阅读说明：本技术 增加胰腺β细胞增殖的方法、治疗方法以及组合物 (Methods, treatment methods, and compositions for increasing pancreatic beta cell proliferation ) 是由 A·F·斯图尔特 C·阿克菲 P·王 B·戴维塔 于 2019-01-05 设计创作，主要内容包括：本文公开了增加胰腺β细胞群中细胞增殖的方法。还公开了治疗受试者胰岛素分泌不足相关病状的方法。还公开了包含DYRK1A抑制剂和GLP1R激动剂的组合物。本公开还描述了使移植患者的胰腺β细胞再生的方法。(Disclosed herein are methods of increasing cell proliferation in a pancreatic beta cell population. Also disclosed are methods of treating conditions associated with insufficient insulin secretion in a subject. Also disclosed are compositions comprising a DYRK1A inhibitor and a GLP1R agonist. The disclosure also describes methods of regenerating pancreatic beta cells in a transplant patient.)

1. A method of increasing cell proliferation in a pancreatic beta cell population, the method comprising:

contacting a population of pancreatic beta cells with a bispecific tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist, wherein said contacting is performed under conditions effective to cause a synergistic increase in cell proliferation in said population of pancreatic beta cells.

2. The method of claim 1, wherein the method is performed ex vivo.

3. The method of claim 1, wherein the method is performed in vivo.

4. The method of any one of claims 1 to 3, wherein the contacting is performed using a composition comprising both the DYRK1A inhibitor and the GLP1R agonist.

5. The method of any one of claims 1-4, wherein said contacting increases the number of proliferating pancreatic beta cells in said population by about 4-6%/day.

6. The method of any one of claims 1-5, wherein said contacting increases the number of proliferating pancreatic beta cells in said population by about 6-10%/day.

7. The method of any one of claims 1-6, wherein the DYRK1A inhibitor is selected from the group consisting of: harmine (harmine), INDY, leucetine-41, 5-iodotubercidin (5-IT), GNF4877, CC-401, a thiadiazine kinase inhibitor, and combinations thereof.

8. The method of any one of claims 1 to 7, wherein the GLP1R agonist is selected from the group consisting of: GLP1 analogs, exendin-4, liraglutide (liraglutide), lissamide (lixisenatide), somaglutide (semaglutide), and combinations thereof.

9. The process of any one of claims 1 to 6, wherein the contacting is performed using harmine and GLP1 (7-36).

10. The method of any one of claims 1 to 9, wherein the pancreatic beta cells are primary human pancreatic beta cells.

11. The method of any one of claims 1-10, wherein the contacting does not induce beta cell death or DNA damage.

12. The method of any one of claims 1-11, wherein said contacting induces beta cell differentiation.

13. The method of any one of claims 1-12, wherein the contacting increases glucose-stimulated insulin secretion.

14. A method of treating a condition associated with insulin hyposecretion in a subject, the method comprising:

administering to a subject in need of treatment for a condition associated with insufficient levels of insulin secretion a bispecific tyrphostin 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist, wherein said administering is performed under conditions effective to cause a synergistic increase in the amount of pancreatic beta cells in said subject, to treat the subject for insufficient levels of insulin secretion.

15. The method of claim 14, wherein the subject is receiving treatment for one or more of type I diabetes ("T1D"), type II diabetes ("T2D"), gestational diabetes, congenital diabetes, adult onset diabetes ("MODY"), cystic fibrosis-related diabetes, hemochromatosis-related diabetes, drug-induced diabetes, or monogenic diabetes.

16. The method of claim 15, wherein the subject is treated for type I diabetes.

17. The method of claim 15, wherein the subject is treated for type II diabetes.

18. The method of any one of claims 14 to 17, wherein the administration is nasal, oral, transdermal, parenteral, subcutaneous, intravenous, intramuscular, or intraperitoneal.

19. The method of any one of claims 14 to 18, wherein the subject is a mammalian subject.

20. The method of any one of claims 14 to 19, wherein the subject is a human subject.

21. The method of any one of claims 14-20, wherein the administration increases the number of proliferating pancreatic beta cells in the subject by about 4-6%/day.

22. The method of any one of claims 14-21, wherein the administration increases the number of proliferating pancreatic beta cells in the subject by about 6-10%/day.

23. The method of any one of claims 14-22, wherein the administration increases glucose-stimulated insulin secretion in pancreatic beta cells of the subject.

24. The method of any one of claims 14 to 23, wherein the DYRK1A inhibitor is selected from the group consisting of: harmine, INDY, leucettine-41, 5-iodotubercidin (5-IT), GNF4877, CC-401, thiadiazine kinase inhibitors, and combinations thereof.

25. The method of any one of claims 14 to 24, wherein the GLP1R agonist is selected from the group consisting of: GLP1 analogs, exendin-4, liraglutide, lisinopeptide, and combinations thereof.

26. The method of any one of claims 14 to 23, wherein the administration is performed using harmine and GLP1 (7-36).

27. A composition, comprising:

a dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) inhibitor, and

glucagon-like peptide-1receptor (GLP1R) agonists.

28. The composition of claim 27, further comprising:

and (3) a carrier.

29. The composition of claim 28, wherein the carrier is a pharmaceutically acceptable carrier.

30. A method of regenerating pancreatic beta cells in a transplant patient, the method comprising:

administering to a transplant patient a bispecific tyrphostin-regulated kinase 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist, wherein said administering is performed under conditions effective to cause a synergistic increase in the amount of pancreatic beta cells in said transplant patient, such that said pancreatic beta cells in said patient are regenerated.

31. The method of claim 30, wherein the transplant patient has undergone a pancreas transplant, islet allograft, islet autograft, or islet xenotransplant.

32. A method of treating a condition associated with insulin hyposecretion in a subject comprising:

administering to a subject in need of treatment for a condition associated with insufficient levels of insulin secretion a bispecific tyrphostin 1A (DYRK1A) inhibitor and a dipeptidyl peptidase IV (DPP4) inhibitor, wherein said administering is performed under conditions effective to cause a synergistic increase in the amount of pancreatic β cells in said subject, to treat the insufficient levels of insulin secretion in said subject.

33. The method of claim 32, wherein the subject is receiving treatment for one or more of type I diabetes ("T1D"), type II diabetes ("T2D"), gestational diabetes, congenital diabetes, adult onset diabetes ("MODY"), cystic fibrosis-related diabetes, hemochromatosis-related diabetes, drug-induced diabetes, or monogenic diabetes.

34. The method of any one of claims 32 to 34, wherein the subject is a mammalian subject.

35. The method of any one of claims 32 to 35, wherein the subject is a human subject.

36. The method of any one of claims 32-36, wherein the administration increases glucose-stimulated insulin secretion in pancreatic beta cells of the subject.

37. The method according to any one of claims 32 to 37, wherein the administering is performed using a composition comprising both the DYRK1A inhibitor and the DPP4 inhibitor.

38. The method of any one of claims 32-38, wherein the DYRK1A inhibitor is selected from the group consisting of: harmine, INDY, leucettine-41, 5-iodotubercidin (5-IT), GNF4877, CC-401, thiadiazine kinase inhibitors, and combinations thereof.

39. The method according to any one of claims 32 to 39, wherein the DPP 4inhibitor is selected from the group consisting of: sitagliptin (sitagliptin), vildagliptin (vildagliptin), saxagliptin (saxagliptin), linagliptin (linagliptin), alogliptin (alogliptin), and combinations thereof.

Technical Field

Methods of increasing cell proliferation in pancreatic beta cell populations, methods of treating conditions associated with insulin hyposecretion in a subject, and compositions comprising a bispecific tyrphostin 1A inhibitor and a glucagon-like peptide-1receptor agonist are described.

Background

Diabetes affects 4 hundred million people worldwide, with increasing prevalence (World Health Organization Global Diabetes Report (World Health Organization Global Report on Diabetes), 2016), and ultimately results from an insufficient number of functional insulin-producing Beta cells (Butler et al, "Beta Cell deficiency and Increased Beta Cell Apoptosis in people with Diabetes mellitus (Beta Cell deficiency and induced Beta Cell Apoptosis in humans with Diabetes mellitus)", "Diabetes mellitus (Diabetes mellitus) 52(1): 102-" 110(2003) and Campbell-Thpson et al, "insulin and Beta Cell mass in the Natural disease History of Diabetes mellitus of Type 1" (insulin and Beta Cell mass of Natural Diabetes mellitus of Type 1) "65 (3): 2016 (2016)).

Both type 1 and type 2diabetes ultimately result from an insufficient number of functional insulin-secreting pancreatic beta cells. Therefore, replacing human beta cell mass and function or regenerating it is a major goal of diabetes research. Unfortunately, induction of adult beta cell replication has proven challenging: after Early childhood, Human beta cells do not replicate at a therapeutically effective (Relevant) rate and have proven recalcitrant to efforts to induce their expansion in response to drugs, growth factors, nutrients or other methods (Greg et al, "Formation of Human beta Cell populations within Islets determined Early in Life" (Formation of a HumanBeta Cell Population with tissue Islets is Set Early in Life), "J.Clin.Endocrinol Metab.) (97) (3197) (2012) and Wang et al," progression and challenge in Proliferation of Human beta cells for Diabetes "(Advances and Charles Cell Proliferation efforts)," Natural evaluation of Endocrinology (Nature. Rev. Endocrinol.) (201511) (212)).

Among the most widely opened diabetic drugs in the world today are those that directly or indirectly activate the glucagon-like peptide-1receptor ("GLP 1R"). The GLP1R agonist family includes GLP1(7-36) itself, the more stable reptile homolog Exenatide (Exenatide), and modified Exenatide analogs such as liraglutide (liraglutide), linatide (lixisenatide), somaglutide (semaglutide), and others (Drucker DJ, "mechanism of Action and Therapeutic Application of Glucagon-Like Peptide-1" Cell metabolism (Cell Metab.) 27(4):740 (2018) and Guo X-H, "Short Acting and Long Acting Glucagon-Like Peptide Agonists have value in Type 2Diabetes management using Experience of Short-Acting and activating-Peptide Agonists (Exampelin. 2. Exampelin. 2. with the pharmaceutical Application of Peptide of interest (Cell metabolism and Peptide of interest). 67-76(2016)). These GLP1R agonists, and additional agents that prevent degradation of endogenous GLP1 by the enzyme Dipeptidyl Peptidase IV ("DPP 4") (examples are sitagliptin, vildagliptin, saxagliptin, and others (Drucker DJ, "mechanism of action and therapeutic application of glucagon-like peptide-1", "cell metabolism 27(4): 740. sup. (2018), and Deacon et al," Dipeptidyl Peptidase-4Inhibitors for treating Type 2Diabetes mellitus: Comparison, Efficacy, and safety (Dipeptidyl Peptidase-4Inhibitors for the Treatment of cell Type 2Diabetes mellitus: compatibility, Efficacy and safety) "," drug therapy opinion (Expert. pharmaceutical surgery 14.). Beta. 15. (2047) but no rodent cell Proliferation of Beta. Cells (Pat. 7), "rodent Proliferation of Human islet Cells for inducing Proliferation of Human islet Cells (Rat Proliferation, Rat Proliferation 2058, et al.)," Proliferation of Human insulin receptor Beta. 7 (Pat 7), "Proliferation of Rat Proliferation" ("Proliferation of Human insulin receptor Beta. 10) (Rat Proliferation). Diabetes mellitus 51(1), 91-100(2008), and Dai et al, "Age-related Human Beta cell proliferation Induced by Glucagon-Like Peptide-1 and Calcineurin Signaling (Age-Dependent Human Beta cell proliferation Induced by Glucagon-Like Peptide-1 and Calcineurin Signaling)", J.Clin.invest. (127 (10), 3835-3844 (2017)). However, the agents have a beneficial "incretin" effect, inducing beta cells to enhance insulin secretion at elevated blood glucose (Drucker DJ, "mechanism of action and therapeutic application of glucagon-like peptide-1", "cell metabolism 27(4): 740-. In the present case, while GLP1R agonists do not induce Human beta cell proliferation, GLP1R has a restricted Tissue distribution and is highly expressed in beta cells in particular (Drucker DJ, "mechanism of action and therapeutic application of glucagon-like peptide-1"; cell. metabolism 27(4):740-756 (2018); Pyke et al, "GLP 1Receptor Localization in Monkey and Human tissues: novel distribution exhibited using well-Validated Monoclonal antibodies (GLP 1Receptor Localization in Monkey and Human Tissue); Endocrinology 155(4):1280-90 (2014); and Amistens et al," Functional Analysis of protein G Receptor in Hantao island "coupled to protein metabolism 391 (protein metabolism) 359 of calcium Tissue) 3 (protein metabolism). Thus, it provides a currently unique degree of beta cell specificity.

Bispecific tyrosine-regulated kinase 1A ("DYRK 1A") is a downstream component of the Signaling cascade capable of inducing proliferation of Human and rodent beta cells according to the following scenario (Wang et al, "High-Throughput Chemical screening exhibiting Harmine-Mediated Inhibition of DYRK1A Increases Human Pancreatic beta cell Replication (A High-Throughput Chemical Screen regenerative harbor-media Inhibition of DYRK1A incorporated Human Pancreatic beta cell Replication)," Nature. Med. 21(4): 383; Gallo et al, "Lymphocyte Calcium Signaling from cell Membrane to nucleus" (Lymphocyte Calcium Signaling from Membrane of cell nucleus), "Nature. notice. 7(1): 25-32"; Human Pancreatic cell Signaling/Growth pathway receptor (NFAT) and Growth regulation of beta cell Signaling pathway NFAT-Signaling pathway and NFAT-Signaling pathway, NFAT-functional regulation of Human Pancreatic beta cell Replication (NFAT-7, 25-32, Calcium Signaling pathway and NFAT-Signaling pathway, NFAT-7, NFAT-4, and NFAT-beta cell Replication, and NFAT-7, and NFAT-4, NFAT-7, and NFAT-Signaling pathway, and NFAT-4, and NFT, nature 443(7109), 345 and 349 (2006); and Goodyer et al, "Neonatal beta cell Development in Mice and Humans is Regulated by Calcineurin/NFaT" (neuronal beta cell Development in rice and somas Regulated by Calcineurin/NFaT), "developmental cells (dev. cell) 23:21-34 (2012)). Increases in intracellular calcium (e.g., induced by glucose, drugs, etc.) cause the sequential activation of calmodulin and calcineurin, which dephosphorylates the cytoplasmic pool of a transcription factor known as nuclear factor of activated T ("NFaT"). This allows NfaT to translocate to the nucleus where it transactivates cell cycle activator genes and represses cell cycle repressor genes, causing beta cell proliferation. The nuclear kinase DYRK1A acts as a "brake" (on this process, re-phosphorylating nuclear NFaT, forcing it out of the nucleus, thereby terminating its mitogenic signal. The family of harmine analogues ("harmalog") inhibits DYRK1A, removing the "brake" of beta cell proliferation, thereby allowing cell cycle entry.

The DYRK1A inhibitor class of drugs includes harmine (Wang et al, "high-throughput chemical screening exhibiting harmine-mediated inhibition of DYRK1A increases human pancreatic beta-cell replication", "Nature medicine" 21(4):383-388(2015)), INDY (Wang et al, "high-throughput chemical screening exhibiting harmine-mediated inhibition of DYRK1A increases human pancreatic beta-cell replication", "Nature medicine" 21(4):383-388(2015)), leucitine (Tahtouh et al, "Selectivity, Co-crystalline Structures and neuroprotective properties of the Protein Kinase inhibitor Family leucitine Derived from spongine B" (Medium-crystalline Structure) and neuroprotective properties of peptides, drugs of peptides, vitamin J.9321. sub.933.12 GNF4877(Shen et al, "Inhibition of DYRK1A and GSK3B Induces Human Beta Cell Proliferation (Inhibition of DYRK1A and GSK3B indexes Human Beta Cell Proliferation)," Nature communication (Nat. Commun.) -6: 8372(2015)), 5-iodotubercidin ("5-IT") (Dirice et al, "Inhibition of DYRK1A Stimulates Human Beta Cell Proliferation (Inhibition of DYRK1A Stitulations Human Beta Cell Proliferation)," diabetes 65(6):1660-1671 (2016)); CC-401(Abdolazimi et al, "CC-401 Promotes Beta cell replication as a result of Pleiotropic Inhibition by DYRK1A/B (CC-401 proteins Beta cell replication via Pleiotropic sequences of DYRK1A/B Inhibition)," Endocrinology "159 (9):3143 3157(2018)), and others (Wang et al," high-throughput screening showing that dehydrocameline-mediated DYRK1A inhibits the increase of human pancreatic Beta cell replication "," Nature. medicine "21 (4):383-, endocrinology 159(9), 3143 and 3157 (2018); aamott et al, "developing a Reliable Automated Screening System to Identify small molecules and Biologics that Promote Regeneration of Human Beta cells (Development of a Reliable Automated Screening System to Identify Small molecules and Biologics that Promote Regeneration of Human Beta cells", journal of physiology, Endocrinology and metabolism in the United states (AJP endo. Metab.) 311: E859-68 (2016); and Wang et al, "Single Cell Mass Cytometry Analysis of human endocrine Pancreas (of human endocrine Pancreas)", "Cell metabolism 24(4):616-626 (2016)). These drugs induce proliferation of human beta cells by their ability to induce nuclear translocation of the nuclear factor of activated T cells ("NFaT"), a transcription factor that subsequently transactivates cell cycle activating genes and represses cell cycle suppressor genes (Wang et al, "high-throughput chemical screening exhibiting dehydroharmine-mediated inhibition of DYRK1A increases human pancreatic beta cell replication", "Nature. medicine" 21(4):383-388 (2015); Shen et al, "inhibition of DYRK1A and GSK3B induces human beta cell proliferation", "Nature. Med. Commun. 6:8372 (2015); and Dirice et al," inhibition of DYRK1A stimulates human beta cell proliferation "," diabetes 65(6): 1670) -1671 (2016)).

Although the peganine analog family has encouraging mitogenic capabilities, two difficulties remain. First, the observed proliferation rate in response to the harmine family was in the range of 1-3%/day as assessed by Ki67 or BrdU/EdU β cell marker index (Wang et al, "high-throughput chemical screening exhibiting harmine-mediated inhibition of DYRK1A increases human pancreatic β cell replication,", Nature & medicine 21(4): 383. cndot. 388 (2015); Shen et al, "inhibition of DYRK1A and GSK3B induces human β cell proliferation"; Nature & communications 6:8372 (2015); Dirice et al, "inhibition of DYRK1A stimulates human β cell proliferation"; diabetes 65(6): 1660. cndot. 1671 (2016); Aat et al, "development of a reliable automated screening system to identify small molecules and biologicals that promote human β cell regeneration"; U.S. journal of physiology & Metabolism & E8568: 2016 & 2016; Wark 311 and Wang et al; Wark 311; Ward et al, "Single cell mass cytometry analysis of human endocrine pancreas" [ cell metabolism ] 24(4):616-626(2016) ]. Although this mimics the physiological proliferation rate described by normal juvenile pancreas in the first year of life (Gregg et al, "formation of human Beta Cell populations within islets determined early in life"; J. Clin. Endocrinology & Metabolism 97(9):3197-3206(2012) and Wang et al, "progression and challenge in proliferation of Beta cells in humans against Diabetes"; Nature review. Endocrinology 11(4):201-212(2015)), this rate is unlikely to cause a rapid filling of Beta Cell mass in humans with Type 1Diabetes with a reduction in Beta Cell mass of 80-99% (Meier et al, "continued Beta Cell Apoptosis in patients with long-standing Type 1 Diabetes?. Indirect Evidence of islet regeneration (Sustanated Beta Cell Apoptosis with islet regeneration with Type 1 Diabetes) and Diabetes mellitus 22248 (Keyed. J. 1. Diabetes mellitus), "residual insulin production and beta cell renewal after 50years of diabetes: joslin media Study (Residual Insulin Production and Cell turnabout after 50 Yeast of Diabetes: Joslin media Study), "Diabetes" 59:2853 (2010)). Therefore, higher proliferation rates are desired. Secondly, the biological effects of the harmine family are not limited to the proliferation of beta cells: this class of drugs causes proliferation of other islet cell types (α cells, etc.) (Wang et al, "high-throughput chemical screening exhibits dehydroharmine-mediated inhibition of DYRK1A to increase human pancreatic β -cell replication"; Nature. medicine 21(4): 383-. Furthermore, Harmine is a CNS active Drug with well known psychotropic and hallucinogenic properties (Brierley et al, "Harmine pharmacological development-south america pharmacy-expressions for Ayahuasca using Drug Dependence Treatment)," neuropsychological and biopsychiatric evolution (prog. neuro-Psychopharmacol biol. psych.). 39: 263. 272(2012), and Heise et al, "south america bark Exposure: 2005. electroconchological Control center association Descriptive Analysis (Ayahuasca exposition: Descriptive Analysis of Call to US Poison Control center) (ayahuacla ex vivo: 20113: 2015. J.8.) (20113: 20113). Therefore, there is a need to identify methods to target or limit mitogenic activity of the harmine class to human beta cells, limiting off-target adverse effects.

Whether combination therapy with GLP1R agonists can further enhance the mitogenic potency of harmine class drugs has not been investigated.

The disclosure provided herein is directed to overcoming the deficiencies in the art.

Disclosure of Invention

One aspect of the disclosure relates to methods of increasing cell proliferation in a pancreatic beta cell population. This method involves contacting a population of pancreatic beta cells with a bispecific tyrphostin-regulated kinase 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist, wherein said contacting is under conditions effective to cause a synergistic increase in cell proliferation in said population of pancreatic beta cells.

Another aspect of the disclosure relates to methods of treating conditions associated with insufficient insulin secretion in a subject. This method involves administering to a subject in need of treatment of a condition associated with insufficient levels of insulin secretion a bispecific tyrphostin 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist, wherein the administration is under conditions effective to cause a synergistic increase in the amount of pancreatic beta cells in the subject, so as to treat the subject with insufficient levels of insulin secretion.

Another aspect of the disclosure relates to compositions comprising a bispecific tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist.

Yet another aspect of the disclosure relates to a method of regenerating pancreatic beta cells in a transplant patient. This method involves administering to a transplant patient a bispecific tyrphostin-regulated kinase 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist, wherein said administering is performed under conditions effective to cause a synergistic increase in the amount of pancreatic beta cells in said transplant patient, such that the pancreatic beta cells in said patient are regenerated.

Another aspect of the disclosure relates to methods of treating conditions associated with insufficient insulin secretion in a subject. This method involves administering to a subject in need of treatment of a condition associated with insufficient levels of insulin secretion a bispecific tyrphostin 1A (DYRK1A) inhibitor and a dipeptidyl peptidase IV (DPP4) inhibitor, wherein said administering is performed under conditions effective to cause a synergistic increase in the amount of pancreatic β cells in said subject, so as to treat insufficient levels of insulin secretion in said subject.

Among the most widely prescribed type 2diabetes drugs are GLP1R agonists and DPP4 inhibitors. Although it increases insulin secretion from beta cells (Reimann et al, "G protein-Coupled Receptors as novel Therapeutic Targets for Type 2Diabetes mellitus", < 6 > Diabetes mellitus > < 59(2) < 229 > 233(2016), which is incorporated herein by reference in its entirety, it fails to increase human beta cell proliferation (Drucker DJ, < 11 > mechanism of action and Therapeutic application of glucagon-like peptide-1 > < 27 > < 4 > < 740 > < 756 > < 2018 > < Parnaud et al, < proliferation of sorted and rat beta cells > < 51 > < 1 > < 91 > < 100 > < 2008 > and < 35 > age-related human beta cell proliferation induced by glucagon-like peptide-1 and calcineurin signaling </35 </10 > < 3844 > < 387 > < 3844 > < 3835 > < 35 > < 3844 >, which is incorporated herein by reference in its entirety). Small molecule inhibitors of DYRK1A represent the first class of drugs capable of inducing proliferation of adult beta cells, but at low rates (about 2%/day) (Wang et al, "high-throughput chemical screening exhibits dehydropeganine-mediated DYRK1A inhibition to increase human pancreatic beta cell replication", "Nature & medicine" 21(4):383- (2015), "Shen et al," inhibition of DYRK1A and GSK3B induces human beta cell proliferation "," Nature & communications "6: 8372(2015)," Dirice et al, "inhibition of DYRK1A stimulates human beta cell proliferation", "diabetes mellitus" 65(6): 1670 (2016); Abdolazimi et al, "CC-401 promotes beta cell replication through the pleiotropic consequences of DYRK1A/B inhibition," (159) 159 [ 9 ], "3157 (318); and" AaZizimi screening et al "automated screening for promoting regeneration of small molecules by DYRK 20138/B, journal of physiology, endocrinology and metabolism 311E 859-68 (2016); and Wang et al, "Single cell Mass Spectrometry flow cytometry analysis of the human endocrine pancreas," cell metabolism 24(4):616-626(2016), which is incorporated herein by reference in its entirety.

The following description will demonstrate that any GLP1R agonist in combination with any DYRK1A inhibitor unexpectedly induces a synergistic rate of human beta cell replication (5-6%/day) and increases the actual number of human beta cells. Synergy requires a combination of DYRK1A inhibition with cAMP increase. Treatment did not cause dedifferentiation of beta cells and provided a previously unachieved degree of human beta cell specificity. These beneficial effects extend from normal human beta cells to beta cells derived from people with type 2 diabetes. The present disclosure demonstrates that these effects are applicable not only to normal human beta cells, but also to beta cells from humans with type 2 diabetes.

As surprisingly disclosed herein, one is able to induce a "rate" or "marker index" of human beta cell replication by combining any one of a large group of currently widely used diabetes drugs that directly (GLP1 analogs) or indirectly (DPP4 inhibitors) activate GLP1R with small molecule DYRK1A inhibitors with oral activity (such as harmine, INDY, leucoettine, 5-IT, GNF4877, or others). These rates exceed the rate of DYRK1A inhibitor alone and are within the range one might expect to need to restore normal beta cell mass in humans with type 2diabetes and perhaps type 1 diabetes. An increase in human beta cell proliferation markers is accompanied by a substantial increase in the number of adult beta cells. The increase in proliferation is synergistic in a strict pharmacological sense and extends even to doses of harmine and GLP1 that have no proliferative effect on their own.

Drawings

Figures 1A-1G demonstrate that combinations of DYRK1A inhibitors with GLP1R agonists produce synergistic increases in human beta cell proliferation. Figure 1A is a graph showing the effect of indicated drug treatment on dispersed human islet cells treated over 96 hours and immuno-labeled against Ki67 and insulin. "DMSO" indicates a 0.1% concentration of control vehicle. Bars indicate mean ± SEM, and numbers within bars indicate the number of human islet preparations studied at each dose. For each slide, a minimum of 1000 beta cells were counted.^*Indication of harmine p relative to harmine alone<0.05, and^**indication of harmine p relative to harmine alone<0.001. Fig. 1B shows an example of Ki67 and the insulin immunolabeling used in fig. 1A. Fig. 1C is a graph showing a negative control, which presents measurements of FACS quantified human beta cells corresponding to a progressively decreasing number of human islets. Fig. 1D is a graph showing a second negative control, which exhibits a reduction in the number of human beta cells in response to treatment with an interleukin (1000 IU/ml each for TNF α, IL1 β, and IFN γ). Figure 1E is a graph showing the increase in the number of human beta cells within 96 hours of treatment in seven of eight human islet preparations treated with vehicle (0.1% DMSO) or a combination of harmine (10 μ M) and GLP1(5 nM). In the case of figures 1C-1E,^*indication p<0.05，^**p<0.001 and^***p<0.02. figure 1F is a graph showing the results of treatment of dispersed human islets with multiple DYRK1A inhibitors with or without GLP1 as indicated. Bars indicate mean ± SEM, and numbers within bars indicate the number of human islet preparations studied at each dose. For each slide, a minimum of 1000 beta cells were counted. In the case of figures 1F-1G,^#indicating inhibitor p relative to DYRK1A alone<0.025, and^**indication of harmine p relative to harmine alone<0.01. Note that proliferation in the case of each DYRK1A inhibitor was normalized to a value of 1, and GLP1 combinations were expressed as a function of the proliferative effects of DYRK1A inhibitors, as detailed in figure 4. The actual Ki 67% values were: harmine 2.1%, INDY 1.7%, leucoettine 2.8%, 5-IT 2.6% and GNF 47882.9%. These values are approximately two-fold when GLP 15 nM is administered. Figure 1G is a graph showing the results of treatment of dispersed human islets with harmine as indicated with or without GLP1R agonist. Data as represented in fig. 1F; the actual Ki 67% value for harmine is 2.2%, so the original Ki67 value from each GLP1R agonist is approximately twice the value for harmine. Bars indicate mean ± SEM, and numbers within bars indicate the number of human islet preparations studied at each dose. For each slide, a minimum of 1000 beta cells were counted. Indications vs harmine p alone<0.05, and^**indication of harmine p relative to harmine alone<0.03。

FIG. 2 shows the data for each donor Ki 67-insulin immunolabeling of FIGS. 1A and 3A. Figure 2 is a representation showing the proliferative response of twenty human islet donors shown in figures 1A and 3A to DMSO (0.1%), harmine (10 μ M), GLP1(5nM), or GLP 1-harmine combination. The thick black bars represent the average of the respective treatment conditions. Standard error and statistical significance are shown in figure 1A. This figure highlights the reactive variability of the difference in responsiveness of different human islet donors that is well known. It also exhibits the enhanced proliferation shown for each human islet donor in response to the harmine-GLP 1 combination.

Figures 3A-3E provide additional evidence of synergistic activation of proliferation by harmine-GLP 1 combinations. Fig. 3A is a graph showing the results of the same experiment as shown in fig. 1A and 2, adjusted such that Ki67⁺Insulin⁺cell/Total insulin⁺The percentage of immuno-labelled cells was normalized to the value in the harmine group defined as "1". This allows for the adjustment of the data sets for all experiments performed in the same human islet donor, since not every human islet preparation can be analyzed at every dose of every drug. This adjustment was used to subsequently display individual plots of Ki67 immunolabeling. Bars indicate mean ± SEM.^*Indication p<0.02 and^**indication p<0.001. The number of islet donors used in each experiment was the same as in fig. 1A. Fig. 3B is a graph showing incorporation of BrdU into beta cells in dispersed islets in response to the conditions shown. BrdU was added to the culture 18 hours before fixation. Bars indicate mean ± SEM. The numbers in or above the bars indicate the number of human islet donors used for each condition. Figure 3C is an image showing an example of BrdU incorporation in human islets treated with a control vehicle or 10 μ M harmine-5 nMGLP1 combination. Figures 3D-3E are graphs showing the same type of experiment as shown in figures 1A and 3A, but using lower doses, 3 μ M (figure 3D) and 1 μ M (figure 3E) harmine. Also, it is evident to note the clear synergy, especially in fig. 3E, where harmine and GLP1 induce little or no proliferation, butThe combination induced proliferation (2.7%) to the same extent as the 10 μ M peganine dose. Bars indicate mean ± SEM. The numbers in or above the bars indicate the number of human islet donors used for each condition.^*Indication p<0.05 and^**indication p<0.01。

Figure 4 is a graph showing unadjusted data for small molecules of the DYRK1A inhibitor family used alone or in combination with GLP 1. These are the same data shown in figure 1C, shown to enable visualization of the effect of each DYRK1A inhibitor alone or in combination with 5nM GLP 1.

Figures 5A-5H demonstrate that peganine-GLP 1 synergy requires inhibition of DYRK1A and an increase in beta cell cAMP. FIGS. 5A-5C are graphs showing the results of experiments in which agents that increase cAMP (forskolin), dibutyryl cAMP and the phosphodiesterase inhibitors Isobutylmethylxanthine (IBMX) and dipyridamole (DPD) have no effect on proliferation when administered alone but may replace GLP1 when administered together with banglastrine FIG. 5D is a graph showing the results of experiments in which PKA inhibitor H89 does not exert an effect on itself but blocks the synergistic proliferation induced by banglastrine and GLP1, thus, the synergistic portion is mediated by PKA. additional evidence for the synergistic effects of PKA and EPAC is seen in FIGS. 5F-5H FIG. 5E is a graph showing the effect of PKA activator 6-bnz-cAMP on bangladine induced proliferation of human beta cells FIG. 5F is a graph showing the effect of EPAC 36 8-CPT-3632-cAMP induced proliferation and the graph of EPAC sorting of human beta cells in EPAC2 FACS is a graph showing the effect of EPAC2 Is a map of the most abundant EPACs among other islet Cell types (see, RNA sequencing data from Wang et al, "insight into the Regeneration of Human Beta cells against Diabetes obtained by Integration of molecular profiles in Human Insulinomas (Insights inter Human Beta Cell Regeneration for Diabetes mellitus of the Integration of molecular Landscapes in Human insulin omas.)" (Nature. communications 8(1):767 (2017)), which is incorporated herein by reference in its entirety). In contrast, EPAC1 is only slightly detectable. Figure 5H is a graph showing the effect of EPAC2 inhibitor ESI-05 on harmine combinations. In fig. 5E, 5F and 5H, bars indicate mean ± SEM. The numbers in or above the bars indicate the number of human islet donors used for each condition.

FIGS. 6A-6F show additional information about the mechanism of synergy of DYRK1A-GLP 1. Fig. 6A is a graph showing the results of experiments in which dispersed human islets were treated as indicated. "GLP 1" indicates 5nM GLP1, "Har" indicates harmine 10 μ M, "con.sh" indicates a control adenovirus expressing shRNA against β -galactosidase, and "DYRK 1A-sh" indicates an adenovirus expressing shRNA against DYRK1A as described previously (Wang et al, "high-throughput chemical screening exhibits harmine-mediated inhibition of DYRK1A increases human pancreatic β -cell replication", "nature medicine" 21(4): 383-2015388 (which is incorporated herein by reference in its entirety) and fig. 5E-5G and 6D-6F (below). Note that adenoviral silencing DYRK1A achieved the same GLP1 synergy observed with GLP1 and harmine. Fig. 6B is a diagram showing the inverse experiment of fig. 6A. Here, DYRK1A cDNA or control (Cre) was overexpressed in human islets in an adenovirus manner (CMV promoter). Note that DYRK1A overexpression blocks the effect of both harmine and harmine-GLP 1 combination. Fig. 6C shows an example of the immunolabeling of insulin and Ki67 in fig. 6A-6B. FIG. 6D is a graph showing the results of quantitative PCR display of adenovirus overexpression of DYRK1A in four dispersed human islets. DYRK1A silencing increased DYRK1A mRNA expression by approximately 25-fold. Fig. 6E shows immunohistochemistry demonstrating DYRK1A overexpression at the protein level in β cells in response to ad. FIG. 6F is a graph showing that adenoviral silencing of DYRK1A in human islets reduces expression of DYRK1A in human islets by about 80%. Bars indicate mean ± SEM. The numbers in or above the bars indicate the number of human islet donors used for each condition.^*Indication p<0.01. In all figures, bars indicate mean ± SEM, and the number of isolated human islets is shown within the bar. p values are as indicated. As in the previous figures, the values of harmine are normalized to 1.0 and the other values are expressed as a function of said values.

FIGS. 7A-7B show the evaluation of vehicle, 5nM GLP1, 10. mu.M harmine and harmine-GLP 1 combinations on cell cycle molecules by qPCRInfluence. Fig. 7A is a graph showing the effect on cell cycle activators 72 hours after exposure to four conditions. Figure 7B shows the effect on cell cycle inhibitors on the same samples. For p15^INK4、p16^INK4、p18^INK4、p19^INK4、p21^CIP、p27^CIPAnd p57^KIPThe gene names of the cell cycle inhibitors are CDKN2B, CDKN2A, CDKN2C, CDKN2D, CDKN1A, CDKN1B, and CDKN1C, respectively. Bars indicate mean ± SEM. The numbers in or above the bars indicate the number of human islet donors used for each condition.^*Indication p<0.05 and^**indication p<0.005。

Figures 8A-8C show that harmine-GLP 1 treatment maintained or enhanced human beta cell differentiation. Figure 8A is a graph showing the effect of control vehicle (DMSO, 0.1%), GLP 15 nM, harmine 10 μ M, or a combination on markers of beta cell differentiation as assessed using qPCR. Bars indicate mean ± SEM, and numbers within or above the bars indicate the number of human islet preparations studied under each condition.^*Indication of treatment p relative to control vehicle<0.05, and^**indication of treatment p relative to control vehicle<0.008. Figure 8B shows images of beta cells immunolabeled against PDX1, MAFA, and NKX6.1 from the experiment shown in figure 8A. Note that in the case of harmine or combination, each is increased in the beta nucleus. Representative experiments in four human islet preparations. Figure 8C is a graph showing insulin secretion from human islets from four different donors in response to low (2.8mM, gray bars) and high (16.8mM, black bars) glucose after 72 hours of treatment with vehicle, GLP1(5nM), harmine (10 μ M), or combination. Data are expressed as fold increase in insulin following high glucose stimulation. Insulin concentrations (mean. + -. SEM) were 19.9. + -. 9.1 pmol/islet in 2.8mM glucose control (DMSO) wells and 33.3. + -. 12.6 pmol/islet at 16.7mM glucose.

Figures 9A-9E show the effect of harmine-GLP 1 combinations on beta cells from humans with type 2diabetes ("T2D"). FIG. 9A is a graph showing harmine pair with and without GLP1 as shown in FIG. 8A for the same differentiationGraph of the effect of the marker. Harmlessness of harmine or harmine-GLP 1 combination to differentiation. Indeed, it appears to increase PDX1, MAFB, NKX6.1, GLUT2, GLP1R and PCSK1 in islets from humans with T2D. Bars indicate mean ± SEM, and numbers within or above the bars indicate the number of human islet preparations studied under each condition.^*Indication of treatment p relative to control vehicle<0.05, and^**indication of treatment p relative to control vehicle<0.008. Fig. 9B presents images showing examples of PDX1, MAFA, and NKX6.1 immunolabeling human T2D islets after the indicated treatment. Note that all three increased at the protein level within the beta cells. For MAFA, even though there was no increase in mRNA levels in fig. 9A, the increase was apparent. Figure 9C is a graph showing insulin secretion in three different T2D islet preparations that were pre-treated with vehicle, GLP1, harmine, or combination for 72 hours in response to low (2.8mM, gray bars) and high (16.8mM, black bars) glucose. Data are expressed as fold increase in insulin following high glucose stimulation. The mean insulin concentration was 18.1. + -. 3.2 pmol/islet in 2.8mM glucose control (DMSO) wells and 32.2. + -. 4.6 pmol/islet at 16.7mM glucose. Error bars indicate mean ± SEM.^*Indicating p relative to low glucose<0.01，^**Indicating high glucose response p relative to vehicle treatment<0.02. Figure 9D is a graph showing cell proliferation in response to vehicle, GLP1, harmine, or combination human T2D β. Bars indicate mean ± SEM, and numbers within or above the bars indicate the number of human islet preparations studied under each condition.^*Indication of treatment p relative to vehicle<0.01, and^**p is indicated as 0.02 relative to harmine alone. Figure 9E shows examples of insulin and Ki67 immunolabeling in beta cells derived from donors with T2D.

Figures 10A-10D show the effect of harmine-GLP 1 combinations on proliferation of non-beta cells and on beta cell death and DNA damage. FIG. 10A is a graph showing β (INS) in response to the processing shown in the inset⁺)、α(GCG⁺)、(SST⁺) And a conduit (CK 19)⁺) In cells, e.g., using BrdUMap of assessed proliferation was labeled. Note that peganine activates proliferation of all four cell types, as previously reported (Wang et al, "high-throughput chemical screening exhibits peganine-mediated DYRK1A inhibition increases human pancreatic beta cell replication", "nature & medicine" 21(4): 383) -388(2015), which is incorporated herein by reference in its entirety), and GLP1 potentiates this proliferation of GLP1 receptor-containing beta cells and ductal cells. Bars indicate mean ± SEM. The numbers below the bars indicate the number of human islet donors used for each condition. Fig. 10B shows an example of BrdU immunolabeling in human islet cell subtypes in response to the indicated agents. Figure 10C is a graph showing the effect of harmine-GLP 1 combinations on cell death as assessed by TUNEL analysis. The cytokine cocktail in the second form was a positive control and contained IFN γ, TNF α and IL1 β as described in the examples (below). Bars indicate mean ± SEM. The numbers within the bars indicate the number of human islet donors used for each condition. Fig. 10D shows an example of TUNEL reaction to the conditions shown in fig. 10C.

Detailed Description

Disclosed are methods of increasing cell proliferation in a pancreatic beta cell population, methods of treating conditions associated with insulin hyposecretion in a subject, and compositions comprising a bispecific tyrphostin 1A inhibitor and an agent that increases glucagon-like peptide-1receptor activity.

One aspect relates to a method of increasing cell proliferation in a pancreatic beta cell population. The method involves contacting a population of pancreatic beta cells with a bispecific tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor and an agent that increases glucagon-like peptide-1receptor (GLP1R) activity, wherein the contacting is performed under conditions effective to cause a synergistic increase in cell proliferation in the population of pancreatic beta cells. Suitable agents that increase GLP1R activity are described below and include, but are not limited to, GLP1R agonists and DPP4 inhibitors.

In performing this and other methods described herein, the pancreatic beta cells can be mammalian cells. Mammalian cells include cells from, for example: mice, hamsters, rats, cows, sheep, pigs, goats, horses, monkeys, dogs (e.g., Canis familiaris), cats, rabbits, guinea pigs, and primates, including humans. For example, the cell may be a human pancreatic beta cell.

According to one embodiment, the "pancreatic beta cells" are primary human pancreatic beta cells.

In one embodiment, the method and other methods described herein are performed ex vivo or in vivo. When performed ex vivo, the cell population may be provided by obtaining cells from the pancreas and culturing the cells in a liquid medium suitable for culturing mammalian cells (particularly human cells) in vitro or ex vivo. By way of example, but not limitation, suitable and non-limiting media can be based on commercially available media, such as RPMI1640 from Invitrogen.

Methods of determining whether a cell has a pancreatic beta cell phenotype are known in the art and include, but are not limited to, incubating the cell with glucose and testing whether insulin expression is increased or induced in the cell. Other methods include testing for the expression of beta cell-specific transcription factors, detection of beta cell-specific gene products by means of RNA quantitative PCR, transplantation of candidate cells in diabetic mice and subsequent testing of the physiological response after said transplantation, and analysis of the cells with a microscope.

Several DYRK1A inhibitors from natural sources and small molecule drug discovery programs have been identified and characterized. Suitable DYRK1A inhibitors include, but are not limited to, harmine (harmane), INDY, leucostatin-41, 5-iodotubercidin (5-IT), GNF4877, harmine analogs, CC-401, thiadiazine kinase inhibitors, and others. Other suitable DYRK1A inhibitors include, but are not limited to, GNF7156 and GNF6324(Shen et al, "inhibit DYRK1A and GSK3B induce human beta cell proliferation," nature communication 6:8372(2015), which is incorporated herein by reference in its entirety). In carrying out the methods of the present invention or forming the compositions of the present invention, a combination of DYRK1A inhibitors may be used. Among all DYRK1A Inhibitors, harmine and its analogs (β -carboline) are the most widely studied of the Inhibitors covered so far and are still the most potent and orally bioavailable class (Becker et al, "Activation, Regulation, and Inhibition of DYRK 1A" (Activation, Regulation, and Inhibition of DYRK1A), "european journal of the biochemical association (FEBS J.) (278 (2): 246-) -256(2011) and Smith et al," Recent Advances in the design, Synthesis, and Biological Evaluation of Inhibitors of DYRK 1A?, a new pathway for Disease-regulating Treatment of Alzheimer's Disease? (Recent Advances in the design, Synthesis, and Biological Evaluation of regulatory therapy of Alzheimer's Disease?, and Biological Evaluation of regulatory mechanisms of DYRK1A, analytical science, and Biological Evaluation of Regulation of neuro? (attention is cited in 3), and is incorporated by new neuro's study, 3.

In addition to harmine, natural products thereof which have been shown to inhibit DYRK1A and other kinases are EGCg and other flavan-3-ols (Guedj et al, "Green tea polyphenols Rescue Brain Defects Induced by The Overexpression of DYRK1A (Green tea polyphenols of Brain Defects Induced by fermentation of DYRK1A)," public science library Integrated services (PLoS One) 4(2): e 6(2009) and Baikin et al, "specificity of Protein Kinase Inhibitors: renewal (The Specificities of Protein Kinase Inhibitors: upstate:" Anertdate), "Biochemical engineering sponges (biochem. J.). 371.) (199) (204) (2003) which are incorporated herein by reference in their entirety," chemoprotectant proteins derived from biochem family of biochem, Tauch et al, "chemoprotectant proteins derived from biochem family 933, and Leete Kinase family 933, 2" (933) 2, and "chemoprotectant family of neurokinase Inhibitors of drugs such as Leucokinase, Tauch 1, 933, 21, and Leete family of chemo family, 933, 2, "leucetine L41, a DYRK1A-preferential DYRK/CLK Inhibitor, Prevents Memory impairment and neurotoxicity Induced by Administration of Oligomeric A β 25-35 peptides in Mice (leucetine L41, a DYRK1A-preferential developmental DYRKs/CLKs Inhibitor, Prevents Memory impairements and chemotherapeutic Induced by Oligomeric A β 25-35 Peptide)," European neuropsychologic (Eur. neuropsychoPharmacol.) 25(11): 0. sup.) "2182 (2015), which is incorporated herein by reference), quinone (quinine) (cozzza et al," Potent inhibitors as Kinase CK2, and Selective inhibitors of Cell permeable quinine 395 (calcium citrate) incorporated herein by reference), biological inhibitors of alizarin (calcium citrate 395, calcium citrate) 3, incorporated herein by reference, (calcium citrate 3, calcium citrate 387) 3, which are incorporated herein by reference, biological inhibitors of calcium citrate 3 and calcium citrate 387 (calcium citrate) 2, which are incorporated herein by reference, and which are incorporated herein by reference, in the title 3,2, a method of "method of inducing inhibition of intracellular inhibition by reference," calcium Kinase 3, which is incorporated herein by reference, "calcium citrate" The Two novel lignin analogs from Acacia nilotica with Kinase Inhibitory Activity (Two novel lignin analogs from Acacia nilotica with Kinase Inhibitory Activity) (plant Med.) (76 (5) 458-460(2010), incorporated herein by reference in its entirety), benzocoumarin (dNBC) (Sarno et al, "basic Structural feature of Selectivity for Kinase inhibitors NBC and dNBC": Nitro Group distinguishes the Role of CK2and DY 1A (Structural feltes undermining the Selectivity of the Kinase inhibitors NBC and dBC) (cell of Nitro Group of the family of KinaseInhibins NBC and dBC) incorporated herein by reference in their entirety as well as the Biocide molecules (RK 460 ) 52 (Biocarbazole), incorporated herein by reference in its entirety Butterfly's mycin (rebeccamycin) and its analogs) (Sanchez et al, "Generation of Potent and Selective kinase inhibitors by the Combinatorial Biosynthesis of Glycosylated Indolocarbazoles by Synthesis of Glycosylated Indocalamus", Chem. Commun. 27:4118-4120(2009), which is incorporated herein by reference in its entirety.

Among other backbone structures (scaffolds) identified by small molecule drug discovery attempts, the following showed potent DYRK1A activity with varying degrees of kinase selectivity: INDY (Ogawa et al, "Development of Novel Selective inhibitors of Down syndrome-Related Kinase Dyrk 1A" (Development of a Novel Selective Inhibitor of the Down synthetic-Related Kinase Dyrk1A), "Nature communication" 1: article No. 86(2010), incorporated herein by reference in its entirety), DANDY (Gourdain et al, "Development of Novel 3,5-Diaryl-7-azaindole DANDY exhibiting Potent DYRK1A Kinase Inhibitory Activity" (Development of DANDY, New 3,5-Diaryl-7-azaindole DANDY), pharmaceutical chemistry (56) (23) 9585 (2019585) 9585 (201953), incorporated herein by reference, and "Selective Inhibition of the Folding of the protein Kinase by the protein Kinase DYRK1 (derived from the protein of the protein Kinase) 1" (Development of pharmaceutical chemistry, 56 (4623) 9585 (2019556) (incorporated herein by reference) and "Selective Inhibition of the Folding of the protein Kinase by the protein RK1 (derived from the protein Kinase K1, 5929)", pharmaceutical chemistry (Development of the protein Kinase, incorporated herein by reference in its entirety), nature. Comm 7:11391(2016), incorporated herein by reference in its entirety), Pyrazolidine-Diones (Koo et al, "QSAR Analysis of Pyrazolidine-3,5-dione Derivatives as Dyrk1A Inhibitors" (QSAR Analysis of Pyrazolidine-3,5-Diones Derivatives ASDyrk1A Inhibitors), "Bioorg. Chem. chem. Lett.) -19 (8):2324-2328 (2009); kim et al, "Putative therapeutics for Learning and Memory Deficits in people with Down Syndrome" (biological organic chemistry and medicinal chemistry communications "16 (14): 3772-3-3776 (2006) incorporated herein by reference in its entirety), amino-quinazolines (Rosenthal et al," Cdc2-Like kinase (Clk), and Specific Isoforms of Dual Specific Tyrosine Phosphorylation regulated Kinases (Dyrk) "Potent and Selective Small Molecule Inhibitors (patent and Selective Small Molecule Inhibitors of protein Kinases of Cdc2-Like kinase (Clk) and Dual Specificity-protein Phosphorylation-regulation kinase (3121) incorporated herein by reference in its entirety, biological chemistry and medicinal chemistry 3121 (3121) incorporated herein by reference in its entirety, Merlins (meriolines) (Giraud et al, "Synthesis of Derivatives of merlins, Protein Kinase Inhibitory Potencies, and in Vitro Antiproliferative Activity (Synthesis, Protein Kinase Inhibitory Potencies, and Andin Vitro Inhibitory Activities of Meridianin Derivatives)", "journal of pharmaceutical chemistry 54(13):4474-4489 (2011); Echalier et al," merlins (3- (Pyrimidin-4-yl) -7-azaindole): Synthesis, Kinase Inhibitory Activity, Cellular Effects, and CDK2/Cyclin A/merlins Complex Structure (Melilins (3- (pyrindin-4-yl) -7-Azaindoles): Synthesis, Kinase Inhibitory Activity, Cellular Effects, and CDK2/Cyclin A/merlins Complex Structure (merins (3- (pyridodin-4-yl) -7-Azaindoles): Synthesis, Kinase Inhibitory visity, Lular Effects, tissue, Struture of Cyclin 2/Cyclin A737.), (2008-2008; and Ak et al.), "Synthesis and Biological Activity of Aminopyrimidinyl-Indoles Structurally Related to Merridins" (Synthesis and Biological Activities of amino-indole structures Related to Merridins), "Bio-organic chemistry and medicine chemistry (bioorg. Med. chem.) -17 (13):4420-4424(2009), which is incorporated herein by reference in its entirety), Pyridine and Pyrazine (Kassis et al," Synthesis and Biological evaluation of New 3- (6-hydroxyindol-2-yl) -5- (Phenyl) Pyridine or Pyrazine V-shaped molecules as Kinase Inhibitors and Cytotoxic Agents "(Synthesis and Biological evaluation of New 3- (6-hydroxy-indol-2-yl) -5- (Phenyl) Pyridine or Pyrazine V-shaped molecules)," Synthesis and Biological assays of amino-pyrimidine V-indole-5- (Phenyl) Pyridine or Pyrazine V-shaped molecules (European J5434.),46), which is incorporated herein by reference in its entirety), benzopyranoindoles (chromenoids) (Neagoie et al, "synthesis of benzopyrano [3,4-b ] indoles as lamellarin D analogs: a Novel Class of DYRK1A inhibitors (Synthesis of Chromeno [3,4-b ] indole asLamellarin D antibodies: A Novel DYRK1A Inhibitor Class), "European journal of medicinal chemistry 49:379-396(2012), which is incorporated herein by reference in its entirety), 11H-indolo [3,2-c ] quinoline-6-carboxylic acid, Thiazolo [5,4-f ] quinazoline (EHT 5372) (foucott et al, "Design and Synthesis of Thiazolo [5,4-f ] quinazoline as a DYRK1A inhibitor, Part I (Design and Synthesis of Thiazolo [5,4-f ] quinazolines asDYRK1A Inhibitors, Part I.)", "Molecules (Molecules) 19(10):15546 15571(2014) and Coutadeur et al, "novel DYRK1A (bispecific tyrosine phosphorylation regulated kinase 1A) inhibitors for the treatment of Alzheimer's disease: in vitro effects on tau and amyloid lesions (A Novel DYRK1A (Dual specific tyrosine Phosphorylation-Regulated Kinase 1A) Inhibitor for the Treatment of Alzheimer's Disease: effect on Tau and Amyloid Pathology In Vitro) "," J.neurochem. (133) (3):440-451(2015), incorporated herein by reference in its entirety) and 5-iodotubercidin (Dirice et al, "inhibition of DYRK1A stimulates human beta cell proliferation", diabetes 65(6):1660-1671(2016) and Annes et al, "Inhibition of Adenosine Kinase Selectively Promotes beta cell Replication in rodents and Porcine islets (Adenosine Kinase Inhibition selection promoters and Islet beta-cell Replication)," 109(10) at Proc. Natl. Acad. Sci.): 3915-.

Suitable thiadiazine kinase inhibitors include, for example, but are not limited to, the inhibitors described in PCT application No. PCT/US2018/062023 filed on 11/20/2018, which is incorporated herein by reference in its entirety. Specific examples include the inhibitors shown in tables 1 and 2.

TABLE 1 Thiadiazine kinase inhibitors

TABLE 2 other thiadiazine kinase inhibitors

As previously described, glucagon-like peptide-1receptor agonists mimic the effect of the incretin hormone GLP-1 released from the intestine in response to food intake. The effects of the agonists include increased insulin secretion, decreased glucagon release, increased satiety, and slowed gastric emptying.

GLP1R agonists suitable for performing the disclosed methods include, but are not limited to, exenatide, liraglutide, exenatide LAR, tasaglutide, lissamide, albiglutide, dulaglutide, and somaglutide. Exenatide and exenatide LAR are synthetic exendin-4 analogs of saliva obtained from heloderma suggestum (lizard). Liraglutide is a GLP-1 acylated analogue that self-assembles into a heptameric structure that delays absorption from the subcutaneous injection site. Tassellutide has 3% homology with native GLP-1 and is completely resistant to DPP-4 degradation. The linatide is a human GLP1R agonist. Abiroglutide is a long-acting GLP-1 mimetic that is resistant to DPP-4 degradation. Dolastatin is a long-acting GLP1 analog. Somaglutide is a GLP1R agonist approved for use in T2D. Clinically useful GLP1R agonists include, for example, exenatide, liraglutide, albiglutide, dulaglutide, lissamide, somaglutide.

In some embodiments, the GLP1R agonist is selected from the group consisting of: GLP1(7-36), exendin-4, liraglutide, lissamide, somaglutide, and combinations thereof.

Other suitable GLP1 agonists include, but are not limited to, disubstituted 7-aryl-5,5-bis (trifluoromethyl) -5,8-dihydropyrimido [4,5-d ] pyrimidine-2,4(1H,3H) -dione compounds and derivatives thereof, such as 7- (4-chlorophenyl) -1, 3-dimethyl-5, 5-bis (trifluoromethyl) -5,8-dihydropyrimido [4,5-d ] pyrimidine-2,4(1H,3H) -dione (see, e.g., Nance et al, "based on 1,3-disubstituted-7-aryl-5,5-bis (trifluoromethyl) -5,8-dihydropyrimido [4,5-d ] pyrimidine-2,4(1H,3H) a Series of Novel Orally bioavailable and CNS-penetrating Glucagon-like Peptide-1Receptor (GLP-1R) noncompetitive antagonists of the diketone Core (Discovery of a Novel Series of organic bioavailablelableend CNS Peptide glucose-like Peptide-1R) -noncompetitive Peptide-1Receptor (GLP-1R) noncompetent antibodies Based on a1, 3-disubstuted-7-aryl-5, 5-bis (trifluoromethyl) -5, 8-dihydrazide [4,5-d ] pyridine-2, 4(1H,3H) -dione) "," journal of pharmaceutical chemistry 60:1611 cori 6(2017), which is incorporated herein by reference in its entirety).

Other suitable GLP1 agonists include positive allosteric modulators of GLP1R ("PAMS"), such as (S) -2-cyclopentyl-N- ((1-isopropylpyrrolidin-2-yl) methyl) -10-methyl-1-oxo-1, 2-dihydropyrazino [ l,2-a ] indole-4-carboxamide; (R) -2-cyclopentyl-N- ((l-isopropylpyrrolidin-2-yl) methyl) -10-methyl-1-oxo-1, 2-dihydropyrazino [ l,2-a ] indole-4-carboxamide; 2-cyclopentyl-N- (((S) -1-isopropylpyrrolidin-2-yl) methyl) -10-methyl-1-oxo-1, 2,3, 4-tetrahydropyrazino [1,2-a ] indole-4-carboxamide; n- (((S) -1-isopropylpyrrolidin-2-yl) methyl) -10-methyl-1-oxo-2- ((S) -tetrahydrofuran-3-yl) -l, 2-dihydropyrazino [ l,2-a ] indole-4-carboxamide; n- (((R) -1-isopropylpyrrolidin-2-yl) methyl) -10-methyl-1-oxo-2- ((S) -tetrahydrofuran-3-yl) -l, 2-dihydropyrazino [ l,2-a ] indole-4-carboxamide; (S) -2-cyclopentyl-8-fluoro-N- ((l-isopropylpyrrolidin-2-yl) methyl) -10-methyl-1-oxo-1, 2-dihydropyrazino [ l,2-a ] indole-4-carboxamide; (R) -2-cyclopentyl-8-fluoro-N- ((1-isopropylpyrrolidin-2-yl) methyl) -10-methyl-1-oxo-1, 2-dihydropyrazino [ l,2-a ] indole-4-carboxamide; (R) -2-cyclopentyl-N- (((S) -1-isopropylpyrrolidin-2-yl) methyl) -10-methyl-1-oxo-1, 2,3, 4-tetrahydropyrazino [1,2-a ] indole-4-carboxamide; (S) -2-cyclopentyl-N- (((S) -1-isopropylpyrrolidin-2-yl) methyl) -10-methyl-1-oxo-1, 2,3, 4-tetrahydropyrazino [1,2-a ] indole-4-carboxamide; (S) -10-chloro-2-cyclopentyl-N- ((l-isopropylpyrrolidin-2-yl) methyl) -1-oxo-1, 2-dihydropyrazino [ l,2-a ] indole-4-carboxamide; (R) -10-chloro-2-cyclopentyl-N- ((1-isopropylpyrrolidin-2-yl) methyl) -1-oxo-1, 2-dihydropyrazino [ l,2-a ] indole-4-carboxamide; (S) -10-bromo-2-cyclopentyl-N- ((l-isopropylpyrrolidin-2-yl) methyl) -1-oxo-1, 2-dihydropyrazino [ l,2-a ] indole-4-carboxamide; (R) -10-bromo-2-cyclopentyl-N- ((1-isopropylpyrrolidin-2-yl) methyl) -1-oxo-1, 2-dihydropyrazino [ l,2-a ] indole-4-carboxamide; (R) -N- ((l-isopropylpyrrolidin-2-yl) methyl) -10-methyl-1-oxo-2-phenyl-1, 2-dihydropyrazino [ l,2-a ] indole-4-carboxamide; (S) -10-cyano-2-cyclopentyl-N- ((1-isopropylpyrrolidin-2-yl) methyl) -1-oxo-1, 2-dihydropyrazino [ l,2-a ] indole-4-carboxamide; (S) -2-cyclopentyl-N- ((1-isopropylpyrrolidin-2-yl) methyl) -1-oxo-10-vinyl-1, 2-dihydropyrazino [ l,2-a ] indole-4-carboxamide; (S) -N- ((1-isopropylpyrrolidin-2-yl) methyl) -10-methyl-2- (l-methyl-lH-pyrazol-4-yl) -1-oxo-1, 2-dihydropyrazino [1,2-a ] indole-4-carboxamide; (R) -N- ((l-isopropylpyrrolidin-2-yl) methyl) -10-methyl-2- (1-methyl-lH-pyrazol-4-yl) -1-oxo-1, 2-dihydropyrazino [1,2-a ] indole-4-carboxamide; (S) -N- ((l-isopropylpyrrolidin-2-yl) methyl) -10-methyl-l-oxo-2- (pyridin-3-yl) -l, 2-dihydropyrazino [ l,2-a ] indole-4-carboxamide; (R) -N- ((l-isopropylpyrrolidin-2-yl) methyl) -10-methyl-1-oxo-2- (pyridin-3-yl) -1, 2-dihydropyrazino [ l,2-a ] indole-4-carboxamide; n- (azetidin-2-ylmethyl) -2-cyclopentyl-10-methyl-1-oxo-1, 2-dihydropyrazino [1,2-a ] indole-4-carboxamide; and 2-cyclopentyl-N- ((1-isopropylazetidin-2-yl) methyl) -10-methyl-1-oxo-1, 2-dihydropyrazino [ l,2-a ] indole-4-carboxamide; or a pharmaceutically acceptable salt thereof (see PCT publication No. WO 2017/117556, which is incorporated herein by reference in its entirety).

In performing the methods described herein, a population of pancreatic beta cells is contacted with a bispecific tyrphostin-regulated kinase 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist.

Contacting a population of pancreatic beta cells with a bispecific tyrphostin 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist can be performed using harmine and GLP1 (7-36).

Contacting a population of pancreatic beta cells with a bispecific tyrphostin-regulated kinase 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist can be performed using harmine and N- (4-fluorobenzyl) -5- (benzo [ d ] imidazol-2 (3H) -one) -6H-1,3, 4-thiadiazin-2-amine.

Contacting a population of pancreatic beta cells with a bispecific tyrphostin 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist can be performed using a single composition comprising both a DYRK1A inhibitor and a GLP1R agonist. Alternatively, contacting a population of pancreatic beta cells with a bispecific tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist can be performed sequentially (serially). For example, a population of pancreatic beta cells can be first contacted with a bispecific tyrphostin 1A (DYRK1A) inhibitor (or a composition comprising a bispecific tyrphostin 1A (DYRK1A) inhibitor) and subsequently contacted with a glucagon-like peptide-1receptor (GLP1R) agonist (or a composition comprising a glucagon-like peptide-1receptor (GLP1R) agonist); or first with a glucagon-like peptide-1receptor (GLP1R) agonist (or a composition thereof) and then with a bispecific tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor (or a composition thereof).

In performing the methods described herein, contacting a population of pancreatic beta cells with a bispecific tyrphostin-regulated kinase 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist can be performed as follows: multiple times a day, once per week, twice per week, once per month, once in two months, once per year, once in half a year, or any amount of time in between. The DYRK1A inhibitor and the glucagon-like peptide-1receptor (GLP1R) agonist may be administered at different administration frequencies. Contacting the pancreatic beta cell population with a DYRK1A inhibitor and a GLP1R agonist can be performed short-term or long-term. For example, the contacting can be conducted for an extended period of time over a period of 1 year, 2 years, 3 years, 4 years, or longer. In some embodiments, administration is performed infrequently.

Contacting a population of pancreatic β cells with a bispecific tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist can be performed to increase the number of proliferating pancreatic β cells in the population by at least about 4%, 5%, 6%, 7%, 8%, 9%, 10% or more.

Contacting a population of pancreatic beta cells with a bispecific tyrphostin-regulated kinase 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist can be performed such that the number of proliferating pancreatic beta cells in the population is increased by about 4-10%/day, or about 4-6%/day, 5-7%/day, 6-9%/day, or 7-10%/day.

Contacting a population of pancreatic β cells with a bispecific tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist increases the number of proliferating pancreatic β cells in the population by about 6-10%/day.

The method of contacting a population of pancreatic β cells with a bispecific tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist can be performed under conditions effective to cause a synergistic increase in cell proliferation in the population of pancreatic β cells, the synergistic increase meaning an increase in the number of proliferating pancreatic β cells in the population, particularly as compared to when the cells are contacted with a DYRK1A inhibitor or a GLP1R agonist alone, or when the cells are not contacted with either a DYRK1A inhibitor or a GLP1R agonist.

In carrying out this and other methods, contacting a population of pancreatic beta cells with a bispecific tyrphostin-regulated kinase 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist may not induce beta cell death or DNA damage in the population of cells. In addition, the contact may induce beta cell differentiation and increase glucose-stimulated insulin secretion.

The method may be performed to enhance cell survival. For example, the methods can be performed to enhance cell survival of a treated pancreatic beta cell population relative to an untreated pancreatic beta cell population. Alternatively, the method can be performed to reduce cell death or apoptosis of a contacted pancreatic β cell population relative to an uncontacted pancreatic β cell population.

Another aspect relates to a method of treating a condition associated with insufficient insulin secretion in a subject. This method involves administering to a subject in need of treatment of a condition associated with insufficient levels of insulin secretion a bispecific tyrphostin 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist, wherein the administration is under conditions effective to cause a synergistic increase in the amount of pancreatic beta cells in the subject, so as to treat the subject with insufficient levels of insulin secretion.

Another aspect of the disclosure relates to methods of treating conditions associated with insufficient insulin secretion in a subject. This method involves administering to a subject in need of treatment of a condition associated with insufficient levels of insulin secretion a bispecific tyrphostin 1A (DYRK1A) inhibitor and a dipeptidyl peptidase IV (DPP4) inhibitor, wherein said administering is performed under conditions effective to cause a synergistic increase in the amount of pancreatic β cells in said subject, so as to treat insufficient levels of insulin secretion in said subject.

As used herein, a condition associated with insufficient levels of insulin secretion means a condition in which the subject produces lower plasma insulin levels than are required to maintain normal glucose levels in the blood, such that a subject with a condition associated with insufficient insulin secretion becomes hyperglycemic. In this condition, the level of insulin secreted by pancreatic beta cells in a diseased subject is insufficient to maintain normal concentrations of glucose present in the blood (i.e., normoglycemia).

One of the pathologies associated with an insufficient level of insulin secretion is insulin resistance. Insulin resistance is a condition in which cells of a subject become less sensitive to the hypoglycemic action of insulin. Insulin resistance in muscle and adipocytes reduces glucose uptake (and thus local storage of glucose in the form of glycogen and triglycerides), while insulin resistance in hepatocytes leads to reduced glycogen synthesis and storage and inability to inhibit glucose production and release into the blood. Insulin resistance generally refers to a decrease in the hypoglycemic effect of insulin. However, other functions of insulin may also be affected. For example, insulin resistance in adipocytes attenuates the normal action of insulin on lipids, and results in decreased uptake of circulating lipids and increased hydrolysis of stored triglycerides. The motorized increase in storage lipids in these cells raises free fatty acids in plasma. Elevated blood fatty acid concentrations, decreased muscle glucose uptake, and increased hepatic glucose production all contribute to elevated blood glucose levels. If insulin resistance is present, more insulin needs to be secreted by the pancreas. If this compensatory increase does not occur, blood glucose concentration is increased and type II diabetes occurs.

One of the pathologies associated with an insufficient level of insulin secretion is diabetes. Diabetes can be divided into two main categories: type I ("T1D") and type II ("T2D"). The term "diabetes" also refers herein to a group of metabolic diseases in which a patient has elevated blood glucose levels, including type I diabetes, type II diabetes, gestational diabetes, congenital diabetes, adult onset diabetes ("MODY"), cystic fibrosis-related diabetes, hemochromatosis-related diabetes, drug-induced diabetes (e.g., steroid diabetes), and several forms of monogenic diabetes.

In certain embodiments, the subject has received or is receiving treatment for one or more of type I diabetes (T1D), type II diabetes (T2D), gestational diabetes, congenital diabetes, adult onset diabetes (MODY), cystic fibrosis related diabetes, hemochromatosis related diabetes, drug-induced diabetes, or monogenic diabetes. For example, the subject has received or is receiving treatment for type I diabetes. Alternatively, the subject has received or is receiving treatment for type II diabetes.

A condition associated with insufficient levels of insulin secretion is metabolic syndrome. Metabolic syndrome is commonly used to define abnormal clusters (constellations) associated with an increased risk of developing type II diabetes and atherosclerotic vascular disease. Related conditions and symptoms include, but are not limited to, fasting hyperglycemia (type II diabetes or impaired fasting glucose, impaired glucose tolerance, or insulin resistance); hypertension; central obesity (also known as abdominal, male or apple-shaped obesity), means overweight and a substantial accumulation of fat around the waist; a decrease in HDL cholesterol; and triglycerides are elevated.

The condition associated with insufficient levels of insulin secretion may be metabolic syndrome or insulin resistance. Thus, the method may be performed to treat a subject suffering from or receiving treatment for metabolic syndrome or insulin resistance.

Other conditions that may be associated with insufficient levels of insulin secretion include, but are not limited to, hyperuricemia, progression of fatty liver (especially in parallel obesity) to non-alcoholic fatty liver disease, polycystic ovary syndrome (in women), and acanthosis nigricans.

Related disorders can also be treated according to the methods of treatment disclosed herein, including, but not limited to, any disease associated with blood or plasma glucose levels outside the normal range, such as hyperglycemia. Thus, the term "associated condition" includes impaired glucose tolerance ("IGT"), impaired fasting glucose ("IFG"), insulin resistance, metabolic syndrome, postprandial hyperglycemia, and overweight/obesity. Such related conditions may also be characterized by abnormal blood and/or plasma insulin levels.

The methods can be performed to treat a subject having beta cell failure or a deficiency related condition. Such conditions include, but are not limited to, type I diabetes (T1D), type II diabetes (T2D), gestational diabetes, congenital diabetes, adult onset diabetes (MODY), cystic fibrosis related diabetes, hemochromatosis related diabetes, drug-induced diabetes, or monogenic diabetes. Drug-induced diabetes relates to conditions caused by the use of drugs (e.g., steroids, antidepressants, second generation antipsychotics, and immunosuppressants) that are toxic to beta cells. Exemplary immunosuppressive drugs include, but are not limited to, cortisone (cortisone) family members (e.g., prednisone and dexamethasone), rapamycin (rapamycin)/sirolimus (sirolimus), everolimus (everolimus), and calcineurin inhibitors (e.g., FK-506/tacrolimus).

Other conditions associated with beta cell deficiency include, but are not limited to, unaware hypoglycemia, unstable (labile) insulin dependent diabetes mellitus, pancreas resection, pancreas transplantation, islet allograft, islet autograft, and islet xenograft.

As used herein, unaware hypoglycemia is a diabetic complication in which the patient does not perceive a decrease in blood glucose depth because it fails to trigger epinephrine secretion, which produces characteristic symptoms of hyperglycemia (e.g., palpitations, sweating, anxiety) that are used to alert the patient to the decrease in blood glucose.

Pancreas transplantation can be performed alone, after kidney transplantation or in combination with kidney transplantation. For example, in patients with serious disabling and potentially life-threatening complications due to undetected hypoglycemia and unstable insulin-dependent diabetes mellitus (and which persist despite optimal medical management), pancreatic transplantation alone may be considered medically necessary. Pancreas transplantation after a previous kidney transplantation can be performed in patients with insulin-dependent diabetes mellitus. Pancreas transplantation can be performed in combination with kidney transplantation in insulin-dependent diabetes patients with uremia. Pancreas re-transplantation may be considered after the first pancreas transplantation failure.

As used herein, islet transplantation is an operation in which only the islets of langerhans, which contain pancreatic endocrine cells, including insulin-producing beta cells and glucagon-producing alpha cells, are isolated and transplanted into a patient. Islet xenotransplantation occurs when islets of langerhans are isolated from one or more human donor pancreases. Islet cells may also be derived from human embryonic stem cells or induced pluripotent stem cells. Islet xenotransplantation occurs when langerhans islets are isolated from one or more non-human donor pancreases (e.g., porcine or primate pancreases). Islet autografting occurs when langerhans islets are isolated from the pancreas of a patient undergoing a pancreatic resection (e.g., for chronic pancreatitis due to gallstone, drug, and/or familial genetic causes), and returned to the same patient subcutaneously by infusion into the portal vein, laparoscopically to the omentum, endoscopically to the stomach wall, or through a small incision. Like pancreas transplantation, islet transplantation can be performed alone, after kidney transplantation, or in combination with kidney transplantation. For example, islet Transplantation alone may be performed to restore the perception of Hypoglycemia, provide glycemic control, and/or protect patients from Severe hypoglycemic events (Hering et al, "Type 1Diabetes Complicated with Severe Hypoglycemia Human islet Transplantation Phase 3 Trial (Phase 3 tertiary of Transplantation of Human Islets in Type 1Diabetes mellitus)", "Diabetes Care (Diabetes Care) 39(7):1230-1240(2016), which is incorporated herein by reference in its entirety).

Islet transplantation can be performed in combination with total pancreatectomy. For example, Islet transplantation may be performed After Total Pancreatectomy to prevent or ameliorate surgically-induced diabetes by preserving beta Cell function (Johnston et al, "Factors Associated With Islet production and insulin independence following Total Pancreatectomy in Chronic Pancreatitis patients and Islet Cell autografting using ex-situ Islet Isolation-Cleveland Clinic Experience (fans Associated With Islet Yield and insulin independence) and chemistry Cell Automation in Patients With Chronic pancreas transplant Experience, journal of chemical endocrinology and metabolism 2015 (J.chem.Endocrinol.Metab.100), (5): 1770 (176176), incorporated herein in its entirety). Thus, islet transplantation can provide sustained long-term insulin independence.

In some embodiments, islet transplantation can be performed in combination with immunosuppressant administration. Suitable immunosuppressive agents include, but are not limited to, daclizumab (Zenapax), sunipine (Zenapax), low dose rapamycin (sirolimus), and FK506 (tacrolimus) (Van Belle et al, "Immunosuppression in Islet Transplantation," journal of clinical research 118(5):1625-1628(2008), which is incorporated herein by reference in its entirety).

In some embodiments, islet transplantation is performed in the context of an encapsulation device to protect the transplanted islet cells from host autoimmune reactions while allowing glucose and nutrients to reach the transplanted islet cells.

The methods described herein can be performed to enhance pancreas, islet xenotransplantation, islet autotransplantation, islet xenotransplantation by regenerating pancreatic beta cells in a patient. For example, the methods of the present application can be used to prevent or ameliorate surgically-induced diabetes by preserving beta cell function, restore the perception of hypoglycemia, provide glycemic control, and/or protect patients from severe hypoglycemic events. Accordingly, another aspect of the present disclosure relates to a method of regenerating pancreatic beta cells in a transplant patient. This method involves administering to a transplant patient a bispecific tyrphostin-regulated kinase 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist, wherein said administering is performed under conditions effective to cause a synergistic increase in the amount of pancreatic beta cells in said transplant patient, such that the pancreatic beta cells in said patient are regenerated.

The method may be performed to treat a subject at risk of developing type II diabetes. Patients at risk for type II diabetes may have pre-diabetes/metabolic syndrome.

Patients at risk for type II diabetes may have been treated with psychoactive drugs including, but not limited to, selective serotonin reuptake inhibitors ("SSRIs") for depression, obsessive compulsive disorder ("OCD"), and the like.

The subject may be a mammalian subject, e.g., a human subject. Suitable human subjects include, but are not limited to, children, adults, and elderly subjects with beta cell and/or insulin deficiency.

The subject may also be non-human, such as bovine, ovine, porcine, feline, equine, murine, canine, rabbit, and the like.

Administration of a bispecific tyrphostin 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist to a subject increases the number of proliferating pancreatic beta cells in the subject by at least about 4%, 5%, 6%, 7%, 8%, 9%, 10% or more.

Administration of a bispecific tyrphostin 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist to a subject increases the number of proliferating pancreatic beta cells in the subject by about 4-10%/day, or by about 4-6%/day, 5-7%/day, 6-9%/day, or 7-10%/day.

Administration of a bispecific tyrphostin 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist to a subject increases the number of proliferating pancreatic beta cells in the subject by about 6-10% per day.

Administration of a bispecific tyrphostin 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist to a subject results in increased glucose-stimulated insulin secretion in pancreatic beta cells of the subject (e.g., as compared to a subject not administered the bispecific tyrphostin 1A (DYRK1A) inhibitor and the glucagon-like peptide-1receptor (GLP1R) agonist).

The DYRK1A inhibitor may be selected from the group consisting of: harmine, INDY, leucettine-41, 5-iodotubercidin (5-IT), GNF4877, CC-401, thiadiazine kinase inhibitors, and combinations thereof. Exemplary DYRK1A inhibitors are described in detail above.

The GLP1R agonist may be selected from the group consisting of: GLP1 analogs, exendin-4, liraglutide, lissamide, somaglutide, and combinations thereof, described hereinabove.

Administration of a bispecific tyrphostin 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist to a subject may be performed using harmine and GLP1 (7-36).

Administration of a bispecific tyrphostin 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist to a subject can be performed using harmine and N- (4-fluorobenzyl) -5- (benzo [ d ] imidazol-2 (3H) -one) -6H-1,3, 4-thiadiazin-2-amine.

Administration of a bispecific tyrphostin 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist to a subject may be performed by a single composition comprising both a DYRK1A inhibitor and a GLP1R agonist. Alternatively, administration of the bispecific tyrphostin 1A (DYRK1A) inhibitor and glucagon-like peptide-1receptor (GLP1R) agonist to the subject may be performed sequentially. For example, a subject may first administer a bispecific tyrphostin-regulated kinase 1A (DYRK1A) inhibitor (or a composition comprising a bispecific tyrphostin-regulated kinase 1A (DYRK1A) inhibitor), and subsequently administer a glucagon-like peptide-1receptor (GLP1R) agonist (or a composition comprising a glucagon-like peptide-1receptor (GLP1R) agonist); or first with a glucagon-like peptide-1receptor (GLP1R) agonist (or a composition thereof) and subsequently with a bispecific tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor (or a composition thereof).

Administration may be multiple times daily, once weekly, twice weekly, once monthly, once bimonthly, once annually, once semiannually, or any amount of time in between. The DYRK1A inhibitor and the glucagon-like peptide-1receptor (GLP1R) agonist may be administered at different administration frequencies. In some embodiments, administration is performed short-term (neutrely) or long-term (chronically). For example, administration may be for a prolonged period of 1 year, 2 years, 3 years, 4 years, or longer. In some embodiments, administration is performed infrequently. As used herein, the term "treatment" means prophylactic or ameliorating or curative treatment. In other words, the method of treatment may be performed to prevent the subject from developing, or prevent the subject from worsening, a condition associated with insufficient insulin secretion. Alternatively, the treatment is performed to ameliorate the condition associated with insufficient insulin secretion in the subject, or to completely cure the condition (i.e., such that the subject no longer has the condition associated with insufficient insulin secretion levels, as determined by a capable health care professional).

The term "treating" means causing the impaired glucose homeostasis of a subject to be repaired, to have a reduced or reduced rate of change. Glucose levels in blood fluctuate throughout the day. Glucose levels are typically low in the morning, before the first meal of the day, and rise for several hours after a meal. Thus, the term "treating" includes controlling the blood glucose level of a subject by increasing or decreasing the blood glucose level of the subject. This may depend on a number of factors, including the subject's condition and/or the particular time of day, as blood glucose levels fluctuate throughout the day.

By "treating" is meant modulating a temporary or sustained reduction in blood glucose levels in a subject suffering from diabetes or a related disorder. The term "treating" can also mean improving insulin release (e.g., release by pancreatic beta cells) in a subject.

It may be desirable to regulate a subject's blood glucose level to normalize or regulate a subject's blood or plasma glucose level with an abnormal level (i.e., a level below or above a known control, median, or average value for a corresponding subject with normal glucose homeostasis). The treatment methods of the present invention can be performed to achieve such effects.

In performing the method of treatment, administering to the subject a bispecific tyrphostin kinase 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist can involve administering a pharmaceutical composition comprising a therapeutically effective amount of the bispecific tyrphostin kinase 1A (DYRK1A) inhibitor or the glucagon-like peptide-1receptor (GLP1R) agonist or both, the therapeutically effective amount referring to an amount of the DYRK1A inhibitor and the GLP1R agonist effective to treat the condition and/or disorder in the subject. Such amounts will generally vary depending on several factors within the purview of one of ordinary skill in the art. These include, but are not limited to, the general health, age, weight, height, general physical condition, medical history of the particular subject, the particular compound used and the vehicle used in formulation and the chosen route of administration, the length or duration of treatment, and the nature and severity of the condition being treated.

Administration typically involves administration of pharmaceutically acceptable dosage forms, which means dosage forms of the compounds described herein, and includes, for example, tablets, dragees, powders, elixirs, syrups, liquid preparations, including suspensions, sprays, inhalant tablets, buccal tablets, emulsions, solutions, granules, capsules and suppositories, and liquid preparations for injection, including liposomal preparations. Techniques and formulations are generally found in Remington's Pharmaceutical Sciences, mark Publishing company (Mack Publishing Co.), Easton, Pa, pennsylvania, latest edition, which is incorporated herein by reference in its entirety.

In practicing the methods of treatment, any suitable amount of DYRK1A inhibitor and GLP1R agonist can be included in any suitable carrier material. The DYRK1A inhibitor and GLP1R agonist may be present in an amount of up to 99% by weight of the total weight of the composition. The compositions may be provided in dosage forms suitable for oral, parenteral (e.g., intravenous, intramuscular), rectal, transdermal (cutaneous), nasal, vaginal, inhalation, dermal (skin) (patch), or ocular routes of administration. Thus, the composition may be in the form of, for example, a tablet, capsule, pill, powder, granule, suspension, emulsion, solution, gel including hydrogel, paste, ointment, cream, plaster, bolus liquid (drench), osmotic delivery device, suppository, enema, injectable, implant, spray, or aerosol.

The pharmaceutical composition can be formulated to release the active DYRK1A inhibitor and the GLP1R agonist substantially immediately after administration or at any predetermined time or period after administration.

Controlled release formulations include (i) formulations that produce a substantially constant drug concentration over an extended period of time within the body; (ii) producing a formulation in the body with a substantially constant drug concentration for an extended period of time after a predetermined lag time; (iii) formulations that maintain drug action during a predetermined period of time by maintaining a relatively constant effective drug level in the body, while minimizing undesirable side effects associated with fluctuations in plasma levels of the active drug substance; (iv) agents that localize the action of the drug by, for example, spatially placing the controlled release composition adjacent to or in the diseased tissue or organ; and (v) formulations that target the action of drugs by using carriers or chemical derivatives to deliver the drugs to specific target cell types.

Administration of a DYRK1A inhibitor and a GLP1R agonist in a controlled release formulation is particularly preferred where the drug has the following characteristics: (i) a narrow therapeutic index (i.e., the difference between the plasma concentration that causes an adverse side effect or toxic response and the plasma concentration that causes a therapeutic effect is small; generally, the therapeutic index ("TI") is defined as the median Lethal Dose (LD)₅₀) With the median Effective Dose (ED)₅₀) The ratio of (a); (ii) a narrow absorption window in the gastrointestinal tract; or (iii) a very short biological half-life, such that frequent dosing during the day is required in order to maintain plasma levels at therapeutic levels.

Any of several strategies may be implemented to achieve controlled release, wherein the release rate of the target DYRK1A inhibitor and/or GLP1R agonist exceeds the metabolic rate. Controlled release can be achieved by appropriate selection of various formulation parameters and ingredients, including, for example, various types of controlled release compositions and coatings. Thus, the drug is formulated with suitable excipients into pharmaceutical compositions (single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, patches, and liposomes) that release the drug in a controlled manner after administration. Thus, administration can be nasal, oral, topical, transdermal, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, by intranasal instillation, by intracavitary or intravesical instillation, intraocular, intraarterial, intralesional, or by application to the mucosa. The compounds may be administered alone or in combination with a suitable pharmaceutical carrier, and may be in solid or liquid form, such as tablets, capsules, powders, solutions, suspensions or emulsions. In certain embodiments, administration is performed nasally, orally, transdermally, parenterally, subcutaneously, intravenously, intramuscularly, or intraperitoneally.

In certain embodiments, administration is performed using an infusion pump to provide, for example, rate-controlled infusion, periodic infusion, and/or one bolus infusion. The infusion pump may be a fixed or mobile (ambulary) infusion pump. Stationary infusion pumps are used primarily at the patient's bedside. Ambulatory infusion pumps are relatively small, at least substantially self-contained devices used to introduce drugs and other infusible substances (e.g., insulin) to a selected subject. Some ambulatory infusion pumps are configured to be worn on a belt, carried in a garment pocket, or otherwise supported within some sort of holder (collectively, "pocket pumps"). Other infusion pumps are configured to adhere to skin in a patch-like manner (referred to as "patch pumps"). Infusion pumps may be used, for example, to introduce (or "infuse") drugs intravenously or subcutaneously, either progressively or even continuously, outside of a clinical environment. Infusion pumps greatly reduce the frequency of subcutaneous access events, such as needle-based injections. In certain embodiments, the infusion pump is a subcutaneous or intravenous infusion pump. For example, the infusion pump may be a ambulatory subcutaneous insulin infusion pump.

Another aspect relates to compositions comprising a bispecific tyrphostin 1A (DYRK1A) inhibitor and a glucagon-like peptide-1receptor (GLP1R) agonist.

Suitable DYRK1A inhibitors are described above and include, for example, harmine, INDY, leucostatin-41, 5-iodotubercidin (5-IT), GNF4877, CC-401, kinase inhibitors, and derivatives thereof.

Suitable GLP1R agonists are described above and include, for example, extentin-4, liraglutide, lisinopeptide, somaglutide, and derivatives thereof.

The composition may further comprise a carrier. Suitable carriers are described above. The carrier may be a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers are described above.

Another aspect relates to a method of increasing cell proliferation in a pancreatic beta cell population. This method involves contacting a pancreatic β -cell population with a bispecific tyrphostin-regulated kinase 1A (DYRK1A) inhibitor and a compound that increases cAMP, wherein said contacting is under conditions effective to cause a synergistic increase in cell proliferation in said pancreatic β -cell population.

Another aspect of the disclosure relates to methods of treating conditions associated with insufficient insulin secretion in a subject. This method involves administering to a subject in need of treatment of a condition associated with insufficient levels of insulin secretion a bispecific tyrphostin 1A (DYRK1A) inhibitor and a compound that increases cAMP, wherein the administering is performed under conditions effective to cause a synergistic increase in pancreatic beta cell mass in the subject, to treat the insufficient levels of insulin secretion in the subject.

Another aspect of the disclosure relates to compositions comprising a bispecific tyrphostin-regulated kinase 1A (DYRK1A) inhibitor and a compound that increases cAMP.

Cyclic adenosine monophosphate (cAMP) is an intracellular second messenger that regulates beta cell replication. In some embodiments, the cAMP increasing compounds for performing the disclosed methods comprise cAMP analogs, including dibutyryl-cAMP and/or 8-chloro-cAMP. Increasing cAMP has been shown to increase beta cell replication in young rodents (Zhao et al, "use of agents that alter cAMP to Promote beta cell replication," (mol. endocrinol.) -28 (10):1682-1697(2014), which is incorporated herein by reference in its entirety). However, as shown in fig. 5A-5H, administration of an agent that increases cAMP has no effect on human beta cell proliferation unless administered in combination with a DYRK1 inhibitor (e.g., harmine). Thus, the present disclosure demonstrates for the first time that increasing cAMP in combination with a DYRK1A inhibitor synergistically increases human beta cell proliferation.

Intracellular cAMP levels are regulated by a balance between the activities of two enzymes: adenylate cyclase ("AC") and cyclic nucleotide Phosphodiesterases (PDEs). Most of the AC passes through and G_sAlpha subunit of protein (alpha)_s) The interaction is activated downstream of a G protein-coupled receptor (GPCR), such as The β adrenergic receptor, which is released from The heterotrimeric α β γ G protein complex upon binding of an agonist ligand to The GPCR (e.g., binding of epinephrine to The β adrenergic receptor in The case of The β adrenergic receptor), and binds to and activates AC (Sassone-cori, "The Cyclic AMP Pathway," Cold Spring harb.perspect.biol.). 4(12 (2012), which is incorporated herein by reference in its entirety).

Division of beta adrenoceptors into beta₁、β₂And beta₃Subtypes, each of which is linked to G_sProtein coupling. Activation of beta on beta cells₂The adrenergic receptors increase intracellular cAMP. Thus, in certain embodiments, cAMP increasing compounds useful for performing the disclosed methods comprise β₂An adrenergic receptor agonist. Suitably beta₂Adrenergic receptor agonists include, but are not limited to, epinephrine, albuterol (albuterol), salbutamol (salbutamol), bitolterol mesylate (bitolterol mesylate), formoterol (formoterol), isoproterenol, levalbuterol, metaproterenol (metaproterenol), salmeterol (salmeterol), terbutaline (terbutaline), and/or ritodrine (rit)odrine)。

Various GPCRs promote beta cell replication by activating cAMP-dependent signaling pathways and intracellular cAMP levels. In certain embodiments, the compound that increases intracellular cAMP levels is a ligand for the GPCR. Suitable (and non-limiting) agonists for increasing cAMP levels are listed in table 3 below.

TABLE 3 exemplary GPCR, ligands and agonists

Other GPCRs are described in Amisten et al, "mapping and functional analysis of G protein-coupled receptors in the islets of Langerhans in human," pharmacology and therapeutics 139(3):359-391(2013), which is incorporated herein by reference in its entirety, and which identifies the 293 GPCRs present in human islets.

Norepinephrine acts as a physiological inhibitor of cAMP synthesis in beta cells and by activating alpha₂Adrenergic receptors block beta cell activation (Zhao et al, "use of agents that modify cAMP to promote beta cell replication", "molecular endocrinology" 28(10):1682-1697(2014), which is incorporated herein by reference in its entirety). Thus, α is suppressed₂Adrenergic receptors increase intracellular cAMP levels. In certain embodiments, cAMP increasing compounds for performing the disclosed methods comprise α₂Adrenergic receptor antagonists including, but not limited to, mirtazapine (mirtazapine).

PDEs catalyze the hydrolysis of cAMP. In humans, there are 21 PDE genes comprising 11 structurally related families (PDE 1-11). Beta cells express several PDE family members, including PDE1, PDE3, PDE4, PDE7, PDE8, PDE10, and PDE 11.

PDE inhibitors increase intracellular cAMP levels by preventing degradation of intracellular second messengers (e.g., cAMP) by PDEs. Thus, cAMP-increasing compounds suitable for use in performing the disclosed methods include, but are not limited to, Phosphodiesterase (PDE) inhibitors. In certain embodiments, the PDE inhibitor is a non-selective inhibitor. For example, the PDE inhibitor may be 3-isobutyl-1-methylxanthine, zadaverin (zardaviramine), and/or trequinsin (trequinin). In certain embodiments, the PDE inhibitor is a PDE1 inhibitor, a PDE3 inhibitor, a PDE 4inhibitor, a PDE7 inhibitor, a PDE8 inhibitor, a PDE10 inhibitor, and/or a PDE11 inhibitor. Suitable PDE3 inhibitors include, but are not limited to, cilostamide (cilostamide) and/or milrinone (milrinone). Suitable PDE 4inhibitors include, but are not limited to, issoradine (irsogladine), glaucine (glaucine), etazolate (etazolate), CGH2466, rolipram (rolipram) and/or bay 19-8004. Suitable PDE5 inhibitors include, but are not limited to, dipyridamole (dipyridamole), vardenafil (vardenafil), and/or tadalafil (tadalafil). Suitable PDE10 inhibitors include, but are not limited to, papaverine (papaverine). In certain embodiments, the PDE inhibitor is selected from the group consisting of: trequinsin (treequinin), zadavirin, cilostamide. In certain embodiments, the PDE inhibitor increases beta cell replication by acting as a PDE4/PDE10 inhibitor. Thus, the PDE inhibitor is dipyridamole.

In some embodiments, the PDE inhibitors are specific for beta cells rather than alpha cells (Zhao et al, "use of agents that modify cAMP to promote beta cell replication"; molecular endocrinology 28(10):1682-1697(2014), which is incorporated herein by reference in its entirety). Thus, in one embodiment, the PDE inhibitor is dipyridamole.

As described above, GLP1R agonists and additional agents that prevent degradation of endogenous GLP1 by the enzyme dipeptidyl peptidase IV (DPP4) have been shown to induce rodent beta cell proliferation, but fail to show activation of beta cell replication in adult islets (DruckerDJ, "mechanism of action and therapeutic application of glucagon-like peptide-1", "cell metabolism" 27(4): 740-. The present disclosure demonstrates that the combination of inhibiting DYRK1A and an agent that increases GLP1 activity synergistically increases human beta cell proliferation.

Accordingly, another aspect of the present disclosure relates to methods of treating conditions associated with insufficient insulin secretion in a subject. The method involves administering to a subject in need of treatment of a condition associated with insufficient levels of insulin secretion a bispecific tyrphostin 1A (DYRK1A) inhibitor and a dipeptidyl peptidase IV (DPP4) inhibitor, wherein said administering is performed under conditions effective to cause a synergistic increase in the amount of pancreatic beta cells in said subject, to treat the subject with insufficient levels of insulin secretion.

As described above, the methods described herein may be performed in vivo. In some embodiments, administering a bispecific tyrphostin 1A (DYRK1A) inhibitor and a dipeptidyl peptidase IV (DPP4) inhibitor is performed using a composition comprising both a DYRK1A inhibitor and a DPP4 inhibitor.

As described above, the subject may be treated for one or more of type I diabetes ("T1D"), type II diabetes ("T2D"), gestational diabetes, congenital diabetes, adult onset diabetes ("MODY"), cystic fibrosis-related diabetes, hemochromatosis-related diabetes, drug-induced diabetes, or monogenic diabetes. In some embodiments, the subject is a mammalian subject. The subject may be a human subject.

As described above, administration of a bispecific tyrphostin-regulated kinase 1A (DYRK1A) inhibitor and a dipeptidyl peptidase IV (DPP4) inhibitor can increase glucose-stimulated insulin secretion in pancreatic beta cells of a subject.

Suitable DYRK1 inhibitors are described in detail above. In some embodiments, the DYRK1A inhibitor is selected from the group consisting of: harmine, INDY, leucettine-41, 5-iodotubercidin (5-IT), GNF4877, CC-401, thiadiazine kinase inhibitors, and combinations thereof.

Suitable DPPR inhibitors include, but are not limited to, sitagliptin, vildagliptin, saxagliptin, linagliptin, alogliptin and combinations thereof (Drucker DJ, "mechanism of action and therapeutic application of glucagon-like peptide-1", "cell metabolism" 27(4): 740-.