Also included are amides formed from alkyl, aromatic, heteroaromatic (in which the heteroaromatic group contains one or more nitrogen, oxygen, or sulfur atoms, alone or in combination) carboxylic acids, or any of the numerous known activated derivatives (such as acid chlorides, active esters, active azolides, and related derivatives), as well as lysine, ornithine, or 2,3-diaminopropionic acid.

[0252] Replacement of acidic amino acids: including aspartic acid, glutamic acid, homoglutamic acid, tyrosine, alkyl-, aryl-, arylalkyl- and heteroaryl sulfonamides of 2,4-diaminopropionic acid, ornithine or lysine and tetrazole-substitution where other alkyl amino acids.

[0253] Substitution of amide residues in the side chain: including asparagine, glutamine and alkyl or aromatic substituted derivatives of asparagine or glutamine.

[0254] Substitution of hydroxyl-containing amino acids: including serine, threonine, homoserine, 2,3-diaminopropionic acid, and alkyl- or aromatic-substituted derivatives of serine or threonine. It should also be understood that amino acids in each of the categories listed above may be replaced by another from the same group.

[0255] For example, the hydropathic index of amino acids can be considered (Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relative hydropathic character of amino acids contributes to the secondary structure of the resulting protein, which in turn determines the interactions of the protein with other molecules. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics (Kyte & Doolittle, 1982): isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).When making conservative substitutions, it is preferable to use amino acids whose hydropathic indices are within the range of +1-2, more preferably within the range of + / -1, and even more preferably within the range of + / - 0.5.

[0256] When substituting amino acids, the hydrophilicity of the amino acid residue can also be taken into account (see U.S. Patent No. 4,554,101). Amino acid residues are assigned the following hydrophilicity values: arginine (+3.0); lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (O); threonine (-0.4); proline (-0.5+-0.1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3,4). It is preferable to replace amino acids with others of similar hydrophilicity.

[0257] Other considerations include the size of the amino acid side chain. For example, it is generally not preferable to replace an amino acid with a compact side chain, such as glycine or serine, with an amino acid with a bulky side chain, such as tryptophan or tyrosine. The effect of different amino acid residues on the secondary structure of the protein should also be considered. The effect of different amino acid residues on the propensity of protein domains to adopt alpha-helical, beta-sheet, or reverse-turn secondary structure has been empirically determined, and this is known in the art (see, e.g., Chou & Fasman, 1974, Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47:251-276; 1979, Biophys. J., 26:367-384).

[0258] Based on the above considerations and careful empirical study, tables of conservative amino acid substitutions have been constructed and are known in the art. For example: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R) gin, asn, lys; Asn (N) his, asp, lys, arg, gin; Asp (D) asn, glu; Cys (C) ala, ser; Gin (Q) glu, asn; Glu (E) gin, asp; Gly (G) ala; His (H) asn, gin, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met, ala, phe, ile; Lys (K) gin, asn, arg; Met (M) phe, ile, leu; Phe (F) leu, val, ile, ala, tyr; Pro(P)ala; Ser(S),thr; Thr(T)ser; Trp (W) phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

[0259] Other considerations for amino acid substitution include whether the residue is located in the interior of the protein or is solvent-exposed. For internal residues, conservative substitutions include: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala and Gly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr; Tyr and Trp. (See, for example, the Rockefeller University PROWL website.) For solvent-exposed residues, conservative substitutions include: Asp and Asn; Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala and Gly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu; Leu and Ile; Ile and Val; Phe and Tyr. Various matrices have been created to facilitate the selection of amino acid substitutions, such as the PAM250 substitution matrix, Dayhoff matrix, Grantham matrix, McLachlan matrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix, Jones matrix, Rao matrix, Levin matrix, and Risler matrix (see above).

[0260] When determining amino acid substitutions, the existence of intermolecular or intramolecular bonds can also be taken into account, such as the formation of ionic bonds (salt bridges) between positively charged residues (e.g. His, Arg, Lys) and negatively charged residues (Asp, Glu) or disulfide bonds between adjacent cysteine ​​residues.

[0261] Methods for replacing any amino acid with any other amino acid in an encoded peptide sequence are well known and are a matter of routine experimentation for those skilled in the art, for example, by using site-directed mutagenesis technology or by synthesizing and assembling oligonucleotides encoding the amino acid replacement and splicing into an expression vector construct.

[0262] The terminology used herein is for the purpose of describing particular variations or embodiments only and is not intended to limit the scope of the disclosed subject matter. The entire contents of all publications referenced herein are incorporated by reference.

[0263] P. Illustrative compositions of the described subject matter of the invention

[0264] According to the present document, compositions having lacritin activity for use in the disclosed methods are described. In some embodiments, a composition is provided comprising a lacritin polypeptide, a bioactive fragment of lacritin, a non-native lacritin peptide, or a lacritin peptidomimetic derivative. In some embodiments, the composition comprises, consists essentially of, or consists of a sequence selected from the group consisting of SEQ ID NOs: 1-60, or a sequence that differs from SEQ ID NOs: 1-60 by 1, 2, 3, 4, or 5 amino acid modifications. In some embodiments, the amino acid modifications are amino acid substitutions, and in some embodiments, the 1, 2, 3, 4, or 5 amino acid substitutions are conservative amino acid substitutions.In some embodiments, the composition comprises, consists essentially of, or consists of a sequence selected from the group consisting of SEQ ID NO: 62, or a sequence that differs from SEQ ID NO: 62 by 1, 2, 3, 4, or 5 amino acid modifications.

[0265] In some embodiments, a composition is provided comprising, consisting essentially of, or consisting of a bioactive fragment of lacritin, wherein the bioactive fragment comprises, consists essentially of, or consists of a sequence selected from the group consisting of SEQ ID NO: 1-60, or a derivative that differs therefrom by one or more amino acid substitutions. In some embodiments, the composition provides the N-94 peptide, which is a synthetic fragment of the lacritin protein that has pro-secretory, pro-survival, and mitogenic properties, is currently in Phase II clinical trials for autoimmune dry eye disease, and is detected in plasma as C-terminal peptides comprising the N-94 sequence. As described herein, islet cells are responsive to lacritin / N-94 and prominently express known elements of the N-94 receptor complex and signaling pathways.Lacritin-based peptides are described in U.S. Patent No. 10393755, the entire contents of which are incorporated herein by reference.

[0266] IIA. Dosage forms

[0267] Thus, in some embodiments, the described subject matter relates to compositions, in some embodiments, to pharmaceutical compositions for use in the described methods. In some embodiments, the pharmaceutical composition comprises an effective amount of the composition described herein.In some embodiments, the pharmaceutical composition comprises a peptide and / or a pharmaceutically acceptable salt thereof and / or a biologically active fragment, analog or derivative thereof, wherein said peptide, pharmaceutically acceptable salt thereof and / or a biologically active fragment, analog or derivative thereof comprises, consists essentially of, or consists of an amino acid sequence comprising, consisting essentially of, or consisting of KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 1), KRFYKRGAELG (SEQ ID NO: 25), KRFYKRGAELGKNRR (SEQ ID NO: 29), KRFYKRGAELGKNRRKNWH (SEQ ID NO: 33), KRFYKRGAELGKNRRKNWHAQLFVL (SEQ ID NO: 21), KKLFGGRNDVLRQMMDRLGPKFNLF (SEQ ID NO: 13), or any combination thereof. In some embodiments, a derivative having SEQ ID NOs: 1, 25, 29, 33, and 21 comprises, consists essentially of, or consists of an amino acid sequence comprising, consists essentially of, or consists of any of SEQ ID NOs: 2-4, 26-28, 30-32, 34-36, and 22-24, respectively.More specifically, the peptides according to the described subject matter of the invention are based on the peptide N-94 having SEQ ID NO: 1 and its derivatives presented in Table 2.

[0268]

[0269]

[0270]

[0271] In some embodiments, the peptide and / or its pharmaceutically acceptable salt, and / or its biologically active fragment, analog, or derivative according to the described subject matter may be presented in a composition that comprises a carrier, in particular a pharmaceutically acceptable carrier, such as, but not limited to, a carrier pharmaceutically acceptable for humans. Any suitable pharmaceutical dosage form can be used to prepare compositions for administration to a subject.

[0272] For example, suitable dosage forms may include aqueous and non-aqueous sterile injection solutions that contain antioxidants, buffers, bacteriostats, bactericidal antibiotics, and solutes that ensure the dosage form is isotonic with the physiological fluids of the intended recipient.

[0273] It should be understood that, in addition to the ingredients specifically mentioned above, the dosage forms according to the described subject matter may contain other agents conventional in the art for the type of dosage form in question. For example, sterile, apyrogenic aqueous and non-aqueous solutions may be used.

[0274] The methods and compositions according to the described subject matter of the invention can be used with additional adjuvants or biological response modifiers, including, but not limited to, cytokines or other immunomodulatory compounds.

[0275] In some embodiments, therapeutic agents can be used as an adjunct therapy when using the compositions described herein, including, but not limited to, cytotoxic agents, antiangiogenic agents, proapoptotic agents, antibiotics, hormones, hormone antagonists, chemokines, drugs, prodrugs, toxins, enzymes or other agents. Drugs useful according to the described subject matter may, for example, have a certain pharmaceutical property and can be selected from the group consisting of antimitotic, antikinase, alkylating agents, antimetabolites, antibiotics, alkaloids, antiangiogenic, proapoptotic agents and combinations thereof.

[0276] In some embodiments, the disclosed compositions and methods may further comprise administering to the subject at least one additional active agent (in some embodiments, an immunosuppressant agent). In some embodiments, the at least one additional immunosuppressant agent is selected from the group consisting of methotrexate, cyclophosphamide, cyclosporine, cyclosporine A, chloroquine, hydroxychloroquine, sulfasalazine (sulfasalazopyrine), a gold salt, D-penicillamine, leflunomide, azathioprine, anakinra, infliximab (REMICADE®), etanercept, a TNFa blocker, a non-steroidal

[0277] anti-inflammatory drug (NSAID) or any combination thereof. In some embodiments, the NSAID is selected from the group consisting of acetylsalicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, cyclooxygenase-2 (Cox-2) inhibitor, tramadol, rapamycin (sirolimus), analogs thereof, or any combination thereof.

[0278] II.B. Introduction

[0279] Suitable methods of administering the compositions of the described subject matter include, but are not limited to, intravenous administration and delivery directly to the target tissue or organ (e.g., the peritoneal cavity and / or pancreas). In some embodiments, the method of administration includes features for regional delivery or accumulation of the compositions of the described subject matter in the area in need of treatment. In some embodiments, the compositions of the described subject matter are delivered directly to the pancreas. In some embodiments, selective delivery of the compositions of the described subject matter is achieved by intravenous injection of the compositions of the described subject matter, whereby they accumulate and / or act in the pancreas, optionally in the islet cells.Other routes of administration that may be used include topical, oral, buccal, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, vaginal, ophthalmic, pulmonary, or rectal routes. Compounds or agents according to the described subject matter may be administered to a subject by one or more of these routes, as appropriate. In some embodiments, the method or methods of administering the compositions according to the described subject matter include intratracheal instillation, insufflation, nebulization, dry powder inhalation, aerosol inhalation, and combinations thereof.In some embodiments, a pharmaceutical composition useful for the methods of the described subject matter may be prepared, packaged, or sold in dosage forms suitable for oral, rectal, vaginal, parenteral, intravenous, topical, pulmonary, intranasal, buccal, ophthalmic, intrathecal, or other administration. Other contemplated dosage forms include engineered nanoparticles, liposomal formulations, secondary compacted red blood cells containing the active ingredient, and immunologically based dosage forms. In some embodiments, an oral dosage form of the described subject matter may be packaged in PEG or, in some embodiments, lyophilized into a pill form.

[0280] If the compositions according to the described subject matter are administered by injection or direct application, the injection or direct application may be in the form of a single dose or multiple doses. If the compositions according to the described subject matter are administered by infusion, the infusion may be a single maintained dose over an extended period of time or multiple infusions.

[0281] The pharmaceutical compositions described herein may be prepared in dosage forms by any method now known or hereafter developed in the art of pharmacology. Generally, such methods of preparation include the step of bringing the active ingredient into contact with a carrier or one or more other accessory ingredients and then, if necessary or desired, shaping or packaging the product into the desired unit dosage form or into a form containing multiple doses.

[0282] The pharmaceutical composition according to the described subject matter may be prepared, packaged, or sold in bulk form, as a single unit dose, or as multiple single unit doses. As used herein, a "unit dose" is a discrete quantity of the pharmaceutical composition containing a predetermined amount of the active ingredient. The quantity of active ingredient is typically equal to the dose of the active ingredient to be administered to the subject, or a convenient fraction of such a dose, such as, for example, one-half or one-third of such a dose.

[0283] The relative amounts of the active ingredient(s), pharmaceutically acceptable carrier, and any additional ingredients in the pharmaceutical composition according to the described subject matter vary depending on the identity, size, and pathological condition of the subject being treated, and further depend on the route of administration of the composition. For example, the composition may contain from 0.1% to 100% (w / w) of the active ingredient.

[0284] In addition to the active ingredient, the pharmaceutical composition according to the described subject matter may further comprise one or more additional pharmaceutically active agents. Specifically provided additional agents include antiemetics and scavengers, such as cyanide and cyanate scavengers.

[0285] Dosage forms of the pharmaceutical composition according to the described subject matter of the invention with controlled or sustained release can be obtained using conventional technologies.

[0286] As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous carriers and solvents; oily carriers and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, softeners; buffers; salts; thickeners; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" that may be included in the pharmaceutical compositions according to the described subject matter are known in the art and are described, for example, in Genaro, ed., 1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., the contents of which are incorporated herein by reference.

[0287] In some embodiments, the proposed composition further comprises one or more stabilizing agents, wherein the one or more stabilizing agents stabilize the peptide, its pharmaceutically acceptable salt, its biologically active fragment, its analog and / or its degradation derivative and / or stabilize the peptide, its pharmaceutically acceptable salt, its biologically active fragment, its analog and / or its derivative in a certain conformation to increase its chemical stability. In some embodiments, the stabilizing agent includes tyloxapol (see, for example, U.S. Patent Application Publication No. 2019 / 0381136, the entire contents of which are incorporated herein by reference).

[0288] II.C. Doses

[0289] An effective dose of a composition according to the described subject matter is administered to a subject in need thereof. An "effective amount for treatment" or "therapeutic amount" is an amount of a therapeutic composition sufficient to provide a measurable response (e.g., a biologically or clinically relevant response in the subject being treated). In some embodiments, activity that suppresses an immune response against a transplant (e.g., transplant rejection) is measured. Actual dose sizes of the active ingredients in the compositions according to the described subject matter can vary so that, upon administration, an amount of the active compound(s) is provided that is effective in achieving the desired therapeutic response for a particular subject.The selected dose depends on the activity of the therapeutic composition, the route of administration, the combination with other drugs or treatments, the severity of the pathological condition being treated, and the condition and medical history of the subject being treated. However, it is common practice in the art to begin administering doses of the compound at levels lower than those necessary to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. The efficacy of the composition may vary, and therefore the "effective therapeutic amount" may vary. However, using conventional analytical methods, those skilled in the art can readily assess the potency and efficacy of a candidate compound according to the described subject matter and tailor the therapeutic regimen accordingly.After reviewing the disclosure of the present invention set forth herein, those skilled in the art can adjust dosages for a specific subject based on the specific dosage form, route of administration of the composition, and the specific disease being treated. Additional dosage calculations can take into account the subject's height and weight, the severity and stage of symptoms, and the presence of additional adverse health factors. Such adjustments or adjustments, as well as the assessment of when and how to make such adjustments or adjustments, are familiar to those skilled in the art.

[0290] Thus, in some embodiments, the compositions according to the described subject matter are in a pharmaceutically acceptable carrier, which in some embodiments may be pharmaceutically acceptable for use in humans.

[0291] Typically, the doses of the compounds according to the described subject matter that can be administered to an animal, preferably a human, are from 1 μg to about 100 g per kilogram of animal body weight. However, the exact dose to be administered will vary depending on any number of factors, including, but not limited to, the type of animal and the type of disease condition being treated, the age of the animal, and the route of administration. In some embodiments, the dose of the compound will be from about 1 mg to about 10 g per kilogram of animal body weight. In some embodiments, the dose will be from about 10 mg to about 1 g per kilogram of animal body weight. In some embodiments, the peptide, its pharmaceutically acceptable salt, and / or its biologically active fragment, analog, or derivative is present in the composition at a concentration of 1.0 nM to 100 μM.

[0292] The proposed compound can be administered to an animal at a frequency of several times per day, or it can be administered less frequently, for example, once per day, once per week, once every two weeks, once per month, or even less frequently, for example, once every few months or even once per year, or less frequently. Those skilled in the art can readily determine the frequency of dosing, and it depends on any number of factors, such as, but not limited to, the type of cancer diagnosed, the type and severity of the pathological condition or disease being treated, the type and age of the animal, etc.

[0293] Suitable preparations include injectables in the form of liquid solutions or suspensions, but solid forms suitable for dissolution or suspension in liquid prior to injection can also be prepared. The preparation can also be in the form of an emulsion or polypeptides encapsulated in liposomes. The active ingredients are often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, aqueous saline, dextrose, glycerol, ethanol, or combinations thereof. Furthermore, if necessary, the vaccine preparation may also contain small amounts of excipients such as wetting or emulsifying agents, pH buffering agents, and / or adjuvants.

[0294] Thus, the described subject matter relates to compositions for use, in some embodiments, to restore pancreatic islet viability and / or cellular proliferation in vitro, ex vivo, and / or in vivo; in some embodiments, for use to restore pancreatic islet viability and / or proliferation in vito, ex vivo, and / or in vivo; in some embodiments, for use to restore glucose-stimulated insulin secretion; in some embodiments, for use to restore the viability and / or cellular proliferation of transplanted islets; in some embodiments, for use to prevent and / or suppress rejection of transplanted islets; in some embodiments, for use in islet transplantation;in some embodiments, for use in treating a symptom of a pathological condition, disorder, or disease associated with an impaired insulin response to glucose in a subject.

[0295] Furthermore, the compositions according to the described subject matter of the invention can also be used in some embodiments for the production of medicinal products.By way of example, and not limitation, the compositions of the described subject matter can be used to prepare a medicament, in some embodiments, to restore pancreatic islet viability and / or cellular proliferation in vitro, ex vivo, and / or in vivo; in some embodiments, to restore glucose-stimulated insulin secretion; in some embodiments, to restore viability and / or cellular proliferation of transplanted pancreatic islets; in some embodiments, to prevent and / or suppress rejection of transplanted islets; in some embodiments, for islet transplantation; and / or in some embodiments, to treat a symptom of a pathological condition, disorder, or disease associated with an impaired insulin response to glucose in a subject.

[0296] In any of the above-mentioned embodiments, the compositions according to the described subject matter of the invention comprise, consist essentially of, or consist of a peptide and / or a pharmaceutically acceptable salt thereof, and / or a biologically active fragment, analog, or derivative thereof, wherein said peptide, pharmaceutically acceptable salt thereof, and / or a biologically active fragment, analog, or derivative thereof comprises, consists essentially of, or consists of an amino acid sequence comprising, consisting essentially of, or consisting of any of SEQ ID NOs: 1-60, or any combination thereof. In some embodiments, the composition is formulated for administration to a subject, optionally a human, by intravenous, intramuscular, oral, intranasal, and / or transdermal delivery routes.

[0297] In some embodiments, the composition is formulated in the form of nanoparticles, nanovesicles, microparticles, microvesicles, liposomes, packaged in PEG, lyophilized in the form of pills, or any combination thereof.

[0298] In some embodiments, the peptide, its pharmaceutically acceptable salt, and / or its biologically active fragment, analog, or derivative comprises at least one modification selected from the group consisting of N- and / or C-terminal amidation, N- and / or C-terminal acylation, N- and / or C-terminal acetylation, attachment of an N- and / or C-terminal cysteine, PEGylation, and combinations thereof. In some embodiments, PEGylation comprises attachment of a PEG group to the N-terminal cysteine, the C-terminal cysteine, or both.

[0299] Methods for PEGylation of peptides and proteins are known in the art and include PEGylation at a reactive cysteine ​​(see, for example, U.S. Patent Nos. 8,329,866 and 9,050,371; the entire contents of each of which are incorporated herein by reference). With respect to the PEGylated compounds of the described subject matter, the PEG covalently attached to the peptide, its pharmaceutically acceptable salt, and / or its biologically active fragment, analog, or derivative has a molecular weight of, in some embodiments, from about 1 kDa to about 40 kDa, in some embodiments, from about 10 kDa to about 40 kDa, and in some embodiments, about 40 kDa.

[0300] In some embodiments, the N-terminal amidation, C-terminal amidation, or both comprise a substituted amide modification. In some embodiments, the N-terminal acylation, C-terminal acylation, or both comprise a substituted acyl group.

[0301] In some embodiments, the proposed composition does not contain any type of enzymatic, chemical or biochemical molecules capable of degrading the said peptide at its ends, which is a sequential degradation of the peptide, its pharmaceutically acceptable salt and / or its biologically active fragment, analog or derivative at its end in the absence of N- and / or C-terminal amidation, N- and / or C-terminal acylation, N- and / or C-terminal acetylation or a combination thereof.

[0302] III. Illustrative methods and application of the described subject matter of the invention

[0303] In some embodiments, the described subject matter also relates to the use of the described compositions in various treatment methods and / or therapeutic methods.

[0304] In some embodiments, the described subject matter relates to methods for restoring glucose-stimulated insulin secretion by contacting pancreatic islet cells in vitro, ex vivo, and / or in vivo with an effective amount of a composition comprising a peptide and / or a pharmaceutically acceptable salt thereof, and / or a biologically active fragment, analog, or derivative thereof, wherein said peptide, pharmaceutically acceptable salt thereof, and / or a biologically active fragment, analog, or derivative thereof comprises, consists essentially of, or consists of an amino acid sequence comprising, consisting essentially of, or consisting of any of SEQ ID NOs: 1-60, or any combination thereof.In this context, the expression "restoration of glucose-stimulated insulin secretion" refers to the activation of pancreatic islet cells that have ceased normal insulin secretion in response to glucose levels to resume their response to ambient glucose levels by secreting insulin. Thus, in some embodiments, the term "restoration" and its grammatical variants refer to the activation of the response of insensitive pancreatic islet cells to ambient glucose levels, in response to which they do not or have not previously secreted insulin, by contacting them with a peptide and / or a pharmaceutically acceptable salt thereof, and / or a biologically active fragment, analog, or derivative thereof according to the described subject matter.In some embodiments, the contacting is performed in vitro, in some embodiments, the contacting is performed ex vivo, in some embodiments, the contacting is performed in vivo, and in some embodiments, the contacting is performed repeatedly in vitro, ex vivo, and / or in vivo.

[0305] In some embodiments, the described subject matter also relates to methods for restoring the viability and / or cellular proliferation of transplanted and / or endogenous pancreatic islets by contacting the pancreatic islets before, simultaneously with, and / or after transplantation or without transplantation in diabetics with an effective amount of a composition comprising a peptide and / or a pharmaceutically acceptable salt thereof, and / or a biologically active fragment, analog, or derivative thereof, wherein said peptide, pharmaceutically acceptable salt thereof, and / or a biologically active fragment, analog, or derivative thereof comprises, consists essentially of, or consists of an amino acid sequence comprising, consisting essentially of, or consisting of any of SEQ ID NOs: 1-60, or any combination thereof, wherein the viability and / or proliferation of the transplanted pancreatic islets or endogenous islets is restored relative to the level of islet cells,not brought into contact with an effective amount of the said composition. In this context, the expression "restoring the viability and / or cellular proliferation of transplanted and / or endogenous pancreatic islets" refers to any manipulation that is carried out in vitro, ex vivo or in vivo, in which, as a result of bringing into contact with a composition containing a peptide and / or its pharmaceutically acceptable salt and / or its biologically active fragment, analogue or derivative according to the described subject matter of the invention, the viability and / or proliferation of pancreatic islets is increased relative to similar parameters of the same pancreatic islet cells that were not brought into contact with the peptide and / or its pharmaceutically acceptable salt and / or its biologically active fragment, analogue or derivative according to the described subject matter of the invention.

[0306] In some embodiments, the disclosed subject matter relates to methods of preventing and / or suppressing the rejection of transplanted pancreatic islets, or preventing further degeneration of endogenous pancreatic islets by contacting isolated pancreatic islets before, simultaneously with, and / or after transplantation, and / or contacting endogenous diabetic islets with an effective amount of a composition comprising a peptide and / or a pharmaceutically acceptable salt thereof, and / or a biologically active fragment, analog, or derivative thereof, wherein said peptide, pharmaceutically acceptable salt thereof, and / or a biologically active fragment, analog, or derivative thereof comprises, consists essentially of, or consists of an amino acid sequence comprising, consisting essentially of, or consisting of any of SEQ ID NOs: 1-60, or any combination thereof,wherein the rejection of transplanted islet cells is prevented and / or suppressed compared to the same parameter of islet cells not brought into contact with an effective amount of said composition.

[0307] In some embodiments, the disclosed subject matter relates to methods of transplanting pancreatic islets by transplanting pancreatic islets into a transplant recipient, wherein the islets have been contacted before, simultaneously with, and / or after the transplantation step with an effective amount of a composition comprising a peptide and / or a pharmaceutically acceptable salt thereof, and / or a biologically active fragment, analog, or derivative thereof, wherein said peptide, pharmaceutically acceptable salt thereof, and / or a biologically active fragment, analog, or derivative thereof comprises, consists essentially of, or consists of an amino acid sequence comprising, consisting essentially of, or consisting of any of SEQ ID NOs: 1-60, or any combination thereof, wherein the rejection of the transplanted pancreatic islet cells is prevented and / or suppressed compared to a similar parameter of pancreatic islet cells,not brought into contact with an effective amount of the said composition.,

[0308] In some embodiments, the described subject matter relates to methods for restoring the healthy status of the nerves innervating the pancreatic islets. As used herein, the phrase "restoring the healthy status of the nerves innervating the pancreatic islets" refers to enhancing any biological activity of the nerves innervating the pancreatic islets, which in some embodiments may be a biological activity that deviates from the normal, compared to the biological activity of the nerves innervating the pancreatic islets in a healthy subject. Thus, in some embodiments, "restoring the healthy status of the nerves innervating the pancreatic islets" refers to enhancing the biological activity of the nerves innervating the pancreatic islets to a level that is approximately equal to the level in a healthy subject.In some embodiments, the disclosed methods comprise contacting pancreatic islet nerves in vitro, ex vivo and / or in vivo with an effective amount of a composition comprising a peptide and / or a pharmaceutically acceptable salt thereof and / or a biologically active fragment, analog or derivative thereof, wherein said peptide, pharmaceutically acceptable salt thereof and / or a biologically active fragment, analog or derivative thereof comprises, consists essentially of, or consists of an amino acid sequence comprising, consisting essentially of, or consisting of any of SEQ ID NOs: 1-60 or any combination thereof.

[0309] In some embodiments, the disclosed subject matter relates to methods of treating a symptom of a condition, disorder, or disease associated with an impaired insulin response to glucose in a subject, wherein said condition, disorder, or disease is not necessarily type 1 or type 2 diabetes, by administering to the subject an effective amount of a composition comprising a peptide and / or a pharmaceutically acceptable salt thereof, and / or a biologically active fragment, analog, or derivative thereof, wherein said peptide, pharmaceutically acceptable salt thereof, and / or a biologically active fragment, analog, or derivative thereof comprises, consists essentially of, or consists of an amino acid sequence comprising, consisting essentially of, or consisting of any of SEQ ID NOs: 1-60, or any combination thereof,wherein the rejection of transplanted pancreatic islets is prevented and / or suppressed compared to that of islets not contacted with an effective amount of said composition. In this context, the expression "a pathological condition, disorder, or disease associated with an impaired insulin response to glucose" refers to any pathological condition, disorder, or disease, at least one symptom and / or consequence of which is caused by an impaired response of a cell, tissue, or organ to a local glucose concentration. In some embodiments, the cell is a pancreatic islet cell, and the impaired response includes an inability to secrete insulin in response to glucose. A specific example of a pathological condition, disorder, or disease associated with an impaired insulin response to glucose is diabetes, including type 1 and type 2 diabetes.

[0310] Thus, in some embodiments, the subject to which the described methods are applicable is a subject with a pathological condition, disorder, or disease associated with an impaired insulin response to glucose, which in some embodiments is a subject with type 1 or type 2 diabetes. In some embodiments, such a subject can be treated with a composition according to the described subject matter in combination with one or more additional antidiabetic therapies. As used herein, the term "antidiabetic therapy" refers to any medically acceptable intervention intended to alleviate at least one symptom or consequence of diabetes. Examples of such antidiabetic therapies include, but are not limited to, immunotherapies, such as, but not limited to, the administration of IgM (seeUS Patent Application Publication No. 2015 / 0265704); administration of one or more calcineurin inhibitors such as, but not limited to, tacrolimus (IUPAC name (1R,9S,12S,13R,14S,17R,18E,21S,23S,24R,25S,27R)-1,14-dihydroxy-12-[(1E)-1-[(1R,3R,4R)-4-hydroxy-3-methoxycyclohexyl]prop-l-en-2-yl]-23,25-dimethoxy-13,19,21,27-tetramethyl-17-(prop-2-en-1-yl)-11,28-dioxa-4-azatricyclo[22.3.1.0. 4 , 9 ]octacoz-18-ene-2,3,10,16-tetron; see, e.g., U.S. Patent Nos. 6,884,433; 8,623,410; 8,911,777; and 9,011,922); administration of a glucagon-like peptide-1 (GLP-1) analog, such as, but not limited to, exendin-4 (amino acid sequence HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS; SEQ ID NO: 63; see, e.g., U.S. Patent Nos. 6,902,744; 8,057,822; and 9,133,260); or any combination thereof. The entire disclosures of each of these U.S. patent application publications and patents are hereby incorporated by reference.

[0311] In some embodiments of the described methods, the composition used is formulated for use in humans, and / or the pancreatic islet cell is a human islet cell, and / or the pancreatic islet cell is in the body of a subject, which in some embodiments is a human. In some embodiments of the described methods, the proposed composition comprises, consists essentially of, or consists of a sequence selected from the group consisting of SEQ ID NO: 62, or a sequence that differs from SEQ ID NO: 62 by 1, 2, 3, 4, or 5 amino acid modifications.

[0312] EXAMPLES

[0313] The following is a more detailed description of the described subject matter with reference to the accompanying examples, which illustrate illustrative embodiments of the described subject matter. However, the described subject matter may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. On the contrary, the embodiments provided are provided so that this description is comprehensive and complete, and fully conveys the scope of the described subject matter to those skilled in the art.

[0314] EXAMPLE 1

[0315] C57BL / 6 islets (see Fig. 3) were grown with 4 μM N-94 peptide (N-94) or control peptide C-95 for different periods of time from 1 to 15 days in DMEM at 37°C. Islet viability was measured by propidium iodide-fluorescein diacetate staining. The results are shown in Fig. 1A.

[0316] C57BL / 6 islets were also incubated overnight with 4 μM peptide N-94 (SEQ ID NO: 1) or peptide C-95, or without additive, followed by glucose-stimulated insulin secretion (GSIS) analysis. The results are shown in Fig. 1B.

[0317] Human islet cells were incubated with the N-94 or C-95 peptide in the same manner for various periods of time, followed by GSIS analysis and viability measurements. Insulin was measured using an insulin enzyme-linked immunosorbent assay kit (Mercodia, Uppsala, Sweden).

[0318] Human islet cells were incubated overnight with 1 μM of N-94 peptide (SEQ ID NO: 1), N-94 / C6 peptide (SEQ ID NO: 62), N104 peptide, Tearpep3 peptide (SEQ ID NO: 21), Tearpep3 / C6 peptide (SEQ ID NO: 33), C-95 peptide, or no treatment, followed by GSIS analysis (see Fig. 2A for structures and Table 2 for sequences of these exemplary peptides). C57BL / 6 islet cells were pretreated with 4 μM of N-94 or C-95 peptide, or saline for 24 hours, and minimal islet mass was then transplanted into syngeneic diabetic recipients. Tail vein blood glucose levels were measured daily. The results are shown in Fig. 2B.

[0319] Islet cells cultured with N-94 peptide retained 61% viability on day 15, compared to islets cultured with the control peptide C-95, which showed only 20% viability (p<0.001). N-94 peptide provided an approximately 2-fold increase in GSIS (91±22 mg / L), compared to islets without treatment (39.86±7.81 mg / L) or after treatment with C-95 peptide (54.9±18.14 mg / L). The viability of human islets incubated with N-94 peptide was 84% ​​on day 6, compared to 60% viability observed with C-95 peptide and 58% with saline. However, the maximum activity of GSIS in human islets with the N-94 peptide was observed only up to day 3.

[0320] After incubation with various N-94 peptide analogs, a comparison of their GSIS responses showed a nearly two-fold increase with N-94 peptide compared to C-95 peptide, with the N104 peptide exhibiting approximately 3.5-fold increase; and with Tearpep3 peptide and Tearpep3 / C6 peptide, >2.5-fold increase. Incubation of islets with N-94 peptide ensured consistent return of post-transplant mice to normoglycemia with glucose levels less than 200 ng / dL for 9 days post-transplantation, with treatment efficacy continuing beyond 40 days post-transplantation. With C-95 peptide treatment, glucose levels on day 12 post-transplantation were <250 mg / dL. Mean blood glucose levels measured between days 22 and 43 were 142.9±17.6 mg / dL for the N-94 peptide group, compared with 275±34.8 mg / dL for the C-95 peptide group.

[0321] EXAMPLE 2

[0322] Thirty-five C57BL / 6 islets per well were cultured with 4 μM of the N-94 peptide analog or the control peptide C-95 for 1, 2, 5, 7, 10, or 15 days in DMEM at 37°C. Islet viability was assessed after PI / FDA staining. Islets cultured with the N-94 peptide retained 95±3, 89±15, 80±17.5, and 61±21% viability, while islets cultured with the C-95 peptide retained 95±3, 87±13, 40±28, and 20±19% viability at days 1, 5, 10, and 15.

[0323] C57BL / 6 islets were incubated overnight with 4 μM N-94 or C-95 peptide, or without additive. Fifty islets per well were used for the glucose-stimulated insulin secretion (GSIS) assay. Islets without treatment or after treatment with C-95 or N-94 peptide and stimulation with a high glucose concentration (28 mM) secreted 39.86 ± 7.81, 54.9 ± 18.14, and 91 ± 22.16 ng / mL of insulin. Peptide N-94 increased insulin secretion by 2-fold compared with the untreated control sample. The viability of human islets incubated with N-94 peptide on days 1, 3, 4, 6, and 9 was 95±4, 95±3, 94±4, 84±9, and 80±15%, while with C-95 it was 94±4, 91±7, 88±8, 66±16, and 65±18%. Viability with saline was 94±4, 94±3, 88±8, 58±19, and ~53. Although the GSIS results were similar to those obtained for mice, the maximum insulin secretory activity with N-94 peptide was observed only on days 1 and 3.

[0324] Fifty human islets were incubated with peptide N-94 or peptide C-95 for 1, 3, 4, 6, and 9 days, followed by GSIS analysis and viability measurement. Insulin was measured using an insulin enzyme-linked immunosorbent assay kit (Mercodia). When comparing insulin secretion in response to 28 mM glucose, insulin secretion with peptide N-94 was approximately double that of peptide C-95, insulin secretion with peptide N104 was approximately 3.5 times greater, and with peptide Tearpep3 and peptide Tearpep3 / C6 it was >2.5 times greater.

[0325] Human islets were incubated overnight with 1 μM N-94 peptide, N-94 / C6 peptide, N104 peptide, Tearpep3 peptide, Tearpep3 / C6 peptide, C-95 peptide, or no treatment and subjected to GSIS analysis.

[0326] 75 C57 B1 / 6 islets were transplanted into C57BL / 6 recipients induced to diabetes by a single injection of STZ (220 mg / kg). The islets were pretreated with 4 μM N-94 or C-95 peptide, or saline 24 hours before transplantation into diabetic mice (n=2 mice / group). Tail vein blood glucose levels were measured daily. Incubation of islets with N-94 peptide ensured a persistent return of transplanted mice to normoglycemia, with glucose levels <200 mg / dL, for 9 days after transplantation. With C-95 peptide treatment, glucose levels were <250 mg / dL on day 12 after transplantation. Mice transplanted with untreated islets remained diabetic.

[0327] EXAMPLE 3

[0328] Fig. 4 is a graph of blood glucose levels for pancreatic islets treated with the indicated peptides and transplanted into diabetic mice, which were observed for 60 days. A graph of blood glucose levels after minimal transplantation of 75 C57B1 / 6 islets treated with the N-94 peptide, treated with the C-95 peptide, or treated with saline under the renal capsule of C57BL / 6 mice in which diabetes was induced before transplantation by a single injection of streptozocin (220 mg / kg; two mice per group) is shown. Pretreatment was carried out for 24 hours. Blood was collected from the tail vein daily for glucose analysis. Each line represents the changing blood glucose level of one diabetic mouse.Six mice received islets treated with the N-94 peptide (black), six others received islets treated with the C-95 peptide (blue), and four mice received islets treated with saline (orange). The N-94 peptide consistently returned mice to normoglycemia after transplantation, with glucose levels <200 ng / dL for 9 days post-transplantation, with efficacy continuing beyond 40 days post-transplantation. With the C-95 peptide, glucose levels on day 12 post-transplantation were <250 mg / dL. Mean blood glucose levels measured between days 22 and 43 were 142.9 ± 17.6 mg / dL for the N-94 group compared with 275 ± 34.8 mg / dL for the C-95 peptide group. This figure shows that pancreatic islets pre-treated with the N-94 peptide transplanted into diabetic mice provided rapid restoration of normoglycemia.

[0329] EXAMPLE 4

[0330] Human islets were treated with various peptides according to the described subject matter of the invention, and viability was assessed twenty days after treatment. The results are shown in Fig. 5. Human islets treated with 4 μM Tearpep3 / N-104 peptide demonstrated excellent viability twenty days after isolation, compared with 4 μM Tearpep3 / C-6 peptide and lacritin N-94 peptide. Synthetic lacritin C-95 peptide (4 μM), derived from the inactive N-terminus of lacritin, was used as a negative control.

[0331] EXAMPLE 5

[0332] Blood glucose levels were tested in NOD mice administered three doses of 200 μg IgM on days 1, 3, and 5, starting from the initial onset of hyperglycemia (e.g., baseline value (BG) 180-340 mg / dL). The results are shown in Fig. 6. Diabetes was reversed in 70% of NOD mice throughout the observation period.

[0333] EXAMPLE 6

[0334] Blood glucose levels were tested in IgM-treated NOD mice. As shown in Fig. 7, IgM therapy delayed the onset of T1D. 80% of saline-treated control NOD mice developed diabetes by 18–20 weeks of age. Mice treated with IgM twice weekly (~50 μg in 100 μL PBS) showed significant protection against T1D when treatment was started early (at 5 weeks of age; p<0.0001). BSA resulted in a 70% T1D incidence, whereas IgG treatment resulted in a 50% reduction in incidence (n=10 / group) at 25 weeks of age. Treatment cessation resulted in diabetes in only 9 of 33 mice 22 weeks after cessation, indicating the development of tolerance.

[0335] IgM treatment was also tested to see if it could reverse the diagnosed disease when combined with islet transplantation. Figure 8 shows that IgM treatment combined with islet transplantation resulted in reversal of the diagnosed disease.

[0336] EXAMPLE 7

[0337] It was tested whether peptides according to the described subject matter of the invention can increase the viability of human sensory neurons exposed to inflammatory cytokines. Sensory neurons were obtained from induced pluripotent stem cells (iPSCs). iPSCs were seeded in 12-well plates coated with Matrigel (1.25 x 10 5cells / well). Once the cells reached confluency, neuronal differentiation was initiated using KSR medium. The culture was supplemented with 100 nM LDN-193189 and 10 mM SB431542 from days 0 to 6 to inhibit SMAD signaling. On day 4, 25% N2 medium was added to the culture, reaching 100% N2 medium on day 10. Sensory neuron induction was initiated on day 2 (days 2–10) by adding 3 mM CHIR 99021, 10 mM SU5402, and 10 mM DAPT. From days 10 to 21, neuronal maturation was promoted using 25 ng / ml p-NGF, BDNF, and GDNF. Cells were fed daily.

[0338] To analyze sensory neuron markers, immunofluorescence analysis of cells was performed on day 0, day 10, and day 21. Cells were incubated overnight with primary antibodies to MAP2 (1:500, ABCAM), Brn3a (1:125, Merck), PMCA (1:500, ABCAM), Tujl (10 mg / ml, R&D Systems), Nestin (1:500, ABCAM), and TRPM8 (1:500, NovusBio). Secondary antibodies Alexa 568 (Thermo) and Alexa 488 (Thermo) were incubated for 2 h at room temperature. DAPI (1:5000, Thermo) was incubated for 5 min at room temperature. Illustrative fluorescence micrographs are shown in Fig. 9A.

[0339] To analyze mRNA levels, total RNA was extracted at day 0, day 10, and day 18 and analyzed by quantitative real-time PCR. The results are shown in Fig. 9B.

[0340] Sensory neurons (21 days old) were incubated with IFN-γ (1000 U / ml)+TNF-α (100 ng / ml) with or without 1 mM N-94 / C-6 peptide or negative control peptide C-95 for 72 hours in a 96-well plate. Alamar blue reagent (10%) was added to each well. Fluorescence was measured after 6 hours (excitation at 545 nm / emission at 590 nm). The results are shown in Fig. 9C.

[0341] Discussion of Examples Peptide N-94 and its analogs and derivatives increase the viability of mouse and human islets, enhance insulin secretion, and prevent islet transplant rejection. Thus, the compositions described herein have utility as interventional agents for enhancing islet survival, for example, but not limited to, after transplantation, as well as for the treatment of type 1 diabetes. Interestingly, the peptides according to the described subject matter also restore the viability of human neurons derived from induced pluripotent stem cells (IPSCs) exposed to interferon-gamma and TNF. Without being limited to any particular mode of action, the positive effect on neurons can be used to enhance the viability of endogenous islets using the compositions and methods according to the described subject matter.

[0342] Type 1 diabetes (T1D), in particular, remains a severe chronic autoimmune disease whose prevalence continues to increase annually. Because T1D is rarely diagnosed before ~70-80% of β-cells are destroyed, developing therapies that facilitate β-cell regeneration after the diagnosis of newly diagnosed diabetes remains an important goal. IgM-based therapy has been shown to play a key role in maintaining β-cell-specific tolerance and reversing diabetes, and that this effect is associated with its ability to both deplete autoreactive B cells and suppress insulin autoantibody production, attenuate inflammation, expand immunoregulatory T cells (Treg), and increase islet cell numbers. Early IgM administration completely suppresses the occurrence of T1D (p<0.0001) in non-obese diabetic (NOD) mice.It restores B-cell homeostasis, regulates T-cell activation, proliferation, and chemotaxis, suppresses insulitis, accelerates islet cell proliferation, and, when combined with islet transplantation, reverses diabetes in mice diagnosed with T1D. It has also been shown that IgM alone reverses the disease in 70% of mice with newly diagnosed diabetes. It is possible that in the 30% of NOD mice that developed diabetes despite IgM treatment, beta-cell destruction was too advanced for IgM therapy to be effective at the time of intervention, and / or immunosuppression with IgM alone was insufficient to reverse the autoimmune response.

[0343] Thus, in some embodiments, a study was conducted of combination treatment of IgM with other short-acting immunosuppressants and / or with an agent that promotes endogenous beta cell regeneration.

[0344] LINKS

[0345] All references listed below, as well as all references cited in this specification, including, but not limited to, all patents, patent applications and their publications, scientific journal articles and database entries (e.g., entries in the GENBANK® and UniProt biosequence databases and all notes cited therein), are incorporated herein by reference in their entirety to the extent that they complement, explain, condition or describe the methodology, technologies and / or compositions used according to this specification.

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[0360] Although the described subject matter has been disclosed with reference to specific embodiments, it should be understood that other embodiments and modifications of the described subject matter can be developed by those skilled in the art without departing from the true spirit of the scope of the described subject matter.

[0361] --->

[0362] SEQUENCE LISTING

[0363] <110> PATENT FUND OF THE UNIVERSITY OF VIRGINIA

[0364] GORDON, LAURIE W.

[0365] BRAYMAN, KENNETH

[0366] CHHABRA, PREETI

[0367] MA, MINGYANG

[0368] KARINA, TEIXEIRA

[0369] GADEK, THOMAS R.

[0370] <120> COMPOSITIONS AND METHODS OF STIMULATING THE VIABILITY OF ISLETS AND

[0371] INCREASE IN INSULIN SECRETION

[0372] <130> 3062 / 118 PCT

[0373] <150> US 62 / 817790

[0374] <151> 2019-03-13

[0375] <160> 63

[0376] <170> PatentIn, version 3.5

[0377] <210> 1

[0378] <211> 19

[0379] <212> Protein

[0380] <213> Artificial sequence

[0381] <220>

[0382] <223> Artificially synthesized peptide

[0383] <400> 1

[0384] Lys Gln Phe Ile Glu Asn Gly Ser Glu Phe Ala Gln Lys Leu Leu Lys

[0385] 1 5 10 15

[0386] Lys Phe Ser

[0387] <210> 2

[0388] <211> 20

[0389] <212> Protein

[0390] <213> Artificial sequence

[0391] <220>

[0392] <223> Artificially synthesized peptide

[0393] <400> 2

[0394] Cys Lys Gln Phe Ile Glu Asn Gly Ser Glu Phe Ala Gln Lys Leu Leu

[0395] 1 5 10 15

[0396] Lys Lys Phe Ser

[0397] 20

[0398] <210> 3

[0399] <211> 20

[0400] <212> Protein

[0401] <213> Artificial sequence

[0402] <220>

[0403] <223> Artificially synthesized peptide

[0404] <400> 3

[0405] Lys Gln Phe Ile Glu Asn Gly Ser Glu Phe Ala Gln Lys Leu Leu Lys

[0406] 1 5 10 15

[0407] Lys Phe Ser Cys

[0408] 20

[0409] <210> 4

[0410] <211> 21

[0411] <212> Protein

[0412] <213> Artificial sequence

[0413] <220>

[0414] <223> Artificially synthesized peptide

[0415] <400> 4

[0416] Cys Lys Gln Phe Ile Glu Asn Gly Ser Glu Phe Ala Gln Lys Leu Leu

[0417] 1 5 10 15

[0418] Lys Lys Phe Ser Cys

[0419] 20

[0420] <210> 5

[0421] <211> 25

[0422] <212> Protein

[0423] <213> Artificial sequence

[0424] <220>

[0425] <223> Artificially synthesized peptide

[0426] <400> 5

[0427] Lys Asn Phe Ile Glu Asn Gly Ser Glu Phe Ala Gln Lys Leu Leu Lys

[0428] 1 5 10 15

[0429] Lys Phe Ser Leu Leu Lys Pro Trp Ala

[0430] 20 25

[0431] <210> 6

[0432] <211> 26

[0433] <212> Protein

[0434] <213> Artificial sequence

[0435] <220>

[0436] <223> Artificially synthesized peptide

[0437] <400> 6

[0438] Cys Lys Asn Phe Ile Glu Asn Gly Ser Glu Phe Ala Gln Lys Leu Leu

[0439] 1 5 10 15

[0440] Lys Lys Phe Ser Leu Leu Lys Pro Trp Ala

[0441] 20 25

[0442] <210> 7

[0443] <211> 26

[0444] <212> Protein

[0445] <213> Artificial sequence

[0446] <220>

[0447] <223> Artificially synthesized peptide

[0448] <400> 7

[0449] Lys Asn Phe Ile Glu Asn Gly Ser Glu Phe Ala Gln Lys Leu Leu Lys

[0450] 1 5 10 15

[0451] Lys Phe Ser Leu Leu Lys Pro Trp Ala Cys

[0452] 20 25

[0453] <210> 8

[0454] <211> 27

[0455] <212> Protein

[0456] <213> Artificial sequence

[0457] <220>

[0458] <223> Artificially synthesized peptide

[0459] <400> 8

[0460] Cys Lys Asn Phe Ile Glu Asn Gly Ser Glu Phe Ala Gln Lys Leu Leu

[0461] 1 5 10 15

[0462] Lys Lys Phe Ser Leu Leu Lys Pro Trp Ala Cys

[0463] 20 25

[0464] <210> 9

[0465] <211> 25

[0466] <212> Protein

[0467] <213> Artificial sequence

[0468] <220>

[0469] <223> Artificially synthesized peptide

[0470] <400> 9

[0471] Lys Gln Phe Ile Glu Asn Gly Ser Glu Phe Ala Asn Lys Leu Leu Lys

[0472] 1 5 10 15

[0473] Lys Phe Ser Leu Leu Lys Pro Trp Ala

[0474] 20 25

[0475] <210> 10

[0476] <211> 26

[0477] <212> Protein

[0478] <213> Artificial sequence

[0479] <220>

[0480] <223> Artificially synthesized peptide

[0481] <400> 10

[0482] Cys Lys Gln Phe Ile Glu Asn Gly Ser Glu Phe Ala Asn Lys Leu Leu

[0483] 1 5 10 15

[0484] Lys Lys Phe Ser Leu Leu Lys Pro Trp Ala

[0485] 20 25

[0486] <210> 11

[0487] <211> 26

[0488] <212> Protein

[0489] <213> Artificial sequence

[0490] <220>

[0491] <223> Artificially synthesized peptide

[0492] <400> 11

[0493] Lys Gln Phe Ile Glu Asn Gly Ser Glu Phe Ala Asn Lys Leu Leu Lys

[0494] 1 5 10 15

[0495] Lys Phe Ser Leu Leu Lys Pro Trp Ala Cys

[0496] 20 25

[0497] <210> 12

[0498] <211> 27

[0499] <212> Protein

[0500] <213> Artificial sequence

[0501] <220>

[0502] <223> Artificially synthesized peptide

[0503] <400> 12

[0504] Cys Lys Gln Phe Ile Glu Asn Gly Ser Glu Phe Ala Asn Lys Leu Leu

[0505] 1 5 10 15

[0506] Lys Lys Phe Ser Leu Leu Lys Pro Trp Ala Cys

[0507] 20 25

[0508] <210> 13

[0509] <211> 25

[0510] <212> Protein

[0511] <213> Artificial sequence

[0512] <220>

[0513] <223> Artificially synthesized peptide

[0514] <400> 13

[0515] Lys Lys Leu Phe Gly Gly Arg Asn Asp Val Leu Arg Gln Met Met Asp

[0516] 1 5 10 15

[0517] Arg Leu Gly Pro Lys Phe Asn Leu Phe

[0518] 20 25

[0519] <210> 14

[0520] <211> 26

[0521] <212> Protein

[0522] <213> Artificial sequence

[0523] <220>

[0524] <223> Artificially synthesized peptide

[0525] <400> 14

[0526] Cys Lys Lys Leu Phe Gly Gly Arg Asn Asp Val Leu Arg Gln Met Met

[0527] 1 5 10 15

[0528] Asp Arg Leu Gly Pro Lys Phe Asn Leu Phe

[0529] 20 25

[0530] <210> 15

[0531] <211> 26

[0532] <212> Protein

[0533] <213> Artificial sequence

[0534] <220>

[0535] <223> Artificially synthesized peptide

[0536] <400> 15

[0537] Lys Lys Leu Phe Gly Gly Arg Asn Asp Val Leu Arg Gln Met Met Asp

[0538] 1 5 10 15

[0539] Arg Leu Gly Pro Lys Phe Asn Leu Phe Cys

[0540] 20 25

[0541] <210> 16

[0542] <211> 27

[0543] <212> Protein

[0544] <213> Artificial sequence

[0545] <220>

[0546] <223> Artificially synthesized peptide

[0547] <400> 16

[0548] Cys Lys Lys Leu Phe Gly Gly Arg Asn Asp Val Leu Arg Gln Met Met

[0549] 1 5 10 15

[0550] Asp Arg Leu Gly Pro Lys Phe Asn Leu Phe Cys

[0551] 20 25

[0552] <210> 17

[0553] <211> 25

[0554] <212> Protein

[0555] <213> Artificial sequence

[0556] <220>

[0557] <223> Artificially synthesized peptide

[0558] <400> 17

[0559] Lys Lys Leu Phe Gly Gly Arg Asn Asp Val Leu Arg Asn Met Met Asp

[0560] 1 5 10 15

[0561] Arg Leu Gly Pro Lys Phe Asn Leu Phe

[0562] 20 25

[0563] <210> 18

[0564] <211> 26

[0565] <212> Protein

[0566] <213> Artificial sequence

[0567] <220>

[0568] <223> Artificially synthesized peptide

[0569] <400> 18

[0570] Cys Lys Lys Leu Phe Gly Gly Arg Asn Asp Val Leu Arg Asn Met Met

[0571] 1 5 10 15

[0572] Asp Arg Leu Gly Pro Lys Phe Asn Leu Phe

[0573] 20 25

[0574] <210> 19

[0575] <211> 26

[0576] <212> Protein

[0577] <213> Artificial sequence

[0578] <220>

[0579] <223> Artificially synthesized peptide

[0580] <400> 19

[0581] Lys Lys Leu Phe Gly Gly Arg Asn Asp Val Leu Arg Asn Met Met Asp

[0582] 1 5 10 15

[0583] Arg Leu Gly Pro Lys Phe Asn Leu Phe Cys

[0584] 20 25

[0585] <210> 20

[0586] <211> 27

[0587] <212> Protein

[0588] <213> Artificial sequence

[0589] <220>

[0590] <223> Artificially synthesized peptide

[0591] <400> 20

[0592] Cys Lys Lys Leu Phe Gly Gly Arg Asn Asp Val Leu Arg Asn Met Met

[0593] 1 5 10 15

[0594] Asp Arg Leu Gly Pro Lys Phe Asn Leu Phe Cys

[0595] 20 25

[0596] <210> 21

[0597] <211> 25

[0598] <212> Protein

[0599] <213> Artificial sequence

[0600] <220>

[0601] <223> Artificially synthesized peptide

[0602] <400> 21

[0603] Lys Arg Phe Tyr Lys Arg Gly Ala Glu Leu Gly Lys Asn Arg Arg Lys

[0604] 1 5 10 15

[0605] Asn Trp His Ala Gln Leu Phe Val Leu

[0606] 20 25

[0607] <210> 22

[0608] <211> 26

[0609] <212> Protein

[0610] <213> Artificial sequence

[0611] <220>

[0612] <223> Artificially synthesized peptide

[0613] <400> 22

[0614] Cys Lys Arg Phe Tyr Lys Arg Gly Ala Glu Leu Gly Lys Asn Arg Arg

[0615] 1 5 10 15

[0616] Lys Asn Trp His Ala Gln Leu Phe Val Leu

[0617] 20 25

[0618] <210> 23

[0619] <211> 26

[0620] <212> Protein

[0621] <213> Artificial sequence

[0622] <220>

[0623] <223> Artificially synthesized peptide

[0624] <400> 23

[0625] Lys Arg Phe Tyr Lys Arg Gly Ala Glu Leu Gly Lys Asn Arg Arg Lys

[0626] 1 5 10 15

[0627] Asn Trp His Ala Gln Leu Phe Val Leu Cys

[0628] 20 25

[0629] <210> 24

[0630] <211> 27

[0631] <212> Protein

[0632] <213> Artificial sequence

[0633] <220>

[0634] <223> Artificially synthesized peptide

[0635] <400> 24

[0636] Cys Lys Arg Phe Tyr Lys Arg Gly Ala Glu Leu Gly Lys Asn Arg Arg

[0637] 1 5 10 15

[0638] Lys Asn Trp His Ala Gln Leu Phe Val Leu Cys

[0639] 20 25

[0640] <210> 25

[0641] <211> 11

[0642] <212> Protein

[0643] <213> Artificial sequence

[0644] <220>

[0645] <223> Artificially synthesized peptide

[0646] <400> 25

[0647] Lys Arg Phe Tyr Lys Arg Gly Ala Glu Leu Gly

[0648] 1 5 10

[0649] <210> 26

[0650] <211> 12

[0651] <212> Protein

[0652] <213> Artificial sequence

[0653] <220>

[0654] <223> Artificially synthesized peptide

[0655] <400> 26

[0656] Cys Lys Arg Phe Tyr Lys Arg Gly Ala Glu Leu Gly

[0657] 1 5 10

[0658] <210> 27

[0659] <211> 12

[0660] <212> Protein

[0661] <213> Artificial sequence

[0662] <220>

[0663] <223> Artificially synthesized peptide

[0664] <400> 27

[0665] Lys Arg Phe Tyr Lys Arg Gly Ala Glu Leu Gly Cys

[0666] 1 5 10

[0667] <210> 28

[0668] <211> 13

[0669] <212> Protein

[0670] <213> Artificial sequence

[0671] <220>

[0672] <223> Artificially synthesized peptide

[0673] <400> 28

[0674] Cys Lys Arg Phe Tyr Lys Arg Gly Ala Glu Leu Gly Cys

[0675] 1 5 10

[0676] <210> 29

[0677] <211> 15

[0678] <212> Protein

[0679] <213> Artificial sequence

[0680] <220>

[0681] <223> Artificially synthesized peptide

[0682] <400> 29

[0683] Lys Arg Phe Tyr Lys Arg Gly Ala Glu Leu Gly Lys Asn Arg Arg

[0684] 1 5 10 15

[0685] <210> 30

[0686] <211> 16

[0687] <212> Protein

[0688] <213> Artificial sequence

[0689] <220>

[0690] <223> Artificially synthesized peptide

[0691] <400> 30

[0692] Cys Lys Arg Phe Tyr Lys Arg Gly Ala Glu Leu Gly Lys Asn Arg Arg

[0693] 1 5 10 15

[0694] <210> 31

[0695] <211> 16

[0696] <212> Protein

[0697] <213> Artificial sequence

[0698] <220>

[0699] <223> Artificially synthesized peptide

[0700] <400> 31

[0701] Lys Arg Phe Tyr Lys Arg Gly Ala Glu Leu Gly Lys Asn Arg Arg Cys

[0702] 1 5 10 15

[0703] <210> 32

[0704] <211> 17

[0705] <212> Protein

[0706] <213> Artificial sequence

[0707] <220>

[0708] <223> Artificially synthesized peptide

[0709] <400> 32

[0710] Cys Lys Arg Phe Tyr Lys Arg Gly Ala Glu Leu Gly Lys Asn Arg Arg

[0711] 1 5 10 15

[0712] Cys

[0713] <210> 33

[0714] <211> 19

[0715] <212> Protein

[0716] <213> Artificial sequence

[0717] <220>

[0718] <223> Artificially synthesized peptide

[0719] <400> 33

[0720] Lys Arg Phe Tyr Lys Arg Gly Ala Glu Leu Gly Lys Asn Arg Arg Lys

[0721] 1 5 10 15

[0722] Asn Trp His

[0723] <210> 34

[0724] <211> 20

[0725] <212> Protein

[0726] <213> Artificial sequence

[0727] <220>

[0728] <223> Artificially synthesized peptide

[0729] <400> 34

[0730] Cys Lys Arg Phe Tyr Lys Arg Gly Ala Glu Leu Gly Lys Asn Arg Arg

[0731] 1 5 10 15

[0732] Lys Asn Trp His

[0733] 20

[0734] <210> 35

[0735] <211> 20

[0736] <212> Protein

[0737] <213> Artificial sequence

[0738] <220>

[0739] <223> Artificially synthesized peptide

[0740] <400> 35

[0741] Lys Arg Phe Tyr Lys Arg Gly Ala Glu Leu Gly Lys Asn Arg Arg Lys

[0742] 1 5 10 15

[0743] Asn Trp His Cys

[0744] 20

[0745] <210> 36

[0746] <211> 21

[0747] <212> Protein

[0748] <213> Artificial sequence

[0749] <220>

[0750] <223> Artificially synthesized peptide

[0751] <400> 36

[0752] Cys Lys Arg Phe Tyr Lys Arg Gly Ala Glu Leu Gly Lys Asn Arg Arg

[0753] 1 5 10 15

[0754] Lys Asn Trp His Cys

[0755] 20

[0756] <210> 37

[0757] <211> 15

[0758] <212> Protein

[0759] <213> Artificial sequence

[0760] <220>

[0761] <223> Artificially synthesized peptide

[0762] <400> 37

[0763] Gly Lys Asn Arg Arg Lys Asn Trp His Ala Gln Leu Phe Val Leu

[0764] 1 5 10 15

[0765] <210> 38

[0766] <211> 16

[0767] <212> Protein

[0768] <213> Artificial sequence

[0769] <220>

[0770] <223> Artificially synthesized peptide

[0771] <400> 38

[0772] Cys Gly Lys Asn Arg Arg Lys Asn Trp His Ala Gln Leu Phe Val Leu

[0773] 1 5 10 15

[0774] <210> 39

[0775] <211> 16

[0776] <212> Protein

[0777] <213> Artificial sequence

[0778] <220>

[0779] <223> Artificially synthesized peptide

[0780] <400> 39

[0781] Gly Lys Asn Arg Arg Lys Asn Trp His Ala Gln Leu Phe Val Leu Cys

[0782] 1 5 10 15

[0783] <210> 40

[0784] <211> 17

[0785] <212> Protein

[0786] <213> Artificial sequence

[0787] <220>

[0788] <223> Artificially synthesized peptide

[0789] <400> 40

[0790] Cys Gly Lys Asn Arg Arg Lys Asn Trp His Ala Gln Leu Phe Val Leu

[0791] 1 5 10 15

[0792] Cys

[0793] <210> 41

[0794] <211> 25

[0795] <212> Protein

[0796] <213> Artificial sequence

[0797] <220>

[0798] <223> Artificially synthesized peptide

[0799] <400> 41

[0800] Lys Arg Phe Tyr Lys Arg Gly Ala Glu Leu Gly Lys Asn Arg Arg Lys

[0801] 1 5 10 15

[0802] Asn Trp His Ala Asn Leu Phe Val Leu

[0803] 20 25

[0804] <210> 42

[0805] <211> 26

[0806] <212> Protein

[0807] <213> Artificial sequence

[0808] <220>

[0809] <223> Artificially synthesized peptide

[0810] <400> 42

[0811] Cys Lys Arg Phe Tyr Lys Arg Gly Ala Glu Leu Gly Lys Asn Arg Arg

[0812] 1 5 10 15

[0813] Lys Asn Trp His Ala Asn Leu Phe Val Leu

[0814] 20 25

[0815] <210> 43

[0816] <211> 26

[0817] <212> Protein

[0818] <213> Artificial sequence

[0819] <220>

[0820] <223> Artificially synthesized peptide

[0821] <400> 43

[0822] Lys Arg Phe Tyr Lys Arg Gly Ala Glu Leu Gly Lys Asn Arg Arg Lys

[0823] 1 5 10 15

[0824] Asn Trp His Ala Asn Leu Phe Val Leu Cys

[0825] 20 25

[0826] <210> 44

[0827] <211> 27

[0828] <212> Protein

[0829] <213> Artificial sequence

[0830] <220>

[0831] <223> Artificially synthesized peptide

[0832] <400> 44

[0833] Cys Lys Arg Phe Tyr Lys Arg Gly Ala Glu Leu Gly Lys Asn Arg Arg

[0834] 1 5 10 15

[0835] Lys Asn Trp His Ala Asn Leu Phe Val Leu Cys

[0836] 20 25

[0837] <210> 45

[0838] <211> 15

[0839] <212> Protein

[0840] <213> Artificial sequence

[0841] <220>

[0842] <223> Artificially synthesized peptide

[0843] <400> 45

[0844] Gly Lys Asn Arg Arg Lys Asn Trp His Ala Gln Leu Phe Val Leu

[0845] 1 5 10 15

[0846] <210> 46

[0847] <211> 16

[0848] <212> Protein

[0849] <213> Artificial sequence

[0850] <220>

[0851] <223> Artificially synthesized peptide

[0852] <400> 46

[0853] Cys Gly Lys Asn Arg Arg Lys Asn Trp His Ala Gln Leu Phe Val Leu

[0854] 1 5 10 15

[0855] <210> 47

[0856] <211> 16

[0857] <212> Protein

[0858] <213> Artificial sequence

[0859] <220>

[0860] <223> Artificially synthesized peptide

[0861] <400> 47

[0862] Gly Lys Asn Arg Arg Lys Asn Trp His Ala Gln Leu Phe Val Leu Cys

[0863] 1 5 10 15

[0864] <210> 48

[0865] <211> 17

[0866] <212> Protein

[0867] <213> Artificial sequence

[0868] <220>

[0869] <223> Artificially synthesized peptide

[0870] <400> 48

[0871] Cys Gly Lys Asn Arg Arg Lys Asn Trp His Ala Gln Leu Phe Val Leu

[0872] 1 5 10 15

[0873] Cys

[0874] <210> 49

[0875] <211> 15

[0876] <212> Protein

[0877] <213> Artificial sequence

[0878] <220>

[0879] <223> Artificially synthesized peptide

[0880] <400> 49

[0881] Ala Gln Lys Leu Leu Lys Lys Phe Ser Leu Leu Lys Pro Trp Ala

[0882] 1 5 10 15

[0883] <210> 50

[0884] <211> 16

[0885] <212> Protein

[0886] <213> Artificial sequence

[0887] <220>

[0888] <223> Artificially synthesized peptide

[0889] <400> 50

[0890] Cys Ala Gln Lys Leu Leu Lys Lys Phe Ser Leu Leu Lys Pro Trp Ala

[0891] 1 5 10 15

[0892] <210> 51

[0893] <211> 16

[0894] <212> Protein

[0895] <213> Artificial sequence

[0896] <220>

[0897] <223> Artificially synthesized peptide

[0898] <400> 51

[0899] Ala Gln Lys Leu Leu Lys Lys Phe Ser Leu Leu Lys Pro Trp Ala Cys

[0900] 1 5 10 15

[0901] <210> 52

[0902] <211> 17

[0903] <212> Protein

[0904] <213> Artificial sequence

[0905] <220>

[0906] <223> Artificially synthesized peptide

[0907] <400> 52

[0908] Cys Ala Gln Lys Leu Leu Lys Lys Phe Ser Leu Leu Lys Pro Trp Ala

[0909] 1 5 10 15

[0910] Cys

[0911] <210> 53

[0912] <211> 15

[0913] <212> Protein

[0914] <213> Artificial sequence

[0915] <220>

[0916] <223> Artificially synthesized peptide

[0917] <400> 53

[0918] Ala Asn Lys Leu Leu Lys Lys Phe Ser Leu Leu Lys Pro Trp Ala

[0919] 1 5 10 15

[0920] <210> 54

[0921] <211> 16

[0922] <212> Protein

[0923] <213> Artificial sequence

[0924] <220>

[0925] <223> Artificially synthesized peptide

[0926] <400> 54

[0927] Cys Ala Asn Lys Leu Leu Lys Lys Phe Ser Leu Leu Lys Pro Trp Ala

[0928] 1 5 10 15

[0929] <210> 55

[0930] <211> 16

[0931] <212> Protein

[0932] <213> Artificial sequence

[0933] <220>

[0934] <223> Artificially synthesized peptide

[0935] <400> 55

[0936] Ala Asn Lys Leu Leu Lys Lys Phe Ser Leu Leu Lys Pro Trp Ala Cys

[0937] 1 5 10 15

[0938] <210> 56

[0939] <211> 17

[0940] <212> Protein

[0941] <213> Artificial sequence

[0942] <220>

[0943] <223> Artificially synthesized peptide

[0944] <400> 56

[0945] Cys Ala Asn Lys Leu Leu Lys Lys Phe Ser Leu Leu Lys Pro Trp Ala

[0946] 1 5 10 15

[0947] Cys

[0948] <210> 57

[0949] <211> 9

[0950] <212> Protein

[0951] <213> Artificial sequence

[0952] <220>

[0953] <223> Artificially synthesized peptide

[0954] <400> 57

[0955] Ala Gln Lys Leu Leu Lys Lys Phe Ser

[0956] 1 5

[0957] <210> 58

[0958] <211> 10

[0959] <212> Protein

[0960] <213> Artificial sequence

[0961] <220>

[0962] <223> Artificially synthesized peptide

[0963] <400> 58

[0964] Cys Ala Gln Lys Leu Leu Lys Lys Phe Ser

[0965] 1 5 10

[0966] <210> 59

[0967] <211> 10

[0968] <212> Protein

[0969] <213> Artificial sequence

[0970] <220>

[0971] <223> Artificially synthesized peptide

[0972] <400> 59

[0973] Ala Gln Lys Leu Leu Lys Lys Phe Ser Cys

[0974] 1 5 10

[0975] <210> 60

[0976] <211> 11

[0977] <212> Protein

[0978] <213> Artificial sequence

[0979] <220>

[0980] <223> Artificially synthesized peptide

[0981] <400> 60

[0982] Cys Ala Gln Lys Leu Leu Lys Lys Phe Ser Cys

[0983] 1 5 10

[0984] <210> 61

[0985] <211> 138

[0986] <212> Protein

[0987] <213> Homo sapiens

[0988] <400> 61

[0989] Met Lys Phe Thr Thr Leu Leu Phe Leu Ala Ala Val Ala Gly Ala Leu

[0990] 1 5 10 15

[0991] Val Tyr Ala Glu Asp Ala Ser Ser Asp Ser Thr Gly Ala Asp Pro Ala

[0992] 20 25 30

[0993] Gln Glu Ala Gly Thr Ser Lys Pro Asn Glu Glu Ile Ser Gly Pro Ala

[0994] 35 40 45

[0995] Glu Pro Ala Ser Pro Pro Glu Thr Thr Thr Thr Ala Gln Glu Thr Ser

[0996] 50 55 60

[0997] Ala Ala Ala Val Gln Gly Thr Ala Lys Val Thr Ser Ser Arg Gln Glu

[0998] 65 70 75 80

[0999] Leu Asn Pro Leu Lys Ser Ile Val Glu Lys Ser Ile Leu Leu Thr Glu

[1000] 85 90 95

[1001] Gln Ala Leu Ala Lys Ala Gly Lys Gly Met His Gly Gly Val Pro Gly

[1002] 100 105 110

[1003] Gly Lys Gln Phe Ile Glu Asn Gly Ser Glu Phe Ala Gln Lys Leu Leu

[1004] 115 120 125

[1005] Lys Lys Phe Ser Leu Leu Lys Pro Trp Ala

[1006] 130 135

[1007] <210> 62

[1008] <211> 19

[1009] <212> Protein

[1010] <213> Artificial sequence

[1011] <220>

[1012] <223> Artificially synthesized peptide

[1013] <400> 62

[1014] Lys Gln Phe Ile Glu Asn Gly Ser Glu Phe Ala Gln Lys Leu Leu Lys

[1015] 1 5 10 15

[1016] Lys Phe Ser

[1017] <210> 63

[1018] <211> 39

[1019] <212> Protein

[1020] <213> Artificial sequence

[1021] <220>

[1022] <223> Artificially synthesized peptide

[1023] <400> 63

[1024] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu

[1025] 1 5 10 15

[1026] Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

[1027] 20 25 30

[1028] Ser Gly Ala Pro Pro Pro Ser

[1029] 35

[1030] <---

Claims

1. A method for restoring glucose-stimulated insulin secretion, comprising contacting pancreatic islet cells with an effective amount of a composition containing a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide or a pharmaceutically acceptable salt thereof comprises an amino acid sequence comprising any of the sequences SEQ ID NO: 1-44, 49-60 or 62, or any of the sequences SEQ ID NO: 45-58 with Q replaced by N.

2. The method according to claim 1, characterized in that the composition is formulated for administration to a human subject.

3. The method according to claim 1 or 2, characterized in that the composition is formulated in the form of nanoparticles, nanovesicles, microparticles, microvesicles, liposomes, packaged in PEG, lyophilized in the form of pills, or is any combination thereof.

4. The method according to any one of claims 1-3, characterized in that the peptide or pharmaceutically acceptable salt thereof contains at least one modification selected from the group consisting of C-terminal amidation, N-terminal acylation, N-terminal acetylation, N-terminal cysteine ​​addition, C-terminal cysteine ​​addition, PEGylation, and combinations thereof.

5. The method of claim 4, wherein the PEGylation comprises attaching a PEG group to the N-terminal cysteine, the C-terminal cysteine, or both.

6. The method of claim 5, wherein the PEG group has a molecular weight of from about 1 kilodalton (kDa) to about 40 kDa.

7. The method according to any one of claims 4-6, characterized in that the C-terminal amidation includes a substituted amide and / or the N-terminal acylation includes a substituted acyl group.

8. The method according to any one of claims 1-7, characterized in that the composition additionally contains one or more of a pharmaceutically acceptable carrier, an excipient, a diluent, tonicity agents, viscosity-increasing agents, and encapsulating agents.

9. The method according to any one of claims 1-8, characterized in that the peptide or its pharmaceutically acceptable salt is present in the composition at a concentration of from 1.0 nM to 100 μM.

10. The method according to any one of claims 1-9, characterized in that contacting the pancreatic islet cells comprises administering the composition to a subject, wherein the subject has a disease, disorder, or pathological condition associated with an abnormal response to glucose.

11. The method according to claim 10, characterized in that the disease, disorder or pathological condition associated with an abnormal response to glucose is type 1 or type 2 diabetes.

12. The method of claim 11, further comprising administering to the subject one or more additional antidiabetic therapies.

13. The method of claim 12, wherein the one or more additional antidiabetic therapies are selected from the group consisting of immune therapy, optionally immune therapy comprising administration of IgM; administration of a calcineurin inhibitor, optionally tacrolimus; administration of a glucagon-like peptide-1 (GLP-1) analogue, optionally exendin-4; and any combination thereof.

14. The method according to any one of paragraphs 10-13, characterized in that the composition is formulated for use by humans, and wherein the subject is a human.

15. The method according to any one of claims 1-14, wherein the peptide or pharmaceutically acceptable salt thereof comprises the amino acid sequence of any one of SEQ ID NOs: 21-44, or any one of SEQ ID NOs: 45-48 with Q replaced by N.

16. The method according to any one of claims 1-14, wherein the peptide or pharmaceutically acceptable salt thereof consists of the amino acid sequence of any one of SEQ ID NO: 21, 22, 23, 24, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44.

17. The method according to any one of claims 1-14, wherein the peptide or pharmaceutically acceptable salt thereof consists of the amino acid sequence SEQ ID NO:

21.

18. The method according to any one of claims 1-14, wherein the peptide or pharmaceutically acceptable salt thereof consists of the amino acid sequence SEQ ID NO:

33.

19. The method according to any one of claims 1-14, wherein the peptide or pharmaceutically acceptable salt thereof consists of the amino acid sequence SEQ ID NO:

37.

20. The method according to any one of claims 1-14, wherein the peptide or pharmaceutically acceptable salt thereof consists of the amino acid sequence SEQ ID NO: 41.

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