WO2008064306A2

WO2008064306A2 - Methods and compositions relating to islet cell neogenesis

Info

Publication number: WO2008064306A2
Application number: PCT/US2007/085378
Authority: WO
Inventors: Claresa S. Levetan; Loraine V. Upham
Original assignee: CureDM Inc
Current assignee: CureDM Inc
Priority date: 2006-11-22
Filing date: 2007-11-21
Publication date: 2008-05-29
Anticipated expiration: 2009-05-22
Also published as: WO2008064306A3; WO2008064306A9; US20090068145A1; US20150010508A1; US8785400B2

Abstract

The present invention provides methods and kits for treating diseases and conditions associated with impaired pancreatic function. The present invention further provides methods of stimulating islet cell neogenesis and stimulating islet cell differentiation from progenitor cells.

Description

A. 1 itle: Methods and Compositions Relating to Islet Cell Ncogcncsis

B. Cross-Refcrencc to Related Applications:

|0001] l his application claims the benefit ol U S Provisional Application Serial

No 60/867 005 filed on November 22. 2006. which is hereb) incorporated bv reference in its entiretv

C. Government Interests: Not applicable

D. Parties to a Joint Research Agreement: Not Applicable

E. Incorporation by Reference of Material submitted on a Compact Disc: Not applicable

F. Background

1. Field of Invention: Not Applicable

2. Description of Related Art: Not Applicable

G. Brief summary of the invention

[00021 Some embodiments of the present invention provide methods for stimulating islet cell neogenesis with the 111P2 peptide and pharmaceutical formulations containing the HIP2 peptide as well as related therapeutic methods for treating patients with diseases or disease conditions related to decreased pancreatic function In one embodiment, the disease is type 1 diabetes, and in another, the disease is type 2 diabetes In another embodiment, the patient has a condition associated with type 1 or type 2 diabetes In various embodiments, the methods of the invention can be practiced by administration of a therapeutically effective amount of 1 IIP2 alone in combination with insulin, in combination with insulin and another agent, and in combination with one or more agents other than insulin

[U003] As exemplified by the preceding paragraph, some embodiments of the present invention provide methods of stimulating islet cell neogenesis in a subject in need thereol, comprising administering to the subject a therapeutical Iv effective amount ol 1 IIP2. or an analog or derivative iheieot, and methods ol stimulating islet cell neogenesis in a subject in need thereof, compiising administering to the subiect a therapeutically eilective amount of 11IP2. or an analog or derivati\e thereof, and a therapeutically effective amount of an agent selected from the group consisting of: an immune therapy agent and an additional agent that stimulates islet cell regeneration. In some embodiments, the total islet number is increased. In some embodiments, the total islet number is increased by up to 50%. In some embodiments, the total islet number is increased by at least 50%. In some further embodiments, the total islet mass is increased. In some embodiments, the total islet mass is increased by up to 100%. In some embodiments, the total islet mass is increased by at least 100%.

[0004] Some embodiments of the present invention in part arise from the discovery that HIP2 is functionally more active, i.e., capable of exhibiting greater activity with respect to one or more of the functional activities associated with HIP or the hamster INGAP peptide.

[0005) Some embodiments of the present invention provide pharmaceutical formulations and unit dose forms of HIP2. In one embodiment, the pharmaceutical formulation provided contains H1P2 alone or in combination with one or more other active pharmaceutical ingredients (APIs) or agents in soluble liposome preparations that allow the HIP2 to be administered by a variety of routes, including subcutaneously, intramuscularly, intravenously, and even orally, depending on the formulation selected. In one embodiment, the formulation is for general systemic administration, but in other embodiments, the formulation comprises a targeting agent for targeted administration to specific locations, receptors, cells, tissues, organs, or organ systems within a subject.

|0006J Those of skill in the art will appreciate that the preceding paragraph exemplifies some embodiments of the invention providing pharmaceutical compositions

.->_ comprising a therapeutically elleeme amount of 11IP2 and one or more pharmaceutical acceptable exupients and or adμnants

I0007J In other embodiments of the

method ot treating a pjtholog} associated with impaired pancreatic function in a subject in need of such treatment the method further comprises the step oi administering one or more agents for stimulating pancreatic islet cell regeneration in addition to HIP2 In one aspect oi this embodiment the agents are selected from a member of the group consisting ot HIP or a HIP-related peptide other than H1P2 amylin/Pramhntide (SYMHN ™) eκendin-4 (r XI NAI IDt ™) GIP, GLP-I, GI P-I receptor agonists, OLP-I analogs, hamster INGAP peptide and related peptides, Liraglutide (NN221 1), and a dipeptidyl peptidase inhibitor, which blocks the degradation of GLP-I

[0008] In another embodiment ot the inventive method ot treating a pathology associated with impaired pancreatic function in a subject in need of such treatment the method further comprises one or more of the steps of (1) intensifying glyccniic control (2) administering oral vitamin D3 (cholecalciferol) to maintain 25-hydroxy vitamin levels above 40 ng/ml, (3) administering one or more immune therapies for protecting new islet cell formation including administration of immunosuppressive agents, (4) administering II1P2 in combination with insulin but decreasing the insulin administered over time and (5) repeatedly administering a therapy for protection of islets on a 3 to 24 month basis, depending on the selected immune therapy in addition to the step ot administering H1P2

[0009] In another embodiment ol the inventive method ol treating a pathology associated with impaired pancreatic (unction in a subject in need of such treatment the method further comprises one or more oi the steps ot (I ) lntensitv ing gljcemie control (2) administering oral vitamin DJ (choleealciierol) to maintain 25-hydro\\ wtamin levels above 40 ng/ml (3) administering an agent ior stimulating pancreatic islet regeneration in addition to HIP2. including but not limited to HlP and HlP analogs other than HIP2. (4) coadministering an agent selected trom the group consisting of amylin/Pramlintide (SYMLlN¹*), exendin-4 (EXLNA l IDb™. BYl I IΛ™), Gastrin, L pidc-rmal Growth Factor and I pidermal Growth Factor analog GIP, GLP-I, GLP-I receptor agonists Gl P-I analogs, INGΛP, Liraglutide (NN221 1), and a dipepttdyl peptidase IV inhibitor, which blocks the degradation of Gl P-I and (5) reducing, or tapering off, administration ot another diabetes therapy

|0010] In another embodiment of the inventive method of treating a pathology associated with impaired pancreatic function in a subject in need of such treatment, the method further comprises, in addition to administering HIP2, the step of administering one or more agents that inhibit, block, or destroy the autoimmune cells that target pancreatic islets Such therapies are termed "immune therapies" above In various aspects of this embodiment, the agents that inhibit, block, or destroy the autoimmune cells that target pancreatic islets are selected from the group consisting of Anti CD-3 antibodies (hOK 13' 1 (Ala-Ala) and ChAglyCD3) that target the immune response and specifically block the T - lymphocytes that cause beta cell death in type 1 diabetes Sirolimus (Rapamycin). t acrolimus (1 K5O6), a heat-shock protein 60 (Diapep277), an anti-Glutamic Acid Decarboxylase 65 (GAD65) vaccine, Myeophenolate Moietil alone or in combination with Dacli/umab the anti-CD20 agent, Rituximab, Campath-111 (Anti-CD52 Antibod} ). lysotylline. Vitamin D, IBC-VSO vaccine, which is a synthetic, metabolicall) inactive lomi ol insulin designed to prevent pancreatic beta-cell destruction, mterferon-alpha, and a \ accιne using C D4 CD25 antigen specific regulator I tells In some embodiments these or similar agents can be used in the combination therapies provided by the invention that utilize regulator) 1 cells either directly or through the use ot immunotherapy to arrest the destruction oi insulin-producing cells

[I)OIl) In another embodiment ot the inventive method of treating a pathology associated with impaired pancreatic (unction in a subject in need of such treatment at least one symptom of the pathology associated with impaired pancreatic function is treated or reduced as a result ot the administration of KIP2 In one aspect of this embodiment, the symptom is selected from a member of the group consisting of low levels ot insulin or insulin activity, insulin resistance, hyperglycemia, hemoglobin AlC level greater than 6 0%, frequent urination, excessive thirst extreme hunger unusual weight loss or gain, being overweight, increased fatigue, irritability, blurry vision, genital itching, odd aches and pains dry mouth, dry or itchy sktn, impotence, vaginal yeast infections, poor healing of cuts and scrapes, excessive or unusual infections, loss or worsening of glycemic control fluctuations in blood glucose, fluctuations in blood glucagon, and fluctuations in blood triglycerides, with hyperglycemia ultimately leading to microvascular and macrovascular complications, which include visual symptoms that lead to blindness, accelerated kidnev impairment that can lead to renal failure necessitating dialysis or kidney transplant and neuropathy leading to loot ulcers and amputations

[0012) In another embodiment of the method ot treating a pathology associated with impaired pancreatic function in a subject in need ot such treatment the pathology associated with impaired pancreatic f unction is any one of type 1 diabetes new onset type 1 diabetes t\ pe 2 diabetes latent autoimmune diabetes oi adulthood pre-diabetes impaired lasting glucose, impaued glucose tolerance insulin resistant ss ndrome meubolic

obesit)

edtmg disorders

cycles and polycystic ovarian s) ndrome

|0013I Some embodiments ol the invention also provide an antibodv which selectively binds to HIP2 In one embodiment the antibody is a monoclonal antibodv In another embodiment, the antibody is a pol) clonal antibod) In some embodiments such antibodies can be used in diagnostic methods provided by the invention, which methods comprise detecting HIP2 levels in the serum or tissue of a mammal In one embodiment, such methods are used to diagnose a disease or condition related to aberrant HIP2 levels In another embodiment the diagnostic method is used to monitor treatment with HIP2 to ensure that therapeutically effective levels are being achieved in a patient receiving such therapy

[0014| Some embodiments of the invention also provide a kit for treating a patient having type 1 or type 2 diabetes or other condition in which there are aberrant insulin levels perturbation in glucose metabolism or insulin resistance, comprising a therapeutically effective dose of HIP2 and optionally at least one agent for stimulating CJI P-I receptors or enhancing Ol P-I levels promoting beta cell regeneration, increased satiety, decreased food intake and weight loss, either in the same or separate packaging and instructions lor Us use 1 urther embodiments of the invention also provide a kit lor measuring 111P2 levels in a sample the kit comprising a HIP2-speeiiϊc antibody and optionally I1IP2 and optionally a labeling means

[0015] I hese and other aspects and embodiments of the imention are described in greater detail below

H. Description of Drawings

|0016| 1 he file oi this patent contains at least one drawing/photograph executed in color Copies of this patent with color dra\ving(s)/photograph(s) will be provided b> the Office upon request and payment ot the necessary fee

[0017] Figure 1 is a bar graph showing increased insulin production in human pancreatic ductal tissue culture after treatment with HIP analogs, as compared with similar treatment with SEQ ID NO 1 and ShQ ID NO 2

[0018] higure 2 is a bar graph showing increased insulin production in human pancreatic islet tissue after treatment with IHP analogs, as compared with similar treatment with Sl-Q ID NO I and SEQ ID NO 5

|0019] Figure 3 is a bar graph showing increased insulin content observed following treatment with HIP analogs, compared with similar treatment with SFQ ID NO 1 and a negative control

[0020] Figure 4A shows a micrograph of a pancreatic ductal tissue fraction culture after six days of culture with SLQ ID NO 4 New islet structure has formed within the cell culture

10021 ] I igure 4 B shows a micrograph oi a pancreatic ductal tissue fraction culture without culture with SFQ ID NO 4

10022 ] 1 igure 4C shows a micrograph ol a higher magnification micrograph of the micrograph shown in I igure 4Λ

|0023] I igure 4D shows a micrograph ol a 10 da\ culture ol ductal tissue tissue lraction culture not treated with Sl Q ID NO 4 [0024] 1 igure 41 shows, a micrograph of a 10 da\ culture ot ductal tissue tissue traction culture treated with SI Q ID NO 4

(0025] 1 igure 5 is a bar graph showing increased insulin production in human pancreatic ductal tissue cultures treated with I 11P2 alter 10 da> s according to the Rosenberg protocol Ihis graph shows the results of treatment with Sl Q ID NO 4 as compared with similar treatment with Sl Q ID NO 1 and Sl Q ID NO 5 Samples are 5 μg total protein in duplicate and measured by M ISA assay

|0026| figure 6 is a bar graph showing increased insulin production in human pancreatic islet tissue cultures treated with HIP2 after 10 days according to the Rosenberg protocol This graph shows the results of treatment with Sl Q ID NO 4, as compared with similar treatment with SFQ ID NO 1 and SLQ ID NO 5 Samples are 0 002 μg total protein in duplicate and measured by PLISA assay

|0027] Figure 7Λ is an inverted micrograph showing human pancreatic progenitor cells, forming a nidus of new insulin producing islets after two days ol treatment with HlP

|0028] 1 igure 7B is an inverted micrograph showing human pancreatic progenitor cells forming insulin producing islet like structure after six days ot treatment with HIP

[0029] 1* igure 8Λ is a bar graph showing increased insulin production in human pancreatic islet tissue cultures treated with two concentrations of HIP2 1 his graph shows the results of treatment with Sl Q ID NO 4, as compared with similar treatment with Sl Q ID NO 1 and Sl Q ID NO 5 Values are mean insulin units (of duplicate samples) as measured by 1 I ISA assa>

|0030| 1 igure 8H is a bar graph showing increased insulin production in human pancreatic ductal tissue cultures treated with two concentrations of H1P2 I his graph shows the results of treatment with SlIQ ID N'ϋ:4. as compared with similar treatment with SHQ ID NO: 1 and SIiQ ID NO:5. Values are mean insulin units (of duplicate samples) as measured by KLISA assay,

[00311 Figure 9 is a graph depicting the insulin requirements in mice rendered diabetic with strepto/.ocin and treated with HIPl , H1P2, H1P3. and hamster INGΛP.

[0032] Figure 10 is a table depicting the increased total islet number and increased total islet mass observed after treatment with H1P2.

[0033] Figure 11 is an image depicting a representative sample of the 900 images taken of the immunofluorescent staining for insulin from the histological evaluation of control versus I HP-treated mice pancreata. etailed Description

(0034] Insulin has been, since 1922, the primary if not the only available therapy lor the treatment of type 1 diabetes and other conditions related to the lack of or diminished efficacy or production of insulin. However, diabetic patients on insulin do not have normal glucose metabolism, because insulin is only part of the missing and aberrant pancreatic function. Despite decades of research and the advent of pancreatic islet transplantation in 1974 and newer claims of success resulting from the Edmonton Protocol for islet transplantation, these approaches have not been very successful in the United States. For example, at lour years post-transplant, fewer than 10% of patients who have received islet transplants remain insulin independent. Additionally, there is an 18% rate of serious side effects.

[0035] Investigators have also researched whether endogenous production of insulin can be stimulated by drug treatment. Fur example, over the past several decades, several therapies have been studied in which a peptide involved in glucose metabolism, or analogs ol such peptides have Ken administered to diabetic patients I hese therapies include the administration ot peptides with amino aud sequences similar to those ol Glucagon 1 ike Pcptιde-1 (Ol P 1) and such peptides include CJI P-I receptor analog;, I

1

l 1 I Λ^{I M} which is demed lrom the GiIa Monster lanm ia ^{l vι} Gastric Inhibitory Peptide'Glucose-Dependent lnsulinoptropic polypeptide (GlP) compounds homologous to GLP- 1 such as I iraglutide (NN221 1), Dφeptidyl Peptidase-4 Inhibitors which inhibit the breakdown ol Gl P-I , Gastrin, 1 pidermal Growth 1 actor and I pidermal Growth Factor Analogs and Hamster derived Islet Neogenesis Associated Peptide (INGΛP)

10036) In addition, hamster INGAP tragments have been identified that ma> beef fective in facilitating pancreatic islet neogenesis In particular, the hamster INGAP peptide identified as SLQ ID NO 1 has been identified as an agent beneficial tor the stimulation of ductal cell proliferation in hamsters However, INGAP is not a human protein and thus proteins and peptides based on its sequence may not be as efficacious as human counterpart proteins and peptides and could even produce an adverse immune response in some subjects U S Patent Application Publication No 2003/0212000 describes a human Reg3a gene that appears to be the human counterpart ol the hamster INGAP gene and identifies a 15-mer (hereinatter referred to as I IIP' α peptide of sequence identified as Sl Q IO NO 2) peptide corresponding to the hamster INGAP peptide sequence but does not demonstrate that the gene product or peptide can stimulate islet cell neogenesis

|0037J U S Patent Application Publication No 20070087971 Al which is hereby incorporated herein by relerence in its entirely discloses methods lor using HlP alone and in combination with other agents to stimulate islet cell neogenesis and treat various disease and disease conditions In addition tin;, patent application discloses related compounds I HPl (SL Q ID NO 3) and 1 I1P2 (Sl Q ID NO 4)

(00381 I or any islet cell neogenesis agent to be ettecUxe the pancreas must be elastic with respect to its ability to generate new islet cells Proof ol the elasticity ol the pancreas with respect to the generation ot new pancreatic islets throughout one's litetime in response to pancreatic islet death or apoptosis has replaced the long held concept that the number ol insulin producing islet structures is fixed at birth and maintained throughout lite whereas the plasticity and ability ot beta cells to proliferate within existing islets has been well established It is currently accepted that pancreatic islet neogenesis occurs lrom preexisting pancreatic cells through differentiation of progenitor cells found amongst both the endocrine and exocrine tractions of the pancreas Data demonstrates that, even decades alter the onset of type I diabetes pancreatic islets can be regenerated

[0039] 1 ^or example, patients with type 1 diabetes are able to make normal levels of C-peptide during pregnancy Several teams have found a paradoxical rise in O-peptide levels during the first trimester ot pregnancy into the normal range in as many as one-third of all pregnant t>pc 1 I his rise in C-pcptide is accompanied by a significant reduction in insulin requirements with some patients being able to discontinue insulin transiently during the first trimester of pregnancy 1 his rise m C-peptide during pregnancy that occurs within 10 weeks of gestation among patients despite no measurable C-peptide prior to pregnane) implies the restoration ol functioning islet structures It is hypothesized that the islet neogenesis that occurs during pregnancy results lrom the concomitant rise in endogenous steroid production and a down regulation ol the immune sy stem preventing immune attack on the tetus which likeh aho

suppression of lymphocyte attack on the islets

I I- Along with immune suppression it is also speculated that there is, an up regulation ol maternal iskt growth promoting factors during pregnant) to compensate lor the lowering ol the maternal glucose setpcnnt tn pregnancy Similarly patients who

been on long term immunosuppression tor kidney transplantation

been observ ed to regenerate insulin producing islets

|0040] Over the past decade clinical trials have been conducted to evaluate the impact of a number oi immune modulators that may arrest the destruction of the beta cells of the pancreas Λnti CD-3 antibodies (hOK 13^* 1 (Λla-Λla and ChAgIyCTO ) that target the immune response and specifically block the T-lymphocytes that cause beta cell death in type 1 diabetes have been utilized tor this purpose, as have treatments involving the administration of Sirohmus (Rapamycin), Tacrolimus (TK.506), a heat-shock protein 60 (D1APEP277^{I M}), an anti-Glutamic Acid Decarboxylase 65 (OAD65) vaccine Mycophenolate Mofetil alone or in combination with Daclimmab, the anti-CD20 agent ly sofylhne, Rituximab, Campath-1H (Anti-CD52 Antibody), Vitamin D, IBC-VSO vaccine, which is a synthetic, mctabolically inactive form of insulin designed to prevent pancreatic beta-cell destruction, and interferon-α vaccination using CD4'CD25' antigen-specific regulatory 1 cells 1 hese therapeutic approaches are intended to utilize regulatory 1 cells either directly or through the use ol immunotherapy to arrest the destruction of insulin- producing cells I he aim ol these trials is to determine the ability ot such agents to preserve islet iunction by preventing turlher immune attack on the beta cells ol the islets ol the pancreas

10041 ) Additionally, recent studies have lound that vitamin U ma\ play an important immune modulating role in the prevention of type 1 diabetes Up to 54 7% of populations in the US regardless of latitude have low 25 h\

le\ Js \ ilamin D deficiency has been demonstrated not only to be associated with the increased risk ol type 1 diabetes and seen at the onset ol t\ pe 1 diagnosis but also is commonK seen among both patients with tvpe 1 and 2 diabetes Maintaining levels above 40 ng/ml are recommended to sustain normal immune lunction No adverse ellects have been seen with doses up to 10 000 IU 'day

[0042) To date however there has been no therapy that has been successfully used to treat the underlying disease mechanisms ol type 1 diabetes, type 2 diabetes or conditions in which there is a lack ot or diminished insulin production and/or alterations in glucose metabolism or insulin secretion including obesity overweight, insulin resistant syndromes and the metabolic syndrome 1 here remains a need tor new treatments, methods and pharmaceutical compositions that address the underlying mechanisms tor the alterations in type 1 diabetes mellitus type 2 diabetes melhtus and other conditions in which there is a decrease in insulin secretion or an increased need lor insulin I specially needed are methods and compositions that can also treat the many other conditions in which the lack of, or diminished insulin production has a causative role or contributes to the symptoms of patients in need ol treatment I he present invention meets the need tor improved therapies lor treating type 1 dtabetes, t> pe 2 diabetes and other conditions

|00431 Some embodiments ol the present invention provide methods and compositions ior stimulating islet cell ncogenesis with Hl P2 a peptide fragment ot the human protein regenerating islet-deπvcd 3 alpha protein (Rl G3Λ) (NM 138937 1 ) also known as pancreatitis associated protein precursor (NP 002571 ) located on chromosome 2pl2 In some embodiments H1P2 induces or stimulates islet neogenesis lrom progenitor cells resident within the pancreas. In sonic embodiments, this neogenesis agent is used in accordance with the methods of the invention to treat diseases associated with low or inadequate levels of insulin or insulin activity resulting in aberrant carbohydrate metabolism that may result from pancreatic islet dysfunction or immune destruction. In some embodiments, these diseases include diabetes mellitus (type 1 diabetes), type 2 diabetes (non-insulin dependent diabetes mellitus and insulin requiring adult onset diabetes, diabetes in childhood and adolescence), and Latent Autoimmune Diabetes in Adults (LADA).

(0044| Some embodiments of the invention also provide pharmaceutical compositions and therapies for the treatment of pancreatic dysfunction, including type 1 and type 2 diabetes, with such compositions. In one embodiment, these compositions comprise HIP2. In another embodiment, these compositions comprise HIP2 and other agents that affect glucose metabolism. In some embodiments, these other agents that affect glucose metabolism are agents that are involved in pancreatic islet neogenesis and agents that inhibit, block, or destroy the autoimmune cells that target pancreatic islet cells. In one embodiment, the therapies of the invention are practiced by administering a therapeutically effective amount of HIP2 to a mammal in need of such therapy. In another embodiment, the therapies of the invention are practiced by administering a therapeutically effective amount of HIP2 to a mammal in need of such therapy in combination with another agent (such as a hormone or compound) that affects glucose metabolism, including but not limited to hormones or compounds that are involved in beta cell regeneration, satiety, and gastric emptying, such as OLP-I, CiIP. GLP-I receptor analogs. CiLP-I analogs, and Dipeptidvl Peptidase-4 Inhibitors, which prevent destruction of CiLP-I, and agents that inhibit, block, or destroy the autoimmune cells that target pancreatic cells. In some embodiments, the I II 1*2 and the other agent nuj be administered separatelv or mav first be admixed to pro\ idc a combination composition ot the invention and administered simultaneously

|00451 ^ VViIl be appreciated by those ot skill in the art the preceding paragraph exemplifies, some embodiments ol the present invention which provide methods ot treating a disease or condition associated with impaired pancreatic function in a subject in need thereof, comprising administering to the subject a therapeutically effective amount ol HIP2 or an analog or derivative thereot and one or more agents selected trom the group consisting of an immune therapy agent and an additional agent that stimulates islet cell regeneration In some embodiments, the immune therapy agent is selected from the group consisting ot anti-CD3 antibodies, sirohmus, tacrolimus a heat-shock protein 60, an anti-glutamic acid decarboxylase 65 vaccine, mycophenolate moletil alone or in combination with daclizumab an anti-CD20 agent, πtuximab, campath-lH, lysolylline, vitamin D, IBC-VSO vaccine interteron-alpha, and a vaccine using CD4'CD25* antigen-speufic regulatory T cells In some embodiments, the additional agent that stimulates islet cell regeneration is selected from the group consisting ot a HIP or a HIP-related peptide, amylin, pramhntide, insulin, e\endin-4 GIP, GI P-I. GI P-I receptor agonists, GI P-I analogs, hamster INGAP peptide and related peptides liraglutidc, and a dipeptidyl peptidase inhibitor which blocks the degiadation ot Gl P-I

|0046] I he following definitions are provided to assist the reader Unless otherwise defined, all terms ot art, notations and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood b> those ol skill in the chemical and medical arts In some cases, teπns with commonly understood meanings are defined herein tor clarity and'or tor ready reference and the inclusion ol such definitions herein should not necessaπlv be construed to represent a substantial dif ference over the definition ot the tenti as generallv understood in the art

(00471 Λs used herein 'treating ' a condition or patient refers to taking steps to obtain beneficial or desired results including clinical results 1 or purposes ol this invention beneficial or desired clinical results include but are not limited to alleviation or amelioration oi one or more symptoms of diabetes dimimshment of extent of disease delay or slowing of disease progression, amelioration, palliation or stabilization ol the disease state, and other beneficial results described below Symptoms ot diabetes include low or inadequate levels of insulin or insulin activity, lrequent urination, excessive thirst extreme- hunger, unusual weight loss, increased fatigue irritability blurry vision genital itching odd aches and pains, dry mouth, dry or itchy skin impotence, vaginal yeast infections poor healing of cuts and scrapes, excessive or unusual infections hyperglycemia, loss ot glycemic control fluctuations in postprandial blood glucose, fluctuations in blood glucagon fluctuations in blood triglycerides Diabetes may be diagnosed by methods well known to one of ordinary skill in the art I or example, commonly diabetics have a plasma blood glucose result of greater than 126 mg/dl of glucose Pre diabetes, which may also be treated by the compositions and methods oi the invention is commonly diagnosed in patients with a blood glucose level between 100 and 125 mg/dl of glucose Other symptoms may also be used to diagnose diabetes, related diseases and conditions and diseases and conditions attected b> diminished pancreatic function

[0048| Λs used herein reduction ol a symptom or svmptoms (and grammatical equivalents ol this phiase) means decreasing ot the severity or lrcqucney ol the s>mptom(s) or elimination ol the svmptom(s) [0049| Vs used herein a pathυlogv associated with impaired pancreatic function is, one in which the pathologv is associated with a diminished capacity in a subject for the pancreas ot the subject to produce and or secrete hormones anώor e>tokιncs Prelerabh this hoimone or cvtokine is insulin Pathologies that are associated with impaired pancreatic lunction include type 1 diabetes new onset type 1 diabetes, type 2 diabetes latent autoimmune diabetes of adulthood, pre-diabetes impaired lasting glucose impaired glucose tolerance insulin resistant syndrome metabolic syndrome, being overweight, obesity, hyperhpidemia hypertriglyceridemia, eating disorders and polycystic ovarian syndrome

(0050| As used herein "administering" or "administration of' a drug to a subject (and grammatical equivalents of this phrase) includes both direct administration including self-administration and indirect administration including the act of prescribing a drug for example as used herein, a physician who instructs a patient to self-administer a drug and/or provides a patient with a prescription for a drug is administering the drug to the patient

(0051) Λs used herein a subject or ' patient ' is a mammal, typically a human but optionally a mammalian animal of veterinary importance including but not limited to horses, cattle sheep, dogs and cats

|0052] As used herein, a "manifestation" ol a disease refers to a symptom sign anatomical state (t g lack ot islet cells) physiological state (e g glucose level), or report (e % triglyceride level) characteristic of a subject with the disease

|0053] As used herein a "therapeutically cllective amount" ol a drug or agent is an amount ol a drug or agent that when adminislcied to a subject with a disease or condition will have the intended therapeutic effect L # alleviation amelioration palliation or elimination ol one or moie manifestations ot the disease or condition in the subject I he lull therapeutic effect does not πecessaπlv occur

administration ot one dose and ma\ occur only alter administration ot a series ol doses 1 hus a therapeuticall) effective amount may be administered in one or more administrations

[0054] As used herein, a "proph\lacticalh effective amount" of a drug is an amount ot a drug that when administered to a subject, will ha\ e the intended prophy lactic ei lect e g preventing or delaying the onset (or reoccurrence) ot disease or symptoms or reducing the likelihood of the onset (or reoccurrence) ol disease or symptoms I he full prophylactic cfiect does not necessarily occur by administration ot one dose and may occur only after administration ot a series of doses Thus, a prophylactically eltective amount may be administered in one or more administrations

(0055| Λs used herein, " I ID", "QD" and "QlIS^"' have their ordinary meanings of "three times a day", "once daily," and "once before bedtime", respectively

[0056] Administration of an agent "in combination with" includes parallel administration (administration of both the agents to the patient over a pcnod-of time, such as administration of a monoclonal antibody and a peptide hormone such as an incretin hormone or analog on alternate days for one month), co-administration (in which the agents arc administered at approximately the same time, e g , within about a few minutes to a few hours ot one another), and co-toπnulation (in which the agents are combined or compounded into a single dosage form suitable tor oral, subcutaneous or parenteral administration)

[0057| DPP-4 Inhibitors are dipeptidyl peptidase-4 inhibitors

[0058] Hamster INGAP is a non-human islet neogenesis associated peptide

|0059| CiIP is Gastric Inhibito Peptide also known as Glucose-Dependent Insulmotiopic PoK peptide

|00601 GI P 1 is Glucagon like- Peptide 1

|0061j HIP (SI Q ID NO 2) is a Human prolslet Peptide in purified synthetic or recombinant torm HIP and Sl Q ID NO 2 are used interchangeably herein

10062] JIIPl (SI Q ID NO 3) is a Human prolslet Peptide in purified synthetic or recombinant iorm HIPl and Sl Q ID NO 3 are used interchangeably herein

[0063| HIP2 (SI Q ID NO 4) is a Human prolslet Peptide in purified synthetic or recombinant form HIP2 and SFQ ID NO 4 are used interchangeably herein

10064] 1 here has been contusing nomenclature in the literature regarding the regenerative processes of the pancreas Often the term islet 'cell" has been used synonymously with beta cells and this distinction is important, as new therapies tor the treatment of diabetes are considered 1 he pancreatic islets are not cells but are structures each of which is composed an estimated 1000 cells of four distinct cell types 1 ) Beta cells that make insulin and amyhn and comprise 65-80% of the islet cells, 2) Alpha cells that release glucagon and make up 15-20% of the cells 3) Delta cells that make somatostatin and 4) Pancreatic polypeptide (PP) cells, sometimes referred to as gamma cells Delta and PP cells comprise less than 10% of the islet structure Islet structures comprise only 1 2% ol the pancreatic mass vet utilize 20% ot the blood suppK to the pancreas and are considered one ol the most vascularized tissues in the bodv

[0065] Ihere is a highly organized arrangement ot the lour tvpes ot cells within the islet structure Ihe deli\ery ol blood flow within each islet is in a centπlugal manner with the beta cells located most centrallj and therelore leceiving the core blood suppK while the alpha delta and pancreatic poh peptide tells are positioned outside the beta tells in a lower state ot perfusion

[0066| In addition to glucose le\e!s> vvhich aitett the beta cells beta cells, are coupled electricalK to other beta cells but not to other islet or pancreas cells 1 his elaborate s> stem ot communication within the islet ma) explain a compensatory rise in alpha cells within an islet when there is a significant decline in the beta cell mass

[0067] I he Human prolslet Peptides are active tragments ol human Rl G3Λ or pancreatitis-associated protein precursor, the gene tor which is located on chromosome 2pl2 1 he R1-G3A protein from which HIP2 is derived is shown in Fable 1 I he domain which provides HIP2 is shown in boldlace

(SLQ ID NO 6)

|0069] HIP is the putative human homologue ot the hamster INGΛP peptide U S

Patent No 5 834 590, incorporated herein by relerence in its entirety, discloses a hamster islet neogenesis associated protein (INGAP) and associated peptides at least 15 amino acids in length A BLAS 12P alignment of human RI G3Λ and hamster INGΛP performed on the NCBl website is shown below, in l able 2

|0071] In boldface in I able 2 above is lhe domain in RI O3Λ lrom which I IIP2 SI Q ID NO 4 is derived and the corresponding hamster sequence in INGΛP In U S Patent Publication No 20040132644. incorporated herein by reference in its entirety, an 1NGΛP peptide s>hown in bold above in lablc 2 is disclosed I his hamster INGAP peptide is purportedly being studied for its efficacy in stimulating islet neogenesis

|0072J Microarray analysis of gene expression in NOD mice has shown the upregulation of the Reg genes specifically in islet neogenesis In addition, Reg genes have been known to upregulate in late fetal development to populate the pancreas of a developing human to maintain its own glucose metabolism postpartum Hao el al , 2006. Nature Medicine 12(3) 310-6 showed that co-transplantation of fetal tissue with non-endocrine pancreatic epithelial cells (NLPHCs) resulted in stimulation ot new islet structures from the NI-PI C population The upregulation of Reg in the co-transplanted fetal material was likely the stimulus lor this eflect

|0073] Hamster INGΛP has been the subject of clinical tπals While hamster INGΛP was apparently well tolerated in Phase 1 and II tπals, a Phase II trial had high drop out of diabetic patients due to discomfort and bruising at the hamster INGΛP injection site 1 ittle ef fectiveness was found lor hamster INGΛP in the Phase Il trial as well I he HIP2- bsaed methods and compositions ol the present invention should not have these problems because the API used in them is domed from human as opposed to hamster sequenςes I urther 111P2 ma\ be administered at an increased number oi doses a day I he number ol dαil) doses rcu\ be one or more including 2 3 4, 5 6 7 8 9 or 10 doses per da\ l he doses ma) be gι\en betore meals to increase effectiveness in some patients I I1P2 stimulates ditlerentiation oi progenitor tells within the pancreas into new islet structures Administration or IIIP2 immediately prior to meals and its presence during hyperglycemia following ingestion of the meal mimics the wild type secretion schedule or Rf G3Λ, which provides more effective treatment to patients

|0074] Despite the adverse effects shown in the Phase II hamster 1NOΛP trials INGAP did show some signs ot effectiveness in the trials Patients treated with 600 mg/dav of hamster INGAP showed an increase in C-peptide secretion In the 300 mg/day treatment group of the Phase II study, 22% ot the patients had a >50% increase in GAD65 antibody titers GAD65 antibody binds to lymphocytes which attack beta cells within the islets Thus a rise in GAD65 antibody titers reflects new beta cell production associated with islet neogenesis stimulated by hamster INGAP Also, hemoglobin AlC fell in type 2 diabetes patients 1 his is correlated to a decrease in glycemic exposure and thus a lower average blood glucose and indicates that hamster INGAP had some positive effect on islet function in patients despite its

effects

|0075| In one embodiment HIP2 is provided by the present invention in purified svnthetic or recombinant form and is administered in accordance with the methods of the invention to induce pancreatic islet neogenesis I IIP2 is advantageous relative to the non- human hamster INCiAP because 1 HP2 does not contain any non human peptide sequence

Urns, there is little chance for immune reaction when H1P2 is administered to humans, as opposed to the hamster INGΛP peptides.

[0076| Further. 1IIP2, ma; be stabl> stored for long periods of time. IIIP2 is stable for months when stored at 20 C in isotonic saline.

|0077) In a specific embodiment, 111P2 is functionally hyperactive, i e . capable of exhibiting greater activity of one or more of the functional activities associated with RliG3A, other HIP peptides, and non-human HlP homologues. such as the hamster INGΛP

[0078] Due to the degeneracy of nucleotide coding sequences, a variety of DNA sequences which encode the same or a substantially similar amino acid sequence as 111P2 may be used in the practice of some embodiments of the present invention to prepare expression vectors for the production of recombinant HIP2. In some embodiments, these include, but are not limited to, nucleic acid sequences comprising all or portions of HIP2 that are altered by the substitution of different codons that encode the same or a functionally equivalent amino acid residue within the sequence, thus producing a silent change. In some embodiments the HIP2 derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of I I1P2 including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity that acts as a functional equivalent, resulting in a silent alteration In some embodiments, substitutes for an amino acid within the sequence ma\ be selected from other member.^, of the class to which the amino acid belongs. In some embodiments, the nonpolar (hjdrophobic) amino acids include alanine, leucine, isoleucine, valine, proline. phenylalanine, to ptophan and nicihioninc. In some embodiments, the polar neutral amino aeids include gh cine. serine, threonine, cysteine, tyrosine, asparagine. and glutamine. In some embodiments, the positively charged (basic) amino aeids include arginine, lysine and histidine. In some embodiments, the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. In some embodiments. IHP derivatives of the invention also include, but are not limited to. those containing, as a primary amino acid sequence, all or part of the amino acid sequence of HlP including altered sequences in which amino acid residues are substituted for residues with similar chemical properties. In a specific embodiment. 1, 2, 3. 4, or 5 amino acids of HIP2 are substituted resulting in analogs and'or derivatives of HIP2.

[0079J In a specific embodiment, chimeric or fusion proteins may be used in the method of the invention. As used herein, a "chimeric protein" or "fusion protein" comprises IIIP2 or an analog or derivative thereof operatively-linked to a noπ-HIP2 polypeptide or an analog or derivative thereof. Within the fusion protein, HIP2 and the non-HIP polypeptide are "operatively-linked", that is they are fused in-frame with one another. In some embodiments, the non-I IIP polypeptide can be fused to the N-terminus or C-terminus of IIIP2. In some embodiments, the fusion protein may be I11P2 containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of II1P2 or an analog or derivative thereof can be increased through use of a heterologous signal sequence. In yet another embodiment, the fusion protein is a HIP2-immunoglobulin fusion protein in which the I 1IP2 sequence is fused to sequences derived from a member of the immunoglobulin protein family. In some embodiments, the I IIP2-immunoglobulin fusion protein can be incorporated into pharmaceutical compositions and administered Io a subject to inhibit an immunological response according to the present invention

(00801 In some embodiments I 1IP2 or an analog or derivative thereof or a I I1P2- chimeπc or iusion protein tor use in the methods ol the invention ma\ be chemically modified tor the purpose ot improv ing bioavailability, and/or increasing etfiuacv , solubility and stability I or example, the protein may be eovalcntly or non-covalently linked to albumin, translerrin or polyethylene glycol (PhG)

[00811 In some embodiments, H1P2 or an analog or derivative thereof or a I IIP2- chimeπc or Iusion protein for use in the method of the invention can be produced by standard recombinant DNA techniques in accordance with the teachings of the invention 1-or example, DNA fragments coding for the different polypeptide sequences may be hgated together in-frame in accordance with conventional techniques, e g , by employing blunt-ended or stagger-ended termini for ligation, restriction en/yme digestion to provide for appropriate termini, filling-in ot cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and en/ymatic ligation In further embodiments the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers Alternatively, PCR amplification ot gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamphfϊed to generate a chimeric gene- sequence Moreover many expression \ectors are commercially available that dlruadv encode a fusion moiety (e ^ . a GS I polypeptide) In some embodiments, a HIP2-encoding nucleic acid can be cloned into such an expression vector such that the Iusion moiety is linked in-frame to 11IP2 In some embodiments the fusion protein can be 11IP2 fused to a His tag or epitope tag (e g \ 3) to aid in the purification and detection ol the recombinant 111P2 or to mask the immune iesponse in a subject I he short amino acid sequences ol IIIP2 and its analogs and deriv atives make sMithetic production ol these valuable peptides readih practicable as well and a \anetv ol automated instruments lor peptide sy nthesis are commereialh a\ailablc. and synthetic methods tor peptide synthesis not requuing automation have long been known and tan be used in accordance with the teachings herein to prepare 111P2 or an analog or denvative thereol

[00821 In some embodiments, I1IP2 or an analog or derivative thereof, or a 111P2- chimeπc or fusion protein can be modified so that it has an extended half-lite ιn \ ι\ o using any methods known in the art For example, the I c fragment ot human IgG or inert polymer molecules such as high molecular weight polyethyleneglycol (PI G) can be attached to H1P2 or an analog or deπvative thereol with or without a multifunctional linker either through site-specific conjugation ol the PLG to the N- or C-terminus oi the protein or via epsilon- amino groups present on lysine residues In some embodiments linear or branched polymer demarcation that results m minimal toss of biological activity will be used I he degree oi conjugation can be closely monitored by SDS-PAGI- and mass spectrometry to ensure proper conjugation ol Pl O molecules to 111P2 or an analog or derivative thereot Unreacted Pl G can be separated trom I 1IP2-P1 G conjugates by si/e-extlusion or by ιon-e\change chromatography Pl G deπvati/ed conjugates can be tested (or in i no elficaey using methods known to those ol skill in the art

[0083| In some embodiments, the present invention provides HIP2-based therapies and methods lor deliver) ol I 1IP2 m the treatment ol diabetes and the various other indications involving impaired pancreatic lunctiυn Λs exemplified by the preceding statement some embodiments ol the. present imcntion provide methods ol treating J disease or condition associated vwth impaired pancreatic function in a subject in need thereot comprising administering to the subject a therapeutically eflectiv e amount ol 11IP2 or an analog or derivative thereol In some embodiments the disease or condition associated with impaired pancreatic lunUion is selected lrom the group consisting oi Upe 1 diabetes new onset type 1 diabetes type 2 diabetes I atent Autoimmune Diabetes in Adults prediabetes impaired fasting glucose tasting hypeπnsulinemid, impaired glucose tolerance, insulin resistant syndrome insulin deficiency metabolic syndrome obesity anorexia, bulimia neuropathic pain pancreatitis pancreatic cancer hyperlipidemia, hypertriglyceridemia eatmg disorders, anovulatory cycles, lack ot or diminished insulin production resulting in aberrant glucose metabolism, and polycystic ovarian syndrome In further embodiments, the disease or condition associated with impaired pancreatic function is selected from the group consisting ot type 1 diabetes, type 2 diabetes, Latent Autoimmune Diabetes in Adults, pre-diabetes and metabolic syndrome

|0084) In one embodiment ol the invention the therapeutically eftective dose ot I IIP2 administered alone or in combination with other agents to treat diabetes or other conditions is in the range ol 0 1 to 100 mg/ day ol 1I1P2 per kg ol patient weight (0 1 to 100 mg/kg/day ) when administered subcutancously In one embodiment, the dose is in the range of 1 to 50 mg/kg/day In one embodiment, the dose is in the range of 5 to 25

In one embodiment the dose is 10 mg/kg/day In other embodiments the IIIP2 is administered bv a route other than subcutaneous administration and the dose is adμisted to equal a dose bιoet]uι\alcnt Io the subcutaneous dose In another embodiment the invention provides unit dose torms of I IIP2 that provide j dose that allov\s the practitioner to practice the method comenienth In some embodiments such unit dose forms oi the invention include those containing 50 100 150 200 2M) SOO IM) 400 450 and 500 mg ot 1IIP2 in unit dose lorm

[0085J I hus some embodiments oi the imention provide a method lor administering a therapeutically elleetive dose ol H1P2 In one embodiment the method lmolvcs administering a therapeuticallv effective dose of II1P2 in the range ol 0 1 to 100 mg/kg/day with or without adjunct therapv (such as administration or a OI P-I receptor agonist, a GL P-I analog, and/or a dipeptidyl peptidase inhibitor) In some embodiments, the dose of H1P2 may be conveniently administered via subcutaneous or intramuscular injection to stimulate islet differentiation from progenitor cells and endogenous insulin production within the pancreas of patients to treat type 1 diabetes type 2 diabetes, prediabetes/impaired fasting glucose and insulin resistant syndromes, polycystic ovarian syndrome and associated infertility obesity, metabolic svndrome, hypertriglyceridemia, hypercholesterolemia, and/or other conditions m which there is a lack of or diminished insulin production and/or alterations in glucose metabolism or insulin secretion

(0086] In one embodiment of the method, a therapeutically effective dose of HIP2 alone or in combination with other agents is administered to a patient via subcutaneous or intramuscular injection 10 minutes to 60 minutes (for example 20 minutes or a halt an hour) prior to each meal and at bedtime In another embodiment of the method ol the invention especially beneficial tor patients with diabetes, a therapeuticallv effective dose ol I11P2 is administered alone or in combination with other agents via subcutaneous or intramuscular injection at least twice daily prior to the two largest meals

|0087| In another embodiment of the method especially beneficial for patients with the metabolic syndrome, poly cystic o\arian syndrome, impaired fasting glucose prediabetes and other conditions, who have previously been untreated with diabetes medications or medications associated with hypoglycemia. H1P2 will be administered subcutaneous!} or intramuscularly 10 minutes to 60 minutes (i.e., 20 minutes) prior to the two largest meals ingested per day. In some embodiments, patients treated pursuant to this method check glucose levels 2 hours after the meals that were preceded by an injection of IIIP2. In some embodiments, if there are two distinct episodes in which the 2-hour postprandial glucose level is less than 70 mg/dL for a given meal (breakfast, lunch or dinner), then the dose prior to that meal will be reduced by half in future administrations. In some embodiments, if there are glucose levels of less than 70 mg/dL after the dose has been reduced by half, HIP2 dosing will be completely discontinued for that meal thereafter.

[00881 In another embodiment of the method especially beneficial for newly diagnosed type 2 diabetes patients, who have previously been untreated with diabetes medications, HIP2 is administered subcutaneously or intramuscularly 10 minutes to 60 minutes (i.e.. 20 minutes) prior to each meal and at bedtime. In some embodiments, patients treated pursuant to this method check glucose levels before and 2 hours after each meal. In some embodiments, if there are two distinct episodes in which the 2-hour postprandial glucose level is less than 70 mg/dL for a given meal, then the dose prior to that meal will be reduced by half in the future. In some embodiments, if there are glucose levels less than 70 mg'dl. after the dose has been reduced by half. HIP2 administration will be complete!} discontinued prior to that meal in the future.

|0089] In another embodiment of the method especial!) beneficial for patients w ith t\ pe 1 and 2 diabetes who are treated with insulin. 111P2 is administered subcutaneoush or intramuseularK as described abo\e. In some embodiments, patients treated per this embodiment of the method will cheek glucose levels immediately before meals. 2 hours after meals, and at bedtime. In some embodiments, to minimi/.e the risk of hypoglycemia when H1P2 therapy is initiated, patients are instructed to reduce their mealtime insulin doses by 10% from their baseline dosage during the first week of therapy. In some embodiments, if any patient has an episode of symptomatic hypoglycemia with a glucose level of less than 70 mg/dL. all of the pre-meal insulin doses will be reduced by 10%. In some embodiments, if there are two distinct episodes in which the 2-hour postprandial glucose level is less than 70 mg/dL for a given meal, the dosage of pre-meal insulin for that meal will be reduced by an additional 10% beginning at the next meal after the episode. In some embodiments, if the fasting glucose is less than 70 mg/dL, the evening long-acting insulin or basal rate, if the patient is on an insulin pump, will be reduced by 10%. In some embodiments, during weeks 2-12 of HIP2 therapy, the pre-meal insulin level will be reduced by an additional 5% each week from the previous dosage before each meal. In some embodiments, if any patient has an episode of symptomatic hypoglycemia with a glucose level of less than 70 mg/dL, all of the pre-meal insulin doses will be reduced by 10%. In some embodiments, if there are two distinct episodes in which the 2-hour postprandial glucose level is less than 70 mg/dL for a given meal, the dose of pre-meal insulin for that meal in the future will be reduced by an additional 10% beginning at the next meal alter the episode. In some embodiments, if the fasting glucose is less than 70 mg/dL, the evening long-acting insulin or basal rate, if the patient is on an insulin pump, will be reduced by 10%. (0090J In another embodiment ot the method especially beneficial lor patients on a combination of insulin and oial medications those patients on prc'-meal jnd or basal insulin will be instructed to reduce their mealtime insulin doses by 10% lrom their baseline dose during the first week ol therapy to minimi/e the risk, ot hypoglycemia when II1P2 therap\ is initiated In some embodiments it any patient has an episode ol symptomatic hypoglycemia vMth a glucose level oi less than 70 mg/dL, the pre-meal insulin doses will be reduced by 10% In some embodiments, it there are two distinct episodes in which the 2-hour postprandial glucose level is less than 70 mg/dl tor a given meal, the dose ol pre-meal insulin tor that meal in the future will be reduced by an additional 10% beginning at the next meal after the episode In some embodiments, it the fasting glucose is less than 70 mg/dL. the evening long-acting insulin will be reduced by 10% In some embodiments during weeks 2-12 of H1P2 therapy, the pre-meal insulin level will be reduced by an additional 5% each week from the previous dose before each meal In some embodiments, it any patient has an episode of symptomatic hypoglycemia with a glucose level of less than 70 mg/dl . all of the pre-meal insulin doses will be reduced by 10% In some embodiments, it there are two distinct episodes in which the 2-hour postprandial glucose level is less than 70 mg/dl lor a given meal, the dose ol pre-meal insulin tor that meal will be reduced

an additional 10% beginning at the ne\t meal alter the episode In some embodiments if the lasting glucose is less than 70 mg,dl the evening long-acting insulin will be reduced by 10%

|0091| In another embodiment ol the method especially beneficial lor patients on sullonvlureas. meglitinides or other diabetic medications that can result in hvpogl) cemia the dose ol such medication will be reduced by 50% when I I1P2 therapv is initiated In Mime embodiments patients will cheek glucose

two-hours alter the meal before which 111P2 has been administered In some embodiments it an\ patient has an episode ol SΛmptomatit hv poglvcemia with a glucose

ol less than 70 mg/dl or if there are two distinct episodes in which the 2-hour postprandial glucose le\el is less than 70 mg dl for a given meal administration ot the oral agent (sulfonylurea meglitinides, and the like) will be discontinued In some embodiments it there is another episode ot symptomatic h>poglycemia or two distinct episodes in which the 2-hour postprandial glucose level is less than 70 mg/dL then dosing ot H1P2 will be discontinued prior to that particular meal in the future

[0092] In another embodiment especially beneficial for patients with new onset type Ia diabetes, HIP2 will be administered over an 18-week period during which time insulin administration is tapered down, and glucose levels are maintained in a narrow window In some embodiments, prior to treatment with HIP2, patients with new onset type Ia diabetes must first optimize their glucose levels in addition to usage of a targeted immune suppressant (such as treatment with anti-CD3 antibody at 5 ug/day intravenously for 5 consecutive days prior to treatment) In some embodiments, newly diagnosed type Ia patients will go through a protocol of intensive glucose management, which may include but is not limited to use of continuous glucose sensing continuous subcutaneous insulin administration and/or pramlintide therapy concomitantly with insulin

[0093) In some embodiments, following the optimization of glucose levels. 11IP2 can be administered over an 18 week evaluation period as described above In some embodiments avoidance of hvpoglyccmia is a primary goal with a target upper limit lor glucose ol 180 mg/dl In some embodiments 1 IIP2 is administered prior to meals and αl bedtime. In some embodiments. HIP2 is dosed al 0.1 to 100 mg kg'dav. such as 1 to 25

or 5 to 15

. In one embodiment, the dail) dose is administered in 4 divided doses. In some embodiments, these doses are given before meals and during the night, for example, at 3 a.m. In some embodiments, the present in\ ention also pro\ ides methods for dosing I UP and 1 HPl to treat the same diseases and conditions amenable to HIP2 treatment. In one embodiment. HIPl is dosed at 0.1 to 100 mg/kg/day, such as 1 to 25 mg/kg/day, or 5 to 15 mg/kg/day, or about 8 mg/kg/day. In some embodiments, the daily dose can be administered in multiple divided doses, including 4 divided doses, as described above. In some embodiments. HIP is dosed at 0.1 to 100 mg/kg/day . such as 1 to 25 mg/kg/day, or 5 to 15 mg/kg/day, or about 10 mg/kg/day, and again, the daily dose can be administered in multiple divided doses, including 4 divided doses, as described above.

[0094] In accordance with the one embodiment of the invention, HIP2 can be administered in combination with insulin. In some embodiments, the dose of insulin will be decreased over time ("tapered"), with close monitoring of stimulated C-peptide levels. In some embodiments, when normal C-peptide levels arc achieved and glucose levels are within the desired target range, insulin administration will be discontinued. In some embodiments, 11IP2 administration can be subsequently discontinued. In some embodiments, immune therapy to protect new islets can be administered in accordance

the method of the invention. Recent studies with humanized anti-C'D3 antibodies have demonslrated that, among newly diagnosed type Ia patients, immune protection may be rendered for as long as 24-months.

|0095] In another embodiment especially beneficial for patients with existing t>pe Ia diabetes and patients with Latent Autoimmune Diabetes of Adulthood (l.ΛDΛ). 1 IIP2 is administered over at least an 18-week period during which time insulin administration is tapered down and glucose le\els are maintained in a narrow window. In some embodiments, prior to treatment with H1P2. patients with existing type Ia diabetes or LΛDΛ will in some instances optimize their glucose levels in addition to use a targeted immune suppressant (such as treatment with anti-CD3 antibody at 5 ug/da> intravenously for 5 consecutive days prior to treatment). In some embodiments, following the optimization of glucose levels, IIIP2 is administered over an 18 week period as mentioned above. In some embodiments, avoidance of hypoglycemia is a primary goal, with a target upper limit of glucose of 180 mg/dL. In some embodiments, HIP2 is administered prior to meals and at bedtime. In some embodiments, I1IP2 will continue to be administered while insulin administration is being tapered, with close monitoring of stimulated C-peptide levels. In some embodiments, when normal C-peptide levels are achieved and glucose levels are within the target range, insulin will be discontinued, and subsequently, H1P2 administration can in some patients be discontinued. In some embodiments, immune therapy to protect new islets will be dosed based upon the therapy type in accordance with the methods of the invention.

[0096| In another embodiment of the method especially beneficial for patients with new onset type 2 diabetes, 11IP2 is administered to aid in replenishing lost beta cells, a loss that is progressive in type 2 diabetes. In some embodiments, to overcome the problem of insulin resistance associated with type 2 diabetes, 1UP2 is administered at the onset of l>pe 2 diabetes, optionally in combination with one or more additional pharmacological agents. to replenish the beta cell population and reduce the risk of lurther beta cell strain from hvperinsuhnemia In some embodiment;., the agents that mav be used in combination with U1P2 m the treatment ot l>pe 2 diabetes include, but are not limited to

CiI P- I . OI P-I analogues ϋipeptidj l Peptιdase-4 Inhibitors, and Pramhntide I UP2 is administered prior to meals and at bedtime In some embodiments HIP2 will be dosed at ϋ I to 100 mg/kg/day, such as 1 to 25 mg/kg/day, or 5 to 25 mg/kg/day, or about ! 6 mg/kg/day. optionally in 4 divided doses, as described above (before meals and in the nighttime, e g 3 a m ) In some embodiments HIPl is dosed at 0 1 to 100 mg/kg/day, such as 1 to 25 mg/kg/day, or 5 to 25 mg/kg/day, or about 14 mg/kg/day, optionally in 4 divided doses, as described above In some embodiments, HIP is dosed at 0 1 to 100 mg/kg/day. such as 1 to 25 mg/kg/day. or 5 to 25 mg/kg/day, or about 18 mg/kg/day optionally in 4 divided doses, as described above

[0097| In another embodtment of the method especially beneficial to patients with pre-existing type 2 diabetes and patients with type Ib diabetes being treated with an oral or injectable anti-diabetic agent, such as insulin, Pramhntide or exenatide. H1P2 is administered to aid in replenishing the beta cell loss, which is progressive in type 1 b and t)pe 2 diabetes In some embodiments, to overcome the problem of insulin resistance associated with type Ib and type 2 diabetes, HIP2 is utilized in the treatment ot patients with existing tvpe 2 diabetes or patients with type I b diabetes In some embodiments HIP2 is administered in conjunction with one or more pharmacological agents as a means ul both replenishing the beta cell population and reducing the risk ol further beta cell strain lrom hvpeπnsulinemta in some embodiments, the agents that may be used in conjunction with I IIP2 in the treatment of tvpe 2 diabetes or t>pc Ib diabetes wherein the patient is treated with oral or injectable anti-diabetic agents, may include, but are not limited to: cxendin-4. ϋl.P-1 , GLP-I analogues. Dipeptidyl Peptidase-4 Inhibitors, and Pramlintide. In some embodiments. I 11P2 is dosed ut 0.1 to 100 mg/kg/day. such as 1 to 25 mg/kg/day . or 5 to 25 mg/kg/day. or about 16 mg/kg/day. optionally in 4 divided doses, as described above. In some embodiments, IHPl is dosed at 0.1 to 100 mg/kg/day . such as 1 to 25 mg/kg/day. or 5 to 25 mg/kg/day. or about 14 mg/kg/day, optionally in 4 divided doses, as described above. In some embodiments, HlP is dosed at 0.1 to 100 mg/kg/day, such as 1 to 25 mg/kg/day. or 5 to 25 mg/kg/day. or about 18 mg/kg/day, optionally in 4 divided doses, as described above. In some embodiments, the mode of delivery and dosage of the other pharmacological agent(s) administered in combination with HlPl . HIP2 and/or HIP will be determined based upon the selected agent.

J0098] In another embodiment of the method especially beneficial to patients with obesity, insulin resistance, metabolic syndrome/dymetabolic syndrome, polysistic ovarian syndrome and anovulatory cycles. HIP2 is administered in combination with other pharmacological agent(s) beneficial in the reduction or reversal of insulin resistance. Because reduced physical activity and dietary indiscretion are associated with obesity, insulin resistance, metabolic syndrome/dysmetabolic syndrome, ploycystic ovarian syndrome and anovulatory cycles, in addition to restoring islet mass, it will be critical in some embodiments to address the problem of insulin resistance, which taxes the beta cell to over secrete insulin to overcome the insulin resistance. In some embodiments. 111P2 is therefore utilized in the treatment of obesity, insulin resistance, metabolic syndrome/dysnielabolie syndrome, ploycystic ovarian syndrome and anovulatory cycles in combination with one or more pharmacological agents beneficial in the reduction or reversal ol insulin resistance In some embodiments the agents that ma; be used in combination with 1 IIP2 include but are not limited to e\endiπ 4 GI P-I Gl P-I analogues Dφeptid\ 1 Peptιdase-4 inhibitors and Pramlintide In some embodiments 11IP2 is delivered either oral]) or subcutaneous!) prior to meals and at bedtime In some embodiments H1P2 is dosed at 0 1 to 100 mg/kg'day such as 1 to 25 mg/kg/day or 5 to 25 mg/kg/day or about 16 mg/kg/dav optionally in 4 divided doses as described above In some embodiments HlPl is dosed at 0 1 to 100 mg/kg/day, such as 1 to 25 mg/kg/day or 5 to 25 mg/kg'dav or about 14 mg/kg/dav optionally in 4 divided doses, as described above In some embodiments HIP is dosed at 0 1 to 100 mg/kg/day such as 1 to 25 mg/kg/day or 5 to 25 mg/kg/day, or about 18 mg/kg/day, optionally in 4 divided doses as described above

|0099] In another embodiment of the method HIP2 is administered in a pharmaceutical composition of the invention in which the HIP2 is encapsulated, alone or in combination with one or more other agents in a soluble liposome formulation In one embodiment this liposome formulation is administered orally

[00100] In one embodiment of the liposome formulation, H1P2 is admixed in a soluble liposome preparation with other agents, wherein those other agents are selected lrom the group consisting of amylin and/or an amylin analog, such as Pramlintide. CiIP GI P-I and/or homologous compounds and analogs, GI P-I receptor analogs which include 1 xendm-4 I iraglutide (NN221 1 ) hamster INGΛP or analogs thereol. another biologically active HlP peptide in addition to 11IP2. and/or a Dipeplidjl Pcptidase-4 inhibitor which delays, the degradation of Gl P-I In some embodiments the other agent mav aflect beta cell regeneration gastric empH iπg, satiety insulin requirements (through their ellec! on the

GLP-I and ann lin receptor sites in the panereas. nueleus accumbens. area postrema. and gut).

|00101] In some embodiments, practice of the methods of the invention can in\olve multiple rounds, or "cycles." of treatment. I^'or example. I UP2 can be administered until no further therapeutic benefit is observed, and then dosing can be discontinued until symptoms of the disease or condition being treated reoccur, at which time dosing can be readministered. In some embodiments, the dose of 111P2 administered can be reduced as evidence of therapeutic benefit appears.

|00102] Those of skill in the art will recognize upon review of the present disclosure that certain agents, when administered in combination with 1I1P2, can enhance the biological activity of HIP2 and therefore when administered in combination with such agents, the 11IP2 dose may in some instances be titrated so as to reach a HIP2 concentration that yields the desired therapeutic effect. Those of skill in the art will also appreciate, in view of the disclosure herein, that the skilled artisan may select particular doses of and agents to be used in combination with Hl P2 based on the disease and condition being treated and the medical status of the patient.

|00103] In one embodiment, the method of the invention is practiced to treat type 1 or type 2 diabetes mellitus and related conditions in which there is a lack of or diminished insulin production in a patient resulting in aberrant glucose metabolism. In some embodiments, the method comprises administering to that patient an agent that stimulates pancreatic islet regeneration and/or differentiation from pancreatic progenitor cells into islet structures. In some embodiments, this agent is I IIP2 or an analog or derivative thereof. In some embodiments, optionally 111P2 or a 11IP2 analog or derivative is administered with the

-.18- simultaneous or contemporaneous administration of an agenl that inhibits the activ it\ ol and or blocks or destroy the autoimmune cells that target pancreatic islet beta cells and optionalh another agent which mav also stimulates pancreatic beta cell regeneiation and or result in elevation of GI P-I or GI P-I receptor stimulation or is a (51 P-I analog or is a Dipeptidvi Peρtidase-4 Inhibitor which inhibits the degradation ot Gl P-I

|00104| Λs exemplified by the preceding paragraph some embodiments of the present invention provide methods ot stimulating islet cell differentiation trom progenitor cells into islet structures in a subject in need thercot, comprising administering to the subject a therapeutically elfective amount of IIIP2 or an analog or derivative thereof and a therapeutically effective amount of one or more agents selected from the group consisting of an immune therapy agent and an additional agent that stimulates islet cell regeneration

(00105] In some embodiments the therapeutic methods provided by the present invention address several different underlying mechanisms that result in either the absence of, or diminished or inadequate amounts of, insulin and other hormones, or which are otherwise produced m aberrant quantities In some embodiments the IIIP2 based combination therapies provided by the present invention can restore more normal glucose metabolism, including achieving and maintaining appropriate levels ol insulin, am) lin postprandial glucose triglycerides and glucagon levels and ameliorate the significant weight gain and increased risk for serious hypoglycemia that is associated with tight glvcemic control using insulin or oral diabetic medications

[00106] Some embodiments of the present invention also provide single agent therapies lor treating insulin deficiency including diabetes and related conditions In some embodiments these single agent the'rapies include methods lor the administration ol I IIP2

V) or an analog or

that stimulate pancreatic islet tell regeneration and or transtυrmation ot new insulin producing islet tells lrom pancreatic progenitor cells located within the adult pancreas In some embodiments the islet cell neogenesis resulting from such administration with H1P2 can be used to treat diabetes and other diseases and conditions relating to aberrant glucose regulation In various embodiments, these methods comprise the administration ot HJP2 alone or in combination with an immune blocking agent and/or co administered with a GI P-I receptor agonist OI P-I, GLP-I analog, or Dipeptidyl peptidase-inhibitor in the case ior type 1 diabetes or H1P2 in combination with Gl P-I receptor agonist, GI P-I, GI P-I analog or dipeptidyl peptidase-inhibitor without the need for an immune blocker in the case ot type 2 diabetes In some embodiments disease conditions amenable to treatment with this methodology include but are not limited to, type

1 and 2 diabetes where these treatments tan be used to improve glycemic control, as measured by hemoglobin Λ1C, and to reduce bolus insulin before meals by at least 10-20%, with reduced fluctuations and decreased postprandial glucose, glucagon, and triglycerides In some embodiments, these methods can also be used to prevent progression of impaired glucose tolerance to diabetes and to prevent progression ot impaired lasting glucose to progression to impaired glucose tolerance and diabetes and to reverse newly diagnosed type

2 diabetes It yet other embodiments, these methods can also be used to treat type 2 diabetes

|00107| 1 \ogenous injectable insulin is a therapy tor patients with tvpe 1 diabetes and other conditions in which insulin is either absent or present in diminished or inadequate amounts relative to the glucose content in the bloodstream Insulin therapv does not treat the underlying mechanisms ot disease resulting in type 1 diabetes and other such conditions m which there is diminished endogenous insulin production I he therapies methods modalities and treatments described herein address the mam laeets ol the causes and complications ot diabetes I he unique therapies prov ided b> the invention encompass diverse aspects ot diabetolog) metabolism and immunology In some embodiments these therapies include those that restore normal levels ot the many different hormones in addition to insulin that are diminished or absent m type I diabetes In some embodiments the methods ol the invention provide lor the regeneration ol new pancreatic islet structures In some embodiments, these new islet structures provide for the proliferation of new insulin producing cells, thereby ameliorating diminishing, or abolishing the need tor exogenous insulin among patients with type 1 diabetes and other conditions associated with inadequate insulin production and secretion

|00108) In type 1 diabetes, there are several underlying mechanisms that result in significant reduction in the production of insulin 1 hese include autoimmune destruction ol the beta cells and reduction in regeneration capacity not only within the beta cells, but an inability of progenitor cells to differentiate into new islets may be due to the altered glucose milieu In some embodiments, the present invention also provides combination treatment methods that are especially elficacious, because when the autoimmune response is blocked b\ the co-administration with IIIP2 or a 1 IIP2 analog or derivative of an immunosuppressant the autoimmune cells that attack the pancreatic islet cells are blocked and as peptides or other compounds that stimulate regeneration of the pancreatic islet cells are administered and exert their therapeutic effect the patient becomes less dependent on insulin administration

|001()9| In some embodiments, the methods of the invention can even render some patients completely free of their dependence on administered insulin for both tv pe 1 and 2 diabetes. Other studies (see the references Levetan et a!., 2002. Dkibeles 51 (supple 2):429. Levetan el ul. Diabetes 2002. 51(suppl. 2):474. Levetan Diabetes 2001 : 50(supple 2):2105 PO. and Levetan el a/., 2003, Diabetes Care 26: 1-8, each incorporated herein b) reference) show that, when diminished hormones other than insulin are replaced, insulin requirements in type 1 patients are significantly diminished with improved glucose control. By stimulating differentiation of new insulin producing islet structures and optionally blocking the immune cells that can destroy their function, the methods of the present invention have even greater promise of therapeutic benefit, because they result in sustained, endogenous production of insulin itself, and other co-secreated hormones such as amylin.

[00110] As exemplified by the preceding paragraph, some embodiments of the present invention provide methods of reducing insulin requirements in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of HIP2. or an analog or derivative thereof. In some embodiments, the present invention provides methods of reducing insulin requirements in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of IIIP2. or an analog or derivative thereof and one or more agents selected from the group consisting of: an immune therapy agent and an additional agent that stimulates islet cell regeneration.

100111 ] There is a demonstrated need for the therapeutic benefits provided by the present invention. There are new insulin formulations and evidence to support that intensive insulin therapy prevents deaths and reduces the rate of blindness, amputations, and kidney failure necessitating dialysis. Also, recent studies have demonstrated both microvascular and macrovascular/cardiovaseular risk reduction among type 1 diabetes patients who have improved glycemic control. However, intensive insulin therapy utilizing modern modalities of multiple insulin injections and continuous insulin delivery via pump therapy is associated with a t\vo-to-three fold increased risk of serious hypoglycemia requiring assistance from another person. In a clinical study setting, despite normalization of glucose in type 1 diabetes patients by means of intravenous insulin and glucose, the standard deviation in glucose levels, both high and low, is significantly wider than non-diabetic study subjects with the same average glucose over a 24-hour period. In some embodiments, the present invention offers an alternate means to achieve the therapeutic benefit of intensive insulin therapy with reduced iatrogenic risk, because the endogenous production of insulin stimulated by the present methods should provide more normal rates of insulin production than can be effectively mimicked by intensive insulin therapy.

[00112] Thus, despite insulin's availability and new technologies, including new formulations of human insulin, self blood glucose monitoring systems, continuous glucose sensors and pump therapy, normal glucose control is not approximated by current therapies. Moreover, the underlying mechanisms causing type 1 diabetes are not impacted by the current therapies available for patients with type 1 diabetes and conditions in which there is no or diminished or inadequate or otherwise aberrant insulin or amylin production and dysregulation of glucagon.

[00113| In some embodiments, the present invention provides new methods and pharmaceutical compositions for stimulating islet ncogenesis, increasing insulin or other pancreatic hormone production in a patient in need thereof, and treating type 1 diabetes mellilus. type 2 diabetes mellitus and other conditions in which the lack of or diminished insulin production is a causative taetor tor the disease svmptoms In some embodiments the methods and compositions of the invention can reverse the underlv ing pathologic mechanisms υt these disease conditions 1 hus in some embodiments the methods ot the invention dimmish and in some cases eliminate the need tor insulin administration to patients formerly m need thereof

|00114| In one embodiment ot this method, an additional agent that stimulates islet regeneration and/or differentiation from pancreatic progenitor cells into insulin producing islet structures is co- administered with HIP2 or an analog or derivative thereof including HlP and INOΛP In some embodiments, other agents that be administered with I I1P2 lor the treatment of type 1 and type 2 diabetes include amylin and/or an analog such as Pramlintide GIP, GI P-I and/or homologous compounds and analogs GLP-I receptor analogs which include I xendin-4, 1 iraglutide (NN2211), hamster INuAP, or HIP analogs thereof any biologically active HIP peptide and/or the Dipeptidyl Peptidase-4 inhibitors, which delay the degradation of GI P-I In some embodiments, the second agent may affect beta cell regeneration gastric emptying, satiety or insulin requirements through impacting the GI P-I and amylin receptor sites in the pancreas nucleus accumbens, area postrema, and gut

(001 IS] In some embodiments a method ot treating type 1 diabetes and other pathologies resulting lrom diminished pancreatic function, includes a five step process In some embodiments these steps include 1 ) Intensive Glycemic Management 2) Achievement and mamtainence ot 25-hvrdro\>vitamrn D levels to ->40 ng/dl via oral cholecalcilerol (Vitamin D^) 3) Immune l herapy 4) 1 I1P2 administration and Insulin

lapenng tollcmed b> discontinuation ol both 1IIP2 and Insulin and 5) Repeated usage ot immune modulation on a quarterl) or annual basis dependent on immune therapv chosen

|001 16| 1 hose ol skill in the art would appreciate that the preceding paragraph exemplifies some embodiments ol the invention providing methods ol treating a disease or condition associated with impaired pancreatic iunction in a subject in need thereol, comprising the steps of intensifying glvcemic control administering oral cholecalcilerol administering one or more immune therapies administering a therapeutically eltective amount of HIP2 in combination with insulin, wherein the insulin administered is decreased over time and repeating the administering of the one or more immune therapies every 3 months to every 24 months

[00117] In some embodiments, another method of the invention includes a two step process for the treatment ol type 2 diabetes, obesity, overweight, insulin resistance, hyperhpidemia, hypertriglyceridemia, and eating disorders In some embodiments this process includes the steps ot 1) Achievement and maintenance ot 25-hyrdroxy vitamin D levels to >40 ng/dl via oral cholccalcifcrol (Vitamin D3), and 2) Administration of HIP2 in combination with a GLP-I or Gl P-I receptor agonist or GLP-I analog or Dipeptidyl Peptidase-4 Inhibitor

[00118| I he first two steps ot the five step process of treating type 1 diabetes and other pathologies resulting from diminished pancreatic function are described in more detail below In some embodiments lor the first step (a three month time period prior to the administration ol I1IP2 or 111P2 analog or derivative administration and prior to or with the simultaneous or contemporaneous administration υl an agent that inhibits the actιvit> ol and or blocks or destrovs the autoimmune cells that target islet beta cells) there will be a period of tighl'intensc glucose optimization. In some embodiments, this period of light/intense glucose optimization ma> include multiple daih doses of insulin administered subeutaneousl) or via continuous subcutaneous administration through an insulin pump and ma) include the administration of s>nthethie amylin/Pramlintide (SymIin^{I M}). which is also absent in t> pe 1 diabetes and aberrantl) secreted in type 2 diabetes. Synthetic amylin/Pramlintide (Symlin™), has been shown to reduce glvcemic excursions in type 1 patients, while reducing insulin requirements before meals.

[00119] Additionally, throughout the period of tight control, immune therapy, and I1IP2 administration, the invention in one embodiment contemplates the administration of vitamin D3, cholecalciferol, at a dose of about 1000-2000 lU/day. Recent studies have demonstrated that up to 54.7% of populations in the US, regardless of latitude, have low 25- hydroxyvitamin D levels. Vitamin D deficiency has been demonstrated not only to be associated with the increased risk of type 1 diabetes and seen at the onset of type 1 diagnosis but also to be commonly seen in patients with diabetes, including both type 1 and type 2 patients, and maintaining levels above 40 ng/ml is recommended to maintain normal immune function in those with and without diabetes. No adverse effects have been seen with doses up to 10,000 IU/day. Vitamin D in doses of 1000-2000 lU/day are continued to maintain 25-hydroxyvitamin D levels > 40 ng/dl for both type I and 2 diabetes patients.

(001201 In one embodiment of the inventive method, prior to the administration of the 111P2 or HIP2 analog or derivative, an immune modulator will be administered in accordance with the methods for which it is otherwise prescribed. In some embodiments, such immune modulators include immunomodulatory proteins and peptides that arrest pancreatic islet cell destruction. In one embodiment, the immune modulator is a monoclonal antibody that can delay the progression ol islet loss or blow or slop the onsel oi type 1 diabetes Λnti-CD3 antibodies constitute a general class ol agents useful in the methods ol the invention 1 or example suitable anti-C D3 antibodies for purposes of the present in\ ention include the 1 RX4 ( Ma-Ala and C h \gly CD3) antibody under

by FolcrRx and the humanized anti-CI)3 antibody described in the reference IIerold ei al 30 May 2002 SUM 346(22) 1692-1698. incorporated herein by reference In one embodiment, the Bluestone humanized anti-CD3 antibody is delivered intravenously at least 14 days per year in a dose of 1-1 42 μg/kg on day 1. 5 67 μg/kg on day 2, 1 1 3 μg/kg on day 3, 22 6 μg/kg on day 4 and 45 4 μg/kg on days 5-14 In some embodiments these therapies can be repeated annually or more frequently contemporaneously or following the administration of H1P2 In some embodiments, HIP2 administration can continue for days, weeks, or months In one embodiment, I1IP2 is administered for 3 months, and in another embodiment, IIIP2 is administered for 6 months In various embodiments, insulin administration is being tapered (decreased over time) as new islet cell formation occurs In some embodiments, during the HIP2 treatment phase Vitamin D and/or the administration of pramhntide/Symlin™ may be continued in some embodiments In some embodiments, following the discontinuation of H1P2 and insulin therapy, immune modulation can be repeated annually or more frequently or as needed For example, for the anti-CD3 antibodies, a recent study has reported efficacy lor as long as 24 months

]001211 In another embodiment the immuno-modulatory compound is a Iy soty lime or a heal shock protein that can arrest or slow islet cell destruction In some embodiments such pi oteins include Dl M'l P277 ^{I M} a heat-shock protein under development bv Develogen ΛCi In one embodiment, DIΛP1 P277^{I M} is delivered subcutaneous!) by administering 1 mg in 40 mg mannitol in vegetable oil subeutaneυusly at baseline and at one month and then at 3 month intervals. In some embodiments. DIΛPHP277™ is continued throughout 1 IIP2 therapy and following I11P2 therapy at quarterly intervals to protect newly generated islets from I11P2 therapy. In one embodiment of the combination therapy of the invention. HIP2 is co-administered with DIΛPHP277^m. In some embodiments, the DIΛPEP277^{I M} is first administered subcutaneously at a dose of about 1 mg, about 30 days prior to the initiation of the HIP2 therapy. In some embodiments, a second administration of the D1ΛPEP277^{I M} is then made at the time (30 days after the first administration) of initiating the IIIP2 therapy. In some embodiments, the HIP2 therapy may be repeated as necessary, and the DIAPKP277^{I M} is administered at a frequency of about every 3 months.

[00122) In some embodiments, the new H1P2 therapeutic methods provided by the present invention address several different underlying mechanisms that result in either the absence of, or diminished or inadequate amounts of insulin and other hormones or which are otherwise produced in aberrant quantities. In some embodiments, the HIP2 based. HIP2 analog or derivative based, or combination therapies provided by the present invention can restore more normal glucose metabolism, including achieving and maintaining appropriate levels of insulin, amylin, postprandial glucose, triglycerides, and glucagon and ameliorate the significant weight gain and increased risk for serious hypoglycemia that is associated with tight glycemic control.

|00123] Those of skill in the art will appreciate in view of the disclosure herein that more than one agent that stimulates islet neogenesis and/or progenitor cell

differentiation and/or which slovvs the degradation of such agents can be used in combination in the methods of the invention.

|00124] Optionally, in the practice of the methods of the invention, the HIP2 or analog or derivative thereof, with or without the co-administration of another selected agent, such as Symlin™/pramlintide GLP-I . a GLP-I receptor agonist, GLP-I agonist, or dipeptidyl-4 peptidase inhibitor, which inhibits the degradation of GLP-I , which may reduce weight, improve satiety, slow gut absorption of glucose, may be used in combination with a specific agent that inhibits, blocks the activity of. or destroys autoimmune cells that target the pancreatic beta cells. In some embodiments, such agents include, for example, peptides, proteins, and synthetic compounds.

[00125] In one embodiment, the agent is a monoclonal antibody, a heat-shock protein, or another compound that specifically delays, prevents, or halts autoimmune destruction of the islet function. Those of skill in the art will appreciate in view of the disclosure herein that more than one agent that blocks autoimmune destruction of pancreatic islet function can be used in combination in the methods of the invention. In some embodiments, agents that inhibit, block the activity of. or destroy autoimmune cells that target the pancreatic islet function include: Λnti CD-3 antibodies (hOKT3^Λ'l Ala-Ala and ChΛglyCD3). Sirolimus (Rapamycin). Tacrolimus (FK.506), a heat-shock protein 60 (DiaPep277) a anti-Glutamic Acid Decarboxylase 65 (GΛD65) vaccine. Mycophenolate Mofctil alone or in combination with Daclizumab. the anti-CD20 agent Rituximab, Campath-l li (Anti-CD52 Antibody), lysofylline, and Vitamin D. IBC-VSO \accine which is a synthetic, metabolically inactive form of insulin designed to prevent pancreatic beta-cell destruction, interferon-alpha. vaccination using CD4 CD25 antigen-specific regulatory T cells or a similar agent, designed to prevent pancreatic beta-cell destruction. In this latter embodiment, interferon-u \accination using CD4 CD25^' antigen-specilϊe regulatory T cells or a similar agent is used in the combination therapy for utilizing regulatory T cells either directly or through the use ofanti-CD3 immunotherapy. This embodiment, which includes an immune agent would specifically be used in type ! diabetes patients to protect newly generated islet cells from immune attack.

[00126] Thus, in some embodiments, the combination therapies and related methods of the invention involve the administration of HIP2 or analogs or derivatives thereof or co-administration of H1P2 or analogs or derivatives thereof with either one or more agents that stimulate islet differentiation from cells in the adult pancreas or with one or more agents that block autoimmune destruction of pancreatic beta cells or both. As used herein, an agent is "co-administered" or "'used in combination" with another agent (also referred to herein as, "compound or "hormone") when the two or three agents are administered as part of the same course of therapy. In one embodiment, a first agent is first administered prior to administration of the second agent, and treatment with both is continued throughout the course of therapy. In another embodiment, the second agent is administered after the initiation or completion of the therapy involving the first agent. In other embodiments, the first agent is administered contemporaneously with the initiation of the therapy with the second agent. In another embodiment, a third agent is administered contemporaneously or before or after the administration of the first or second agent or both. In one embodiment, a therapy involving one or more agents to block or kill autoimmune cells that target pancreatic beta cells, which make insulin and amylin. is first administered prior to administration of the therapy that stimulates islet differentiation from progenitor cells in the adult pancreas In another embodiment treatment with the specific autoimmune blocker is continued alter the cessation ol treatment with agents that stimulate islet differentiation In some embodiments prior to or contemporaneous!) administration ol immune modulating agents there will be a period ot intensified 'tight ghcemic control which ma\ include multiple dailv injections oi insulin insulin pump therapy and/or usage ot pramhntide/Symlin™ and'or vitamin D therapy in doses ot 1000-2000 lU/da> to maintain a 25-hydroxvitamin D level above 40 ng/ml In some embodiments, this period may last tor

or months, in one embodiment, the period is tor at least 3 months

|00127] As noted above, practice or the methods ot the invention can involve multiple rounds or "cycles," of treatment For example, an administration of an agent that stimulates islet differentiation from progenitor cells together with an administration of an agent that blocks autoimmune cells that target pancreatic beta cells can be viewed as one cycle ot the method of the invention that involves co-administration ot both types ot agents Alternatively in some embodiments, each administration ot an islet differentiation agent can be viewed as a cycle of treatment, and if an autoimmune cell blocking agent is administered, it may be administered in only a subset of such cycles, or after the last administration of the islet differentiation agent I or example only two DIAMYD™ injections ot aluminum formulated human recombinant GΛD65 delivered 4 weeks apart subcutaneously have been needed in some instances to stave oft further beta cell destruction in patients with autoimmune diabetes A single course ot anti-C D3 monoclonal anttbodv hOk 1 3gainmal(Λla-ΛIa) results in improvement in C-peptide responses and clinical parameters for at least 2 jears after onset ol type 1 diabetes in some patients I hus depending on the selected immune blocker the cvclicity ot therapv may vary to protect new islets from immune attack. It will be understood that the above examples are lor illustration onh and not intended to limit the invention in any fashion. Those of skill in the art will also appreciate that, in man}' cases, the schedule of co-administration may differ in the first or a later therapeutic cycle for the convenience of the patient.

|00128j The combination therapies and related methods of the invention uniquely target the underlying pathologic mechanisms of type 1 diabetes with agents that regenerate new islet structures and/or differentiate pancreatic progenitor cells in combination with agents that provide targeted immune therapy. In some embodiments, this combination therapy can reverse, wholly in some patients and partially in others, the underlying mechanisms of type 1 diabetes, which is an autoimmune phenomenon in which anti-self antibodies attack the pancreas. Current therapies for type 1 diabetes that rely on the administration of insulin do not reverse the underlying defects in type 1 diabetes. Moreover, current immune therapies for type 1 diabetes are based upon rejection of pancreatic beta cells and do not impact the differentiation of new fully functional islet structures containing new alpha, beta, delta, and polypeptide cells within each new islet.

|00129] Among patients with type 2 diabetes, an immune blocking agenl will not be necessary as the basis of the disease is not immune destruction, although there may be some type 2 diabetic patients that will benefit from such combination therapy. Recent studies have pointed to a potentially important role of vitamin D deficiency in type 1 diabetes. Λ recent study found that at the time of diagnosis, more patients with type 2 diabetes are vitamin D deficient than type 1 diabetes, and maintaining levels above 40 ng/ml is recommended to maintain normal immune function. No adverse effects have been

been with \ itamin D doses up to 1C 000 IU dav l hus in some embodiments the methods of the invention that in\ol\e utαmin D co-therapv are beneficial to t\pe 2 diabetic patients

[00130] In some embodiments the new methods provided by the present invention reverse the underlying pathologic mechanisms ot type 2 diabetes and diseases and conditions resulting trom decreased insulin production due to an imbalance between destruction, regeneration, and sustenance ol beta cells via the differentiation of new islet structures which contain fully functional new beta cells Λs exemplified by the preceding statement some embodiments of the present invention provide methods of stimulating islet cell differentiation from progenitor cells in a subject in need thereol, comprising administering to the subject a therapeutically effective amount ot HIP2 or an analog or derivative thereof In some embodiments, the methods and compositions ot the invention can reduce the insulin and diabetes medication requirements of patients currently taking insulin due to having type 2 diabetes or another disease or condition and tan improve glucose control in such patients In some patients, treatment in accordance with the methods of the invention can ameliorate or obviate the need for administered insulin fhe following section describes a variety of diseases and conditions that the methods and compositions of the present invention can be used to treat with therapeutic benefit

[001311 In some embodiments the 11IP2 or I IIP2 analog or derivative therapies or combination therapies of the present invention can be used to treat an> mammal, including humans and animals, suffering from a disease symptom or condition related to a diminished production or secretion of insulin due to the loss of or diminished beta cell function or the need lor greater insulin production than can be prov ided to the sub|cct \ ia

differentiation of new islet structures from progenitor cells utilizing H1P2 compounds and methods of treatment.

[00132| In some embodiments, such diseases and conditions include type 1 diabetes mellitus. type 2 diabetes, pre-diabetes, impaired fasting glucose, lasting hyperinsulinemia, including but not limited to patients with type Ia diabetes patients or patients with Latent Autoimmune Diabetes of Adulthood who may manifest antibodies (anti-OAD65 antibodies, anti-islet antibodies, or anti-insulin antibodies) or those patients with type 1 diabetes with insulin deficiency without autoimmunity directed toward the beta cells (type Ib diabetes). Moreover, some embodiments of the present invention can be practiced with therapeutic benefit for patients newly diagnosed as having type 1 diabetes, the siblings and first degree relatives of patients with type 1 diabetes, and people with positive antibodies and other autoimmune conditions that indicate a predilection to type 1 diabetes. In one embodiment, the methods of the invention are practiced to reverse type 1 diabetes in a patient in need of such treatment.

|00133| In some embodiments, the combination therapies and related methods and compositions of the invention can also be employed as adjunctive therapy to insulin therapy in type 1 diabetes in children and adults, to ameliorate glucose swings in patients with diabetes, and in patients with poorly controlled diabetes, hypoglycemic unawareness, and recurrent hypoglycemia in type 1 diabetes.

|()0134| In some embodiments, the I IIP2 or I IIP2 analog or derivative therapies and related methods and compositions of the invention can be used to treat patients having

diagnosed type 2 diabetes, type 2 diabetes in children and adults with hy perglycemia, type 2 diabetes being concurrently treated with insulin, oral diabetic or other subcutaneous diabetic therapies, and poorly controlled type 2 diabetes. In some patients, both children and adults, the methods and compositions of the imention can reverse tv pe 1 and 2 diabetes. In some embodiments, the methods and compositions of the invention can also be used to treat both children and adults having aty pical forms of diabetes and patients having the conditions of postprandial hyperglycemia.

[0013S) In some embodiments, the 11IP2 or I IIP2 analog or derivative therapies and related methods and compositions of the invention can also be used to treat patients who are children, as well, as adult patients, in need of weight loss, reduction in triglycerides, LDL cholesterol, including but not limited to achieve weight loss or treat obesity, overweight in patients having diabetes as well as those who do not have type 1 or 2 diabetes. In one embodiment, the methods and compositions of the invention are used to treat a patient having morbid obesity. In other embodiments, the methods and compositions of the invention are used to treat a patient having morbid obesity or patients having anorexia, bulimia, or other eating disorders.

[00136] In some embodiments, the single agent HIP2 therapies and related methods and compositions of the invention can also be used to treat children and adults having dysmetabolie syndrome or metabolic syndrome, as well as patients exhibiting the conditions of neuropathic pain syndromes secondary to altered glucose metabolism, and those with hypertriglyceridemia with and without diabetes, and postprandial hypertriglyceridemia. In one embodiment, these methods are practiced to treat polycystic ovarian syndrome in a patient in need of such treatment.

[00137] In some embodiments, other patients lhat can benefit from the I HI'2 or I IIP2 analog or derivative therapies and related methods of the invention include children and adult patients diagnosed as ha\ ing conditions such as fasting hv pergh cemia. prediabetes, impaired lasting glucose, impaired glucose tolerance, and

conditions generally.

(00138] In some embodiments, the 1 I1P2 or H1P2 analog or deri\ati\e therapies and related methods and compositions of the invention can also be used to treat patients having neuropathic pain syndromes and neuropathy, regardless of whether the patient is diagnosed as diabetic.

[00139| In some embodiments, the 111P2 or IIIP2 analog or derivative therapies and related methods and compositions of the invention can also be used to treat patients having recurrent pancreatitis or pancreatic cancer and can be used in all modalities aimed at achieving new islet structures derived from progenitor cells in the pancreas.

[00140] The following sections describe the agents useful in some embodiments of the methods of the invention. Those of skill in the art will appreciate, in view of the disclosure herein, that the skilled artisan may select particular agents based on the disease and condition being treated and the health and medical status of the patient.

(001411 In one embodiment of the methods of the invention, the agent that stimulates islet differentiation from pancreatic progenitor cells into insulin producing islet structures is selected from the group consisting of I IIP2 or an analog or derivative thereof. In another embodiment, a combination of 111P2 and another agent may be administered to stimulate islet cell neogenesis. In some embodiments, this additional agent can be. for example, amylin and/or an analog, including but not limited to Pramlintidc (SYM LIN ™), CiI.P-1 receptor analogs, exendin-4 (i:XHNΛT!DH^lM). I.iraglutide (NN221 1 ). GI.P-1. GLP- 1 analogs CiIP. CiLP-I . hamster INGAP, other increlin-mimetic hormones, and/or similar!} acting compounds and agents and agents thai extend the hali-lile or increase lhe le\el or activity ot an> oi the foregoing compounds and agents, such us tor example dipeptidv l peptidase-4 inhibitors, which delay the degradation of GLP-I I here are numerous OI P-I mimeties that act via direct agonist acli\ it\ on the GLP-I receptors or by inhibiting the degradation oi Gl P-I I hese agents are usetul in the methods ot the invention In some embodiments, GI P-I mimeties can be used in conjunction with HIP and/or targeted immune therapy for the treatment ol type 1 diabetes, and as provided by the present invention they can be used to improve ghcemic control, increase satiety, delay gut glucose absorption and lead to a reversal oi the undcrlj ing mechanisms resulting in type 1 diabetes In some embodiments, these agents and methods may prevent progression ot impaired glucose tolerance in diabetes, to prevent pre-diabetes, progression of impaired tasting glucose to impaired glucose tolerance and diabetes, to reverse newly diagnosed type 2 diabetes, to treat type 2 diabetes, and to treat or prevent overweight, obesity, polycystic ovarian syndrome and neuropathic pain syndromes

[001421 In some embodiments, methods, agents, and pharmaceutical formulations uselul in the practice of the present invention to achieve pancreatic islet differentiation from progenitor cells in the adult pancreas include those described for other purposes in the following references, each ol which is incorporated herein by reference Rosenberg el ul 1992. 4ώ Exp Vial Biol 321 95-104. Mar 1996. Diabelologui 39(3) 256-62 JuI 1996 Panu ens /J( I ) 38-46 and

2004, Inn Sw g 240(5) 875-84 Vimk tV u/ Jun 1997 I form Meiab Res 29(6) 278-93 In some embodiments the successful stimulation ol islet regeneration or dillerenltation ol pancreatic progenitor cells can be shown through lhe increased production and'or secietion of insulin in α subject [00143| In one embodiment of the imcntion. amylin or an analog of amylin such as Symlin™ or Pramlintidc is employed prior to administration or in concomitant administration with HIP2. In some embodiments, amylin may be administered prior to islet regeneration and continued through the islet regeneration period administration in accordance with the teachings of the reference Young e/ a!.. 1997. Curr. Opin. Enchain. Diabetes 4: 282-290, incorporated herein by reference. In one embodiment of the invention, amylin and/or an analog, including but not limited to Pramlintide, is administered subcutaneously to optimize glycemic control prior to the initiation of II1P2 and may then be used alone or in conjunction with other islet stimulating peptides, such as HIP2 or a HIP2 analog or derivative. In one embodiment, arnylin or Pramlintide is dosed at 0.3-0.8 micrograms per kilogram patient weight. In one embodiment, this dose is administered subcutaneously before meals, for example, QHS and 3 AM. In one embodiment, the therapeutically effective dose is delivered subcutaneously or via an infusion device/pump and/or a transdermal, intranasal, buccal, microneedle delivery system, oral encapsulation method. In another embodiment, the therapeutically effective dose is administered utilizing sustained release formulations requiring administration by injection or other delivery method no more frequently than once a week, once every 2 weeks, or once monthly. Λs noted above, in some embodiments, amylin or Pramlintide is co-administered with another islet stimulating agent.

[001-141 In one embodiment of the invention, a CJI-P- 1 receptor analog, including exendin-4 or an analog of exendin 4 is employed in the method w ith I IIP2 at doses of 5- 10 meg with meals. In some embodiments, exendin-4 can be formulated and administered for purposes of the present invention in accordance with the teachings of the following references each of which is incorporated herein bv reference Mcantara el ul 1998 C f// BwJiem I unci /(5( 1 ) 51-6 Duprc el al 2004 / C Un Lndoa m \lelab H9(l) 1469-71 I

?/ t// 1999 Diabetes 4H 86 93 and Xu <?/ <// , 1999 Diabetes -tfi 2270-76 In one embodiment exendin-4 is dosed in the range ol 5-10 micrograms betore meals In one embodiment e\endιn-4 is administered subcutaneously alone or in conjunction with HIP2 and/or other islet stimulating peptides In one embodiment, the therapeutically effective dose is administered subcutaneously In another embodiment delivery ot exendin-4 is via transdermal, buccal, oral encapsulation methods, intranasal or microneedle delivery systems In another embodiment, the therapeutically effective dose is contained in a sustained release formulation that requires administration no more frequently than once a week, once every 2 weeks, or once monthly In one embodiment, exendin-4 is coadministered with HIP2 or another islet cell neogenesis or progenitor cell transformation agent among patients with type 1 or 2 diabetes, or those with obesity, overweight, insulin resistant syndrome, impaired tasting glucose, pre-diabetes, polycystic ovarian syndrome, the metabolic syndrome or eating disorders

[00145| CJIP and Gl P-I belong to the incretin family of growth hormones and in one embodiment of the invention an incretin hormone or analog with or without the concomitant usage of 11IP2 is employed in the method to stimulate differentiation to islets from progenitor cells in the adult pancreas

|00146| In various embodiments of the invention CiIP or a GIP analog is employed with I I1P2 In some embodiments GlP can be formulated and administered tor purposes ol the present invention in accordance with the teachings ol the tollowing references, each ol which is incorporated herein by relercnee Λndcrsen el al 1978 I C Im Imesi 62 152 161 t reut/leldt a al reb 1980 Diabetes 29(2) 140-5 Dupre c/ ti/ 1973 / (. Im l ndou m \klub J^" 826-828 I bert i/ u/ 1980 C ltmial Utsti oeniei ology S>(3) 679-98 1 UYn et ui 1979 Im J Ph) W)I 23^" L 185 1 191 and 1994 Retζitlatυn I'epiiJi J/( l ) 63-74 KidTup et al Jun 1983 I (. Im tndoii m \ktub _>β(6> 1306 12 Krarup W al 1987 Metabolism 36(1) 677-82 krarup ttf _// 1988 kto l/t'</ Sttrøt/ 223(5) 437-41 I ynn ef u/ , 2003, I ASLB H 19-93 Meir t/ a/ 2002, Regulatory Peptides 70" 1-3 and Nauk ι'/ tf/ , 1993, / ( /;/? fncloa m Metab ^"W) 912-7

[001471 I" ^one embodiment, GIP is administered intravenously or subcutaneonsly in combination with HIP2 or an analog or derivative thcreoi and dosed at 2-10 nanograms per kilogram patient weight to provide a 30-mmule continuous infusion by either intravenous or subcutaneous delivery time beginning 3-5 minutes beiore meals, before bedtime and beginning at 3 AM In one embodiment GIP is administered subcutaneously before meals, QHS, and 3AM In one embodiment, GIP is administered orally or using an infusion device or a transdermal, buccal intranasal or microneedle delivery systems In another embodiment, a sustained release formulation requiring administration no more lrequently than once ever) week, once every 2 weeks or once monthly injections is employed Suitable compositions for administering GlP in accordance with some embodiments ol the methods ol the invention are described lor other purposes in the reterence Jones et al 6 Nov 1989 Diabetes Res C Im PiaU 14) 263-9

[00148] In various embodiments ol the invention, Gl P-I or an analog, or a GI P 1 receptor agonist or a Dipeplidvl Peptιdase-4 Inhibitor is employed in combination with 11IP2 or an analog or derivative thereol in the method to stimulate islet dilterentialion lrom progenitor cells In some embodiments Gl P-I Gl P 1 receptor agonists Gl P-I analogs and DPlM inhibitors tan be iormulated and administered tor purposes of the present invention in accordance with the teachings oi lhe following references each of which is incorporated herein b\ reference l lάhi cl al 1994 Regulawn Pt pink s Jl( I ) 6^"?-74 Gutniak <7 «/ 1994 Diabetes C aie T 1039-44 kieymann el al 1987 /urn.*,/ J: 1300 1304 l arsen i^ α/ 1996. Duώeies 45(Supρl 2) 233 \ ( \bstract) I arsen e/ «/ 2001 Diabetes C are 24(S) 1416-21 , L ist e/ «/ 2004 Am J Phy siol LnJouin Metab 2X6(6) 1 875-81, 1 ugaπ e/ tf/ , 2000. Hυi m Melab Λ« 32 424 428 Marque/ et al Mar 1998 C ell Biochem I tint t /6(1 ) 51-6 Meier et al March 2004, ( i itiial C uie Medicine 32(3) 848-851 , Meneilly el al , 2003 Diabetes C are 26 2835-41 Nauk el al . 1996. Diabetologia 39(12) 1546-53, 1 horens el al , Dec 1995 Diabetes Melab 21(5) 31 1-8 Vilsboll el al , 2003, J C Un kndocrm Metab 88(6) 2706-13. Wang el al , 1997, J C Im Im en 99 2883-2889, and Zander et al , 2002 Lancet 359 824-30

|00149) In various embodiments of the invention, GLP- 1 , a GLP- 1 receptor agonist, or a GLP 1 analog is administered subcutaneously or DPP-4 inhibitors are given orally in combination with HIP2 or an analog or derivative thereof and dosed in the range ol 400-800 mg per day at 8-20 mg per kilogram patient weight In one embodiment GI P-I is administered orally or subcutaneously before meals or QHS In one embodiment, GI P-I is administered using a continuous subcutaneous infusion device at a rate of 1-30 ng/kilogram bodj weight 'minute or a transdermal buccal, or microneedle delivery system to provide a 30-minute continuous infusion bv cither intravenous or subcutaneous deliver* tune beginning 3-5 minutes before meals before bedtime and beginning at 3 ΛM In another embodiment a sustained release formulation requiring administration no more Irequ-nth than once every week once every 2 weeks or once monthl) injections is employed [OUlSO] In one embodiment I iraglutidc (NN221 1 ) is administered subcutaneouslv in combination with HIP2 or an analog or derivative thcreoi in doses, ol 10-40 micrograms per kilogram body weight In another embodiment 1 iraglutide is administered subtutancously before meals QHS and 3 AM In another embodiment I iraglutide is administered using an infusion device or a transdermal, buccal, or microneedle delivery s> stem to provide a 30 minute continuous infusion by either intravenous or subcutaneous delivery time beginning 3-5 minutes before meals, before bedtime and beginning at 3 am In another embodiment, a sustained release formulation requiring administration no more frequently than once every week, once every 2 weeks, or once monthly injections is employed

[00151] In one embodiment ol the combination therapies of the invention, Liraglutide or NN221 1 is administered at a dose of about 20 micrograms per kg of patient weight daily in combination with 11IP2 In some embodiments, this dose will provide patients the ability to reduce bolus insulin before meals by 10-20% with reduced fluctuations and decreased postprandial glucose, glucagon and triglycerides Administration of I iraglutide in accordance with some embodiments of the methods of the invention can be used to improve glycemic control as measured, tor example and without limitation by hemoglobin Λ1C, in type 1 diabetes, to prevent progression of impaired glucose tolerance in diabetes to prevent progression of impaired fasting glucose to impaired glucose tolerance and diabetes to reveise newly diagnosed tv pe 2 diabetes and to treat tv pe 2 diabetes

JOOl 52] In an embodiment ol the combination therapy of the invention I iraglutide or NN221 1 is administered at a dose ol about 20 micrograms per kg ol patient weight to an adult patient in the morning about 4 houis before lood intake and at bedtime tor three consecutive weeks during IIIP2 therapv In some embodiments, tor patients initiating treatment with C-peptide levels lower than about 1 0 ng/ml C -peptide levels are monitored and when the) rise above 0 5 ng'nil the antibodv hOK. I IgI (ala-ala) is administered tor 12 consecutive days

[00153] In the combination therapies ol some embodiments ol the invention exendin-4 or synthetic exendin-4 or another Gl P-I analog, OLP-I receptor agonist or Dipeptidjl Peptidase-4 lnhibibtor is administered prior to meals alone or with H1P2 or another islet differentiation agent to improve glycemic control prior to or during the iniaition of HIP2 therapies In some embodiments, such agents, when delivered prior to meals may result in a reduction in the need for insulin of at least 20% and appropriate tapering of insulin and diabetic medications will be conducted while H1P2 is administered In some embodiments, as HIP2 and/or other agents are delivered in both type 1 and type 2 patients, careful tapering of insulin and other diabetes medications will take place to protect against hypoglycemia as new islet cells are differentiated from progenitor cells In some embodiments, insulin and diabetes medications, including HIP2, will be ultimately tapered off, as the pancreas is repopulated with new functional islets In some embodiments, tor patients initiating treatment with C-peptide levels lower than about 1 0 ng/ml , C-peptide levels are monitored and when ιhe> rise above 0 5 ng/ml caretul monitoring and tapering of exogenous insulin doses will occur

[00154| In some embodiments, among patients with type 1 diabetes, immune therapy will be administered to protect newly tormed islets prior to initiation ot 111P2 and'or other peptide compounds (SYMI IN ^{I M} hamster ING ΛP, GI P-I , GI P-I receptor agonists Gl. P-I analogs. DPIM inhibitors are used with (preceding, during, or follow ing). In some embodiments, for example, the untibod) hOK I^'3gl (ala-ala) is administered for 12 consecutive days with its efficacy demonstrated following the first treatment out to 24 months, whereas a similar humanized monoclonal antibody, ChAg]) CD3 maj be administered for 6 consecutive days, then repeated yearly. In some embodiments. Oiarmd s GAD65 compound is delivered in two subcutaneous injections, one month apart. In some embodiments. DIΛPKP277^{I M}, a heat shock protein 60. has demonstrated success among newly diagnosed diabetes patients utilizing a subcutaneous injections of 1 mg with 40 mg mannitol in vegetable oil at study entry. 1 month, and 6 months. In some embodiments, the cyclicity of treatment will be determined based upon the immune modulator selected. In another embodiment, D1ΛPEP277™. a heat shock protein 60 vaccine, and IBC-VSO vaccine, which is a synthetic, metabolically inactive form of insulin designed to prevent pancreatic beta-cell destruction, interferon-alpha. or vaccination using CD4'CD25' antigen- specific regulatory T cells or a similar agent is used in the combination therapy. In another embodiment, immunomodulation agents, including, but not limited to. anti-CD3 immunotherapy agents, are used in combination with H1P2. In some embodiments, such agents also include: Sirolimus (Rapamyciπ), Tacrolimus (I K.506), a heat-shock protein 60 (DIΛPI-P277™), anti-GIutamic Λcid Decarboxylase65 (GΛD65) vaccine, Mycophenolate Mofetil alone or in combination with Daclizumab, the anti-CD20 agent Rituximab. Campath-I I I (Λnti-CD52 Antibody) and/or Vitamin D.

|00155] Some autoimmune cells target pancreatic beta cells and so play a causative role in some of the diseases and conditions treatable in accordance with the methods of the invention. [00156] Prior methods oi tieatment iinoh ing the introduςtion ot immune agents among patients with type 1 diabetes protOLt only those islet cells which ha\e \et been destroy ed

immune attack and do not address to need to repopulale the pancreas with new islet structures with fully functionally beta cells In some embodiments methods of the present invention combine generalised and specific immune modulation aimed at reducing destruction ol beta cells and a methodology of differentiating new islet cells trom progenitor cells within the adult pancreas

(00157) In some embodiments the methods of the present invention may employ agents that specifically inhibit the activity of or block or destroy the autoimmune cells that target pancreatic beta cells that produce insulin, amylin, or glucagon In some embodiments such agents include immunomodulatory peptides that arrest pancreatic islet cell destruction In some embodiments for example, one such agent is a monoclonal antibody that can delay the progression of islet cell loss or slow or stop the onset of type 1 diabetes In some embodiments anti-CD3 antibodies constitute a general class of agents useful in the methods ot the invention In some embodiments, for example, suitable anti-CD3 antibodies lor purposes of the present invention include the IRX4 (Ala-Ala and ChΛglyCD3) antibod) under development by I olcrRx and the humanized anti-CD3 antibody described in the reference Ilerold el cil 30 May 2002, Nt JM 546(21) 1692-1698, incorporated herein bv reference In one embodiment the humanized anti-C D3 antibody is delivered intravenously 14 days per year in the dosage of 1-1 42 μg'kg on day 1 5 67 μg/kg on dav 2 1 1 3 μg/kg on day 3 22 6 μg/kg on day 4 and 45 4 μg/kg on days 5-14 In some embodiments, these therapies may be repealed annually following lht 3-6 month usage of I IIP2 while insulin is being tapered as new islet cell formation occurs During the I 11P2 treatment phase in some embodiments Vitamin I) and the usage ot PramliiUidc S\mlm^{I M}

be continued I allowing the discontinuation ol H1P2 and insulin therapv in some embodiments immune modulation may be repealed annualh lor the anti-C D^~> antibodies though recent stud) has found their etficac) to continue lor as long as 24 months

|00158| In another embodiment the immuno-modulatorv compound is a heat shock protein that can arrest or slow islet cell destruction In some embodiments such proteins include DIΛP1 P277^{I M}, a heat-shock protein under development by Develogen ΛG In one embodiment. D1API-P277^{I M} is delivered subcutaneously by giving 1 mg in 40 mg mannitol in vegetable oil subcutaneously at baseline and at one month and then twice at 3 month intervals In one embodiment ot the combination therapy of the invention HIP2 or a HIP2 analog or derivative is co-administered with DlAPhP277^{I M} as follows In some embodiments, the DIAPEP277^{I M} is first administered subcutaneously at a dose of about 1 mg about 30 days prior to the initiation of the HIP2 or analog or deπvati\e-based therapy In some embodiments, a second administration ot the DIAPEP277^{1 M} is then made at the time (90 days after the first administration) of initiating the HIP2 or analog or deπvative- based therapy

[00159] In some embodiments the I11P2 or analog or derivative thereof may be delivered via subcutaneous injection orally via hepatic targeted vesicle, or other liposomal agent, or via 24 hour continuous subcutaneous mlusion at a therapeutically etlective dose, as described above In one embodiment the daily dose is about 5 to 20 mg per kg of patient body weight per 24 hours In one embodiment the daily dose is -600-800 mg In some embodiments the I IIP2 or analog or deπv ative-based therapy is continued lor a V6 month period and monitored closely bv C -peptide production In some embodiments the immune therap) will be

cyclical!) based upon the immune agent selected. In some embodiments, for example, the DIΛPKP277 ^M is administered at 3 month intervals tor a total of 6 months, and would initially be delhered 3 months prior to I IIP2 or analog or derivathe-based therapy.

[00160] In some embodiments, the immuno-modulatory agents useful in the methods of the invention can be formulated, administered, and dosed as known in the art or as described herein. Pharmaceutical formulations and additional dosing and administration protocols for practice of some embodiments of the methods of the invention are described below.

[00161] In some embodiments, compositions of HIP2 or an analog or derivative thereof, e.g., and pharmaceutically acceptable salts and esters thereof, are synergistically or additively effective to differentiate progenitor cells into new islet cells in treating diabetes or similar disorders when combined with various other compounds. In some embodiments, these compounds include I IIP2 and analogs or derivatives thereof, amylin and/or an analog, including but not limited to Symlin/Pramlintide, GLP-I , GLP-I receptor agonists, such as exendin-4. Liraglutide (NN221 1 ), GLP-I analogs. Dipeptidyl Peptidase-4 Inhibitors. GIP, hamster 1NGΛP, and other incretin-mimelic hormones, and/or similarly acting compounds and agents, and agents that extend the half-life or increase the level or activity of any of the foregoing compounds and agents, such as. for example, dipeptidyl peptidase inhibitors, which delay the degradation of GLP-I . and agents that inhibit, block, or destroy the autoimmune cells that target beta cells including but not limited to: anti CD- 3 antibodies lhOKT3"-l Λla-Λla and ChΛglyCD3). Sirolimus (Rapamycin). Tacrolimus (I K506). a heat- shock protein 60 (l)IΛPl'P277^{I M}) a anti-Glutamic Λcid Decarboxylase 65 (GΛD65) vaccine M>cophenolate Mofetil alone or in combination with Dacli/umab the anti-C D20 agent Rituximab C ampath-I H (Λnti-CD52 Λntibodv ) hsofy lline and Vilamin D IBC- VSϋ vaccine v\hich is a synthetic metabolically inactive torm of insulin designed to prevent pancreatic beta-cell destruction and intcrteron-α vaccination using C D4 CD25 antigen-bpeciilc regulatory 1 cells or a similar agent designed to prevent pancreatic beta- cell destruction In another embodiment interteron-α vaccination using CD4 C D25^* antigen-specific regulatory f cells or a similar agent is used in the combination therapy tor utilizing regulatory I cells either directly or through the use ol anti-CD3 immunotherapy

|00162] In some embodiments compounds such as Sirohmus (Rapamyun) Iacrohmus (rK506), TRX4 antibody, humanized anti-CD3 antibody, DYΛMID™ anti- GAD65 antibody, and DIAPEP277™ are also synergistically or additnely etiective when added to usage of HIP2 or an agent to differentiate progenitor cells into new islet cells in treating diabetes or a similar disorders

|00163] Synergy is defined as the interaction of two or more agents so that their combined effect is greater than the sum ol their individual effects For example if the effect of drug Λ alone in treating a disease is 25% and the effect of drug B alone in treating a disease is 25%, but when the two drugs are combined the effect in treating the disease is 7S°o the effect of A and B is synergistic

(00164) Λdditivity is defined as the interaction of two or more agents so that their combined effect is similar to the average ot their individual effects I or example, it the ef fect ol drug A alone in treating a disease is 25% and the effect ot drug B alone in treating a disease is 25%, but when the two drugs are combined the eliect in treating the disease is about 50% or at least greater than 25% ιlκ effect ot A and B is additive |00165| Λn improvement in a drug therapeutic regimen can be obtained bv the combined administration of two agents having therapeutic effect, if the interaction of the two or more agents is such that their combined effect reduces the incidence of adverse event (AIi) of either or both agents used in the co-therapy. I his reduction in the incidence of adverse effects can be a result of, e #., administration of lower doses of either or both agent used in the co-therapv. l-'or example, if the effect of drug Λ alone is 25% and has an adverse event incidence of 45% when used at the labeled dose; and the effect of drug B alone is 25% and has an adverse event incidence of 30% when used at the labeled dose, but when the two drugs are combined at lower than labeled doses of each, if the overall effect is 35% and the adverse incidence rate is 20%, there is an improvement in the drug therapeutic regimen. The combination therapies provided by the present invention include those exhibiting such improvements.

[00166] In some embodiments, dosing and administration of the agents useful in the methods of the invention as described herein provide accelerated islet differentiation from adult progenitor cells to optimise an individual's ability to secrete insulin from endogenous, newly formed islet structures with used in conjunction with immune therapy or therapies, which give the lowest toxicity while providing protection of the new islets from destruction. In some embodiments, pharmaceutical compositions of the invention provide for kinetic delivery of these agents, ease of delivery, and enhanced efficacy.

[00I67| In one embodiment, IIIP2 peptide is dosed subeutaneouslv or intramuscularly, at a daily dose of ϋ. l to 100 mg/kg. which daily dose is subdiv ided and dosed four times daih . pre-prandial Iv. before each meal and a dose at bedtime. In another embodiment. 11IP2 peptide is dosed at a daily dose of about 5 to 25 mg/kg, which daih dose ib subdn ided and dosed lour times dail\ pre-prandiall) betorc each meal and a dose at bedtime In another embodiment, H1P2 peptide is dosed at a dail> dose ot 10 to 15mg/kg which dose is

and delivered in tour separate subcutaneous injections

[00168| In other embodiments the 1I1P2 peptide is administered onl) once twice or thrice daily and in another embodiment I IIP2 peptide is administered by continuous infusion

[00169| The agents uselul in the methods of the invention can be administered by a variety of routes Known agents useful in the methods ot the invention can be administered by routes and using pharmaceutical lormulations previously developed for other indications Such delivery routes include, at least for most known agents, oral delivery targeted and untargeted liposomal drug delivery systems tor oral or subcutaneous delivery, which may include the hepatic-directed vesicle (AMDG/SDG) attached to HIP2 or compounds used in the methodologies described herein, topical delivery, including micelle and nanosphere topical delivery systems subcutaneous delivery including pump-assisted continuous infusion by either intravenous or subcutaneous delivery and disposable micro-pumps and micro-needles (including but not limited to those available from Λnimas torp ) and buccal delivery

[00170| the particular route of administration and pharmaceutical formulation of an agent used in the practice of the methods of the invention will be selected by the practitioner based on a patient s disease or condition being treated and the agent employed Λ wide vaπetv of pharmaceutical compositions can be emploved in the methods ot the invention In some embodiments extended use preparations can be used lor ease ot administration and increased ef ficacy [00171] In one embodiment, one or more of the agents employed in the method is formulated as a micelle. In some embodiments, ease of administration is best aehie\ ed by oral delivery. While small molecule pharmaceutical agents can often be readily formulated for oral delivery, peptide and protein-based pharmaceutical agents can be more difficult to formulate for oral delivery. However, suitable formulation technology exists, and in one important aspect, the present invention provides pharmaceutical compositions of proteins and peptides formulated for oral delivery. In one embodiment, the pharmaceutical compositions useful in the methods of the invention suitable for oral delivery are formulated generally in accordance with known TKCHNOSPHERI:™ technology developed by MannKind Corp., HLIGEN® Technology developed by F-misphere, a nasal delivery systems developed by Nastech. an oral liposome with specificity to the liver (HDV) developed by AMDG/SDG.

[00172| Other oral delivery and encapsulation technology suitable for use in making the pharmaceutical compositions of some embodiments of the invention includes the hepatic delivery vesicle (HDV). In some embodiments, pancreatic delivery vesicle (PI)V) technology may be used in accordance with the methods of the invention to prepare pharmaceutical formulations of the invention containing HIP2 or HIP2 in combination with GLP-I for delivery of I1IP2 directly to the pancreas. In some embodiments. HDV technology can be used to deliver compounds directly to the liver, and the present invention provides pharmaceutical compositions of 1I1P2 optionally in combination with GI. P-I in I IDV liposomes targeted directly to the liver.

[00173| In some embodiments, agents that can be formulated for oral

and employed in the methods of the invention include I IIP2 or an analog or derivative thereof. SWlI lN '" pramlintide 1 \cndin 4 I iraglutide (NN221 1 ). OI P-I receptor agonists GI P- 1 GI P-I analogs hamster INGAP and its analogs GlP Dipeptid\ 1 peptidase-4 inhibitors and peptide and proteins or non-peptidie mimcties with similar action or homolog) to the preceding agents In some embodiments these agents can be used in accordance with the methods ol the invention with monoclonal antibodies and other specific and general immune agents designed to dela> the progression ot beta cell loss or prevent the onset ot t>pe 1 diabetes in both children and adults In some embodiments, these include, but are not limited to, anti CD-3 antibodies (hOK 13^* 1(AIa-AIa and ChAglyCD3) that target the immune response and specifically block the 1 -lymphocytes that cause islet cell death in type 1 diabetes as well as Sirolimus (Rapamycm) T acrolimus (!^• K.506) a heat-shock protein 60 (DIAPfcP277^IM), an anti-Glutamic Acid Decarboxylase 65 (GAD65¹) vaccine Mycophenolate Mofetil alone or in combination with Dachzumab, the anti-CD20 agent, Rituximab, Campath-111 (Anti-CD52 Antibody), lysotylhne, Vitamin D IBC-VSO vaccine which is a synthetic, metabolically inactive torm ol insulin designed to prevent pancreatic beta-cell destruction, interferon-alpha vaccination using CD4*CD25 antigen-specific regulatory F cells or similar agents In some embodiments, these agents are used in the combination therapies ol the invention to utili/e regulatory 1 cells either directly or through the use of immunotherapy to arrest the destruction ol insulin-producing cells

|00174| In some embodiments, the invention further relates to kits tor treating patients having tvpe 1 or type 2 diabetes or other glucose metabolism disorders in children and adults including pre-diabctes impaired fasting glucose insulin resistant svndromcs the metabolic syndrome, obesity overweight polvevsistie ovarian syndrome hvperhpidemia hvperlriglj ceridemia comprising one or more therapeutically effective methods ol 11IP2 or an analog or dernatn e modes ot treatment thereof OptionalK the kit mas also contain other agents as described above tor use in the combination therapies ol the invention cither in the same or separate packaging and instructions ior use

[00175| Λs exemplified b> the preceding paragraph some embodiments ol the present invention provide kits lor treating a disease or condition associated with impaired pancreatic function in a subject in need thereol, comprising a therapeuticall) ef fective amount of 1 IIP2 in a first dosage unit optionall) a therapeutically eliective amount of one or more immune therapies, optionally a therapeutically effective amount of one or more additional agents that stimulate islet cell regeneration in a third dosage unit, and instructions for use

|00176] Any techniques known in the art can be used in synthesizing and purifying HIP2 or an analog or derivative thereof, including, but not limited to de novo chemical synthesis and purification by precipitation, adsorption (e g , column chromatography, membrane adsorbents, radial flow columns, batch adsorption, high-performance liquid chromatography, ion exchange chromatography, inorganic adsorbents, hydrophobic adsorbents immobilized metal affinity chromatography, affinity chromatography) or gel filtration, electrophoresis, liquid phase partitioning, detergent partitioning, organic sol\ent extraction, and ultrafiltration During purification, the biological activit) ot 111P2 or an analog or derivative thereof may be monitored by one or more in utiυ or in u\o assays l he purity of 11IP2 or an analog or derivative thereof can be assayed by any methods known in the art such as but not limited to gel electrophoresis See Scopes siφia In some embodiments HIP2 or an analog or

thereot employed in a composition ol the invention can be in the iange of 80 to 100 percent ol the total mg protein or at least 80" o at least 83°υ at least 90° o at least 95% or at least 98% of the total mg protein In OIK embodiment 1I1P2 or an analog or derivative thereof emplo\ed in a composition of the invention is at least 99% of the total piotein In another embodiment I1IP2 or an analog or deπ\atι\e thereof is purified to apparent homogeneity as assayed, e g by sodium dodecv l sulfate polyacrylamide gel electrophoresis In one embodiment, I11P2 is svnthesi/ed and tested by I IPLC to a punt) greater than 95%

[00177J Methods known in the art can be utilized to produce HIP2 or an analog or deπvative thereof recombinantly A nucleic acid sequence encoding I I1P2 or an analog or derivative thereof can be inserted into an expression vector for propagation and expression in host cells

|00178) An expression construct, as used herein, refers to a nucleic acid sequence encoding a HIP2 or an analog or derivative thereof operably associated with one or more regulatory regions that enable expression of HIP2 or an analog or deπvative thereol in an appropriate host cell Operably-associated reters to an association m which the regulatory regions and the HIP2 or an analog or derivative thereof to be expressed are joined and positioned in such a way as to permit transcription, and ultimately translation

[00179J In some embodiments the regulatory regions that are necessary for transcription oi I IIP2 or an analog or derivative thereof can be prouded by the expression vector In some embodiments a translation initiation codon (A KJ) may also be provided if a 11IP2 or an analog or derivative thereol gene sequence lacking its cognate initiation codon is to be expressed In a compatible host-construct system cellular transcriptional lactors such as RNΛ poKmerase vvill bind to the regulatorv regions on the expression construct to effect transcription of the I IIP2 sequence in the host organism 1 he precise nature of the regulator) regions needed for gene expression may \ar\ from host cell to host cell. General!) , a promoter is required which is capable of binding RNA poly merase and promoting the transcription of an operabh -associated nucleic acid sequence. Such regulator) regions may include those 5^" non-coding sequences involved with initiation of transcription and translation, such as the TΛTΛ box, capping sequence. CΛAT sequence, and the like. The non-coding region 3^" to the coding sequence may contain transcriptional termination regulatory sequences, such as terminators and polyadenylation sites.

[001801 In some embodiments, in order to attach DNΛ sequences with regulatory functions, such as promoters, to a I IIP2 or an analog or derivative thereof gene sequence or to insert a IIIP2 or an analog or derivative thereof gene sequence into the cloning site of a vector, linkers or adapters providing the appropriate compatible restriction sites may be ligated to the ends of the cDNΛs by techniques well known in the art. In some embodiments, cleavage with a restriction enzyme can be followed by modification to create blunt ends by digesting back or filling in single-stranded DNA termini before ligation. Alternatively, a desired restriction enzyme site can be introduced into a fragment of DNA by amplification of the DNA using PCR with primers containing the desired restriction enzyme site.

(001811 An expression construct comprising a 11IP2 or an analog or derivative thereof sequence operably associated with regulatory regions can be directly introduced into appropriate host cells for expression and production of a HIl'2 or an analog or derivative thereof without further cloning. The expression constructs can also contain DNA sequences that facilitate integration of a HI1^J2 or an analog or

into the genome of the host cell, e g . via homologous recombination. In this instance, it is not necessarv to emplov an expression vector comprising a replication origin suitable tor appropriate host cells to propagate and express IHl" or an analog or deiι\ati\e thereol in the host tells

(00182] A \aπet\ ot expression vectors nwj be used including but not limited to plasmids cosnuds phage phagemids or modified viruses Such host-expression s> stems represent vehicles b> which the coding sequences of a HIP2 or an analog or derivative thereoi gene may be produced and subsequently purified, but also represent cells which may. when translormed or transfected with the appropriate nucleotide coding sequences, express I IIP2 or an analog or derivative thereof m situ I hese include, but are not limited to, microorganisms such as bacteria (e g , t LOII and B subtilis) transformed with recombinant bacteriophage DNA plasmid DNA or cosrnid DNA expression vectors containing HIP2 or an analog or derivative thereof coding sequences, yeast (e g , Saccharomyces, Ptthia) transformed with recombinant expression vectors containing H1P2 or an analog or derivative thereof coding sequences, insect cell systems infected with recombinant virus expression vectors (e g , baculovirus) containing H1P2 or an analog or derivative thereof coding sequences, plant cell systems infected with recombinant v irus expression vectors (e g , cauliflower mosaic virus, CaMV tobacco mosaic virus, 1 MV) or transformed with recombinant plasmid expression vectors (e g , I i plasmid) containing IIIP2 or an analog or derivative thereol coding sequences, or mammalian cell systems (e g . COS, CI lO. Bl IK 293, NSO, and 3 13 cells) harboring recombinant expression constructs containing promoters derived from the genome ol mammalian cells {e g , metallothionem promoter) or lioin mammalian viruses (e t; the adenovirus late promoter the vaccinia virus 7 ^K promoter) Preferably bacterial cells such as / st/ιc/ it luu coll and eukαrvolic cells are used for the expression ol a recombinant III PU or an analog or derivative thereof I or example mammalian tells such as Chinese hamster o\ar\ tells (CHO) tan be used with a \ettor bearing promoter element trom major intermediate early gene ot t>tomegalo\ irus tor ellettive expression oi a 11IP2 or an analog or deriv ative thereol sequente

|00183] In battenal systems a number ol expression \etlors may be ad\ antageously selected depending upon the use intended tor the IIIP2 or an analog or derivative thereof being expressed For example, when a large quantity of a HIP2 or an analog or derivative thereof is to be produced, tor the generation ol pharmateutital compositions of a HIP2 or an analog or derivative thereof, vectors that dirett the expression ot high levels of fusion protein products that are readily purified may be desirable Vettors include, but are not limited to, the k coll expression vector pCR2 1 1 OPO (Invitrogen), pIN vectors, and the like Series of vectors like pH AG (Sigma), pMAL (Nl B), and pi- V (Novagen) may also be used to express the foreign proteins as fusion proteins with H AG peptide, malt-, or CBD- protein 1 hese recombinant proteins may be directed into peπplasmic space for torrect folding and maturation I he fused part can be used for affinity puπfitation of the expressed protein Presence ol cleavage sites tor spctific proteases like cnterokinase allows one to tlea\e olf the I1IP2 or an analog or derivative thereot l he pGl X vtttors may also be used to express loreign proteins as fusion proteins with glutathione 5-transferase (GS I ) In general suth fusion proteins are soluble and tan easily be purified from lystd tells by adsorption and binding to matrix glutathione agarose beads followed bv elution in the presente ol lret glutathione 1 he pGI X vettors are designed to intlude thrombin or lattor λa protease tleavage sites so that the tloncd target gene produtt tan be released lrom the CiS 1 moiety 100184] In an insect sv stem many vectors to express loreign genes tan be used t Sj liiiogiapha califυinica nuclear polvhedrosis virus (AcNPV) can be used as a \ ector to express foreign genes 1 he \ irus grow s in cells like Spoilopu i a fi uyipt. i da cells Λ Hll'2 or an analog or derι\ati\e thereot coding sequence may be cloned individually into nonessential regions (e g , the polyhedπn gene) ol the virus and placed under control ol an AcNPV promoter (e g the polyhednn promoter)

[00185] In mammalian host cells, a number of viral-based expression systems may be utilized In cases where an adenovirus is used as an expression vector, a HIP2 or an analog or derivative thereof coding sequence ot interest may be hgated to an adenovirus transcription/translation control complex, e g the late promoter and tripartite leader sequence I his chimeric gene mav then be inserted in the adenov irus genome by m \ ilro or in \ (V o recombination Insertion in a non-essential region of the viral genome (e g region 1 1 or b3) will result in a recombinant virus that is viable and capable of expressing IIIP2 or an analog or derivative thereof in infected hosts Specific initiation signals may also be required for efficient translation of inserted HIP2 or an analog or derivative thereof coding sequences These signals include the A 1 G initiation codon and adjacent sequences I urthermore, the initiation codon must be in phase with the reading trame ol the desired coding sequence to ensure translation ol the entire insert I hese exogenous translational control signals and initiation codons can be of a vaπetv ot origins both natural and svnthettc I he elficieney of expression may be enhanced by the inclusion of appropriate transcription enhancer elements transcription terminators and the like

[00186] In addition a host cell strain may be chosen which modulates the expression ol the inserted sequences, or modifies and processes the gene product in the specific fashion desired Such modification!) (t # glvcosv lation) and processing (c g cleavage) ot protein products can be important lor the function ol the protein Dilterent host cells have characteristic and specific mechanisms lor the post-translational processing and modification ol proteins and gene products Appropriate cell lines or host s> stems can be chosen to ensure the correct modification and processing ol the foreign protein expressed I o this end, eukaryotic host cells that possess the cellular machincr> for proper processing ot the primary transcript and post-translational modification of the gene product e tξ glvcosylation and phosphorylation ot the gene product, may be used Such mammalian host cells include but are not limited to PC12 CHO, Vl RY BHK, HeI a COS, MDCK 293, 3 13, W 138, B 1483, Hs5781^", H fB2, Bl 20 and T47D, NSO (d murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, and HsS78Bst cells Expression in a bacteπal or yeast system can be used if post-translational modifications are found to be non-essential for a desired activity of HIP2 or an analog or derivative thereof

|00187) For long-term, high-yield production ot properly processed HIP2 or an analog or derivative thereof, stable expression in cells is preferred Cell lines that stably express 1 IIP2 or an analog or derivative thereof may be engineered by using a vector that contains a selectable marker B) way ol example and not limitation following the introduction of the expression constructs engineered cells ma> be allowed to grow lor 1-2 davs in an enriched media, and then are switched to a selective media l he selectable marker in the expression construct confers resistance to the selection and mav depending on the vector construct and host cell

allow cells to stablv integrate the expression construct into their chromosomes and to grow in culture and to be expanded into cell lines Such cells can be cultured for a long period of time while IIIP2 or an analog or derivative thereof is expressed continuously .

|00188] Λ number of selection systems ma) be used, including but not limited to. antibiotic resistance (markers like Neo. which confers resistance to geneticine. or G-418; Zeo. for resistance to Zeocin; and Bsd. for resistance to blasticidin): antimetabolite resistance: gpt. which confers resistance to mycophenolic acid; and hygro, which confers resistance to hygromycin. In addition, mutant cell lines including, but not limited to. tk-. hgprt- or aprt- cells, can be used in combination with vectors bearing the corresponding genes for thymidine kinase, hypoxaπthine, guanine- or adenine phosphoribosyl-transferase. Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel el al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, Λ Laboratory Manual, Stockton Press, NY (1990); Chapters 12 and 13, Dracopoli el a!, (eds), of Current Protocols in Human Genetics. John Wiley & Sons, NY (1994); and Colbcrre-Garapin <?/ «/., 1981 , J. MoI. Biol. 150:1.

[00189) The recombinant cells may be cultured under standard conditions of temperature, incubation time, optical density and media composition. 1 lowever, conditions for growth of recombinant cells may be different from those for expression of HIP2 or an analog or derivative thereof. Modified culture conditions and media may also be used to enhance production of 11IP2 or an analog or derivative thereof. Any techniques known in the art may be applied to establish the optimal conditions for producing 111P2 or an analog or deriv atiλc thereof. [001901 Λn alternative to producing 1 I1P2 or a fragment thereol by recombinant techniques or purification lroni natural sources is peptide svnthesis I or

an entire HIP2 or an analog or deriv ative thereoi or a protein corresponding to a portion ol 1 IIP2 or an analog or derivative thereof can be synthesized by use ot a peptide sy nthesizer Conventional peptide synthesis or other synthetic protocols well known in the art ma> be used

[00191] Proteins having the amino acid sequence of HIP2 or an analog or derivative thereof or a portion thereof may be synthesized by solid-phase peptide synthesis using procedures similar to those described by Merrifield, 1963, J Am C hem Soc , 85 2149 During synthesis, N-α-protected amino acids having protected side chains are added stepwise to a growing polypeptide chain linked by its C-terminal and to an insoluble polymeric support, * e . polystyrene beads The proteins are synthesized by linking an amino group of an N-α-deprotected amino acid to an α-carboxyl group of an N-α-protected amino acid that has been activated by reacting it with a reagent such as dicyclohexylcarbodnmide 1 he attachment of a free amino group to the activated carboxyl leads to peptide bond formation fhe most commonly used N-α-protecting groups include Boc. which is acid labile, and 1 moc, which is base labile Details of appropriate chemistries, resins, protecting groups protected amino acids and ieagents are well known in the art and so are not discussed in detail herein

|00192] Purification ol the resulting 11IP2 or an analog or derivative thereol is accomplished using conventional procedures, such as preparative HPI C using gel permeation partition and/or ion exchange chromatography I he choice ol appropriate matrices and buffers are well known in the art and so are not described in detail herein |00193] 1 he embodiments ot the methods described above can be combined in am manner I hus> features trom one embodiment can be combined with features lrom an\ other embodiment I urthcr

modifications ot the in\ention in addition to those described herein, will be apparent to those skilled in the art trom the foregoing description Such modifications are also intended to tall within the scope of the appended claims Lach reference (including, but not limited to.joumal articles, U S and non-U S patents patent application publications, international patent application publications, gene bank accession numbers, and the like) cited in the present application is incorporated herein by reference in its entirety

[00194] With the foregoing detailed description of the reagents and methods of the invention, the following Fxamples are provided to illustrate vaπous aspects of the invention

EXAMPLE 1

100195] HIP2, HIPl, HIP controls and progenitor tractions were cultured over 10 days according to standard protocol Briefly pancreata from adult human cadaveric organ donors were obtained through the local organ procurement organization Islets were isolated according to established protocols described by Bonner-Weir and Jamal (Bonner- Weir el id Pediatric Diabetes 2004.S(SuPpI 2) 16-22 Jamal el al C ell Death Differ 2005 Jul,12(7) 702 12)

|00196] Briefly, following removal ot the organ, cold ischemia time was no more than 8 hours prior to islet isolation 1 he main pancreatic duct was cannulaled and perfused with I iberase I II (Roche Diagnostics) I he perf used organ was placed in a closed svstem (Ricordi Apparatus) and heated to M X to activate the en/ytne blend hollowing the appearance of tree islets in samples the s\ stem was cooled and tree tissues were collected and washed 1 issues were applied to a continuous densih gradient created using 1 icoll (Biochrom K.G) in a cell processor (C OBl- ) 1 rec islets with diameters ranging trom 75 to 400 μm. determined to be greater than 90% pure bv staining with dithi/one (Sigma) a /inc chelater were collected and washed IHC to detect the presence of amylase and cwokeratin was negative, consistent with the absence ol progenitor and exocπne tissue I he progenitor fraction from this separation was also collected for culture

(00197] Isolated islets were embedded in a type 1 collagen matnx at a density of 2000 islet equivalents/25 cm² and cultured in DMrM/F12 containing 10% HiS, 1 μM dexamethasone, 10 ng/ml FGI , 24 mU/ml insulin and 100 ng/ml cholera toxin

100198) Medium was changed every other day On day 10, culture was continued in the above medium, without the cholera toxin, with HIP2, HIP, HIPl and INGAP in concentrations of 1 0 uM and 3 3uM into 2 ml cultures for final concentrations of 500 nM, 167 nM and 50 nM Medium was changed every other day Collagen-embedded cultures were harvested by incubating with 0 25 g/I collagenase XI (Sigma) for 30 minutes at 37 ⁰C

[00199| Λiter culture, the human pancreatic islet and progenitor fractions were then treated in a blinded study with either I I1P2, HlP, HlPl, the hamster 1NGΛP sequence as a positive control (SL Q ID NO 1 ) or a scrambled peptide sequence that was synthesized by Bachem BioScience (95% pure, research grade) (Sl Q ID NO 5) Duplicate cultures were treated on Day 10 and Day 12 and then lysed for detection of insulin content on Day 14 During 10 day culture the insulin production decreases to negligible amounts and after tieattnem with peptides insulin is produced again

|00200) Insulin levels were detected b\ Radioimmunoassa\ (RIΛ) from cultures treated w ith saline

sermnbled peptide. 111P2, 1 HPl . I IIP and hamster 1NGΛI'. 1 he results for human ductal tissue fraction are shown in Figure 1 and for human islet tissue in Figure 2. Both fractions contain progenitor cells, which are the nidus for new islet structures and upon which HIP peptides exerts stimulator)' effect. Figure 4 shows the ductal tissue culture fraction after H1P2 treatment, just before l>sis and measurement by RIΛ. Morphological changes show islet like structure. Consistently, greater induction of new islets is observed from the cells cultured from the ductal fraction of the pancreatic tissue This observation is consistent with the notion that fewer progenitor cells are among the islet tissue fraction after the isolation process.

EXAMPLE 2

[00201] HIP2, HIPl, HlP, controls and progenitor fractions were cultured over 10 days, according to standard protocol. Briefly, pancreata from adult human cadaveric organ donors were obtained through the local organ procurement organization. Islets were isolated according to established protocols described by Bonner-Weir and Jamal. (Bonner-Weir el ul , Pediatric Diabetes 2004:5(Suppl 2):16-22. Jamal et a! . Cell Death Differ 2005 Jul;12(7):702-12). IIIP2, HIPl, HlP and 1NGΛP were synthesized by Bachem BioScience. Malvern, PΛ and were quality tested by I IPCL to be greater than 95% pure. Hach peptide was provided in lyophili/ed powder and was resuspended in isotonic saline solution. Stock solutions were made so that only 1-2 ul aliquots were added to primary cell cultures to reach the final concentrations provided below. Stock solutions were fro/en and thawed a total of three times for the six

treatment. HPI.C data confirmed that the peptides remained stable in the isotonic saline after the free/.e/lhaw c>cles. |00202] Pancreata horn adult human cada\eπe organ donor;, v\ere oblaincd through the local organ procurement organization Islets were isolated according to established protocols BriefK lollo\\ing remo\dl ot the organ cold ischemia time

no more than 8 hours prior to islet isolation 1 he main pancreatic duct v\as cannulated and perlused with I iberase III (Roche Diagnostics) I he pertused organ was placed in a closed system (Ricordi Apparatus) and heated to 37 ⁰C to activate the en/yme blend Following the apptarance ol tree islets in samples, the system was cooled and lree tissues were collected and washed I issues were applied to a continuous density gradient created using I icoll (Biochrom KG) in a cell processor (COBh) Free islets with diameters ranging from 75 to 400 μm determined to be greater than 90% pure by staining with dithi/one (Sigma) a 7inc chelater were collected and washed IHC to detect the presence ot amylase and eytokeratm was negative, consistent with the absence of progenitor and exocrine tissue The progenitor fraction from this separation was also collected for culture

|00203| In an effort to grow less differentiated cells isolated islets were embedded in a type 1 collagen matrix at a density of 2000 islet equivalents/25 cm² and cultured in DMLM/I 12 containing 10 % FBS, 1 μM dexamethasone, 10 ng/ml I GI-, 24 mil/ml insulin and 100 ng/ml cholera toxin In an effort to promote the death of more differentiated cells 1 NI -α was added to cultures to stimulate nitric oxide induced apoptosis and hydro colloid dextran was added to cultures to disrupt cell adhesions Finally calcium levels were limited to 0 001 0 9 πiM I inalK calcium levels were limited to 0 001-0 9 mM

|00204 J Medium was changed ever) other day On

10 cultures were continued in their medium without the cholera toxin and with the addition ol one ot the following peptides 111P2 I HP I HPl and INGΛP in concentrations of 1 0 μM and 1 3 μM into 2 ml cultures lor final concentrations of 500 iiM. 167 nM and 50 nM. Medium was changed every other day. Collagen-embedded cultures were haπ csted b> incubating w ith 0.25 g'l. collagenase XI (Sigma) for 30 minutes at 37 ⁰C.

|00205] After culture, the human pancreatic islet and progenitor fractions were then treated in a blinded study with either HIP2, HIP. I HPl. the hamster INOAP sequence as a positive control (SIiQ ID NO:1 ) or isotonic saline only (s/0). Duplicate cultures were treated on Day 10 and Day 12 and then lysed for detection of insulin content on day 14. During 10 day culture, the insulin production decreases to negligible amounts and, after treatment with peptides, insulin is produced again.

[00206| Insulin levels were detected by Radioimmunoassay (RIA) from cultures treated with saline only (s/0), UIP2, HIPl , HIP and hamster INGAP. The results are shown in Figure 3. Results here indicate that the human cell cultures (here mixed with islets and ductal tissue cultures as prepared as in Kxample 1 ) are somewhat more stimulated by the human homologs than the native hamster sequence. Specifically, the results indicate that HIP2 demonstrates an increased bioactivity ( 1.50-3.33 fold increase) over the other agents used in this experiment.

EXAMPLK 3

|00207) I Iuman pancreatic tissue was treated as described in Kxample 1 and

Insulin production was measured by HLISA assay. Figures 5 and 6 show the results of this experiment to show a dose response and to again compare the effect of ( IIP2 on the two different fractions of tissue as compared to the hamster INGAP sequence and a scrambled negative peptide sequence. Lane 1 shows results from I lud 270 cells human ductal cells isolated as described in Kxample 1. treated with I 1IP2. Lane 2 shows cells treated with a peptide with the INGΛP sequence (Sl Q ID NO 1 ) I ane ϊ shows tells treated with a scrambled peptide (Sf Q ID NO 5) I he results in 1 igurc 5 were generated using 5 μg ol each peptide while the results in 1 igure 6 were acquired using 0 002 μg ol each peptide 1 igure 8Λ shows results tor cells cultured from the islet traction and treated with 1 μM and 1 inM ot each peptide, while 1 igurc 8B shows results tor cells cultured trom the ductal fraction and treated with 3 μM and I mM of each peptide

[00208] In each ot the examples 1I1P2 induced insulin production more effectively than INGΛP or scrambled peptide and the higher concentration of HIP2 produced a more profound ef fect in ductal cultures in which progenitor cells are more concentrated In the islet cultures, the limiting factor for the degree to which the cultures are able to produce more insulin is not the concentration of the peptide, but the number ot progenitor cells per culture

EXAMPLE 4

(00209) A human ductal tissue fraction was isolated and cultured as described in

I xample 1 After 10 days of culture, cells were treated with HIP2 for four days and observed using inverted microscopy I igure 4Λ 4B and 4C shows cultures treated with I1IP2 and 4D shows the negative control ductal tissue treated with no peptide I igure 7Λ shows human pancreatic progenitor tissue cultures at day 12 (day 2 ol treatment with IIIP2) Islets have lormed what has previously been described as ductal epithelial cvsts and are starting to bud at one end where a progenitor cell resides 1 igure 8B shows human pancreatic progenitor tissue cultures at day 18 (da\ 6 of treatment w ith 1 IIP2) In this panel the darkening ol the budding portion ol the ductal epithelial c\ st indicates the

differentiation of cells consistent with previously shown changes thai occur with hamster INGΛP treatment in vitro.

EXAMPLE 5

[0021U] The non-obese diabetic (NOD) mouse strain has long been studied as an excellent model of type 1 diabetes because it spontaneously develops a disease that is very similar to the human condition. Diabetes in NOD mice is mediated by inflammatory autoreactive T cells that recognize pancreatic islet antigens and escape central and peripheral tolerance. In a parental colony of NOD mice, incidence of diabetes in female NOD mice is typically 75-90% by 30 weeks but may exceed 90% in some cohorts. In this example, 50 female NOD mice, ranging from 13-14 weeks old at the outset of the study, are used to test the effectiveness of HIP2 as compared to other test agents. Due to the unexpected variability of the degree of onset of disease, the number used in this example (50 female mice) is considered to be the minimum needed to result in a sufficient number of mice that will onset with disease within the timeframe of the study.

[00211 ] Animals were group housed in compliance with the National Research Council "Guide for the Care and Use of Laboratory Animals". The animals received 12 hours light/12 hours dark, except when room lights were turned on during the dark cycle to accommodate study procedures. Room temperature was maintained between 18 to 26⁰C at all times. Relative humidity was maintained between 30-70% at all times. All animals were given access to 1 larlan Teklad Rodent Diet or equivalent and tap water via water bottle. Animals were allowed to acclimate to their new environment for a minimum of 7 da\ s prior Io first dosing. [00212| Procedure. Mice were randomly divided and assigned to stud) groups. Hach stud) group was assigned ireatment with one of 5 test agents (see Treatment section below). Prior to being treated, each mouse was tested tor diabetes via monitoring their blood glucose levels. After confirmation that the mice were diabetic (blood glucose levels greater than 16.7 mmol/L (300mg/dl.) for two consecutive days), the mice were dosed.

|00213] Treatment. Kach study group received doses of one of the following test agents: I1IP2, HIPl, HIP, Hamster 1NGΛP (SHQ ID NO: 1 ) or isotonic saline "Vehicle^" (0.9% NaCl) (negative control). Mice were given doses of their respective agent interperitoneally twice daily (am and pm, +/- 2 hours for each adminstration) at 250 ug/dose, in 100 ul dose volume, for 28 consecutive days. Test agents were prepared once daily by dissolving the appropriate amount of test agent in isotonic saline to result in a 2.5 mg/ml solution. Concurrent to the treatment of the test agent, the mice were treated with an immunosuppressant (anti-CD3 antibody) at 5 ug/day intravenously for 5 consecutive days. Blood glucose levels were tested every 3 days, at the same time each day. using a glucometer. If the blood glucose level in any animal became too high, the animal was treated with insulin. At the end of the treatment, all surviving animals were euthanized by CO2 asphyxiation following terminal blood collection. Blood samples were collected under CO2 cardiocentesis. The plasma was separated and then frozen at -8O⁰C. l-^'or histological analysis, the pancreas from each mouse was removed and snap-fro/en.

|00214] Study Assessment. Blood glucose levels are measured every 3 days and tracked to evaluate differences in glucose

a. Results: NOl) mice taking Hl 1*2 display a pronounced reduction in blood glucose levels and decreased showing of insulinitis in their pancreases as compared to the MOD mice taking the other test agents. EXAMPLE 6

[00215] 1 he strepto/oc in-induced diabetic mouse is a com cnicnt animal model of t\pe 1 diabetes because researchers are able to chemieall) induce diabetes under controlled conditions rather than waiting tor mice to become diabetic naturally or b> forcing diabetes through prescribed obesity. Based on experience with the strepto/ocin model in the rat. a high degree of variability was anticipated for the induction of the disease in mice and therefore 60 mice were induced with streptozocin. Mice were injected intraperitoneally with streptozocin at 40 mg/kg in citrate buffer, pH4.5, on 5 consecutive days in an attempt to render them diabetic. Λ mouse was considered diabetic once its blood glucose level was greater than 16.7 mmol/L (300 mg/dl.) for at least 1 week. If the blood glucose level of any animal became too high (>400 mg/dL) the animal was treated with an effective amount of insulin. In this example, 60 male mice, ranging from 6-8 weeks old at the outset of the study, were treated with streptozocin as described above. Those mice that were declared diabetic were then used to test the effectiveness of I IIP2 as compared to other test agents.

[00216| Animals were group housed in compliance with the National Research Council "Guide for the Care and Use of Laboratory Animals". The animals received 12 hours light/12 hours dark, except when room lights were turned on during the dark cycle to accommodate study procedures. Room temperature was maintained between 18 to 26⁰C at all times. Relative humidity was maintained between 30-70% at all times. All animals were

access to Ilarlan Teklad Rodent Diet or equivalent and lap water via water bottle Animals were allowed to acclimate to their new environment for a minimum of 7 days prior to first dosing.

|00217] Procedure. Mice were randomly dh ided and assigned to stud> groups, l.ach study group was assigned treatment with one of 5 test agents (see Frealmunl section below). Prior to being treated, each mouse was tested for diabetes via monitoring their blood glucose levels. After confirmation that the mice were diabetic (blood glucose levels greater than 16.7 mmol/L (300mg/dL) for at least 1 week) the mice were dosed with their respecth e test agent.

(00218J Treatment. I:ach study group received doses of one of the following test agents: I 1IP2, IHPl. HIP, Hamster INGΛP (SFQ ID NO:1 ) or isotonic saline "Vehicle" (0.9% NaCl) (negative control). Mice were given doses of their respective agent interperitoneally twice daily (am and pm, +/- 2 hours for each adminstration) at 250 ug/dose, in 100 ul dose volume, for 28 consecutive days. Test agents were prepared once daily by dissolving the appropriate amount of test agent in isotonic saline to result in a 2.5 mg/ml solution. Blood glucose levels were tested every 3 days, at the same time each day, using a glucometer. If the blood glucose level in any animal became too high, the animal was treated with insulin. At the end of the treatment, all surviving animals were euthanized by CO2 asphyxiation following terminal blood collection. Blood samples were collected under CO2 cardiocentesis. The plasma was separated and then frozen at -8O⁰C. For histological analysis, the pancreas from each mouse was removed and snap-frozen.

|00219] Study Assessment. Blood glucose levels are measured every 3 days and tracked to evaluate differences in glucose levels. a. Results: Strcptozocin-induced diabetic mice taking IIIP2 display a pronounced reduction in blood glucose levels and decreased showing of insulinitis in the pancreas as compared to the streptoΛOciπ-induced diabetic mice taking the other test agents.

|00220] Although the present invention has been described in detail with reference to specific embodiments, those of skill in the art will recognize that modifications and improvements are within the scope and spirit of the invention, as set forth in the claims which iυllow Λll publications and patent documents (patents published patent applications and unpublished patent applications) cited herein are incorporated herein b\ reference as il each such publication or document was specificalh and indi\ lduall) indicated to be incorporated herein bv relerence C nation ot publications and patent documents is not intended as an admission that any such document is pertinent prior art nor does it constitute any admission as to the contents or date ot the same I he invention ha\ ing now been described by way of written description and example, those of skill in the art will recogni/e that the invention can be practiced in a vaπety ot embodiments and that the foregoing description and examples are tor purposes ot illustration and not limitation ot the following claims

[00221| Significant reductions in both the insulin requirements and the rate ot decrease in insulin requirements were seen among all HlP2-treated mice The HIP2 treated mice were completely insulin-free by day 21 There was a significant reduction in mean insulin requirements among HIP-treated mice compared to placebo Overall insulin requirements were 32% lower in the IllP-trealed group and 14% lower in the INOΛP treated groups compared to controls

[00222] 1 here was also a significant reduction in M1P2 -treated mice in glucose levels from baseline compared with control I here was a 9 5% reduction in mean glucose in the placebo group a mean reduction in glucose in the I IIP treatment groups ot 26 9% and hamster-derived INGΛP demonstrated a 20% reduction in glucose

KXAMPLE 7

[00223] Quantitative immunohistochemistrv image anaUsis of the pancreata ot the placebo controlled and I IIP treated mice showed total islet mass ol the I IIP treated group was 153% greater (p- 0.05) than total islet mass of the placebo group. The islet mass of the placebo group was 854,362 μm(2) compared with 2.161.782 μm(2) in HIP-treated mice. While the islet sizes between the two groups were not statistically different, the number of islets counted in the pancrcata of the IiIP treatment groups was 62% greater than the placebo group (p- 0.022) with 280 islets counted in the placebo group compared to 454 in the HIP-treated group.

[00224] This data supports the hypothesis that production is not resulting from increased stimulation of insulin production or beta cell proliferation, but a function of increased islet number supporting the proposed mechanism that HIP stimulates new islet formation.

(00225) Figure 11 demonstrates a representative sample of the 900 images taken of the immunofluorescent staining for insulin from the histological evaluation of control versus HIP-treated mice pancreata. Islet size was not different between HlP treated and control group, whereas, islet number and mass was increased.

Claims

C1.Λ1MS What is claimed is:

1. Λ method of stimulating islet cell neogenesis in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of 111P2.

2. The method of claim 1. wherein the total islet number is increased by at least 50%.

3. The method of claim I. further comprising administering a therapeutically effective amount of an agent selected from the group consisting of: an immune therapy agent and an additional agent that stimulates islet cell regeneration.

4. The method of claim 3. wherein said immune therapy agent is selected from the group consisting of: anti-CD3 antibodies, sirolimus. tacrolimus, a heat-shock protein 60, an anti-glutamic acid decarboxylase 65 vaccine, mycophenolate mofetil alone or in combination with daclizumab, an anti-CD20 agent, riluximab. campath- I H. lysofylline, vitamin D. IBC-VSO vaccine, interferon-alpha, and a vaccine using CD4⁴CD25^ antigen-specific regulatory' T cells.

5. The method of claim 3, wherein said additional agent that stimulates islet cell regeneration is selected from the group consisting of: a UIP or a I IlP-related peptide, amylin, pramlintide, insulin, exendin-4, GIP, Gl. P-I . GLP-I receptor agonists. GLP- I analogs, hamster INGAP peptide and related peptides, liraglutide. and a dipeptidyl peptidase inhibitor which blocks the degradation of GLP- I .

6. The method of claim 3. wherein the total islet number is increased by at least 50%.

7. A method of treating a disease or eondition associated with impaired pancreatic function in a subject in need thereof, comprising administering to said subject a therapeutically elϊecli\e amount of 11IP2.

8. The method of claim 7 wherein said disease or condition associated with impaired pancreatic function is selected from the group consisting of: type 1 diabetes, new onset type 1 diabetes, type 2 diabetes. Latent Autoimmune Diabetes in Adults, prediabetes, impaired fasting glucose, fasting hyperinsulinemia. impaired glucose tolerance, insulin resistant syndrome, insulin deficiency, metabolic syndrome, obesity, anorexia, bulimia, neuropathic pain, pancreatitis, pancreatic cancer, hyperlipidemia. hypertriglyceridemia, eating disorders, anovulatory cycles, lack of or diminished insulin production resulting in aberrant glucose metabolism, and polycystic ovarian syndrome.

9. The method of claim 8, wherein said disease or condition associated with impaired pancreatic function is selected from the group consisting of: type 1 diabetes, type 2 diabetes. Latent Autoimmune Diabetes in Adults, pre-diabetes, and metabolic syndrome.

10. The method of claim 7. further comprising administering a therapeutically effective amount of an agent selected from the group consisting of: an immune therapy agent and an additional agent that stimulates islet cell regeneration.

1 1. The method of claim 10. wherein said immune therapy is selected from the group consisting of: anti-CD3 antibodies, sirolimus, tacrolimus, a heat-shock protein 60, an anti-glutamic acid decarboxylase 65 vaccine, mycophenolate mofetil alone or in combination with daclizumab. an unti-CD20 agent, rituximab. campath-111. lysofylline. vitamin D. IBC-VSO vaccine, interferon-alpha. and a vaccine using CD^Q^S¹ antigen- specific regulatory T cells.

12. The method of claim 10. wherein said additional agent that stimulates islet cell regeneration is selected from the group consisting of: a I UP or a I H P-related peptide. amylin. pramlinlide. insulin, cxendin-4. GlP. GLP- I . GI. P-I receptor agonists. (JI. P- I analogs, hamster INGΛP peptide and related peptides, liraglutide. and a dipcptidyl peptidase inhibitor which blocks the degradation of GLP- 1.

13. The method of claim 10. wherein said disease or condition associated with impaired pancreatic function is selected from the group consisting of: type 1 diabetes, new onset type 1 diabetes, type 2 diabetes. Latent Autoimmune Diabetes in Adults, prediabetes, impaired fasting glucose, fasting hypcrinsulinemia, impaired glucose tolerance, insulin resistant syndrome, insulin deficiency, metabolic syndrome, obesity, anorexia, bulimia, neuropathic pain, pancreatitis, pancreatic cancer, hypcrlipidemia, hypertriglyceridemia, eating disorders, anovulatory cycles, lack of or diminished insulin production resulting in aberrant glucose metabolism, and polycystic ovarian syndrome.

14. The method of claim 13, wherein said disease or condition associated with impaired pancreatic function is selected from: type 1 diabetes, type 2 diabetes, Latent Autoimmune Diabetes in Adults, pre-diabetes, and metabolic syndrome.

15. A method of reducing insulin requirements in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of I 11P2.

16. The method of claim 15, further comprising administering a therapeutically effective amount of an agent selected from the group consisting of: an immune therapy agent and an additional agent that stimulates islet cell regeneration.

17. The method of claim 16. wherein said immune therapy is selected from the group consisting of: anli-CD3 antibodies, sirolimus. tacrolimus, a heat-shock protein 60. an anti-glutamic acid decarboxylase 65 vaccine, mycophenolatc mofctil alone or in combination with dacli/.umab. an anti-CD2U agent, rituximab. campalh- 111. lysofylline. vitamin D. IBC-VSO vaccine, interferon-alpha. and a vaccine using CD4 CD25' antigen- specific regulator) T cells.

18. The method of claim 16. wherein said additional agent that stimulates islet cell regeneration is selected from: a I IIP or a 11 IP-related peptide. am\ lin. pramlintide. insulin, exendin-4. GIP. GI. P-I . GLP-I receptor agonists. GI. P-I analogs, hamster INGΛP peptide and related peptides, liraglutide. and a dipeptid) 1 peptidase inhibitor which blocks the degradation of Gl. P-I .

19. Λ method of treating a disease or condition associated with impaired pancreatic function in a subject in need thereof, comprising the steps of: intensifying glycemic control: administering oral cholecalciferol; administering one or more immune therapies; administering a therapeutically effective amount of I IIP2 in combination with insulin, wherein the insulin administered is decreased over time; and repeating said administering of said one or more immune therapies every 3 months to every 24 months.

20. A method of stimulating islet cell differentiation from progenitor cells into islet structures in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of I HP2.

21. Λ kit for treating a disease or condition associated with impaired pancreatic function in a subject in need thereof, comprising: a therapeutically effective amount of I IIP2 in a first dosage unit; optionally a therapeutically effective amount of one or more immune therapies: optionally a therapeutically effective amount of one or more additional agents that stimulate islet cell regeneration in a third dosage unit; and instructions for use.

22. Λ pharmaceutical composition lor stimulating islet cell neogenesis comprising a therapeutically effective amount of I Il P2.