Targeted delivery of small molecules to pancreatic islet cells

Abstract

Provided herein are compounds comprising a therapeutic molecule conjugated to a beta cell targeting motif, that can specifically deliver therapeutic molecules to the pancreas. Also provided herein are methods of making and using these compounds for the treatment of pancreatic diseases.

Description

TITLETARGETED DELIVERY OF SMALL MOLECULES TO PANCREATIC ISLET CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and priority to US Provisional Application No. 63 / 583,414, filed September 18, 2023, and US Provisional Application No. 63 / 621,324, filed January 16, 2024, the entire contents of which are hereby incorporated by reference in their entirety.ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant No. DK069710 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND1. Field

[0003] The present disclosure relates to compounds for targeted delivery of small molecules to the pancreatic islet cells. Also provided are therapeutic compositions and methods for delivering the same to a subject.2. Background

[0004] The pancreatic islet beta cell plays an essential role in regulating blood glucose homeostasis by releasing insulin. Functional declining of islet beta cell or loss of beta cell mass compromise insulin secretion capacity of pancreatic islets. When the insulin secretion no longer meets metabolic demands, the blood glucose level keeps increasing till frank diabetes is diagnosed.

[0005] Despite extensive efforts in developing therapeutic targets, regular administration of insulin remains the only viable solution to the management of Type 1 Diabetes (T1D). Accumulating evidence indicate that small molecule drugs may preserve p-cell function and survival. However, the therapeutic potential of these small molecules in diabetes is limited by their short half-life in circulation, and by the lack of targeting specificity to p-cells in vivo. Therefore, there is an unmet need to develop novel delivery strategies to overcome the aforementioned limitations. Targeted drug delivery to islet beta cells holds great promise for treating diabetes, including both type 1 diabetes (T1D) and type 2 diabetes (T2D). Such targeted delivery increases the local drug concentration within pancreatic islets, hence improving the therapeutic index by allowing a lower drug dosage to minimize the off-target effect while maintaining the therapeutic efficacy in islet beta cells.SUMMARY

[0006] In an aspect, the current disclosure encompasses a compound comprising a therapeutic molecule conjugated to a Beta-cell Targeting Motif (BTM) comprising a structure of Formula I or a derivative thereof:wherein each A is independently one or more therapeutic molecules or absent; n is 1 , 2, or 3;R1-R5 are each independently hydrogen, Ci-Ce linear alkyl, Ci-Ce branched alkyl, - ORe, - NReR?, -COOH, -C(O)NReR7, or a linker connected to An; and wherein R6and R? are each independently H, Ci-Ce linear alkyl, or Ci-Ce branched alkyl group.

[0007] In some aspects, the linker comprises one or more ofwhereinRs and R9 are each independently H, C1-C3 linear alkyl, or C1-C3 branched alkyl; and n= 0-6.

[0008] In an aspect, the BTM specifically binds the ZnT8 transporter protein. In an aspect, the therapeutic molecule is a drug, or a prodrug. In an aspect, the therapeutic molecule is effective against a pancreatic disease. In an aspect, the pancreatic disease may be hyperglycemia, pre-diabetes, impaired glucose tolerance, diabetes type I, diabetes type II, syndrome X, a pancreatic cancer, acute pancreatitis, chronic pancreatitis, cholangitis, cholecystitis, hereditary pancreatitis, alcohol related pancreatitis, or a combination thereof.

[0009] In an exemplary aspect, the therapeutic molecule is phenylbutyrate (PB) (Formula la or Formula lb),or a derivative or a prodrug thereof. In one aspect, the compound is BTPB-1 or a derivative thereof. In an aspect, the compound comprises a structure as provided in Formula II or a derivative thereof:Formula II (BTPB-1).

[0010] In an aspect, BTPB-1 has increased concentration in the islet beta cells in comparison to PB when administered in equivalent amounts to a subject.

[0011] In an aspect, the compound is BTPB-2 or a derivative thereof. In an aspect, the compound comprises a structure as provided in Formula VII or a derivative thereof:Formula VII (BTPB-2).

[0012] In an aspect, the compound is BTPB-3 or a derivative thereof. In an aspect, the compound comprises a structure as provided in Formula VIII or a derivative thereof:Formula VIII (BTPB-3).

[0013] In an aspect, the compound is BTPB-4 or a derivative thereof. In an aspect, the compound comprises a structure as provided in Formula IX or a derivative thereof:Formula IX (BTPB-4).

[0014] In an aspect, the compound is BTPB-5 or a derivative thereof. In an aspect, the compound comprises a structure as provided in Formula X or a derivative thereof:Formula X (BTPB-5).

[0015] In an aspect, the compound is BTPB-6 or a derivative thereof. In an aspect, the compound comprises a structure as provided in Formula XI or a derivative thereof:Formula XI (BTPB-6).

[0016] In an aspect, the compound is BTPB-7R or a derivative thereof. In an aspect, the compound comprises a structure as provided in Formula XI la or a derivative thereof:Formula Xlla (BTPB-7R).

[0017] In an aspect, the compound is BTPB-7S or a derivative thereof. In an aspect, the compound comprises a structure as provided in Formula Xlla or a derivative thereof:Formula Xllb (BTPB-7S).

[0018] In an aspect, the compound is BTPB-8 or a derivative thereof. In an aspect, the compound comprises a structure as provided in Formula XIII or a derivative thereof:Formula XIII (BTPB-8).

[0019] In an aspect, the compounds as disclosed herein are for treatment of a pancreatic disease. In an aspect, the compound as disclosed herein is for use in the treatment of diabetes.

[0020] In an aspect, the current disclosure also encompasses a pharmaceutical composition comprising a compound as disclosed herein and a pharmaceutically acceptable excipient. In an aspect, the pharmaceutically acceptable excipient may be a liquid or solid filler, a diluent, a binder, a buffering agent, a pH modifying agent, a disintegrant, a dispersant, a preservative, a lubricant or wetting agent, taste-masking agent, an antioxidant, carrier, adjuvant, stabilizing agent, emulsifying agent, solution promoter, salt, solubilizer, antifoaming agent, surfactant, a flavoring agent, a coloring agent, solvent or encapsulating material or any combination thereof. In an aspect, the pharmaceutical composition is for administering by any one of parenteral, oral, intraarterial, intraarticular, intradermal, intramuscular, intraperitoneal, intravenous, intravascular, liposomal, local, mucosal, subcutaneous, sublingual, topical, trans buccal, and transdermal route. In an aspect, the pharmaceutical composition may further comprise additional therapeutic molecules.

[0021] In an aspect, the current disclosure also encompasses a method of making a BTM conjugate, comprising making of one or more of intermediates of BTM, wherein the intermediates of BTM comprise a structure of Formula III and / or Formula IV:Formula III Formula IV.

[0022] In an aspect, the current disclosure also encompasses a method of making BTPB- 1 , comprising conjugating a Beta-cell Targeting Motif (BTM) of Formula I or a derivative thereof to phenylbutyrate (PB) or a derivative thereof, using one or more intermediates of BTM comprising a structure of Formula III and / or Formula IV, and one or more intermediates of PB comprising a structure of Formula V and / or Formula VI:Formula V; wherein Boc is a tert-butyloxycarbonyl protecting group, orFormula VI.

[0023] In an aspect, the current disclosure also encompasses a method for treating a pancreatic disease, comprising administering to a subject in need thereof, a compound or a pharmaceutical composition as disclosed herein. In an aspect, the pancreatic disease is hyperglycemia, pre-diabetes, impaired glucose tolerance, diabetes type I, diabetes type II, syndrome X, a pancreatic cancer, acute pancreatitis, chronic pancreatitis, cholangitis, cholecystitis, hereditary pancreatitis, alcohol related pancreatitis, or a combination thereof. In an exemplary aspect, the pancreatic disease is diabetes, and the compound is BTPB-1 or a derivative thereof. In an aspect, the subject in need thereof is a mammal.

[0024] In an aspect, the current disclosure also encompasses a kit comprising a disclosed compound or a disclosed pharmaceutical composition and a container for administration of the composition.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The following drawings form part of the present specification and are included to further demonstrate certain embodiments of the present disclosure. Certain embodiments can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0026] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0027] FIG. 1A represents ZIGIR structure and Beta cell targeting motif (BTM) in accordance with some embodiments of the present disclosure.

[0028] FIG. 1 B represents the structure of BODIPY-BTM.

[0029] FIG. 1C represents an exemplary confocal image of in vivo islet beta cells selectively and efficiently labeled with BODIPY-BTM in accordance with some embodiments of the present disclosure.

[0030] FIG. 1D represents an exemplary schematic showing synthesis of BODIPY-BTM in accordance with some embodiments of the present disclosure.

[0031] FIG. 2A represents exemplary immunofluorescence images of insulin or ZnT8 in mouse pancreas tissue sections in accordance with some embodiments of the present disclosure.

[0032] FIG. 2B represents exemplary data for the total cellular zinc contents of mouse islets measured by inductively coupled plasma mass spectrometry (ICP-MS) in accordance withsome embodiments of the present disclosure.

[0033] FIG. 2C represents exemplary confocal fluorescence images of dispersed islet cells labeled with BODIPY-BTM in accordance with some embodiments of the present disclosure.

[0034] FIG. 2D represents exemplary data showing quantification of islet cell uptake of BODIPY-BTM by LC-MS in accordance with some embodiments of the present disclosure.

[0035] FIG. 3A represents an exemplary schematic showing synthesis of BTPB-1 in accordance with some embodiments of the present disclosure.

[0036] FIG. 3B represents an exemplary graph showing quantification of BTPB-1 uptake in vitro by dispersed mouse islet cells isolated from wild type C57BL6 / J mouse (ZnT8+ / +) or ZnT8 knockout mouse (ZnT8_ / _) in accordance with some embodiments of the present disclosure. Cellular uptake of BTPB-1 is normalized against the protein content of cell lysate (nmol BTPB- 1 per mg protein).

[0037] FIG. 3C represents an exemplary graph showing quantification of BTPB-1 uptake in vitro by dispersed mouse islet cells isolated from wild type C57BL6 / J mouse (ZnT8+ / +) or ZnT8 knockout mouse (ZnT8_ / in accordance with some embodiments of the present disclosure. Cellular uptake of BTPB-1 is normalized against the cell number (fmol BTPB-1 per cell).

[0038] FIG. 3D represents an exemplary graph showing quantification of BTPB-1 uptake in vivo by C57BL6 / J mouse islet endocrine cells and pancreatic exocrine cells in accordance with some embodiments of the present disclosure. BTPB-1 (8 mg / Kg) was administered through tail vein injection. Mouse pancreata were harvested 15 min for islet isolation and LC- MS analysis

[0039] FIG. 4A represents an exemplary graph showing BTPB-1 delays and prevents diabetes in NOD mice in accordance with some embodiments of the present disclosure. Kaplan-Meier plot of diabetes incidence of mice treated with vehicle, PB or BTPB-1 (n = 12 mice per group. Mann-Whitney test, ** p < 0.01) is provided.

[0040] FIG. 4B represents an exemplary graph showing BTPB-1 delays and prevents diabetes in NOD mice in accordance with some embodiments of the present disclosure. Average non-fasting blood glucose is provided for mice treated with vehicle, PB or BTPB-1 (n = 12 mice per group. Mann-Whitney test, ** p < 0.01).

[0041] FIG. 4C represents an exemplary graph showing body weight of mice (n = 12 mice per group. Mann-Whitney test, ** p < 0.01) treated with vehicle, PB or BTPB-1 in accordance with some embodiments of the present disclosure.

[0042] FIG. 5A represents an exemplary graph showing the treatment of diabetic femaleNOD mice with BTPB-1 (n = 5) in accordance with some embodiments of the present disclosure.

[0043] FIG. 5B represents an exemplary graph showing the treatment of diabetic female NOD mice with PBS (n = 7) in accordance with some embodiments of the present disclosure.

[0044] FIG. 5C represents exemplary graphs showing that BTPB-1 (n = 5), but not PB (n = 7), reversed diabetes in NOD mice. Kaplan-Meier plot of diabetic NOD mice survival after BTPB-1 or PB treatment.

[0045] FIG. 6 represents phenylbutyrate (PB) prodrugs containing the beta cell targeting motif (BTM) in accordance with some embodiments of the present disclosure.

[0046] FIG. 7 represents an exemplary schematic showing synthesis of BTPB-7R in accordance with some embodiments of the present disclosure.

[0047] FIG. 8 shows a schematic of the synthesis of BTPB-4 in accordance with some embodiments of the present disclosure.

[0048] FIG. 9A represents an exemplary graph showing cellular uptake of BTPB-4 in INS1 cells normalized by cellular protein content (left axis) or by cell number (right axis, cellular concentration of BTPB-4 was calculated based on the assumption of an average cell volume of 1000 pm3) in accordance with some embodiments of the present disclosure.

[0049] FIG. 9B represents an exemplary graph showing cellular uptake of BTPB-4 by primary islet cells isolated from wild type (ZnT8+ / +) or ZnT8 knockout (ZnT8' / _) mice in accordance with some embodiments of the present disclosure.

[0050] FIG. 10A shows mode of action of BTM-containing prodrugs. The insulin granule acts as a drug depot to store the prodrug, releasing PB in beta cells over time.

[0051] FIG. 10B shows a schematic representing the hydrolysis of BTPB-4 yields BTPB-4m (metabolite of BTPB-4) and the parent drug PB.

[0052] FIG. 10C represents an exemplary graph showing quantification of BTPB-4 hydrolysis by LC-MS in accordance with some embodiments of the present disclosure.

[0053] FIG. 11 A represents an exemplary graph illustrating BTPB-4 protects beta cells from lipotoxicity in accordance with some embodiments of the present disclosure.

[0054] FIG. 11 B represents an exemplary graph illustrating treating INS1 cells with BTPB- 4 (20 pM) for only 1 hr still protect beta cells from lipotoxicity in accordance with some embodiments of the present disclosure.

[0055] FIG. 11C represents an exemplary graph illustrating treating BTPB-4 protects betacells from ER stress induced by Thapsigargin (Tg) in accordance with some embodiments of the present disclosure.

[0056] FIG. 11D represents an exemplary graph illustrating treating INS1 cells with BTPB- 4 (20 pM) for only 1 hr still protect beta cells from ER stress in accordance with some embodiments of the present disclosure.

[0057] FIG. 11E represents an exemplary graph illustrating BTPB-4 protects beta cells from inflammatory cytokines in accordance with some embodiments of the present disclosure.

[0058] FIG. 11 F represents an exemplary graph illustrating effect of BTPB-4 on expression of Bip in accordance with some embodiments of the present disclosure.

[0059] FIG. 11G represents an exemplary graph illustrating effect of BTPB-4 on expression of CHOP in accordance with some embodiments of the present disclosure.

[0060] FIG. 11H represents an exemplary graph illustrating effect of BTPB-4 on expression of TXNIP in accordance with some embodiments of the present disclosure.

[0061] FIG. 12 represents an exemplary graph illustrating BTPB-4 showed no toxicity to beta cells in accordance with some embodiments of the present disclosure.

[0062] FIG. 13A represents exemplary graphs illustrating biodistribution of BTPB-4 and BTPB-4m in accordance with some embodiments of the present disclosure

[0063] FIG. 13B represents exemplary graphs illustrating in vivo cellular uptake and metabolism of BTPB-4 in mouse islets and acinar cells (Aci.) in accordance with some embodiments of the present disclosure.

[0064] FIG. 14A represents an exemplary workflow of drug dosing and mouse characterizations in accordance with some embodiments of the present disclosure.

[0065] FIG. 14B represents exemplary graphs illustrating Kaplan-Meier plots of mice treated with vehicle, PB, and BTPB-4 in accordance with some embodiments of the present disclosure.

[0066] FIG. 14C represents exemplary graphs illustrating blood glucose measurements of individual NOD mice that received vehicle (left), PB (middle), or BTPB-4 (right) in accordance with some embodiments of the present disclosure.

[0067] FIG. 14D represents exemplary graphs illustrating average blood glucose of each mice that received vehicle, PB, or BTPB-4 in accordance with some embodiments of the present disclosure.

[0068] FIG. 14E represents exemplary graphs illustrating average body weight of each micethat received vehicle, PB, or BTPB-4 in accordance with some embodiments of the present disclosure.

[0069] FIG. 14F represents exemplary graphs illustrating Intraperitoneal glucose tolerance test (IPGTT) performed in the 10-wk old NOD mice in accordance with some embodiments of the present disclosure.

[0070] FIG. 14G represents exemplary graphs illustrating area under the curve analysis of graphs in FIG. 14F in accordance with some embodiments of the present disclosure.

[0071] FIG. 14H represents exemplary graphs illustrating IPGTT performed in the 21-wk old NOD mice in accordance with some embodiments of the present disclosure.

[0072] FIG. 141 represents exemplary graphs illustrating area under the curve analysis of graphs in FIG. 14H in accordance with some embodiments of the present disclosure.

[0073] FIG. 14J represents exemplary graphs illustrating first phase insulin release at 0-5 min of NOD mice that received vehicle, PB, or BTPB-4 solution injection at 21 wk of mouse age in accordance with some embodiments of the present disclosure.

[0074] FIG. 14K represents exemplary graphs illustrating GSIS of NOD islets (20 islets / assay) between low glucose (LG) and high glucose (HG) in accordance with some embodiments of the present disclosure.

[0075] FIG. 14L represents exemplary graphs illustrating stimulation index (L) in accordance with some embodiments of the present disclosure.

[0076] FIG. 14M represents exemplary graphs illustrating BTPB-4 reduced the expression of ER stress marker genes in NOD mouse islets in accordance with some embodiments of the present disclosure.

[0077] FIG. 15A represents an exemplary workflow of drug dosing and mouse characterizations in accordance with some embodiments of the present disclosure.

[0078] FIG. 15B represents exemplary graphs illustrating average blood glucose of young female NOD mice (<12 wk old) treated with vehicle, PB, or BTPB-4 in accordance with some embodiments of the present disclosure.

[0079] FIG. 15C represents exemplary graphs illustrating IPGTT performed in the 12-wk old NOD mice in accordance with some embodiments of the present disclosure.

[0080] FIG. 15D represents exemplary graphs illustrating area under the curve analysis of graphs in FIG. 15C in accordance with some embodiments of the present disclosure.

[0081] FIG. 15E represents exemplary graphs illustrating blood inulin level of 12-wk oldNOD mice 5 mins following an intraperitoneal injection of glucose (2g / kg) in accordance with some embodiments of the present disclosure.

[0082] FIG. 15F represents an exemplary Western Blot in accordance with some embodiments of the present disclosure.

[0083] FIG. 15G represents exemplary graphs illustrating quantification of Western Blot of FIG. 15F in accordance with some embodiments of the present disclosure.

[0084] FIG. 15H represents exemplary insulin immuno-stained images of pancreatic sections of 12-wk old NOD mice after receiving vehicle (left), PB (middle), or BTPB-4 (right) for 8 wks in accordance with some embodiments of the present disclosure.

[0085] FIG. 151 represents exemplary graphs illustrating quantification of immuno-stained images of FIG. 15H in accordance with some embodiments of the present disclosure.

[0086] FIG. 15J represents exemplary graphs illustrating quantification of immuno-stained images of FIG. 15H in accordance with some embodiments of the present disclosure.

[0087] FIG. 15K represents exemplary insulin immuno-stained images of pancreatic sections of 16-wk old NOD mice after receiving vehicle (left), PB (middle), or BTPB-4 (right) for 12 wks in accordance with some embodiments of the present disclosure

[0088] FIG. 15L represents exemplary graphs illustrating quantification of immuno-stained images of FIG. 15K in accordance with some embodiments of the present disclosure.

[0089] FIG. 15M represents exemplary graphs illustrating quantification of immuno-stained images of FIG. 15K in accordance with some embodiments of the present disclosure.

[0090] FIG. 16A represents an exemplary workflow of drug dosing and mouse characterizations in accordance with some embodiments of the present disclosure.

[0091] FIG. 16B represents exemplary graphs illustrating average blood glucose level over the course of treatment using vehicle, BTPB-4 alone, aCD3 alone or aCD3 + BTPB-4 in CY- induced NOD mice in accordance with some embodiments of the present disclosure.

[0092] FIG. 16C represents exemplary Kaplan-Meier plot of diabetes incidence over the course of treatment using vehicle, BTPB-4 alone, aCD3 alone or aCD3 + BTPB-4 in CY- induced NOD mice analyzed by Mann-Whitney test; *p < 0.05 in accordance with some embodiments of the present disclosure.

[0093] FIG. 16D represents exemplary graphs illustration random fed blood glucose of individual mice in each group treated with vehicle in accordance with some embodiments of the present disclosure.

[0094] FIG. 16E represents exemplary graphs illustration random fed blood glucose of individual mice in each group treated with BTPB-4 in accordance with some embodiments of the present disclosure.

[0095] FIG. 16F represents exemplary graphs illustration random fed blood glucose of individual mice in each group treated with aCD3 in accordance with some embodiments of the present disclosure.

[0096] FIG. 16G represents exemplary graphs illustration random fed blood glucose of individual mice in each group treated with aCD3 + BTPB-4 in accordance with some embodiments of the present disclosure.

[0097] The drawing figures do not limit the present disclosure to the specific aspects disclosed and described herein. The drawings are not necessarily to scale, emphasis instead being placed on clearly illustrating principles of certain aspects of the present disclosure.DETAILED DESCRIPTION

[0098] The following detailed description references the accompanying drawings that illustrate various aspects of the present disclosure. The drawings and description are intended to describe aspects and aspects of the present disclosure in sufficient detail to enable those skilled in the art to practice the present disclosure. Other components can be utilized, and changes can be made without departing from the scope of the present disclosure. The following description is, therefore, not to be taken in a limiting sense.

[0099] The present disclosure is based, in part of the surprising discovery that a fluorescent zinc sensor (ZIGIR) is capable of enhanced cellular uptake in the pancreata. Derivates of ZIGIR can thus be used to specifically target therapeutic molecules to the pancreatic milieu. Exploiting the beta cell-targeting motif (BTM) derived from ZIGIR, whose zinctropic property and ZnT8 interaction account for its selective uptake in islet cells both in vitro and in vivo, therapeutic molecules such as phenylbutyrate that were heretofore limited in their use can now be used as antidiabetic drugs. The disclosure enables the development of such compositions and provides characterization of beta cell-targeting phenylbutyrate (BTPB) derivatives as a potential drug candidate. Examples provided herein show highly efficient, cellular zinc-dependent and ZnT8-dependent uptake of BTPB by islet beta cells, with >100 times uptake selectivity by pancreatic islets than by exocrine acinar cells in vivo. Most importantly, in vivo test showed that BTPB protected NOD mice from developing diabetes with a remarkable efficacy (75% diabetes free with BTPB-1 vs. 25% with vehicle control or equimolar PB, p<0.01). Collectively, through targeted delivery of the prodrug to p-cells, thereported benefits of PB on reducing ER stress and promoting cell survival were augmented, ultimately resulting in the preservation of p-cell function and restoration of physiological glycemic control.

[0100] Thus, in an aspect, the current disclosure details compositions and method of making and using compositions for treating pancreatic diseases. In an aspect, the current disclosure provides a drug delivery platform to target islet p-cells. In an aspect, the disclosed platform can be used to target therapeutic molecules to islet cells of the pancreas. In an aspect, the current disclosure also provides methods to apply the platform to enable selective and efficient delivery of an exemplary drug, chemical chaperone and a HDAC inhibitor, phenylbutyrate and it’s derivates to islet cells to mitigate ER stress and inhibit the HDAC activities, and inflammation, and to curb autoimmune attack on p-cells during the pre-diabetic or the early stage of Type 1 Diabetes (T1 D) development.I. Terminology

[0101] The phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. For example, the use of a singular term, such as, “a” is not intended as limiting of the number of items. Also, the use of relational terms such as, but not limited to, “top,” “bottom,” “left,” “right,” “upper,” “lower,” “down,” “up,” and “side,” are used in the description for clarity in specific reference to the figures and are not intended to limit the scope of the present disclosure or the appended claims.

[0102] Further, as the present disclosure is susceptible to aspects of many different forms, it is intended that the present disclosure be considered as an example of the principles of the present disclosure and not intended to limit the present disclosure to the specific aspects shown and described. Any one of the features of the present disclosure may be used separately or in combination with any other feature. References to the terms “aspect,” “aspects,” and / or the like in the description mean that the feature and / or features being referred to are included in, at least, one aspect of the description. Separate references to the terms “aspect,” “aspects,” and / or the like in the description do not necessarily refer to the same aspect and are also not mutually exclusive unless so stated and / or except as will be readily apparent to those skilled in the art from the description. For example, a feature, structure, process, step, action, or the like described in one aspect may also be included in other aspects but is not necessarily included. Thus, the present disclosure may include a variety of combinations and / or integrations of the aspects described herein. Additionally, all aspects of the present disclosure, as described herein, are not essential for its practice. Likewise, other systems, methods, features, and advantages of the present disclosure will be, or become, apparent to one with skill in the art upon examination of the figures and the description. It isintended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be encompassed by the claims.

[0103] Any term of degree such as, but not limited to, “substantially” as used in the description and the appended claims, should be understood to include an exact, or a similar, but not exact configuration. For example, “a substantially planar surface” means having an exact planar surface or a similar, but not exact planar surface. Similarly, the terms “about” or “approximately,” as used in the description and the appended claims, should be understood to include the recited values or a value that is three times greater or one third of the recited values. For example, about 3 mm includes all values from 1 mm to 9 mm, and approximately 50 degrees includes all values from 16.6 degrees to 150 degrees. For example, they can refer to less than or equal to ± 5%, such as less than or equal to ± 2%, such as less than or equal to ± 1 %, such as less than or equal to ± 0.5%, such as less than or equal to ± 0.2%, such as less than or equal to ± 0.1 %, such as less than or equal to ± 0.05%.

[0104] The terms "comprising," "including" and "having" are used interchangeably in this disclosure. The terms "comprising," "including" and "having" mean to include, but not necessarily be limited to the things so described.

[0105] Lastly, the terms “or” and “and / or,” as used herein, are to be interpreted as inclusive or meaning any one or any combination. Therefore, “A, B or C” or “A, B and / or C” mean any of the following: “A,” “B” or “C”; “A and B”; “A and C”; “B and C”; “A, B and C.” An exception to this definition will occur only when a combination of elements, functions, steps, or acts are in some way inherently mutually exclusive.

[0106] The term “treating,” as used herein refers to reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or ameliorating one or more symptoms of such condition or disorder. The term “treatment,” or “therapy” of a subject refers to any type of intervention, or the administration of a compound as disclosed herein, to a subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down, or preventing the onset, progression, development, severity or recurrence of a symptom, complication, condition, or biochemical indicia associated with a disease. In some aspects, the disease is a pancreatic disease.

[0107] The term “pancreatic disease” as used herein refers to any disease, disorder or condition that may afflict the pancreas of an animal or a human subject. In an aspect, the term encompasses genetic, infectious, or injury-based afflictions of the pancreas. In an aspect, the term pancreatic disease encompasses any disease, disorder or condition that can benefit fromtargeted delivery of a therapeutic molecule to the pancreas. Examples of pancreatic diseases include but are not restricted to hyperglycemia, pre-diabetes, impaired glucose tolerance, diabetes type I, diabetes type II, syndrome X, a pancreatic cancer, acute pancreatitis, chronic pancreatitis, cholangitis, cholecystitis, hereditary pancreatitis, alcohol related pancreatitis, or a combination thereof.

[0108] The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce a toxic, allergic, or similar untoward reaction, such as gastric upset, dizziness, and the like, when administered to a human. Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. or European Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0109] The language “effective amount” of the compound is that amount necessary or sufficient to treat or prevent a pancreatic disease, disorder, or condition. One of ordinary skill in the art would be able to study the factors contained herein and make the determination regarding the effective amount of the compounds described herein without undue experimentation.

[0110] The phrase “pharmaceutically acceptable excipient” includes any pharmaceutically acceptable material, composition, or vehicle, suitable for administering the compounds described herein to mammals. The excipient includes liquid or solid filler, a diluent, a binder, a buffering agent, a pH modifying agent, a disintegrant, a dispersant, a preservative, a lubricant or wetting agent, taste-masking agent, an antioxidant, carrier, adjuvant, stabilizing agent, emulsifying agent, solution promoter, salt, solubilizer, antifoaming agent, surfactant, a flavoring agent, a coloring agent, solvent or encapsulating material or any combination thereof. Each excipient must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. The amount and types of excipients utilized to form pharmaceutical compositions may be selected according to known principles of pharmaceutical science. In each of the aspects described herein, a composition of the disclosure may optionally comprise one or more additional drug or therapeutically active agent in addition to the at least one factor disclosed herein. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for treatment of the disease, disorder, or condition.

[0111] As used herein, the term derivative of a disclosed molecule encompasses all optical stereoisomers, as well as racemic mixtures where such isomers or mixtures exist, unless the specific isomer or diastereomer is noted. In an aspect, the current disclosure encompassespharmaceutically acceptable derivatives of the compounds including but not restricted to salts, esters, enol ethers, enol esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, clathrates, solvates or hydrates thereof. Such derivatives may be readily prepared by those of skill in this art using known methods for such derivatization. Pharmaceutically acceptable salts include, but are not limited to, amine salts, such as but not limited to N,N'- dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N- benzylphenethylamine, 1-para-chlorobenzyl-2-pyrrolidin-T-ylmethylbenzimidazole, diethylamine and other alkylamines, piperazine and tris(hydroxymethyl)aminomethane; alkali metal salts, such as but not limited to lithium, potassium and sodium; alkali earth metal salts, such as but not limited to barium, calcium and magnesium; transition metal salts, such as but not limited to zinc; and inorganic salts, such as but not limited to, sodium hydrogen phosphate and disodium phosphate; and also including, but not limited to, salts of mineral acids, such as but not limited to hydrochlorides and sulfates; and salts of organic acids, such as but not limited to acetates, lactates, malates, tartrates, citrates, ascorbates, succinates, butyrates, valerates, mesylates, and fumarates. Pharmaceutically acceptable esters include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, aralkyl, and cycloalkyl esters of acidic groups, including, but not limited to, carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids and boronic acids. Pharmaceutically acceptable enol ethers include, but are not limited to, derivatives of formula C=C(OR) where R is alkyl, alkenyl, alkynyl, aryl, aralkyl and cycloalkyl. Pharmaceutically acceptable enol esters include, but are not limited to, derivatives of formula C=C(OC(O)R) where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl and cycloalkyl. In an aspect, the derivative may be a haloalkyl or heteroaryl derivative. Pharmaceutically acceptable solvates and hydrates are complexes of a compound with one or more solvent or water molecules, or 1 to about 100, or 1 to about 10, or one to about 2, 3 or 4, solvent or water molecules. Some exemplary derivatives

[0112] The term “alkyl” as used herein means a straight or branched hydrocarbon radical having from 1 to 10 carbon atoms and includes, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, iso-pentyl, n-hexyl, and the like.

[0113] The term “alkenyl” as used means straight and branched hydrocarbon radicals having from 2 to 8 carbon atoms and at least one double bond and includes, but is not limited to, ethenyl, 3-buten-1-yl, 2-ethenylbutyl, 3-hexen-1-yl, and the like. The term “alkenyl” includes cycloalkenyl, and heteroalkenyl in which 1 to 3 heteroatoms selected from O, S, N, or substituted nitrogen may replace carbon atoms.

[0114] The term “alkynyl” as used means straight and branched hydrocarbon radicalshaving from 2 to 8 carbon atoms and at least one triple bond and includes, but is not limited to, ethynyl, 3-butyn-1-yl, propynyl, 2-butyn-1-yl, 3-pentyn-1-yl, and the like.

[0115] The term “cycloalkyl” as used means a monocyclic or polycyclic hydrocarbyl group having from 3 to 8 carbon atoms, for instance, cyclopropyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclobutyl, adamantyl, norpinanyl, decalinyl, norbornyl, cyclohexyl, and cyclopentyl. Such groups can be substituted with groups such as hydroxy, keto, amino, alkyl, and dialkylamino, and the like. Also included are rings in which 1 to 3 heteroatoms replace carbons. Such groups are termed “het-erocyclyl,” which means a cycloalkyl group also bearing at least one heteroatom selected from O, S, N, or substituted nitrogen. Examples of such groups include, but are not limited to, oxiranyl, pyrrolidinyl, piperidyl, tetrahydropyran, and morpholine.

[0116] The term “alkoxy” as used herein means a straight or branched chain alkyl groups having 1-10 carbon atoms and linked through oxygen. Examples of such groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentoxy, 2-pentyloxy, isopentoxy, neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy, and 3- methylpentoxy. In addition, alkoxy refers to polyethers such as — O — (CH2)2 — O — CH3, and the like.

[0117] The alkyl, alkenyl, alkoxy, and alkynyl groups described herein are optionally substituted, prefer-ably by 1 to 3 groups selected from NR4R5, phenyl, substituted phenyl, thio C1-C6 alkyl, C1-C6 alkoxy, hydroxy, carboxy, C1-C6 alkoxycarbonyl, halo, nitrile, cycloalkyl, and a 5- or 6-membered carbocyclic ring or heterocyclic ring having 1 or 2 heteroatoms selected from nitrogen, substituted nitrogen, oxygen, and sulfur. “Substituted nitrogen” means nitrogen bearing C1-C6 alkyl or (CH2)pPh where p is 1 , 2, or 3. Perhalo and polyhalo substitution is also included.

[0118] Examples of substituted alkyl groups include, but are not limited to, 2-aminoethyl, 2- hydroxyethyl, pentachloroethyl, trifluoromethyl, 2-diethylaminoethyl, 2-dimethylaminopropyl, ethoxycarbonylmethyl, 3-phenylbutyl, methanylsulfanylmethyl, methoxymethyl, 3- hydroxypentyl, 2-carboxybutyl, 4-chlorobutyl, 3-cyclopropylpropyl, pentafluoroethyl, 3- morpholinopropyl, piper-azinylmethyl, and 2-(4-methylpiperazinyl)ethyl.

[0119] Examples of substituted alkynyl groups include, but are not limited to, 2- methoxyethynyl, 2-ethylsulfanylethynyl, 4-(1-piperazinyl)-3-(butynyl), 3-phenyl-5-hexynyl, 3- diethylamino-3-butynyl, 4-chloro-3-butynyl, 4-cyclobutyl-4-hexenyl, and the like.

[0120] Typical substituted alkoxy groups include aminomethoxy, trifluoromethoxy, 2- diethylaminoethoxy, 2-ethoxycarbonylethoxy, 3-hydroxypropoxy, 6-carboxhexyloxy, and the like.

[0121] Further, examples of substituted alkyl, alkenyl, and alkynyl groups include, but are not limited to, dimethylaminomethyl, carboxymethyl, 4-dimethylamino-3-buten-1-yl, 5- ethylmethylamino-3-pentyn-1-yl, 4-morpholinobutyl, 4-tetrahydropyrinidylbutyl, 3-imidazolidin- 1-ylpropyl, 4-tetrahydrothiazol-3-yl-butyl, phenylmethyl, 3-chlorophenylmethyl, and the like.

[0122] The term “anion” as used herein means a negatively charged counterion such as chloride, bromide, trifluoroacetate, and triethylammonium.

[0123] The term “acyl” as used herein means an alkyl or aryl (Ar) group having from 1-10 carbon atoms bonded through a carbonyl group, i.e. , R — C(O) — . For example, acyl includes, but is not limited to, a C1-C6 alkanoyl, including substituted alkanoyl, wherein the alkyl portion can be substituted by an amine, amide, carboxylic, or heterocyclic group. Typical acyl groups include acetyl, benzoyl, and the like.

[0124] The term “aryl” as used herein refers to an aromatic monocyclic hydrocarbon ring system or a polycyclic ring system where at least one of the rings in the ring system is an aromatic hydro-carbon ring and any other aromatic rings in the ring system include only hydrocarbons. In some embodiments, a monocyclic aryl group can have from 6 to 14 carbon atoms and a polycyclic aryl group can have from 8 to 14 carbon atoms. The aryl group can be covalently attached to the defined chemical structure at any carbon atom(s) that result in a stable structure. In some embodiments, an aryl group can have only aromatic carbocyclic rings, e.g., phenyl, 1-naphthyl, 2-naphthyl, anthracenyl, phenanthrenyl groups, and the like. In other embodiments, an aryl group can be a polycyclic ring system in which at least one aromatic carbocyclic ring is fused (i.e., having a bond in common with) to one or more cycloalkyl or cycloheteroalkyl rings. Examples of such aryl groups include, among others, benzo derivatives of cyclopentane (i.e., an indanyl group, which is a 5,6-bicyclic cycloalkyl / aromatic ring system), cyclohexane (i.e., a tetrahydronaphthyl group, which is a 6,6- bicyclic cycloalkyl / aromatic ring system), imidazoline (i.e., a benzimidazolinyl group, which is a 5,6-bicyclic cycloheteroalkyl / aromatic ring system), and py-ran (i.e., a chromenyl group, which is a 6,6-bicyclic cycloheteroalkyl / aromatic ring system). Other examples of aryl groups include benzodioxanyl, benzodioxolyl, chromanyl, indolinyl groups, and the like.

[0125] In an aspect, a derivative may comprise a halogen. The terms “halogen” or “halo” as used herein means fluorine, bromine, chlorine, and iodine.

[0126] The term “haloalkyl” refers to an alkyl group having one or more halogen substituents. In some embodiments, a haloalkyl group can have 1 to 10 carbon atoms (e.g., from 1 to 8 carbon at-oms). Examples of haloalkyl groups include CF3, C2F5, CHF2, CH2F, CCI3, CHCI2, CH2CI, C2CI5, and the like. Perhaloalkyl groups, i.e., alkyl groups wherein all ofthe hydrogen atoms are replaced with halogen atoms (e.g., CF3 and C2F5), are included within the definition of “haloalkyl.” For example, a C1-10 haloalkyl group can have the formula — CjH2i+i-jXj, wherein X is F, Cl, Br, or I, i is an integer in the range of 1 to 10, and j is an integer in the range of 0 to 21 , provided that j is less than or equal to 2i+1.

[0127] The term “heteroaryl” as used herein refers to an aromatic monocyclic ring system containing at least one ring heteroatom selected from O, N, and S or a polycyclic ring system where at least one of the rings in the ring system is aromatic and contains at least one ring heteroatom. A heteroaryl group, as a whole, can have from 5 to 14 ring atoms and contain 1-5 ring heteroatoms. In some embodiments, heteroaryl groups can include monocyclic heteroaryl rings fused to one or more aromatic carbocyclic rings, non-aromatic carbocyclic rings, or non-aromatic cycloheteroalkyl rings. The heteroaryl group can be covalently attached to the defined chemical structure at any heteroatom or carbon atom that results in a stable structure. Generally, heteroaryl rings do not contain O — O, S — S, or S — O bonds. However, one or more N or S atoms in a heteroaryl group can be oxidized (e.g., pyridine N-oxide, thiophene S-oxide, thiophene S,S-dioxide). Examples of such heteroaryl rings include pyrrolyl, furyl, thienyl, pyridyl, pyrimidyl, pyri-dazinyl, pyrazinyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, isothiazolyl, thiazolyl, thiadiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, indolyl, isoindolyl, benzofuryl, benzothienyl, quinolyl, 2-methylquinolyl, isoquinolyl, quinoxalyl, quinazolyl, benzotriazolyl, benzimidazolyl, benzothiazolyl, benzisothiazolyl, benzisoxazolyl, benzoxadiazolyl, benzoxazolyl, cinnolinyl, 1 H-indazolyl, 2H-indazolyl, indolizinyl, isobenzofuyl, naphthyridinyl, phthalazinyl, pteridinyl, purinyl, oxazolopyridi-nyl, thiazolopyridinyl, imidazopyridinyl, furopyridinyl, thienopyridinyl, pyridopyrimidinyl, pyri- dopyrazinyl, pyridopyrdazinyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl groups, and the like. Further examples of heteroaryl groups include 4,5,6,7-tetrahydroindolyl, tetrahydroquino-linyl, benzothienopyridinyl, benzofuropyridinyl groups, and the like.

[0128] The term “lower alkenyl” as used herein refers to alkenyl groups which contains 2 to6 carbon atoms. An alkenyl group is a hydrocarbyl group containing at least one carboncarbon double bond. As defined herein, it may be unsubstituted or substituted with the substituents described herein. The carbon-carbon double bonds may be between any two carbon atoms of the alkenyl group. It is preferred that it contains 1 or 2 carbon-carbon double bonds and more preferably one carbon-carbon double bond. The alkenyl group may be straight chained or branched. Examples include but are not limited to ethenyl, 1 -propenyl, 2- propenyl, 1-butenyl, 2-butenyl, 2-methyl-1-propenyl, 1 ,3-butadienyl, and the like.

[0129] The term “lower alkynyl” as used herein, refers to an alkynyl group containing 2-6 carbon atoms. An alkynyl group is a hydrocarbyl group containing at least one carbon-carbontriple bond. The carbon-carbon triple bond may be between any two carbon atom of the alkynyl group. In an embodiment, the alkynyl group contains 1 or 2 carbon-carbon triple bonds and more preferably one carbon-carbon triple bond. The alkynyl group may be straight chained or branched. Examples include but are not limited to ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl and the like.

[0130] The term “carbalkoxy” as used herein refers to an alkoxycarbonyl group, where the attachment to the main chain is through the carbonyl group, e.g., — C(O) — . Examples include but are not limited to methoxy carbonyl, ethoxy carbonyl, and the like.

[0131] The term “oxo” as used herein refers to a double-bonded oxygen (i.e. , =0). It is also to be un-derstood that the terminology C(O) refers to a — C=O group, whether it be ketone, aldehyde or acid or acid derivative. Similarly, S(O) refers to a — S=O group.

[0132] The term “cycloalkyl” as used herein refers to a non-aromatic carbocyclic group including cyclized alkyl, alkenyl, and alkynyl groups. A cycloalkyl group can be monocyclic (e.g., cyclohexyl) or polycyclic (e.g., containing fused, bridged, and / or spiro ring systems), wherein the carbon atoms are located inside or outside of the ring system. A cycloalkyl group, as a whole, can have from 3 to 14 ring atoms (e.g., from 3 to 8 carbon atoms for a monocyclic cycloalkyl group and from 7 to 14 carbon atoms for a polycyclic cycloalkyl group). Any suitable ring position of the cycloalkyl group can be covalently linked to the defined chemical structure. Examples of cyclo-alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcaryl, adamantyl, and spiro[4.5]decanyl groups, as well as their homologs, isomers, and the like.

[0133] The term “heteroatom” as used herein refers to an atom of any element other than carbon or hydrogen and includes, for example, nitrogen, oxygen, sulfur, phosphorus, and selenium.

[0134] The term “cycloheteroalkyl” as used herein refers to a non-aromatic cycloalkyl group that contains at least one (e.g., one, two, three, four, or five) ring heteroatom selected from O, N, and S, and optionally contains one or more (e.g., one, two, or three) double or triple bonds. A cyclo-heteroalkyl group, as a whole, can have from 3 to 14 ring atoms and contains from 1 to 5 ring heteroatoms (e.g., from 3-6 ring atoms for a monocyclic cycloheteroalkyl group and from 7 to 14 ring atoms for a polycyclic cycloheteroalkyl group). The cycloheteroalkyl group can be cova-lently attached to the defined chemical structure at any heteroatom(s) or carbon atom(s) that results in a stable structure. One or more N or S atoms in a cycloheteroalkyl ring may be oxidized (e.g., morpholine N-oxide, thiomorpholine S-oxide, thiomorpholine S,S-dioxide). Cyclohet-eroalkyl groups can also contain one or more oxo groups, such as phthalimidyl, piperidonyl, ox-azolidinonyl, 2,4(1 H,3H)-dioxo-pyrimidinyl, pyridin-2(1 H)-onyl, and the like. Examples of cyclo-heteroalkyl groups include, among others, morpholinyl, thiomorpholinyl, pyranyl, imidazolidinyl, imidazolinyl, oxazolidinyl, pyrazolidinyl, pyrazolinyl, pyrrolidinyl, pyrrolinyl, tetrahydrofuranyl, tet-rahydrothienyl, piperidinyl, piperazinyl, azetidine, and the like.

[0135] The compounds described herein may contain chiral centers and therefore may exist in different enantiomeric and diastereomeric forms. One aspect described herein encompasses all optical isomers or stereoisomers of the compounds described herein both as racemic mixtures and as individual enantiomers or diastereoisomers, or mixtures thereof, and to all pharmaceutical compositions or methods of treatment described herein that contain or employ them, respectively. Individual isomers can be obtained by known methods, such as optical resolution, optically selective reaction, or chiral chromatographic separation in the preparation of the final product or its intermediate.

[0136] The compounds described herein can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms, including hydrated forms, are equivalent to unsolvated forms and are intended to be encompassed within the scope described herein.

[0137] Compounds described herein also include isotopically labelled compounds, which are identical to those described herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds described herein include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine and chlorine, such as2H,3H,13C,11C,14C,15N,180,170,31P,32P,35S,18F, and36CI, respectively. Compounds described herein, prodrugs thereof, and pharmaceutically acceptable salts of said compounds or of said prodrugs which contain the aforementioned isotopes and / or other isotopes of other atoms are within the scope described herein. Certain isotopically labelled compounds described herein, for example those into which radioactive isotopes such as3H and14C are incorporated, are useful in drug and / or substrate tissue distribution assays. Tritiated, i.e.,3H, and carbon-14 i.e.,14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e.,2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically labelled compounds described herein and prodrugs thereof can generally be prepared bycarrying out the procedures disclosed in the Schemes and / or in the Examples and Preparations below, by substituting a readily available isotopically labelled reagent for a non- isotopically labelled reagent.

[0138] Compounds described herein also includes compounds conjugated with probes including but not restricted to photocrosslinkers, dyes, biotin etc.II. Compounds and Compositions

[0139] In an aspect, the current disclosure encompasses a compound comprising a therapeutic molecule conjugated to a Beta-cell Targeting Motif (BTM) comprising a structure of Formula I or a derivative thereof:wherein each A is independently one or more therapeutic molecules or absent; n is 1, 2, or 3;R1-R5 are each independently hydrogen, Ci-Ce linear alkyl, Ci-Ce branched alkyl, -ORe, - NReR?, -COOH, -C(O)NReR7, or a linker connected to An, or absent; and wherein Re and R?are each independently H, Ci-Ce linear alkyl, or Ci-Ce branched alkyl group.

[0140] In an aspect, the linker compriseswherein Rs and R9 are each independently H, C1-C3 linear alkyl, or C1-C3 branched alkyl; and n= 0 -6.

[0141] In an aspect, the BTM moiety can comprise a molecule as provided in Formula VII or any derivative or manufacturing intermediate thereof that is able to specifically target a pancreatic cell:Formula VII (BTPB-2).Additional BTM moieties may include homologues of the BTM defined above:n= 1 , 2, or 3 X= F, Cl, Me, OMeY = H, F, Cl, Me, OMeBTM homologues

[0142] In an aspect, the current disclosure encompasses a BTM conjugated to a therapeutic molecule. Thus, in an aspect, the current disclosure also encompasses a compound comprising a BTM moiety as disclosed herein conjugated to a therapeutic molecule. In an aspect, the term therapeutic molecule encompasses any molecule can be effectively targeted to the pancreas using the disclosed BTM platform. In an aspect, the therapeutic molecule can be a small molecule, a known drug, a new drug, a biomolecule for example a nucleotide, a nucleic acid, a nucleic acid sequence, a natural amino acid, a nonnatural amino acid, a peptide, a vitamin, or any derivates thereof. In an aspect, the therapeutic molecule is a drug for treatment or potential treatment of a pancreatic disease.

[0143] In an aspect, the therapeutic molecule is phenylbutyrate or a derivative thereof. Phenyl butyrate (PB) is a clinically approved drug that has been used for many years in both adults and children for treating urea-cycle disorders and cystic fibrosis, at dose of 450-600 mg / Kg of body weight per day in children (up to 20 grams per day). PB has been known as a chemical chaperone for reducing ER stress. Studies have shown targeting ER stress pathway in islet p-cells represents a promising therapeutic strategy for combating type 1 diabetes (T1 D). PB is also an established inhibitor of histone deacetylase, inhibition of which can account for the anti-inflammation activity of PB in reducing cytokine production as well as inhibition of cytokine post-receptor signaling. Besides T1 D, chemical chaperones including PB reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes. Hence, the pleotropic actions of PB provide multiple protections and benefits to islet p-cells, including human p-cells, especially in the context of autoimmunity or chronic inflammation. In an aspect, the current disclosure encompasses a BTM conjugated PB or a derivative thereof. In an aspect the conjugate is BTPB-1 or a derivative thereof. In an aspect, the BTPB-1 comprises a structure as provided here as Formula II, or a derivative thereof:Formula II (BTPB-1)

[0144] In an aspect, the BTM conjugated PB comprises a structure as provided in FormulaVI, VII, VIII, IX, X, XI, XI la, XI lb, XII or a derivative thereof.Formula X (BTPB-5);Formula XIII (BTPB-8).

[0145] In an aspect, any one of the conjugates any one of the BTPB-1-BTPB-8, or a derivative thereof can selectively target pancreatic islet cells. In an aspect, any one of the any one of the conjugates BTPB-1-BTPB-8, or a derivative thereof may bind to the ZnT8 protein. ZnT8 is a zinc transporter abundantly expressed in the pancreatic islet cells especially beta cell. ZnT8 is localized to the limiting membrane of the insulin granule and is responsible for importing Zn2+into the insulin granule, reaching a total granular zinc concentration of ~ 10 mM. Since BTM binds Zn2+with a Kd of 0.4 pM, the high granular zinc content of insulin granule attracts BTM and traps compounds containing BTM in the insulin granule of beta cells. Thus, in an aspect, any one of the compounds BTPB-1-BTPB-8 may effectively targets PB orderivatives thereof to the pancreatic milieu. In an aspect, the BTPB-1 has increased concentration in the islet beta cells in comparison to PB when administered in equivalent amounts to a subject. In an aspect, the compositions comprising any one of the BTPB-1 to BTP-8, or a derivative thereof as disclosed herein may be effective in the treatment of a pancreatic diseases, for example diabetes.Pharmaceutical composition(i) Pharmaceutically acceptable carriers and excipients

[0146] As used herein a “pharmaceutical composition” refers to a preparation of a disclosed composition with one or more other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0147] Hereinafter, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” are interchangeably used herein to refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

[0148] In certain aspects, compositions disclosed herein may further compromise one or more pharmaceutically acceptable diluent(s), excipient(s), and / or carrier(s). As used herein, a pharmaceutically acceptable diluent, excipient, or carrier, refers to a material suitable for administration to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. Pharmaceutically acceptable diluents, carriers, and excipients can include, but are not limited to, physiological saline, Ringer’s solution, phosphate solution or buffer, buffered saline, and other carriers known in the art.

[0149] In some aspects, pharmaceutical compositions herein may also include stabilizers, anti-oxidants, colorants, other medicinal or pharmaceutical agents, carriers, adjuvants, preserving agents, stabilizing agents, wetting agents, emulsifying agents, solution promoters, salts, solubilizers, antifoaming agents, antioxidants, dispersing agents, surfactants, or any combination thereof. Herein, the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.

[0150] In certain aspects, pharmaceutical compositions described herein may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries to facilitate processing of genetically modified endothelial progenitor cells into preparations which can be used pharmaceutically. In some aspects, any of the well- known techniques, carriers, and excipients may be used as suitable and / or as understood in the art.

[0151] In certain aspects, pharmaceutical compositions described herein may be an aqueous suspension comprising one or more polymers as suspending agents. In some aspects, polymers that may comprise pharmaceutical compositions described herein include: water-soluble polymers such as cellulosic polymers, e.g., hydroxypropyl methylcellulose; water-insoluble polymers such as cross-linked carboxyl-containing polymers; mucoadhesive polymers, selected from, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylic acid / butyl acrylate copolymer, sodium alginate, and dextran; or a combination thereof. In some aspects, pharmaceutical compositions disclosed herein may comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% total amount of polymers as suspending agent(s) by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of polymers as suspending agent(s) by total weight of the composition.

[0152] In certain aspects, pharmaceutical compositions disclosed herein may comprise a viscous formulation. In some aspects, viscosity of composition herein may be increased by the addition of one or more gelling or thickening agents. In some aspects, compositions disclosed herein may comprise one or more gelling or thickening agents in an amount to provide a sufficiently viscous formulation to remain on treated tissue. In some aspects, pharmaceutical compositions disclosed herein may comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% total amount of gelling or thickening agent(s) by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of gelling or thickening agent(s) by total weight of the composition. In some aspects, suitable thickening agents for use herein can be hydroxypropyl methylcellulose, hydroxyethyl cellulose, polyvinylpyrrolidone, carboxymethyl cellulose, polyvinyl alcohol, sodium chondroitin sulfate, sodium hyaluronate. In other aspects, viscosityenhancing agents can be acacia (gum arabic), agar, aluminum magnesium silicate, sodium alginate, sodium stearate, bladderwrack, bentonite, carbomer, carrageenan, Carbopol, xanthan, cellulose, microcrystalline cellulose (MCC), ceratonia, chitin, carboxymethylated chitosan, chondrus, dextrose, furcellaran, gelatin, Ghatti gum, guar gum, hectorite, lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, maize starch, wheat starch, rice starch, potato starch, gelatin, sterculia gum, xanthum gum, gum tragacanth, ethyl cellulose, ethylhydroxyethyl cellulose, ethylmethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl cellulose, poly(hydroxyethyl methacrylate), oxypolygelatin, pectin, polygeline, povidone, propylene carbonate, methyl vinyl ether / maleic anhydride copolymer (PVM / MA), poly(methoxyethyl methacrylate), poly(methoxyethoxyethyl methacrylate), hydroxypropyl cellulose, hydroxypropylmethyl-cellulose (HPMC), sodium carboxymethyl-cellulose (CMC), silicon dioxide, polyvinylpyrrolidone (PVP: povidone), Splenda® (dextrose, maltodextrin and sucralose), or any combination thereof.

[0153] In certain aspects, pharmaceutical compositions disclosed herein may comprise additional agents or additives selected from a group including surface-active agents, detergents, solvents, acidifying agents, alkalizing agents, buffering agents, tonicity modifying agents, ionic additives effective to increase the ionic strength of the solution, antimicrobial agents, antibiotic agents, antifungal agents, antioxidants, preservatives, electrolytes, antifoaming agents, oils, stabilizers, enhancing agents, and the like. In some aspects, pharmaceutical compositions disclosed herein may comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% total amount of one or more agents by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more agents by total weight of the composition. In some aspects, one or more of these agents may be added to improve the performance, efficacy, safety, shelflife and / or other property of the muscarinic antagonist composition of the present disclosure. In some aspects, additives may be biocompatible, without being harsh, abrasive, and / or allergenic.

[0154] In certain aspects, pharmaceutical compositions disclosed herein may comprise one or more acidifying agents. As used herein, “acidifying agents” refers to compounds used to provide an acidic medium. Such compounds include, by way of example and without limitation, acetic acid, amino acid, citric acid, fumaric acid and other alpha hydroxy acids, such as hydrochloric acid, ascorbic acid, and nitric acid and others known to those of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic acid may beused. In some aspects, pharmaceutical compositions disclosed herein may comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% total amount of one or more acidifying agents by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more acidifying agents by total weight of the composition.

[0155] In certain aspects, pharmaceutical compositions disclosed herein may comprise one or more alkalizing agents. As used herein, “alkalizing agents” are compounds used to provide alkaline medium. Such compounds include, by way of example and without limitation, ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium bicarbonate, sodium hydroxide, triethanolamine, and trolamine and others known to those of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic base can be used. In some aspects, pharmaceutical compositions disclosed herein may comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% total amount of one or more alkalizing agents by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more alkalizing agents by total weight of the composition.

[0156] In certain aspects, pharmaceutical compositions disclosed herein may comprise one or more antioxidants. As used herein, “antioxidants” are agents that inhibit oxidation and thus can be used to prevent the deterioration of preparations by the oxidative process. Such compounds include, by way of example and without limitation, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophophorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite and other materials known to one of ordinary skill in the art. In some aspects, pharmaceutical compositions disclosed herein may comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% total amount of one or more antioxidants by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more antioxidants by total weight of the composition.

[0157] In certain aspects, pharmaceutical compositions disclosed herein may comprise a buffer system. As used herein, a “buffer system” is a composition comprised of one or more buffering agents wherein “buffering agents” are compounds used to resist change in pH upon dilution or addition of acid or alkali. Buffering agents include, by way of example and without limitation, potassium metaphosphate, potassium phosphate, monobasic sodium acetate and sodium citrate anhydrous and dihydrate and other materials known to one of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic buffer can be used. In some aspects, pharmaceutical compositions disclosed herein may comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% total amount of one or more buffering agents by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more buffering agents by total weight of the composition.

[0158] In some aspects, the amount of one or more buffering agents may depend on the desired pH level of a composition. In some aspects, pharmaceutical compositions disclosed herein may have a pH of about 6 to about 9. In some aspects, pharmaceutical compositions disclosed herein may have a pH greater than about 8, greater than about 7.5, greater than about 7, greater than about 6.5, or greater than about 6.

[0159] In certain aspects, pharmaceutical compositions disclosed herein may comprise one or more preservatives. As used herein, “preservatives” refers to agents or combination of agents that inhibits, reduces or eliminates bacterial growth in a pharmaceutical dosage form. Non-limiting examples of preservatives include Nipagin, Nipasol, isopropyl alcohol and a combination thereof. In some aspects, any pharmaceutically acceptable preservative can be used. In some aspects, pharmaceutical compositions disclosed herein may comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% total amount of one or more preservatives by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more preservatives by total weight of the composition.

[0160] In certain aspects, pharmaceutical compositions disclosed herein may comprise one or more surface- acting reagents or detergents. In some aspects, surface-acting reagents or detergents may be synthetic, natural, or semi-synthetic. In some aspects, compositions disclosed herein may comprise anionic detergents, cationic detergents, zwitterionicdetergents, ampholytic detergents, amphoteric detergents, nonionic detergents having a steroid skeleton, or a combination thereof. In some aspects, pharmaceutical compositions disclosed herein may comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% total amount of one or more surface-acting reagents or detergents by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more surface-acting reagents or detergents by total weight of the composition.

[0161] In certain aspects, pharmaceutical compositions disclosed herein may comprise one or more stabilizers. As used herein, a “stabilizer” refers to a compound used to stabilize an active agent (for example a composition as disclosed herein) against physical, chemical, or biochemical process that would otherwise reduce the therapeutic activity of the agent. Suitable stabilizers include, by way of example and without limitation, succinic anhydride, albumin, sialic acid, creatinine, glycine and other amino acids, niacinamide, sodium acetyltryptophonate, zinc oxide, sucrose, glucose, lactose, sorbitol, mannitol, glycerol, polyethylene glycols, sodium caprylate and sodium saccharin and others known to those of ordinary skill in the art. In some aspects, pharmaceutical compositions disclosed herein may comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% total amount of one or more stabilizers by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more stabilizers by total weight of the composition.

[0162] In some aspects, pharmaceutical compositions disclosed herein may comprise one or more tonicity agents. As used herein, a “tonicity agents” refers to a compound that can be used to adjust the tonicity of the liquid formulation. Suitable tonicity agents include, but are not limited to, glycerin, lactose, mannitol, dextrose, sodium chloride, sodium sulfate, sorbitol, trehalose and others known to those or ordinary skill in the art. Osmolarity in a composition may be expressed in milliosmoles per liter (mOsm / L). Osmolarity may be measured using methods commonly known in the art. In some aspects, a vapor pressure depression method is used to calculate the osmolarity of the compositions disclosed herein. In some aspects, the amount of one or more tonicity agents comprising a pharmaceutical composition disclosed herein may result in a composition osmolarity of about 150 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 350 mOsm / L, about280 mOsm / L to about 370 mOsm / L or about 250 mOsm / L to about 320 mOsm / L. In some aspects, a composition herein may have an osmolality ranging from about 100 mOsm / kg to about 1000 mOsm / kg, from about 200 mOsm / kg to about 800 mOsm / kg, from about 250 mOsm / kg to about 500 mOsm / kg, or from about 250 mOsm / kg to about 320 mOsm / kg, or from about 250 mOsm / kg to about 350 mOsm / kg or from about 280 mOsm / kg to about 320 mOsm / kg. In some aspects, a pharmaceutical composition described herein may have an osmolarity of about 100 mOsm / L to about 1000 mOsm / L, about 200 mOsm / L to about 800 mOsm / L, about 250 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 350 mOsm / L, about 250 mOsm / L to about 320 mOsm / L, or about 280 mOsm / L to about 320 mOsm / L. In some aspects, pharmaceutical compositions disclosed herein may comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% total amount of one or more tonicity modifiers by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more tonicity modifiers by total weight of the composition.(ii) Dosage formulations

[0163] In certain aspects, the present disclosure provides compositions formulated for one or more routes of administration. Suitable routes of administration may, for example, include oral, rectal, transmucosal, transnasal, intestinal, and / or parenteral delivery. In some aspects, compositions herein formulated can be formulated for parenteral delivery. In some aspects, compositions herein formulated can be formulated intramuscular, subcutaneous, intramedullary, intravenous, intraperitoneal, and / or intranasal injections.

[0164] In certain aspects, one may administer a composition herein in a local or systemic manner, for example, via local injection of the pharmaceutical composition directly into a tissue region of a patient. In some aspects, a pharmaceutical composition disclosed herein can be administered parenterally, e.g., by intravenous injection, intracerebroventricular injection, intra-cisterna magna injection, intra-parenchymal injection, or a combination thereof. In some aspects, a pharmaceutical composition disclosed herein can administered to subject as disclosed herein. In some aspects, a pharmaceutical composition disclosed herein can administered to human patient. In some aspects, a pharmaceutical composition disclosed herein can administered to a human patient via at least two administration routes.

[0165] In certain aspects, pharmaceutical compositions of the present disclosure may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping orlyophilizing processes.

[0166] In certain aspects, pharmaceutical compositions for use in accordance with the present disclosure thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. For injection, the active ingredients of a pharmaceutical composition herein may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, physiological salt buffer, or any combination thereof.

[0167] In certain aspects, pharmaceutical compositions described herein may be formulated in the form of a nanoparticle. The nanoparticle may have a monolayer enclosing the nanoparticle core, wherein the siRNA molecule is disposed within the nanoparticle core. In an aspect, the nanoparticle core includes a solid lipid (i.e., lipid that remains solid at room temperature and body temperature) or a liquid lipid (i.e., oil, which remains liquid at room temperature and body temperature, for example, vegetable oil or a lipid extracted from human adipose tissue). In particular, aspects of the present disclosure include nanoparticles and compositions for the controlled and / or sustained release (e.g., release at a predetermined rate to maintain a certain concentration for a certain period of time) of an agent, such as a snoRNA from the nanoparticle.

[0168] In certain aspects, pharmaceutical compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection herein may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. In some aspects, compositions herein may be suspensions, solutions or emulsions in oily or aqueous vehicles, and / or may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0169] The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4th ed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill / Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment.III. Methods of Making

[0170] In an aspect, the current disclosure also encompasses method of making the compounds and pharmaceutical compositions as disclosed herein. In an aspect, the BTM moiety as disclosed herein can be activated through one or more intermediates to allow for conjugation with a suitable therapeutic molecule. In an aspect, the intermediates and method of activation depend on the therapeutic molecule to be conjugated with BTM. For examples the intermediates of BTM may comprise one or more of the Formula III and / or Formula IV:Formula IV.

[0171] In an aspect, the drug is phenylbutyrate (PB). In an aspect, conjugating a Beta-cell Targeting Motif (BTM) of Formula I or a derivative thereof to phenylbutyrate (PB) or a derivative thereof comprises using one or more intermediates of BTM comprising a structure of Formula III and / or Formula IV as disclosed herein, and one or more intermediates of PB comprising a structure of Formula V and / or Formula VI:Formula V; wherein Boc is a tert-butyloxycarbonyl protecting group,Formula VI.

[0172] In an aspect, the BTPB-1 is synthesized using one or more of the steps as provided in FIG. 3A. In an aspect, one or more intermediates as provided may be obtained and added at an appropriate stage of synthesis.

[0173] In an aspect, the method of synthesis may comprise the steps as provided herein. The steps provided herein can be modified by a person of ordinary skill in the art depending on various factors for example availability of reagents and intermediates, tools, devices available, thermal and pressure requirements. In an exemplary aspect the steps may comprise: V. Methods of treatment and use

[0174] In some aspects, the current disclosure encompasses a method for treating a subject in need thereof, the method comprising administering to the subject, a therapeutically effective amount of a compounds or pharmaceutical composition as disclosed herein. In some aspects, the current disclosure encompasses a method of treating a pancreatic disease, condition or disorder in a subject, the method comprising administering to the subject, a therapeutically effective amount of a compounds or pharmaceutical composition as disclosed herein. Nonlimiting examples of pancreatic diseases or conditions include hyperglycemia, pre-diabetes, impaired glucose tolerance, diabetes type I, diabetes type II, syndrome X, a pancreatic cancer, acute pancreatitis, chronic pancreatitis, cholangitis, cholecystitis, hereditary pancreatitis, alcohol related pancreatitis, or a combination thereof. In some aspects, the current disclosure encompasses a method of reducing the blood glucose level of a subject in need thereof, the method comprising administering to the subject, a therapeutically effective amount of a compound or pharmaceutical composition as disclosed herein. In some aspects, the subjectis suspected of having, or diagnosed to have diabetes. In some aspects, the subject is suspected of having, or diagnosed to have type 1 diabetes, type 2 diabetes, pre-diabetes or hyperglycemia.

[0175] As used herein, the term "subject" may include an animal, or human, to whom treatment according to the methods of the present disclosure is provided. More particularly, the term subject can include animals used in assays such as those used in preclinical testing including but not limited to mice, rats, monkeys, dogs, pigs and rabbits; as well as domesticated swine (pigs and hogs), ruminants, equine, poultry, felines, bovines, murines, canines, and the like. Human and veterinary applications are anticipated by the present disclosure. The term includes but is not limited to birds, reptiles, amphibians, and mammals, e.g., humans, other primates, pigs, rodents, such as mice and rats, rabbits, guinea pigs, hamsters, horses, cows, cats, dogs, sheep, chickens, and goats. In some aspects, the subjects are humans, chickens, or mice. In some aspects, the subject is a human. Both pediatric and adult subjects are included. For example, in any of the methods described herein, the subject can be at least 6 months old (e.g., 6 months or older, 12 months or older, 18 months or older, 2 years or older, 4 years or older, 6 years or older, 10 years or older, 13 years or older, 16 years or older, 18 years or older, 21 years or older, 25 years or older, 30 years or older, 35 years or older, 40 years or older, 45 years or older, 50 years or older, 60 years or older, 65 years or older, 70 years or older, 75 years or older, 80 years or older, 85 years or older, 90 years or older, or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16 ,18, 20, 21 ,24, 25, 27, 28, 30, 33, 35, 37, 39, 40, 42, 44, 45, 48, 50, 52, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, or more years old).

[0176] As provided herein, the composition as disclosed herein may be administered to humans and other animals by any suitable route of administration, including parenteral, oral, intraadiposal, intraarterial, intraarticular, intracranial, intradermal, intralesional, intramuscular, intranasal, intrapleural, intraprostatical, intrarectal, intrathecal, intratracheal, intratumoral, intraumbilical, intravaginal, intravenous, intravascular, intravitreal, liposomal, local, mucosal, subcutaneous, sublingual, topical, trans buccal, and transdermal route. In some particular aspects, the route of administration for the compositions of the present disclosure may be selfadministered subcutaneous injection, e.g., by use of a syringe or a pen device, or by continuous subcutaneous infusion therapy with a pump device, though intravenous, intradermal, or intraperitoneal routes may also be used. In some aspects, the present disclosure also provides an article of manufacture comprising the composition as disclosed herein. In certain aspects, the article of manufacture is a multi-use vial. In other aspects, the article of manufacture is a multi-use pre-filled cartridge. In other aspects, the article ofmanufacture is a re-usable pen injector. In other aspects, the article of manufacture is a disposable pen device.

[0177] Regardless of the route of administration selected, the compositions described herein, which may be used in a suitable hydrated form and / or are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Actual dosage levels of the active ingredients in the compositions described herein may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject. The selected dosage level will depend upon a variety of factors including the activity of the composition described herein, the route of administration, the time of administration, other drugs, compounds and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well known in the medical arts. In some aspects, when used in the treatment of diabetes, the composition may require daily dosing. If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

[0178] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds described herein employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0179] While it is possible for a compound described herein to be administered alone, it may be administered in combination with other active composition. By the term “combination” is meant either a fixed combination in one dosage unit form, or a kit of parts for the combined administration where a composition described herein and a combination partner may be administered independently at the same time or separately within time intervals that especially allow that the combination partners show a cooperative, e.g., synergistic, effect, or any combination thereof. The compounds described herein may be administered, simultaneously or sequentially, with glucagon, GLP-1 , an anti-inflammatory, antiproliferative, antibiotics, NSAIDs, painkillers, chemotherapeutic agent, immunosuppressant, other anti-cancer drugs, cytotoxic agent or salt thereof.

[0180] In some aspects, the current disclosure also encompasses use of the compositions disclosed herein for laboratory studies and preclinical testing.

[0181] In some aspects, the current disclosure encompasses a method for treating a subject in need thereof, the method comprising administering to the subject, a combination of a therapeutically effective amount of a compounds or pharmaceutical composition as disclosed herein and anti-CD3 antibody. Examples of anti-CD3 antibody include but are not limited to Teplizumab, Tzield, or the like.

[0182] Teplizumab, a humanized monoclonal antibody to CD3 on T cells, is approved by the Food and Drug Administration to delay the onset of clinical type 1 diabetes (stage 3) in patients 8 years of age or older with preclinical (stage 2) disease. In some embodiments, the subject may be treated for Type 1 diabetes.

[0183] Type 1 diabetes is an autoimmune disease characterized by T-cell mediated destruction of insulin-producing pancreatic p-cells. The loss of endogenous p-cell function necessitates administration of exogenous insulin for metabolic control and survival. Disease progression is most rapid in children.IV. Kits

[0184] In some aspects, the current disclosure encompasses a kit comprising the composition as disclosed herein and instructions for use. In some aspects, the present disclosure provides a kit for administering a composition as disclosed herein. Such a kit may comprise a means for holding and / or administering compositions as disclosed herein.

[0185] In some aspects, kits disclosed herein can have a medical container, which holds the composition in a safe, stable and durable way. In some examples, kits disclosed herein may also comprise a means to administer the composition, such as a needle, tube, a spatula, or combinations thereof.

[0186] Any of the kits may further comprise an instruction manual providing guidance for using the kit for treatment. The manual may be written with the physician or the liver specialist as the intended reader.

[0187] Those skilled in the art will appreciate that the presently disclosed aspects teach by way of example and not by limitation. Therefore, the matter contained in this description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. The following claims are intended to cover all generic and specific features described herein, as well as all statements of the scope of the method and assemblies, which, as a matter of language, might be said to fall there between.EXAMPLES

[0188] The following examples are included to demonstrate preferred aspects of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0189] This disclosure provides compositions for targeted delivery of small molecules, including therapeutic agents, to islet beta cells by conjugating a biologically active molecule (drug) to a chemical scaffold that functions as a Beta-cell Targeting Motif (BTM). The resulting conjugate, Drug-BTM, after being administered to a live animal, is taken up by the islet beta cell rapidly and efficiently to accumulate in islet beta cells to a high concentration. The targeted delivery increases the local concentration of the drug in the islet beta cell to enhance the efficacy of the drug within beta cells while reducing the potential off-target side effects on other tissues, hence improving the therapeutic index of the drug-BTM conjugate. The Examples provide exemplary embodiments of the compositions and methods disclosed herein.Example 1: ZIGIR derivates as Beta-cell Targeting Motif (BTM) scaffolds

[0190] ZIGIR, a fluorescent zinc sensor, is a Zn2+binding motif consisting of a 2- pyridylmethyl-[2-(2-pyridyl)ethyl]amine linked to carboxyrhodamine through its 5-amino substituent. FIG. 1A provides an example of a BTM derived from ZIGIR. The BTM binds zinc ion (Zn2+) with a dissociation constant (Kd(Zn2+)) of 0.4 pM. ZIGIR is cell membrane permeable, and labels insulin granules with exquisite selectivity both in vitro and in vivo. To demonstrate the general utility of the BTM in targeting beta cells, BTM was conjugated with another fluorescent dye, BODIPY, to generate BODIPY-BTM (FIG. 1B). Similar to ZIGIR, BODIPY-BTM labeled islet beta cells with both high efficiency and with good selectivity against exocrine cells in vivo (FIG. 1 C).

[0191] Synthesis of BODIPY-BTM BODIPY-BTM was synthesized according to the scheme outlined in FIG. 1D. All reagents were purchased from Aldrich or VWR. Anhydrous solvents were stored over activated molecular sieves (3A° or 4A°). TLC was performed on precoated silica gel 60F-254 glass plates (EM Science). Reaction products were purified by low-pressure flash chromatography (FC) using silica gel 60 (63 - 200 pm; EM Science).1H- NMR spectra were acquired on a Varian 400-MHz or 500- MHz spectrometer. Chemical shifts(5, ppm) were reported against tetramethylsilane (0 ppm). LC-MS was performed on an Agilent llltivo triple quadrupole mass spectrometer equipped with a quaternary pump, an autosampler, and a diode array detector. Samples were separated on a EclipsePlus C18 column (2.1 x 60 mm) running a gradient of mobile phase consisting of water (0.1 % formic acid) and acetonitrile (0.1% formic acid).

[0192] Step a: Synthesis of Intermediate 1 (FIG. 1D). 2-[2-(2-Pyridyl)ethyl-(2- pyridylmethyl)amino]acetaldehyde (200 mg, 783 pmol) and tert-butyl N-(3- aminophenyl)carbamate (195 mg, 940 pmol) were mixed in MeOH (1 mL) and H2O (1 mL). HOAc (141 mg, 2.35 mmol, 134 pL) was added until pH 4. NaBH(OAc)3 (498 mg, 2.35 mmol) was then added at 0 °C. The mixture was stirred at 25 °C for 2 hours. The reaction mixture was filtered to give a residue. The residue was purified by reversed-phase HPLC (0.1% formic acid, acetonitrile / water) to give the title compound (150 mg, 41.9% yield) as a brown oil. LC- MS: [M + H]+calcd for C26H34N5C 448.26; found: 448.2.

[0193] Step b: Synthesis of Intermediate 2 (FIG. 1 D). To a solution of intermediate 1 (150 mg, 335 pmol) in DCM (8 mL) was added HCI / dioxane (4 M, 1 mL). The mixture was stirred at 25 °C for 1 hour. The reaction mixture was concentrated under reduced pressure to give the title compound (155 mg, crude) as a yellow solid. The crude product was used for the next step directly without further purification. LC-MS: [M + H]+calcd for C2iH2eNs+348.21 ; found: 348.0.

[0194] Step c: Synthesis of BODIPY-BTM (FIG. 1D). To a solution of 3-(2,2-difluoro-10,12- dimethyl-1-aza-3-azonia-2-boranuidatricyclo[7.3.0.03’7]dodeca-3,5,7,9,11-pentaen-4- yl)propanoic acid (BODIPY acid, 20 mg, 68.5 pmol) in DMF (2 mL) was added HATU (39 mg, 103 pmol) and DIEA (44.2 mg, 342 pmol) at 0 °C. Intermediate 2 (31.5 mg, 82.2 pmol, HCI salt) was then added. The mixture was stirred at 25 °C for 2 hours. The reaction mixture was filtered, and the filtration was purified by reverse-phase HPLC (0.1 % formic acid, acetonitrile / water) to give the title compound (11 .0 mg, 25.3% yield) as a brown solid. LC-MS: [M + H]+calcd for C36H39BF2N7O+622.32; found: 622.5.1H NMR (400 MHz, DMSO-d6) 5 = 9.74 (s, 1 H), 8.48 - 8.45 (m, 2H), 7.71 (s, 1 H), 7.66 - 7.60 (m, 2H), 7.25 - 7.17 (m, 4H), 7.10 (d, J = 3.6 Hz, 1 H), 6.97 - 6.93 (m, 2H), 6.73 (br d, J = 7.6 Hz, 1 H), 6.40 (d, J = 4.0 Hz, 1 H), 6.32 (s, 1 H), 6.21 (br d, J = 8.8 Hz, 1 H), 5.39 (t, 1 H), 3.78 (s, 2H), 3.17 (br t, J = 7.6 Hz, 2H), 3.05 - 3.04 (m, 2H), 2.92 - 2.89 (m, 4H), 2.75 - 2.69 (m, 4H), 2.49 (br s, 3H), 2.27 (s, 3H).

[0195] T o further investigate the mechanism of BTM-mediated targeted delivery to islet beta cells, it was proposed that the ZnT8 protein is required. ZnT8 is a zinc transporter abundantly expressed in the pancreatic islet cells especially beta cell. ZnT8 is localized to the limiting membrane of the insulin granule and is responsible for importing Zn2+into the insulin granule,reaching a total granular zinc concentration of ~ 10 mM. Since BTM binds Zn2+with a Kd of 0.4 pM, the high granular zinc content of insulin granule attracts BTM and traps compounds containing BTM in the insulin granule of beta cells. T o confirm the involvement of ZnT8 in BTM uptake and enrichment in beta cells, beta cell accumulation of BODIPY-BTM between wild type mouse islet beta cells expressing ZnT8 (ZnT8+ / +) and ZnT8 knockout mouse islet cells (ZnT8- / -) were compared. Both ZnT8+ / + and ZnT8- / - islet cells contain a similar amount of insulin based on immunofluorescence signal of insulin (FIG. 2A), but the cellular zinc content of ZnT8+ / + islet cells is more than 13-fold higher than that of ZnT8- / - cells (FIG. 2B, zinc content measured by inductively coupled plasma mass spectrometry, or ICP-MS). Fluorescence imaging of mouse islet cells loaded with BODIPY-BTM (1 pM, 15 min) showed highly intense BODIPY fluorescence signal in ZnT8+ / + beta cells, but negligible fluorescence in ZnT8+ / + beta cells (FIG. 2C). Cellular uptake of BODIPY-BTM was further quantified by LC- MS, which showed a >7-fold enrichment of BODIPY-BTM in ZnT8+ / + islet cells relative to ZnT8- / - islet cells (FIG. 2D).Example 2: Preparation of BTM phenylbutyrate drug conjugates

[0196] To apply BTM to deliver therapeutic agents to islet beta cells for treating diabetes, a BTM conjugate of phenylbutyrate (PB) was developed. PB is a clinically approved drug that has been used for many years in both adults and children for treating urea-cycle disorders and cystic fibrosis, at a dose of 450-600 mg / Kg of body weight per day in children (up to 20 grams per day). PB is known as a chemical chaperone for reducing ER stress. Recent studies have shown targeting ER stress pathway in islet b-cells represents a promising therapeutic strategy for combating type 1 diabetes (T 1 D). PB is also an established inhibitor of histone deacetylase, inhibition of which can account for the anti-inflammation activity of PB in reducing cytokine production as well as inhibition of cytokine post-receptor signaling. In fact, inhibition of histone deacetylase limits islet infiltration and protects female NOD mice from diabetes; and leads to remission of new onset diabetes in NOD mice. These results are mediated, at least in part, by the direct beneficial effects of the drugs on b-cells, as in vitro administration of histone deacetylase inhibitors to cultured islets or b-cells have been shown to decrease b-cell death and improve b-cell function after I L-1 b exposure, to reduce islet cytokine production, to inhibit IL-1b-induced NO production in the islets; and to normalize basal insulin secretion in cultured islets. Besides T1 D, chemical chaperones including PB reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes. Hence, the pleotropic actions of PB provide multiple protections and benefits to islet b-cells, including human b-cells, especially in the context of autoimmunity or chronic inflammation.Synthesis of BTPB-1

[0197] BTPB-1 was developed herein as a Beta cell Targeted prodrug of PB. It was prepared in 8 steps according to the scheme outlined in FIG. 3A.

[0198] Step a: Intermediate 3 (FIG. 3A). To a solution of 2-[2-(2-pyridyl)ethyl-(2- pyridylmethyl)amino]acetaldehyde (1.2 g, 4.70 mmol) and methyl 3-aminobenzoate (781 mg, 5.17 mmol) in MeOH (10 mL) and H2O (2 mL) was added HOAc (847 mg, 14.1 mmol) and then NaBH(OAc)3 (2.99 g, 14.1 mmol) at 0 °C. The mixture was stirred at 25 °C for 2 hours. The reaction mixture was used for the next step directly without any work-up. LC-MS: [M + H]+calcd for C23H27N4C 391.21 ; found: 391.1.

[0199] Step b: Intermediate 4 (FIG. 3A). THF (10 mL) was added to the crude product mixture of Intermediate 3. UOH.H2O (2.15 g, 51.2 mmol) was then added. The mixture was stirred at 25 °C for 12 hours, filtered and concentrated under reduced pressure to give a residue. The residue was purified by reverse-phase HPLC (0.1 % formic acid, acetonitrile / water) to give the title compound (350 mg, 35.6% yield for 2 steps) as a yellow solid. LC-MS: [M + H]+calcd for C22H25N4O2+377.19; found: 377.3.

[0200] Step c: Intermediate 5 (FIG. 3A). To a solution of tert-butyl (2- hydroxyethyl) carbarn ate (5.40 g, 33.5 mmol) and 4-phenylbutanoic acid (5 g, 30.5 mmol) in DCM (150 mL) was added EDCI (8.76 g, 45.7 mmol). The mixture was stirred at 25 °C for 12 hours. The reaction mixture was diluted with H2O (200 mL) and extracted with DCM (150 mL x 3). The combined organic layers were washed with brine (150 mL x 3), dried over Na2SC>4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by FC (Petroleum ether / Ethyl acetate, 1 / 0 to 1 / 1), and re-purified by reverse-phase HPLC (0.1% formic acid, acetonitrile / water) to give the title compound (2 g, 21.2% yield) as a yellow oil. LC-MS: [M + Na]+calcd for Ci7H2sNO4Na+330.17; found: 330.2.1H NMR (400 MHz, DMSO-d6) 5 = 7.30 - 7.27 (m, 2H), 7.19 (d, J = 6.8 Hz, 2H), 6.91 (t, J = 4.4 Hz, 1 H), 3.99 (t, J = 5.6 Hz, 2H), 3.15 (q, J = 5.6 Hz, 2H), 2.58 (t, J = 7.6 Hz, 2H), 2.29 (t, J = 7.2 Hz, 2H), 1.85 - 1.78 (m, 2H), 1.36 (s, 9H).

[0201] Step d: Intermediate 6 (FIG. 3A). To a solution of intermediate 5 (100 mg, 325 pmol) in DCM (8 mL) was added HCI / dioxane (4 M, 2 mL). The mixture was stirred at 25 °C for 2 hours. The reaction mixture was concentrated under reduced pressure to give the title compound as a brown oil, which was used for the next step without further purification. LC- MS: [M + H]+calcd for CI2HI8NO2+208.13; found: 208.0.

[0202] Step e: BTPB-1 (FIG. 3A). To a solution of intermediate 4 (100 mg, 266 pmol) in DMF (2 mL) was added HATU (101 mg, 266 pmol) and DIEA (114 mg, 885 pmol, 154 pL). 2- Aminoethyl 4-phenylbutanoate (intermediate 6, 44.0 mg, 181 pmol) was added at 0 °C. Themixture was stirred at 25 °C for 2 hours. The reaction mixture was filtered, and the filtrate was purified by HPLC (Waters xbridge 150 x 25 mm, 10 pm. Mobile phase: water / NH4HCO3- acetonitrile) to give the title compound (38 mg, 37.9% yield) as a yellow oil.1H NMR (400 MHz, DMSO-d6) 5 = 8.45 (t, J = 4.0 Hz, 2H), 8.37 (br t, J = 5.2 Hz, 1 H), 7.69 - 7.58 (m, 2H), 7.21 (br s, 11 H), 7.01 - 6.94 (m, 2H), 6.64 (dd, J = 1.6, 8.2 Hz, 1 H), 5.55 (t, J = 4.8 Hz, 1 H), 4.13 (t, J = 5.6 Hz, 2H), 3.78 (s, 2H), 3.46 (q, J = 5.6 Hz, 2H), 3.13 - 3.05 (m, 2H), 2.94 - 2.85 (m, 5H), 2.72 (br t, J = 6.4 Hz, 2H), 2.57 - 2.55 (m, 3H), 2.29 (t, J = 7.2 Hz, 2H), 1.80 (quin, J = 7.6 Hz, 2H). LC-MS: [M + H]+calcd for C34H40N5CV 566.31 ; found: 566.5.

[0203] Consistent with BODIPY-BTM, islet cell uptake and accumulation of BTPB-1 also depends on ZnT8 expression. FIG. 3B shows quantification of BTPB-1 uptake in vitro by dispersed mouse islet cells isolated from wild type C57BL6 / J mouse (ZnT8+ / +) or ZnT8 knockout mouse (ZnT8_ / _). Cellular upatke of BTPB-1 was normalized against the protein content of cell lystae (nmol BTPB-1 per mg protein). FIG. 3C shows quantification of BTPB-1 uptake in vitro by dispersed mouse islet cells isolated from wild type C57BL6 / J mouse (ZnT8+ / +) or ZnT8 knockout mouse (ZnT8_ / j. Cellular upatke of BTPB-1 was normalized against the cell number (fmol BTPB-1 per cell). In dispersed mouse islet cells, BTPB-1 accumulation in wild type islet cells (ZnT8+ / +) was an order of magnitude higher than that of ZnT8' / _islet cells (FIGS. 3B and 3C, BTPB-1 uptake normalized against protein content or cell number, respectively). Moreover, when normalized against the cell number, the average cellular concentration of BTPB-1 reached ~ 134 pM (assuming an average cell volume of 1.5 picoliter, or 1.5 pL). Since BTPB-1 was administered to cells at 1 pM, this suggested a 134- fold enrichment of BTPB-1 in islet cells after a 15-min incubation, highlighting the extraordinary efficiency and the general utility of BTM in enhancing islet cell uptake of diverse small molecules such as PB (BTPB-1), BODIPY (BODIPY-BTM), and tetramethyl rhodamine (ZIGIR). To assess the efficiency and selectivity of islet cell delivery of BTPB-1 in vivo, BTPB- 1 was administered to C57BL6 / J mice via tail vein injection. Mouse pancreases were harvested 15 min later, and digested to separate islet endocrine cells from exocrine cells. LC- MS analysis of BTPB-1 showed that islet cells took up BTPB-1 more than 100 times more efficiently than exocrine cells (FIG 3D), confirming the outstanding selectivity and efficiency of in vivo delivery of BTPB-1 to islet endocrine cells.Example 2: Therapeutic activity of BTPB-1 for treating diabetes in mouse model

[0204] To assess the therapeutic activity of BTPB-1 in preventing or treating diabetes, BTPB-1 was tested in a T1 D mouse model, the nonobese diabetic (NOD) mouse, a widely used animal model in preclinical diabetes research. Female NOD mice (NOD / ShiLtJ mice, Strain #001976, Jackson Laboratory) were used because of their higher spontaneousdiabetes incidence as compared to males. 12 Female NOD mice per group were treated with vehicle control, PB, or BTPB-1 starting at 5-wk of age. Drugs were given to the mice 5 times a week during weekdays, administered once daily through intraperitoneal injection (IP injection). In the control groups receiving either vehicle solution or the parent drug PB (2.32 mg / Kg or 14.1 pmol / Kg), mice started to develop diabetes (morning non-fast blood glucose > 350 mg / dL) at 13-wk of age, and -80% of these mice developed diabetes by 21-wk of age (FIGS. 4A and 4B). In contrast, female NOD mice receiving BTPB-1 (8 mg / Kg or 14.1 pmol / Kg) did not start to develop diabetes till 16-wk old, and less than 25% of mice developed diabetes by 21-wk of age (FIGS. 4A and B). FIG. 4A shows Kaplan-Meier (Mann-Whitney test, **p<0.01) plot of diabeters incidence of mice treated with vehicle, PB, or BTPB-1. Hence, compared to the vehicle control or the parent drug PB (non-targeted), beta cell targeted BTPB- 1 was effective in preventing T1 D in the NOD animal model. It delayed T1 D onset by 3 weeks and reduced T1 D incidence by more than 50%. In addition, BTPB-1 did not exhibit any sign of gross toxicity to the treated mice. Over the course of treatment, mice receiving BTPB-1 appeared and behaved the same as the control mice receiving only vehicle solution, and their body weights were no different from those of the control group (FIG. 4C), suggesting little toxicity of BTPB-1 at the dosage used in this experiment.Example 3: Therapeutic efficacy of BTPB-1 post diagnosis

[0205] While the above results confirmed BTPB-Ts efficacy in preventing diabetes in NOD mice, it was further evaluated if BTPB-1 could treat or reverse diabetes after the NOD mice became diabetic. In this experiment, administration of BTPB-1 to NOD mice was commenced after the diagnosis of diabetes. Among five diabetic NOD mice treated with BTPB-1 , two of them returned to normoglycemia within 1 week (FIG. 5A). One of these two mice remained diabetes free for up to 16 weeks during which BTPB-1 was administered daily on weekdays. The other mouse remained diabetes free for 5 weeks before relapse. The remaining 3 mice remained diabetic, with one showing a transient episode of euglycemia after two weeks of treatment. Overall, BTPB-1 was able to reverse diabetes in -20% of diabetic NOD mice. In striking contrast, among seven diabetic NOD mice treated with the parent drug PB, none of them responded to PB and their blood glucose levels remained well above 350 mg / dL (FIG. 5B). All these seven mice died within 9 weeks after they had been diagnosed with diabetes (FIG. 5C). By comparison, NOD mice treated with BTPB-1 lived much longer, and 40% of these mice lived at least 16 weeks after BTPB-1 treatment (drug treatment was halted and terminated the experiment when the mice reached 31-wk old).

[0206] Taken together, both the diabetes prevention study (FIGS. 4A-4C) and the diabetes treatment study (FIG. 5) unequivocally demonstrated the superior efficacy of beta cell targetedprodrug BTPB-1 over its parent drug PB in restoring euglycemia in NOD mice. Both fluorescence imaging (FIG. 1 and FIG. 2) and LC-MS quantification (FIG. 3) confirmed that compounds containing the BTM are taken up by pancreatic islet cells with high efficiency and good selectivity against exocrine cells. FIG. 6 lists additional examples of beta cell targeted PB prodrugs containing the BTM.Example 4

[0207] Assaying in vitro islet cell uptake of BODIPY-BTM or BTPB-1 by LC-MS Mouse islets (C57BL / 6J or ZnT8 knock-out mouse (C57BL / 6J background), 12-15 weeks male) were isolated from mouse pancreata following the standard protocol of collagenase A digestion. The digested material was centrifuged in Histopaque to enrich the islet fraction, which were further purified by hand-picking. The purified islets were allowed to recover overnight in RPMI medium supplemented with 10% FBS, Pen / Strep (100 U / rnL and 100 pg / mL), 2 mM L- glutamine.

[0208] To quantify cellular uptake of BODIPY-BTM by LC-MS, -200 islets were dispersed into single cells by first washing them with PBS before digesting them with 0.05 % trypsin in PBS. During islet digestion, cells were gently pipetted up and down every 5 min during a 15- min incubation at 37 °C. The dispersed islet cells were kept in a pre-warmed secretion assay buffer (SAB, 114 mM NaCI, 4.7 mM KCI, 1.2 mM KH2PO4, 2.5mM CaCI2, 1.16 mM MgSO4, and 20 mM Hepes, pH 7.4) containing 3 mM glucose, 0.5% BSA, and 0.1 mg / mL DNase I. To load cells with BODIPY-BTM, dispersed islet cells (-250,000 per tube) were incubated with 1 pM of BODIPY-BTM in 0.5 mL SAB at 37 °C for 15 min. Cells were centrifuged and washed twice with PBS, and quenched with 100 pL of 80% methanol / water (vol / vol). The samples were then subjected to 3 cycles of freeze-thaw between liquid nitrogen and 37 °C water bath. After the last thaw, the sample was vortexed for 1 min and divided into two equal portions. One was centrifuged at 21 ,000 g for 10 min at 4 °C. The supernatant was then analyzed by LC-MS / MS on an Agilent llltivo triple quadrupole mass spectrometer, employing an Agilent Poroshell HPH-C18 (4.6 x 150 mm, 2.7 pm) column with gradient elution. The mobile phase for liquid chromatography consisted of 0.1 % formic acid in water and mobile phase B consisting of 0.1% formic acid in acetonitrile. The other portion of cell lysate was used for protein content assay using a Pierce BCA protein assay kit (Thermo Scientific Cat. No. 23225). Cellular uptake of BODIPY-BTM (nmol) was quantified from its MS signal against the standard calibration curve of BODIPY-BTM, and normalized against the protein content of the corresponding cell lysate sample. Islet cell uptake of BTPB-1 (1 pM, 15-min incubation at 37 °C) was quantified by LC-MS / MS following the same procedure as BODIPY-BTM.

[0209] Assaying in vivo islet cell uptake of BTPB-1 by LC-MS / MS All protocols formouse use and euthanasia were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center. C57BL / 6J mice were maintained in 12-h dark / light cycle with ad libitum access to diet (Teklad 2016) and water. BTPB-1 (prepared as 1 mg / mL injection solution in 1 % Tween-80, 5% DMSO and 94% PBS) was administered to mice (male, 12-15 weeks) at 8 mg / Kg by tail veil injection. After 15 min, mice were euthanized and pancreata were harvested. Mouse islets were isolated and lysed with 80% methanol / water following the same procedure as before. Exocrine tissue was collected from the pelleted material from the Histopaque centrifugation, suspended in SAB, washed twice with PBS before quenching with 100-200 pL of 80% methanol / water. The quenched exocrine cells were further processed by three cycles of freeze thaw, and analyzed by LC-MS following the same procedure as that of endocrine islet cells.

[0210] Preventing diabetes in female NOD mice with BTPB-1 orPB Female NOD / ShiLtJ mice (NOD / ShiLtJ mice, Strain #001976, Jackson Laboratory) 4-wk of age were ordered from Jackson Laboratory. The mice were randomly assigned into 3 groups (N=12 per group) and housed at the UT Southwestern animal facility for 1 week before drug treatment. Drugs or vehicle control was given once daily via IP injection during weekdays (5 times a week). PB injection solution was prepared at 0.29 mg / mL in 1% Tween-80, 5% DMSO and 94% PBS and was administered to a NOD mouse at 2.32 mg / Kg / day (14.1 pmol / Kg / day). BTPB-1 injection solution was prepared at 1 mg / mL in 1 % Tween-80, 5% DMSO and 94% PBS and was administered to a NOD mouse at 8 mg / Kg / day (14.1 pmol / Kg / day). The vehicle control injection applied equ-volume of 1% Tween-80, 5% DMSO and 94% PBS. Body weight and blood glucose levels were measured at least once a week starting at 5-wk of age. Blood glucose concentrations were determined using a Contour next glucometer (Ascensia Diabetes Care). Diagnosis of diabetes was based on morning non-fast blood glucose reaching or exceeding 350 mg / dL, confirmed again 24 hours later.

[0211] Treating diabetic female NOD mice with BTPB-1 or PB In the treatment study, BTPB-1 and PB injection solutions were prepared and dosed the same as in the prevention study. Treatment started immediately when a female NOD mouse was diagnosed diabetes and continued until mice died or reached 30 weeks of age. Body weight and blood glucose levels were monitored weekly.Example 5: Synthesis of BTPB-7R

[0212] BTPB-7R was synthesized according to the scheme outlined in FIG. 7.

[0213] Step a, (R)-1-((tert-butoxycarbonyl)amino)propan-2-yl 4-phenylbutanoate:EDCI (2.10 g, 10.9 mmol) in DCM (5 mL) was added dropwise to a solution of 4-phenylbutanoic acid (1.50 g, 9.14 mmol), tert-butyl N-[(2R)-2-hydroxypropyl]carbamate (1.92 g, 10.9 mmol) and DMAP (111 mg, 913 pmol) in DCM (15 mL). The mixture was stirred at 25 °C for 2 hours. The reaction mixture was quenched by addition of H2O (20 mL) and extracted with DCM (20 mL x 3). The combined organic layers were dried over Na2SC>4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=15 / 1 to 10 / 1) to give the title compound (2.5 g, 82.59% yield, 97% purity) as a colorless oil. LC-MS: [M+Na]+calcd for CI8H27NO4+344.19; found: 344.1.

[0214] Step b, (R)-1-aminopropan-2-yl 4-phenylbutanoate: HCI / dioxane (8 M, 4 mL) was added to a solution of the above product (800 mg, 2.49 mmol) in DCM (8 mL). The mixture was stirred at 25 °C for 1 hour. On completion, the reaction mixture was concentrated under reduced pressure to give the title compound (800 mg, crude) as a white solid, which was used for the next step directly without further purification. LC-MS: [M+H]+calcd for Ci3HigNO2+222.14; found: 222.2.

[0215] Step c, BTPB-7R: (R)-1-aminopropan-2-yl 4-phenylbutanoate (445 mg, 1.73 mmol) was added to a solution of 3-[2-[2-(2-pyridyl)ethyl-(2-pyridylmethyl)amino]ethylamino]benzoic acid (500 mg, 1.33 mmol), HATU (555 mg, 1.46 mmol) and DIEA (686 mg, 5.31 mmol) in DMF (5 mL) at 0 °C, the mixture was stirred at 25 °C for 2 hours. On completion, the reaction mixture was filtered and the filtrate was purified by reverse phase HPLC (0.1 % formic acid, acetonitrile / water gradient) to give the title compound (200 mg, 25% yield for 2 steps) as a brown gum solid. LC-MS: [M+H]+calcd for CssH^NsC 579.32; found: 580.4.Example 6:Synthesis of BTPB-4

[0216] BTPB-4 was synthesized according to the scheme outlined in FIG. 8.

[0217] BTPB-4 was synthesized from intermediate 2 (FIG. 1 D) and 4-phenylbutanoic acid. 4-Phenylbutanoic acid (200 mg, 1.22 mmol) was added in DMF (1 mL) containing HATU (694 mg, 1.83 mmol), DIEA (472 mg, 3.65 mmol, 636 pL) and intermediate 2 (467 mg, 1.22 mmol, HCI salt) at 0 °C. The mixture was stirred at 25 °C for 2 hours. The reaction mixture was filtered under reduced pressure and the filtrate was purified by reverse phase HPLC (0.1 % ammonium hydrogen carbonate, acetonitrile / water gradient) to give the title compound (130 mg, 21.2% yield, 98% purity) as a yellow gum.1H NMR (400 MHz, CDCI3) 6 = 8.58 - 8.48 (m, 2H), 7.59 (br t, J = 7.6 Hz, 1 H), 7.51 (br t, J = 7.6 Hz, 1 H), 7.35 - 7.29 (m, 3H), 7.23 (br d, J = 6.4 Hz, 4H), 7.16 - 7.09 (m, 5H), 7.08 - 7.04 (m, 1 H), 7.01 (br s, 1 H), 6.65 (br d, J = 7.6 Hz, 1 H), 6.30 (br d, J = 8.0 Hz, 1 H), 3.84 (s, 2H), 3.14 (br t, J = 5.6 Hz, 2H), 3.01 (s, 4H), 2.87 (brt, J = 5.6 Hz, 2H), 2.77 - 2.71 (m, 2H), 2.35 (t, J = 7.6 Hz, 2H), 2.14 - 2.05 (m, 2H). LC-MS: [M+H]+calcd for C32H36N4O 492.29; found: 494.4.BTPB-4 cellular uptake

[0218] FIG. 9A depicts cellular uptake of BTPB-4 in INS1 cells normalized by cellular protein content (left axis) or by cell number (right axis, cellular concentration of BTPB-4 was calculated based on the assumption of an average cell volume of 1000 pm3). INS cells in 12-well plate (500,000 INS cells / well ) were incubated with 1 mL of 1 pM BTPB-4 in SAB3G buffer (114 mM NaCI, 4.7 mM KCI, 1.2 mM KH2PO4, 2.5mM CaCI2, 1.16 mM MgSO4, 20 mM Hepes, pH 7.4, and 3 mM glucose) for 15 mins at 37 °C, then washed twice with SAB3G. Cells were then immediately lysed with methanol. BTPB-4 in the cell lysate was quantified by Liquid Chromatography Mass Spectrometry (LC-MS). Data are the mean ± SEM, N=3.

[0219] FIG. 9B depicts cellular uptake of BTPB-4 by primary islet cells isolated from wild type (ZnT8+ / +) or ZnT8 knockout (ZnT8_ / _) mice. Mouse islets (-100 islets per group) were dispersed into single cells and loaded with 1 pM BTPB-4 in SAB3G at 37 °C for 15 mins. Cells were washed with SAB3G, quenched with methanol and analyzed by LC-MS. Data are the mean ± SEM, N=3. **, p < 0.01 (two-sided Student’s f-test). The amount of BTPB-4 in islet cells were replotted as ng / mg protein (left axis, normalized against cellular protein content) or as pM (right axis, normalized against cell number and volume, assuming an average cell volume of 1000 pm3).

[0220] In both cultured cell line INS1 beta cells (FIG. 9A) and in primary mouse islet cells (FIG 9B), BTPB-4 was rapidly taken up by beta cells. After incubating cells with BTPB-4 (1 pM) for 15 min at 37 °C, intracellular BTPB-4 concentration was analyzed by LC-MS. Remarkably, cellular BTPB-4 concentrations reached 150 pM and 373 pM in INS1 and wild type (ZnT8+ / +) mouse islet cells, respectively. Since BTPB-4 was added to cells at 1 pM, the results showed a cellular enrichment of BTPB-4 by more than two orders of magnitude just after 15 min incubation.

[0221] ZnT8 is a zinc transporter that is selectively and abundantly expressed in pancreatic islet cells. ZnT8 is localized to the insulin granule of islet beta cells, and is responsible for importing zinc ion (Zn2+) into the insulin granule. Compared to the wild type mouse islet cells expressing ZnT8 protein (ZnT8+ / +), islet cells of ZnT8 knockout mice (ZnT8_ / ) took up BTPB-4 much less efficiently (FIG. 9B), demonstrating that cellular uptake of BTPB-4 is zinc / ZnT8 dependent. The results show that BTPB-4 is rapidly and efficiently taken up by islet beta cells to become enriched in beta cells.BTPB-4 metabolism

[0222] As a beta cell-targeted prodrug of phenyl butyrate (PB), BTPB-4 is expected to be hydrolyzed by cellular esterases to regenerate PB and BTPB-4m. FIG. 10A shows the mode of action BTM-containing prodrugs. The insulin granule acts as a drug depot to store the prodrug, releasing PB in beta cells over time. FIG. 10B shows the hydrolysis of BTPB-4 yields BTPB-4m (metabolite of BTPB-4) and the parent drug PB.

[0223] BTPB-4 metabolism was followed in INS-1 beta cells by LC-MS. INS cells in 12-well plate (500,000 cells / well) were incubated with 1 mL of 1 pM BTPB-4 in SAB3G for 15 mins at 37 °C, washed twice and incubated in 1 mL SAB3G for 0 min, 10 min or 30 min. Cells were then quenched with methanol and cell lysates were analyed by LC / MS. Data shows in FIG. 10C are the mean ± SEM, N=3. After loading INS1 cells with BTPB-4 (1 pM, 15 min), cells were washed to remove extracellular compound and assayed intracellular BTPB-4 at different times. BTPB-4 showed a time-dependent metabolism to release BTPB-4m. In addition, PB was detected both in the extracellular medium and in the cell lysate, confirming the regeneration of the parent drug PB from BTPB-4 prodrug.

[0224] FIGS. 11A-11H show BTPB-4 protect beta cell against a variety of insults and ameliorate ER stress at pM concentration.

[0225] FIG. 11A illustrates BTPB-4 protects beta cells from lipotoxicity. INS-1 cells were exposed to 0.5 mM palmitate (PA) alone, or in conjunction with PB derivatives at the indicated concentrations for 24 h. Cytotoxicity was assessed by MTT assay. Data are the mean ± SEM, N=3 and analyzed by one-way ANOVA. * p < 0.05 relative to PA, + p < 0.05, relative to 1 pM compound, # p < 0.05, relative to 5 pM compound.

[0226] FIG. 11B shows treating INS1 cells with BTPB-4 (20 pM) for only 1 hr still protect beta cells from lipotoxicity. “Withdrawal +” indicates PB or BTPB-4 was only applied to cells during the first hour; “withdrawal -” indicates PB or BTPB-4 was present in the medium for 24h. INS-1 cells were stressed with 0.5 mM PA for 24 hr. After the first hour, the medium was aspirated and replaced with medium containing no drug (“withdrawal +”), or with medium still containing PB or BTPB-4 (withdrawal -). After another 23 hr, MTT assay was performed to assess cytotoxicity. Data are the mean ± SEM, N=3 and analyzed by one-way ANOVA. * p < 0.05 relative to PA, + p < 0.05 relative to no withdrawal, # p < 0.05 relative to PB withdrawal.

[0227] FIG. 11C shows BTPB-4 protects beta cells from ER stress induced by Tg. Cytotoxicity in INS-1 cells were measured by MTT assay following 24 h treatment with 2 pM thaspsigarin. Data are the mean ± SEM, N=3 and analyzed by one-way ANOVA. * p < 0.05 compared with medium control, + p < 0.05 relative to 1 pM BTPB-4, # p < 0.05 relative to 5 pM.

[0228] FIG. 11D shows treating INS1 cells with BTPB-4 (20 pM) for only 1 hr still protect beta cells from ER stress. INS-1 cells were stressed with 2 pM Tg for 24 hr. After the first hour, the medium was aspirated and replaced with medium containing no drug (“withdrawal +”), or with medium still containing PB or BTPB-4 (withdrawal). Cytotoxicity was assessed by MTT assay. Data are the mean ± SEM, N=3 and analyzed by one-way ANOVA. * p < 0.05 relative to 2 pM thapsigargin, + p < 0.05 relative to no withdrawal.

[0229] FIG. 11 E shows BTPB-4 protects beta cells from inflammatory cytokines. Cultured INS-1 were challenged with a cocktail of pro-inflammatory cytokines consisting of 50 ng / ml TNFa, 10 ng / ml IL-ip and 50 ng / ml IFNy, in the presence of BTPB-4 or PB. After 48h, cytotoxicity in INS-1 was studied by MTT assay. Data are the mean ± SEM, N=3 and analyzed by one-way ANOVA. * p < 0.05 compared with medium control.

[0230] FIGS. 11F-H show Effect of BTPB-4 on expression of BIP (F), CHOP (G) and TXNIP (H) in INS-1. Total RNAs were extracted from INS-1 cells challenged with a cocktail of pro- inflammatory cytokines consisting of 50 ng / ml TNFa, 10 ng / ml IL-1 p and 50 ng / ml IFNy, in the absence or presence of BTPB-4 or PB. Data are the mean ± SEM, N=3-5 and analyzed by one-way ANOVA. *, p < 0.05 and **, p < 0.01.

[0231] As shown in FIGS. 11A-H, compared to its parent drug PB, beta cell-targeted prodrug BTPB-4 boosts cytoprotection by >100-fold, and exhibits prolonged biological activity once taken up by beta cells.

[0232] To compare the cytoprotective activity of BTPB-4 with its parent drug PB, a cell viability assay of beta cells stressed with either a fatty acid palmitate (lipotoxicity), or thapsigargin (Tg, ER stress), or inflammatory cytokines (inflammation) was employed. The presence of palmitic acid (0.5 mM complexed with albumin) resulted in a significant reduction in INS-1 viability. PB was found to protect INS-1 cells from lipotoxicity, however, only at mM concentration range (FIG. 11 A). In contrast, BTPB-4 protected beta cells at a concentration as low as 5 pM and, at 20 pM, it exhibited the same level of protection of beta cells against lipotoxicity as 2500 pM (2.5 mM) PB (FIG. 11 A). Similarly, BTPB-4 protected beta cells stressed under Tg (FIG. 11C) or inflammatory cytokines (50 ng / ml TNFa, 10 ng / ml IL-1 p and 50 ng / ml IFNy, FIG. 11E) at 20 pM or below, while PB showed no effect at 20 pM. These results confirmed a > 100-fold improvement in the cytoprotective activity of BTPB-4 over its parent drug PB.

[0233] In the above experiment, INS-1 beta cells were incubated with either PB or BTPB-4 throughout a 24 h palmitate or Tg challenge. Since this class of beta cell-targeted prodrugs BTPB-4 is expected to accumulate in the insulin granule and to release PB over time (i.e. theinsulin granule acts as a drug depot, cf. FIG. 10A), whether a transient BTPB-4 incubation could have a long-lasting effect on beta cells was studied. In this experiment, only PB or BTPB- 4 were included in the first hour of the 24 h palmitate (or T g) challenge, and withdrew the drug after the first hour so the INS-1 cells were bathed in the drug-free medium for the remaining 23 h (FIG. 11B for palmitate challenge, and FIG. H D forTg challenge, “withdrawal +” indicates PB or BTPB-4 was only applied to cells during the first hour; “withdrawal -” indicates PB or BTPB-4 was present in the medium for 24h). While PB lost its beta cell protection after withdrawal, BTPB-4 withdrawal still produced a significant protection of INS-1 cells against lipotoxicity (FIG. 11 B) or Tg (FIG. 11 D). The results suggest that once-daily dosing of BTPB- 4 or similar BTM-prodrugs is likely to produce a more favorable, lasting pharmacological effect owing to the accumulation of BTPB-4 in beta cells and subsequent release of the parent drug PB over time.

[0234] To elucidate the molecular mechanism of BTPB-4 protection of beta cells, real-time PCR (RT-PCR) was performed to quantify the expression of genes regulated by unfolded protein response (UPR) and ER stress. In INS1 beta cells, inflammatory cytokines (50 ng / ml TNFa, 10 ng / ml IL-1 p and 50 ng / ml IFNy) stressed beta cells to cause a drastic induction of Bip (FIG. 11 F), Chop (FIG. 11G) and TXNIP (FIG. 11H) mRNA expression. BTPB-4 (20 pM) reduced the expression of these genes not only compared to the cytokine cocktail control, but also to the PB treatment (20 pM or 2.5 mM). These results suggest that BTPB-4 treatment protected beta cells and improved cell viability at least in part by ameliorating ER stress when cells are exposed to inflammatory cytokines, a condition mimicking autoimmune attack on beta cells in type 1 diabetes.BTPB-4 cytotoxicity

[0235] When cells were incubated with BTPB-4 at concentration from 1 pM to 50 pM, cells showed normal morphology and proliferated similarly as control cells without BTPB-4. Quantification of cell viability by MTT assay confirmed that BTPB-4 (1-50 pM) did not compromise cell viability. FIG. 12 illustrates that no apparent cytotoxicity of BTPB-4 in INS1 beta cells was observed.In vivo uptake and metabolism of BTPB-4 in mice

[0236] To examine the biodistribution of BTPB-4 in vivo, 16 mg / kg of BTPB-4 was administered to CD1 male mice. Various organs were harvested 5 min later for the LC-MS quantification of BTPB-4 and its metabolite BTPB-4m. BTPB-4 showed the highest uptake in the mouse pancreas, followed by the mouse liver. FIG. 13A depicts biodistribution of BTPB- 4 and BTPB-4m in brain, spleen, kidney, pancreas and liver at 5 min post intraperitoneal (IP)injection BTPB-4 in male CD1 mice. Data are the mean ± SEM, N=3 mice / group.

[0237] BTPB-4m, the hydrolyzed metabolite of BTPB-4, was also found in different organs. To quantify the distribution of BTPB-4 and its metabolites BTPB-4m and PB in the mouse pancreas, 31 pmol / kg of BTPB-4 was injected through the tail vein of BALB / c mice. The mouse pancreas was harvested 15 min later, and separated islet cells from exocrine cells by Ficoll gradient centrifugation after pancreas digestion. FIG. 13B shows in vivo cellular uptake and metabolism of BTPB-4 in mouse islets and acinar cells (Aci.). Islet isolation of mice were performed at 15 min post i.v. injection of BTPB-4 (31 pmol / kg in BALB / c mice and then analyzed by LC-MS / MS. Data are the mean ± SEM, N=3 mice / group, and analyzed by two- sided Student’s t-test. ***, p < 0.001 and ****, p < 0.0001. Accumulation of BTPB-4 and its metabolite, BTPB-4m, were over 100-fold higher in islet than exocrine acinar tissue (FIG. 13B, cellular drug concentration was normalized against the corresponding cellular protein content). In addition, the parent drug PB was detected in the islet cells at a concentration nearly 20-fold higher than that of exocrine cells. These results confirm the extraordinarily high selectivity of BTPB-4 prodrug in targeting the pancreatic islet endocrine cells over the pancreatic exocrine cells in vivo in mice, and further support the general utility of the beta cell targeting motif (BTM) for the targeted delivery of therapeutic agents to islet beta cells.BTPB-4 delayed diabetes onset, improved beta cell function and reduced ER stress in NOD mice.

[0238] In a type 1 diabetes mouse model (NOD mouse), BTPB-4 delayed diabetes onset by improving beta cell function, reducing beta cell loss, and decreasing insulitis (FIG. 14 & FIG. 15). The anti-diabetic activity of BTPB-4 female NOD mice was assessed. Vehicle (solution made of 1 % Tween-80, 5% DMSO and 94% PBS), PB or equimolar BTPB-4 (706 nmol / mouse) dissolved in the same vehicle solution was given to NOD mice beginning at 9 weeks of age on every weekday through intraperitoneal injection (IP injection of 0.4 mL solution, N = 10-13 mice per group). FIG. 14A shows a workflow of drug dosing and mouse characterizations.

[0239] Untreated or vehicle-treated female NOD mice started to develop diabetes (blood glucose > 350 mg / dL) around 14 weeks of age. By 21 wks of age, the cumulative number of diabetic animals in the vehicle- and PB-treated groups reached 60%. In contrast, only 10% mice treated with BTPB-4 developed diabetes (FIGS. 14B and C), and the average blood glucose of BTPB-4 treated mice was significantly lower than that of vehicle or PB treated group (FIG. 14D).

[0240] FIG. 14B shows Kaplan-Meier plot of mice treated with vehicle, PB, and BTPB-4.Data were analyzed by Mann-Whitney test.

[0241] All three groups of mice gain body weight similarly as these animals age (FIG. 14E), suggesting no gross toxicity of BTPB-4 in vivo. Compared to mice treated with vehicle and parent PB, BTPB-4 treated mice showed improved metabolic profiles, including significantly improved profile of intraperitoneal glucose tolerance test (IPGTT) both at 10-wk of age (FIGS. 14F, G) and at 21-wk of age (FIGS. 14H and 141). In addition, when challenged with 2g / Kg glucose (i.p. injection), BTPB-4 treated mice showed an improved first phase insulin release than mice treated with vehicle or PB (FIG. 14J). To further confirm that BTPB-4 improved islet beta cell function, islets were isolated from 13-wk old mice (4-wk post-treatment) and performed GSIS in vitro using these freshly isolated islets. The GSIS stimulation index of islets from BTPB-4 treated mice was more than 2-fold higher than vehicle-treated mouse islets (FIGS. 14K and 14L). Consistent with the mode of action of PB as a chemical chaperone to reduce ER stress, islets from BTPB-4 treated mice showed attenuated expression of ER stress marker genes including Bip, Chop and Txnip (FIG. 14M).

[0242] The focus of another study was on charactering young, prediabetic female NOD mice between 4-wk and 12-wk of age by starting dosing mice when they were 4-wk old.

[0243] FIGS. 15 A-M show in prediabetic NOD mice 12-wk old or younger, BTPB-4 improved glucose homeostasis and beta cell function, reduced ER stress and insulitis, and decreased beta cell loss.

[0244] FIG. 15A shows workflow of drug dosing and mouse characterizations.

[0245] FIG. 15B shows average blood glucose of young female NOD mice (<12 wk old) treated with vehicle, PB or BTPB-4. Data are the mean ± SEM. N= 9-10 mice / group.

[0246] FIGS. 15C and 15D show IPGTT of 12-wk old NOD mice and corresponding area under the curve analysis respectively. Data are the mean ± SEM. N= 9-10 mice / group and analyzed by one-way ANOVA. ***, p<0.001.

[0247] FIG. 15E shows blood inulin level of 12-wk old NOD mice 5 mins following an intraperitoneal injection of glucose (2g / kg). Data are the mean ± SEM, *, p <0.05 one-way ANOVA.

[0248] FIGS. 15F and G show BTPB-4 ameliorated ER stress of NOD islet cells by Western Blot analysis. Islets were isolated from 4-wk, 8-wk and 12-wk NOD mice treated with vehicle or BTPB-4 for Western Blot. Data are the mean ± SEM and analyzed by one-way ANOVA. N = 5. *, p<0.05 relative to 4-week; #, p<0.05 relative to vehicle.

[0249] FIGS. 15H and J show BTPB-4 reduced beta cell loss and decreased insulitis in 12-wk old NOD mice. Insulin immunostaining (brown, H, scale bar, 100 pm) of pancreatic sections of 12-wk old NOD mice received vehicle, PB or BTPB-4 for 8 wks. Beta cell areas and insulitis were quantified in (I) and (J), respectively. Data are the mean ± SEM, N = 3 mice / group, analyzed by one-way ANOVA. *, p<0.05.

[0250] FIGS. 15K - M show BTPB-4 reduced beta cell loss and decreased insulitis in 16- wk old NOD mice. Insulin immunostaining (brown, FIG. 15K, scale bar, 500 pm) of pancreatic sections of 16-wk old NOD mice received vehicle or BTPB-4 for 12 wks. Beta cell areas and insulitis were quantified in (FIG. 15L) and (FIG. 15L), respectively. Data are the mean ± SEM, N = 3 mice / group, analyzed by two-side Student’s / test. **, p<0.01.

[0251] Female NOD mice have been reported to start developing insulitis as early as around 4-wk old, yet they typically do not become diabetic until after turning 13-wk old. These prediabetic mice were euglycemic as judged from their random fed blood glucose (FIG. 15B). IPGTT of these non-diabetic female NOD mice revealed a clear improvement in glucose tolerance in mice treated with BTPB-4 (FIGS. 15 C and 15D). Moreover, BTPB-4 treated mice showed higher blood insulin level 5 min post glucose stimulation (first phase insulin release, FIG. 15E), supporting improved beta cell function by BTPB-4. To investigate the effect of BTPB-4 on beta cell ER stress, ER stress proteins in mouse islets was examined by western blots. Compared to 4-wk NOD islets, XBP1s (spliced XBP1), CHOP and GRP78(Bip) were significantly elevated in 12-wk NOD islets; and BTPB-4 reduced the protein level of XBP1s and CHOP in 12-wk old NOD mice compared to the vehicle control (FIGS. 15F and 15G). Treating NOD mice with BTPB-4 for 8 weeks (from 4-wk old to 12-wk old) markedly preserved beta cell mass and suppressed insulitis compared to the vehicle control or the parent drug PB (FIGS. 15H-J). Treating this cohort of mice additional 4 weeks (from 4-wk old to 16 wk old) with BTPB-4 further reduced beta cell loss relative to the vehicle control (FIGS. 15K and 15L), and decreased insulitis severity (FIG. 15M).

[0252] Collectively, both in vitro and in vivo data support a potent cytoprotective activity of this class of beta cell targeted prodrugs such as BTPB-4. This class of targeted PB prodrugs appear to act through, at least in part, ameliorating ER stress of islet beta cells under metabolic challenge (lipotoxicity, insulin resistance) or immune insult. The beta cell targeting motif (BTM) invented by us is applicable to delivering different classes of therapeutic agents including antioxidants, anti-inflammation, anti-apoptosis, anti-ferroptosis compounds, or any other agents that may help to maintain, improve or restore beta cell function and survival.Treating type 1 diabetes (T1D) with a combination therapy.

[0253] T1 D is a complex disease involving a dynamic dialogue between the immune systemand the islet beta cell. A two-pronged therapeutic strategy - suppressing the immune system on one hand (with an anti-CD3 antibody for example), and protecting beta cells with a targeted agent such as BTPB-4 on the other - would represent the most efficacious therapeutic strategy for preventing or treating T1 D.

[0254] The anti-CD3 therapy (Teplizumab or Tzield) was recently approved as the first disease modifying therapy to delay the progression to T1 D in at-risk subject. Compared to the placebo, Teplizumab delays progression to T1 D with a median of ~2.5 years in people at high risk of developing T1 D. Teplizumab is believed to deactivate p-cell autoreactive T lymphocytes, so its mode of action should complement that of BTM-prodrugs which act on beta cells to alleviate cell stress and to improve beta cell survival / function.

[0255] To investigate the synergistic effect between BTPB-4 and anti-CD3 for treating new onset T1 D, a mouse model of cyclophosphamide (CY) induced T1 D (FIG. 16A) was adopted. In this model, two doses of CY (200 mg / kg, i.p) were given to female NOD mice, one dose at 8-wk of age, and the other at 10-wk of age. Immediately after the second dosage of CY, therapeutic drugs were administered to the mice: (1) aCD3-only: administering only an anti- CD3 antibody (F(ab')2 fragment, BioXCell, clone 145-2C11) once daily for 5 days (10 pg / mouse / day, i.v.); (2) BTPB-4 only: administering BTPB4 once daily (706 nmol / mouse / day) during weekdays (5 times a week) for 4 weeks; (3) aCD3+BTPB-4 (combination therapy): administering an anti-CD3 antibody once daily for 5 days and BTPB-4 for 4 weeks. The combination therapy involving both BTPB-4 and aCD3 was superior in preventing and treating T1 D than either agent alone (FIGS. 16B-16G). The result supports a two-pronged therapeutic strategy of combining aCD3 therapy (Teplizumab) with BTPB-4 (or other beta cell targeted, BTM-containing agents) for treating type 1 diabetes.

[0256] FIGS. 16A-G show a combination therapy applying both aCD3 and BTPB-4 was superior to either agent alone in preventing / treating diabetes in a type 1 diabetes mouse model.

[0257] FIG. 16A shows the Workflow of drug dosing and mouse characterizations.

[0258] FIG. 16B shows average blood glucose level over the course of treatment using vehicle, BTPB-4 alone, aCD3 alone or aCD3 + BTPB-4 in CY-induced NOD mice. Data are the mean ± SEM. N= 9-10 mice / group and analyzed by one-way ANOVA. *, p<0.05 relative to vehicle.

[0259] FIG. 16C shows Kaplan-Meier plot of diabetes incidence , analyzed by Mann- Whitney test; *, p < 0.05.

[0260] FIG. 16D shows random fed blood glucose of individual mice in each group treated with vehicle. FIG. 16E shows random fed blood glucose of individual mice in each group treated with BTPB-4. FIG. 16F shows random fed blood glucose of individual mice in each group treated with aCD3. FIG. 16G shows random fed blood glucose of individual mice in each group treated with aCD3 + BTPB-4. In each group, the number of mice was 9-10.

Claims

CLAIMSWhat is claimed is:

1. A compound comprising a therapeutic molecule conjugated to a Beta-cell Targeting Motif (BTM) comprising a structure of Formula I or a derivative thereof:Formula I; wherein each A is independently one or more therapeutic molecules or absent; n is 1 , 2, or 3;R1-R5 are each independently hydrogen, Ci-Ce linear alkyl, Ci-Ce branched alkyl, -ORe, - NReR?, -COOH, -C(O)NReR7, a linker connected to An, or absent; and wherein Re and R? are each independently H, Ci-Ce linear or branched alkyl group.

2. The compound of claim 1 , wherein the linker comprises:wherein Rs and R9 are each independently H, C1-C3 linear alkyl, or C1-C3 branched alkyl; and n= 0 -6.

3. The compound of claim 1 , wherein the BTM specifically binds ZnT8 transporter protein.

4. The compound of claim 1 , wherein the one or more therapeutic molecules is a drug or a prodrug.

5. The compound of claim 4, wherein the one or more therapeutic molecules is effective against a pancreatic disease.

6. The compound of claim 5, wherein the pancreatic disease is hyperglycemia, prediabetes, impaired glucose tolerance, diabetes type I, diabetes type II, syndrome X, a pancreatic cancer, acute pancreatitis, chronic pancreatitis, cholangitis, cholecystitis, hereditary pancreatitis, alcohol related pancreatitis, or a combination thereof.

7. The compound of claim 4, wherein the one or more therapeutic molecules is phenylbutyrate (PB) of Formula la or Formula lb, or a derivative or a prodrug thereof.

8. The compound of claim 7, comprising a structure as provided in any one of Formula II, Formula VII, Formula VIII, Formula XI, Formula X, of Formula XI or a derivative thereof:,Formula XIII (BTPB-8).

9. The compound of claim 8, wherein BTPB-1 has increased concentration in islet beta cells in comparison to PB when administered in equivalent amounts to a subject.

10. Use of the compound of any one of claims 1-9 for treatment of a pancreatic disease.11 . Use of the compound of claim 10, for the treatment of diabetes.

12. A pharmaceutical composition comprising a compound of any one of claims 1-9 and a pharmaceutically acceptable excipient.

13. The pharmaceutical composition of claim 12, wherein the pharmaceutically acceptable excipient is a liquid or solid filler, a diluent, a binder, a buffering agent, a pH modifying agent, a disintegrant, a dispersant, a preservative, a lubricant or wetting agent, tastemasking agent, an antioxidant, carrier, adjuvant, stabilizing agent, emulsifying agent, solution promoter, salt, solubilizer, antifoaming agent, surfactant, a flavoring agent, a coloring agent, solvent or encapsulating material or any combination thereof.

14. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition is for administering by any one of parenteral, oral, intraarterial, intraarticular, intradermal, intramuscular, intraperitoneal, intravenous, intravascular, liposomal, local, mucosal, subcutaneous, sublingual, topical, trans buccal, and transdermal route.

15. The pharmaceutical composition of claim 12, further comprising additional therapeutic molecules.

16. A method of making a BTM conjugate, comprising making of one or more of intermediates of BTM, wherein the intermediates of BTM comprise a structure of Formula III and / or Formula IVL. NI Formula III, I Formula IV.

17. A method of making BTPB-1 , comprising conjugating a Beta-cell Targeting Motif (BTM) of Formula I or a derivative thereof to phenylbutyrate (PB) or a derivative thereof, using one or more intermediates of BTM comprising a structure of Formula III and / or Formula IV, and one or more intermediates of PB comprising a structure of Formula V and / or Formula VI:Formula V; wherein Boc is a tert- butyloxycarbonyl protecting group,Formula VI.

18. A method for treating a pancreatic disease, comprising administering to a subject in need thereof, the compound of any one of claims 1 , or 7-9 or pharmaceutical composition of any one of claims 12-15.

19. The method of claim 18, wherein the pancreatic disease is hyperglycemia, prediabetes, impaired glucose tolerance, diabetes type I, diabetes type II, syndrome X, a pancreatic cancer, acute pancreatitis, chronic pancreatitis, cholangitis, cholecystitis, hereditary pancreatitis, alcohol related pancreatitis, or a combination thereof.

20. The method of claim 18, wherein the pancreatic disease is diabetes, and the compound is BTPB-1 or a derivative thereof.

21. The method of any one of claims 18-20, wherein the subject in need thereof is a mammal.

22. A kit comprising a compound of any one of claims 1, or 7-9 or a pharmaceutical composition of any one of claims 12-15, and a container for administration of the composition.

23. A method of treating a pancreatic disease, comprising administering to a subject in need thereof, a combination of the compound of any one of claims 1 , or 7-9 or pharmaceutical composition of any one of claims 12-15 and an anti-CD3 antibody.

24. The method of claim 23, wherein the anti-CD3 antibody comprises aCD3.

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