WO2009108777A2

WO2009108777A2 - Use of nitrite to enhance viability, recovery, and function of transplant specimens

Info

Publication number: WO2009108777A2
Application number: PCT/US2009/035280
Authority: WO
Inventors: Rakesh P. Patel; Juan L. Contreras; Devin Eckhoff; John D. Lang; Anupam Agarwal
Original assignee: The Uab Research Foundation
Priority date: 2008-02-28
Filing date: 2009-02-26
Publication date: 2009-09-03
Also published as: WO2009108777A3

Abstract

Provided herein are methods of preparing a transplant specimen and methods for improving the viability of a cellular transplant specimen in culture. Methods for improving the viability of an organ or tissue for transplantation are also provided. Also provided herein are methods for enhancing the survival of pancreatic islet cells for transplantation and methods of treating diabetes in a subject by transplanting to the subject an islet cell population made by the provided methods.

Description

Use of Nitrite to Enhance Viability., Recovery, and Function of Transplant Specimens

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No. 61/032,178, filed February 28, 2008, which is incorporated by reference herein in its entirety.

BACKGROUND

Problems in donor organ transplantation include the detrimental effects of brain-death on cells, tissues and organs in cadaveric or brain-dead organ donors, which negatively affects the outcome of organ and cell transplantation. Decreased recovery, viability and function of cells, tissues and organs from brain-dead donors is a result of multiple interrelated events, such as massive release of pro-inflammatory cytokines (e.g., TNF-α, IL- lβ, and IFN-γ), hemodynamic instability, endocrine abnormalities, hypothermia, coagulopathy, pulmonary dysfunction, and electrolyte imbalances.

Problems in donor organ transplantation also include the storage and preservation of organs between the time of harvest from a donor and the time of transplantation into a recipient. The amount of time that can lapse between the two events is quite limited because the cells and tissues of the donor organ deteriorate over time, even if they are stored at refrigerated temperatures. Once harvested, cells and tissues are deprived of the oxygen that is required to maintain internal metabolism and cell volume integrity. To counteract the ill effects of low oxygen, standard techniques for organ preservation involve the exposure of a harvested organ to preservation solutions at cold temperatures not below 0⁰C. Although colder temperatures are a solution to oxygen deprivation in donor organ tissue, they present their own problems.

Cold or hypothermic conditions may lead to swelling, cell death and cellular damage including a reduced ability to generate energy and to maintain cell volume integrity. Several preservation solutions aimed at minimizing tissue damage in organ transplants during hypothermal storage have been developed. While such solutions have been effective in extending the cold preservation time of organs intended for transplantation, tissue injury during cold storage still occurs. Attorney Docket No. 20674-081 WOl

SUMMARY OF THE DISCLOSURE

Provided herein are methods of preparing a transplant specimen and methods for improving the viability of a cellular transplant specimen in culture. For example, provided herein is a method comprising administering nitrite to a transplant donor and isolating the transplant specimen from the donor. Methods for improving the viability of an organ or tissue for transplantation are also provided, which include contacting the organ or tissue with nitrite.

Also provided herein are methods for enhancing the survival of pancreatic islet cells for transplantation, for example, by administering nitrite to the cells and methods of treating diabetes in a subject by transplanting to the subject an islet cell population made by the provided methods.

The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims. DESCRIPTION OF DRAWINGS

Figure 1 is a graph showing nitrite levels in male Lewis rats after brain death or sham (control) surgery for two (2) hours.

Figure 2 is a graph showing nitrite improves islet recovery (IEQ) in brain dead rats. Adult male Lewis rats underwent brain death for two (2) hours. Ten (10) minutes after induction of brain death, the rats received a continuous intravenous (FV) injection of PBS (BD) or NO₂ ^' (BD w/ NO₂ ^") at a dose of 0.05 mg/kg/hr for two (2) hours.

Figure 3 is a graph showing nitrite protects the kidney during brain death. Adult male Lewis rats were subjected to brain death (BD) for two (2) hours or subjected to brain death for 2 hours together with receiving nitrite (0.05 mg/kg/hour for 2 hours; BD + Nitrite). Kidneys from these animals were then transplanted into syngeneic rats. Creatinine was measured as a function of time post transplantation in transplant recipients to assess kidney function.

DETAILED DESCRIPTION Provided herein are in vivo and in vitro methods of enhancing the viability, recovery and function of transplant specimens, such as, for example, cells, tissues and organs, for transplantation. Thus, provided herein are in vitro methods for improving Attorney Docket No. 20674-081WO1

the viability of a transplant specimen (organ, tissue or cell). Transplant organs include, by way of example, heart, kidney, liver, lung, small bowel, spleen and pancreas. Tissues for transplantation include, by way of example, bone tissue, tendons, cornea, skin, blood vessels and heart valves. The transplant specimen is optionally a cellular population. The cellular population optionally comprises pancreatic islet cells. The cellular population optionally comprises bone marrow cells, heart cells, lung cells, corneal cells, kidney cells, skin cells, liver cells, and spleen cells.

Recently strides have been made in human islet transplantation for the treatment of diabetes. The lack of suitable donor pancreata is a major obstacle in the widespread use of islet cell transplants. Furthermore, this strategy faces limitations due to the large number of transplant islet cells required to achieve long-term insulin independence. Often, two and usually more donor organs are needed to accumulate enough islet cells for a single complete transplant. The islet cells are often severely compromised during storage or transport causing apoptosis of the cells. Provided herein are methods of enhancing the viability, recovery and function of, for example, islet cells for transplantation.

Specifically, provided herein are in vitro methods. Such methods are useful, for example, to enhance the number and health of pancreatic islet cells in a culture, including for example, in a culture of cells to be transplanted into a transplant recipient. Pancreatic islet cells are isolated from a pancreas but could also include pancreatic islet cells from a cell line or from stem cells expanded and/or differentiated in culture. Furthermore, pancreatic islet cells also include cells genetically modified to express insulin or to over express insulin. Thus, provided herein is a method for enhancing the survival of pancreatic islet cells for transplantation comprising administering to the pancreatic islet cells nitrite under conditions that promote the survival of the pancreatic islet cells. In one aspect, the pancreatic islet cells are contacted with nitrite during isolation of the pancreatic islet cells from a transplant donor. Methods of differentiating stem cells into pancreatic islet cells are known, and are described in, for example, Kahan et al., Diabetes 52:2016-2024 (2003), which is incorporated by reference herein in its entirety. See also U.S. Patent Publication No. Attorney Docket No. 20674-081WO1

2005/0260749; and Jiang et al., Cell Research 18:s28 (2008), which are incorporated by reference herein in their entireties. Pancreatic islet cells can be detected, for example, by confirming expression of pancreatic duodenal homeobox 1 (PDXl), and expression of early marker proteins such as peptide YY (YY), a pancreatic polypeptide (PP)-related neuropeptide, and islet amyloid polypeptide (IAPP).

The pancreatic islet cells are derived from any number of sources. The cells are derived, for example, from living donors or cadaveric (e.g., brain-dead) donors. The donor is the same species as the recipient (e.g., allogeneic) or a different species than the recipient (e.g., xenogeneic). In some cases the donor and the recipient are the same individual (e.g., autologous). Islet cells optionally are surgically removed from the pancreas of the transplant donor. The cells, however, are optionally derived from cells lines or genetically modified cells or tissues.

Provided are also in vivo methods. Specifically, methods of preparing a transplant specimen containing the steps of administering nitrite to a transplant donor and isolating the transplant specimen from the donor are provided. As used herein, a transplant donor includes a living donor and a cadaveric donor. As used herein, a cadaveric or brain-dead donor is a deceased or brain-dead subject whose organs, tissues or cells are available for harvest and transplantation into another subject. The nitrite is administered to the transplant donor from about one to forty-eight hours prior to isolating the transplant specimen (organ, tissue or cellular population including, e.g., pancreatic islet cells) from the donor. The nitrite is optionally administered for at least two hours prior to isolating the transplant specimen from the donor. The method optionally further comprises the step of contacting the isolated transplant specimen with nitrite under conditions that promote viability of the transplant specimen. Thus, the provided method includes enhancing the survival or viability of an organ, tissue or cellular population.

In one aspect, provided are methods for enhancing the survival of pancreatic islet cells for transplantation wherein the pancreatic islet cells are located in a transplant donor or a transplant recipient are also provided. The method includes the step of administering nitrite to the transplant donor or transplant recipient in an amount effective for enhancing the survival of the pancreatic islet cells. Attorney Docket No. 20674-081 WOl

Due to biochemical stresses inherently associated with biologic preservation of transplant specimens, such as, cells, tissues and organs in liquid, a significant level of cellular death is initiated during and following the preservation period. Consequently, failure of the transplant specimen ensues due to this preservation- initiated cell death. Thus, provided are methods of improving the viability of an organ or tissue for transplantation comprising contacting the organ or tissue with nitrite under conditions that promote viability of the organ or tissue. The organ or tissue is contacted with nitrite in vitro or in vivo. Thus, the organ or tissue is located in a transplant donor or a transplant recipient. Optionally, the organ or tissue is in culture. The organ or tissue is optionally contacted with nitrite while being stored under hypothermic, pulsatile hypothermic or normothermic conditions prior to transplantation of the organ or tissue. Normothermic, hypothermic and cryopreservative maintenance and storage of cells, tissues and organs is traditionally achieved through suspension in a liquid preservation medium. These media include, but are not limited to, simple saline solution, cell culture media, and preservation solutions including University of Wisconsin (UW) solutions such as, VIASPAN®, (DeraMed Inc., Brooks, KY); HTK solution (CUSTODIOL®, Odyssey Pharmaceuticals, Inc., East Hanover, NJ); Eurocollins (EC) solutions; and HYPOTHERMOSOL® (Bio Life Solutions, Inc., Ewing, NJ.). Organs suitable for preservation treatment include, but are not limited to, heart, kidney, liver, lung, small bowel, spleen and pancreas. Tissues suitable for preservation treatment include, but are not limited to, bone tissue, tendons, cornea, skin, blood vessels and heart valves. Methods of treating diabetes (type 1 or type 2) are also provided herein. The method contains the steps of preparing a pancreatic islet cell population for transplantation according to the methods provided herein and transplanting the islet cell population to the subject to be treated. The method optionally comprises administering nitrite to the subject to be treated. The method optionally is combined with administration of insulin and/or with administration of sulfonylureas, meglitinides, biguanides (e.g., metformin), thiazolidinediones, DPP -4 inhibitors, and/or α-glucosidase inhibitors. The compositions herein are administered in a number of ways. Transplant compositions are frequently administered intrahepatically, for example. In some cases, several transplants are necessary. One of Attorney Docket No. 20674-08 IWOl

skill in the art, however, readily determines the concentration of cells to include in the transplant composition and recognizes the need for a second or subsequent transplant based on such clinical signs as hyperglycemia and the like.

As used herein, subject includes, but is not limited to, a vertebrate, and more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent). The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. The term subject includes human and veterinary subjects. As used herein the terms treatment, treat or treating refers to a method of reducing the effects of a disease or condition or at least one symptom of the disease or condition. Thus, in the disclosed method, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% reduction in the severity of an established disease or condition or symptom of the disease or condition. For example, the method for treating diabetes is considered to be a treatment if there is a 10% reduction in one or more symptoms or clinical signs of the disease in a subject as compared to control.

Thus the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% or any percent reduction in between 10 and 100 as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition or symptoms of the disease or condition. In the methods provided herein, the nitrite in culture or administered to a transplant donor or transplant recipient is optionally a pharmaceutically acceptable salt of nitrite. Nitrites are often highly soluble, and can be oxidized to form nitrates or reduced to form nitric oxide or ammonia. Nitrite may form salts with alkali metals, such as sodium (NaNO₂, also known as nitrous acid sodium salt), potassium and lithium, with alkali earth metals, such as calcium, magnesium and barium, with organic bases, such as amine bases, for example, dicyclohexylamine, pyridine, arginine, lysine and the like. Other nitrite salts may be formed from a variety of organic and inorganic bases. Many nitrite salts are commercially available, and/or readily produced using conventional techniques. Pharmaceutically acceptable salts of nitrite include, but are not limited to, sodium nitrite, potassium nitrite and arginine nitrite. When administered to a transplant donor or recipient, the nitrite is optionally administered as saline buffered sodium nitrite. Attorney Docket No. 20674-081WO1

Nitrites, including their salts, are administered to a subject in accordance to methods provided herein. Nitrite is optionally administered as a pharmaceutically acceptable fluid composition. The nitrite is usually dissolved or suspended in a physiologically acceptable carrier, and the composition optionally comprises minor amounts of one or more non-toxic auxiliary substances, such as emulsifying agents, preservatives, pH buffering agents and the like. For suitable formulations and various routes of administration of therapeutic compounds, see, e.g., Remington: The Science and Practice of Pharmacy (21st ed.) eds. A.R. Gennaro et al., University of the Sciences in Philadelphia 2005. By way of example, delivery of nitric oxide is described in U.S. Patent No. 5,648,101. Nitrite is administered topically, parenterally, peritoneally, intraperitoneally, intravenously, subcutaneously, by inhalation, intramuscularly, parenterally, or orally. The nitrite is optionally administered by continuous intravenous injection. Sustained release or slow release systems such that a constant dosage is maintained are useful in the methods taught herein. Inhaled or aerosolized nitrite is also useful in the provided methods. For example, use of inhaled

NO and nitric oxide-releasing compounds inhaled as solids or liquids in an aerosol to treat pulmonary vasoconstriction and asthma is described in U.S. Pat. No. 5,823,180. Nitrite is optionally administered by nebulization, and appears to exhibit a wide therapeutic-to-safety margin, with limited systemic hemodynamic changes and methemoglobin production.

Administration of the nitrites in accordance with the present disclosure is in a single dose, in multiple doses, and/or in a continuous or intermittent manner. The administration of the nitrites is continuous over a preselected period of time or in a series of spaced doses. Effective concentrations of nitrite for use in vitro in the provided methods is determined by those of skill in the art. Typically, about 0.1 μM to about 20 μM of nitrite is used in culture. Optionally, 10 μM of nitrite is used in culture.

Effective dosages and schedules for administering nitrite in vivo are determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect. The dosage should not be so large as to cause adverse side effects in a transplant recipient, such as unwanted cross-reactions, anaphylactic Attorney Docket No. 20674-08 IWOl

reactions, and the like. Generally, the dosage varies with the age, condition, sex and extent of the disease in the transplant recipient, route of administration, or whether other drugs are included in the regimen, and are determined by one of skill in the art. The dosage is adjusted by the individual physician in the event of any contraindications. A typical dosage of nitrite administered to transplant recipients is from about 0.01 to about 0.1 mg/kg/hr of nitrite. Optionally, 0.05 mg/kg/hr of nitrite is administered to the transplant recipient. Living donors would be treated with similar conditions and doses, whereas brain-dead donors could receive higher levels, so long as such levels are non-toxic to the organ, tissue, or cells to be transplanted. As described herein, cells, tissues or organs contacted with or administered nitrite have enhanced or improved viability or survival as compared to cells, tissue or organs that have not been contacted with or administered nitrite. As used herein, enhancing or improving the viability or survival of a cell, tissue or organ refers to a change as compared to a control. For example, improving the viability of an organ refers to an increase in organ viability compared to a control. Enhancing the survival of a cell refers to an increase in cell survival time or an increase in the percentage of surviving cells as compared to an untreated control. Increased, improved or enhanced includes a statistically significant increase over control. A control is defined as the standard by which a change is measured. For example, a control is not subjected to the experiment, but is instead subjected to a defined set of parameters, or a control is based on pre- or post-treatment parameters.

Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, and the like of these materials are disclosed that, while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular modification of an assay step or method is disclosed and discussed and a number of modifications that can be made to the assay step or method are discussed, each and every combination and permutation of the assay step and method are specifically contemplated unless specifically indicated to the contrary. Attorney Docket No. 20674-081 WOl

Likewise, any subset or combination of these subsets is also specifically contemplated and disclosed.

A number of aspects have been described. Nevertheless, it will be understood that various modifications may be made. Furthermore, when one characteristic or step is described it can be combined with any other characteristic or step herein even if the combination is not explicitly stated. Accordingly, other aspects are within the scope of the claims.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure.

Examples Example 1. Nitrite increases pancreatic islet cell recovery and function. To determine if nitrite levels increase after brain death, male Lewis rats underwent brain death or sham (control) surgery for 2 hours followed by measurement of nitrite levels. Figure 1 shows that, in a rat model of brain death, nitrite levels in the pancreas decreased. Figure 2 shows that infusion of nitrite 5 minutes post induction of brain death significantly improved islet recovery and function after 2 hours. Adult male Lewis rats underwent brain death for 2 hours. Ten minutes after induction of brain death, the rats received a continuous intravenous injection of PBS (BD) or NO₂ ^" (BD w/ NO₂ ^") at a dose of 0.05 mg/kg/hr for 2 hours. Islet recovery in animals receiving NO₂ ^" was significantly enhanced (3 -fold) compared to BD alone. Example 2. Organ Preservation Therapy

Following standard techniques for organ procurement, individual organs (lungs, heart, liver, pancreas, kidneys, small bowel, spleen, and blood vessels) are placed in University of Wisconsin Solution (UWS) (VIASPAN®, DeraMed Inc., Brooks, KY) supplemented with sodium nitrite , at 4°C. For example, organs from rats are placed in separate sterile, conical 50 mL tubes, each containing 20 mL of supplemented UWS. Organs are preserved for 6 hours followed by transplantation (lungs, heart, kidneys, liver, spleen, small bowel and blood vessels) or islet isolation Attorney Docket No. 20674-081WO1

(pancreas). Alternative preservation solutions include HTK solution (CUSTODIOL®, Odyssey Pharmaceuticals, Inc., East Hanover, NJ) supplemented with similar concentrations of sodium nitrite at 4°C.

As shown in Figure 3, nitrite administration during brain death (BD) protects the kidney resulting in improved function post kidney transplantation. Adult male

Lewis Rats were subjected to brain death for two (2) hours or subjected to brain death for 2 hours together with receiving nitrite (0.05mg/Kg/hour) for 2 hours (BD + Nitrite). Kidneys from these animals were then transplanted into syngeneic male Lewis rats. Creatinine was measured as a function of time post transplantation in transplant recipients to assess kidney function. The data presented in Figure 3 shows that, in the BD group (i.e., no nitrite treatment), creatinine levels are high at day 1, increasing slightly at day 2 and then decreasing thereafter. These data are consistent with an initial decreased renal function (due to injury caused from brain death and transplantation) with recovery of function with time post transplantation. However, renal function was significantly improved, as indicated by lower creatinine, in transplanted kidneys isolated from brain dead rats that received nitrite therapy. These data show that nitrite administration during brain death preserves organ function post transplantation.

Example 3. Pancreas Processing for Islet Isolation A 24-guage angiocath is placed on the main bile duct and the pancreas. The pancreas is then injected with 5 mL of cold Ml 99 medium containing 1.5 mg/ml rodent Liberase® (Roche Diagnostics Inc., Indianapolis, IN) supplemented with sodium nitrite.

Example 4. Islet Cell Culture and Isolation Islets are cultured in RPMI 1640 medium (11 mmol/1 glucose), supplemented with 10% FCS (Invitrogen, Carlsbad, CA), 100 units/ml penicillin, and 0.1 mg/ml streptomycin (Invitrogen, Carlsbad, CA), 25 mmol/1 HEPES (Invitrogen, Carlsbad, CA) and sodium nitrite at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Islet cells are typically cultured for 24-48 hours prior to transplantation. Example 5. Transplant Recipient Therapy

Ten minutes before organ or cell implantation, a continuous intravenous infusion of sodium nitrite (0.05 mg/kg/hr) is given to the recipient for 24-48 hours. Attorney Docket No. 20674-081WO1

Immediate post-transplant organ or cell function is assessed by standard techniques. For example, in transplant recipients receiving a pancreas or islet cells, diabetes reversal is measured. Heartbeats are measured in heart transplant recipients. Pulmonary function is tested in lung transplant recipients. Creatinme/BUN/creatinine clearance is measured in kidney transplant recipients. AST, ALD, LDH, total bilirrubin, direct and indirect bilirrubin and lactic acid is measured in liver transplant recipients.

Claims

Attorney Docket No. 20674-081 WOlWHAT IS CLAIMED IS:

1. A method of preparing a transplant specimen comprising the steps of

(a) administering nitrite to a transplant donor and

(b) isolating the transplant specimen from the donor.

2. The method of claim 1 , wherein the transplant specimen is a cellular population.

3. The method of claim 1, wherein the cellular population comprises pancreatic islet cells.

4. The method of claim 1, wherein the transplant specimen is an organ or tissue.

5. The method of claim 1, wherein the organ or tissue is selected from the group consisting of a heart, lung, kidney, cornea, skin, liver, heart valves, tendon, small bowel, spleen, bone, bone marrow, blood vessels and pancreas.

6. The method of claim 1, wherein the nitrite is administered from about one to forty-eight hours prior to isolating the transplant specimen from the donor.

7. The method of claim 1 , wherein the nitrite is administered for at least two hours prior to isolating the transplant specimen from the donor.

8. The method of claim 1, wherein about 0.01 to about 0.1 mg/kg/hr of nitrite is administered to the transplant donor.

9. The method of claim 1, wherein 0.05 mg/kg/hr of nitrite is administered to the transplant donor.

10. The method of claim 1, wherein 0.05 mg/kg/hr of nitrite is administered to the transplant donor for two hours.

11. The method of any one of claims 1-10, wherein the nitrite is a pharmaceutically acceptable salt of nitrite.

12. The method of claim 11, wherein pharmaceutically acceptable salt of nitrite comprises as the cation sodium, potassium or arginine.

13. The method of any one of claims 1-10, wherein the nitrite is administered as sodium nitrite.

14. The method of any one of claims 1-10, wherein the nitrite is administered as saline buffered sodium nitrite. Attorney Docket No. 20674-081 WOl

15. The method of any one of claims 1-10, wherein the nitrite is administered peritoneally, intravenously, subcutaneously, by inhalation, intramuscularly, parenterally, or orally.

16. The method of any one of claims 1-10, wherein the nitrite is administered intravenously.

17. The method of claim 16, wherein the nitrite is administered continuously.

18. The method of any one of claims 1-10, wherein the transplant donor is brain dead.

19. A method for improving the viability of a cellular transplant specimen comprising contacting the specimen in culture with nitrite.

20. The method of claim 19, wherein the transplant specimen is a cellular population.

21. The method of claim 19, wherein the cellular population comprises pancreatic islet cells.

22. The method of claim 19, wherein the nitrite is a salt of nitrite.

23. The method of claim 22, wherein salt of nitrite comprises as the cation sodium, potassium or arginine.

24. The method of claim 19, wherein the nitrite is administered as sodium nitrite.

25. The method of any one of claims 19-24, wherein the pancreatic islet cells are contacted with about 0.1 μM to about 20 μM of nitrite.

26. The method of any one of claims 19-24, wherein the pancreatic islet cells are contacted with 10 μM of nitrite.

27. A method for enhancing the survival of pancreatic islet cells for transplantation comprising administering to the pancreatic islet cells nitrite under conditions that promote the survival of the pancreatic islet cells.

28. The method of claim 27, wherein the nitrite is a pharmaceutically acceptable salt of nitrite.

29. The method of claim 28, wherein pharmaceutically acceptable salt of nitrite comprises as the cation sodium, potassium or arginine.

30. The method of claim 28, wherein the nitrite is administered as sodium nitrite.

31. The method of any one of claims 27-30, wherein the pancreatic islet cells are contacted with about 0.1 μM to about 20 μM of nitrite.

32. The method of any one of claims 27-30, wherein the pancreatic islet cells are contacted with 10 μM of nitrite. Attorney Docket No. 20674-081 WOl

33. The method of any one of claims 27-30, wherein the pancreatic islet cells are located in a transplant donor.

34. The method of any one of claims 27-30, wherein the pancreatic islet cells are located in a transplant recipient.

35. The method of any one of claims 27-30, wherein the pancreatic islet cells are located in culture.

36. The method of any one of claims 27-30, wherein the pancreatic islet cells are contacted with nitrite during isolation of the pancreatic islet cells from a transplant donor.

37. A method of treating diabetes in a subject comprising

(a) preparing a pancreatic islet cell population for transplantation according to the method of claim 1; and

(b) transplanting the islet cell population to the subject to be treated.

38. The method of claim 37, wherein the subject is a mammal.

39. The method of claim 37, wherein the subject is a human.

40. The method of claim 37, further comprising administering nitrite to the subject to be treated.

41. The method of claim 37, wherein the nitrite is a pharmaceutically acceptable salt of nitrite.

42. The method of claim 41, wherein pharmaceutically acceptable salt of nitrite comprises as the cation sodium, potassium or arginine.

43. The method of claim 41, wherein the nitrite is administered as sodium nitrite.

44. The method of any one of claims 37-43, wherein about 0.01 to about 0.1 mg/kg/hr of nitrite is administered to the transplant donor.

45. The method of claim 44, wherein 0.05 mg/kg/hr of nitrite is administered to the transplant donor.

46. The method of claim 1, further comprising contacting the isolated transplant specimen with nitrite under conditions that promote viability of the transplant specimen.

47. The method of claim 46, wherein the transplant specimen is a cellular population.

48. The method of claim 47, wherein the cellular population comprises pancreatic islet cells. Attorney Docket No. 20674-081WO1

49. The method of claim 46, wherein the transplant specimen is an organ or tissue.

50. The method of claim 49, wherein the organ is selected from the group consisting of the heart, kidney, liver, lung, small bowel, spleen and pancreas.

51. The method of claim 49, wherein the tissue is selected from the group consisting of bone tissue, tendons, cornea, skin, blood vessels and heart valves.

52. The method of claim 49, wherein the organ or tissue is being stored under hypothermic conditions prior to transplantation of the organ or tissue.

53. The method of claim 49, wherein the organ or tissue is being stored under pulsatile hypothermic or normo thermic conditions prior to transplant of the organ or tissue.

54. The method of any one of claims 46-53, wherein the nitrite is a pharmaceutically acceptable salt of nitrite.

55. The method of claim 54, wherein pharmaceutically acceptable salt of nitrite comprises as the cation sodium, potassium or arginine.

56. The method of any one of claims 46-53, wherein the nitrite is administered as sodium nitrite.

57. The method of any one of claims 46-53, wherein the pancreatic islet cells are contacted with about 0.1 μM to about 20 μM of nitrite.

58. The method of claim 57, wherein the pancreatic islet cells are contacted with 10 μM of nitrite.