WO2008008507A2 - Methods of treatment of diabetes - Google Patents

Abstract

The present invention relates to the treatment, of diabetes using heme oxygenase- 1 and heme degradation products.

Description

METHODS OF TREATMENT OF DIABETES

RELATED APPLICATIONS

This application claims priority to U.S. Application Serial No. 60/830486, entitled "Methods of Treatment of Diabetes", filed on July 13, 2006, the contents of which are hereby incorporated by reference.

TECHNICALFIELD

The present invention relates to the treatment of diabetes.

BACKGROUND Heme oxygenase-1 (HO-I) catalyzes the first step in the degradation of heme.

HO-I cleaves the α-meso carbon bridge of b-type heme molecules by oxidation to yield equimolar quantities of biliverdin IXa, carbon monoxide (CO), and free iron. Subsequently, biliverdin is converted to bilirubin via biliverdin reductase, and the release Of Fe²⁺ from heme induces the expression of the Fe²⁺ sequestering protein ferritin, which acts as an anti-oxidant by limiting the ability OfFe²⁺ to participate in the generation of free radicals through the Fenton reaction.

Diabetes is caused by occurrence of abnormal metabolisms of glucose, protein, and lipids due to a deficiency or insufficiency of the actions of insulin. Typical signs of diabetes include an abnormal increase in the serum glucose level over the normal range of the glucose level, and an excretion of glucose in the urine.

Several clinical subclasses are recognized, including Type 1 (insulin-dependent diabetes mellitus or IDDM), Type 2 (non-insulin-dependent diabetes mellitus), maturity- onset diabetes of the young (MODY) and gestational diabetes. They differ in etiology, pathology, genetics, age of onset, and treatment. Type 1 diabetes, the most severe form of diabetes, accounts for 5 to 10 percent of diabetes and occurs most often in children and young adults. In this form of diabetes the body does not produce any insulin. Without regular injections of insulin, the sufferer lapses into a coma and dies. Individuals suffering from Type 1 diabetes are totally insulin dependent.

Type 2 diabetes, the most prevalent type of diabetes, is usually characterized by gradual onset and occurs mainly in people over 40. Type 2 diabetes is a metabolic disorder resulting from the body's inability to make enough insulin or to properly use insulin to meet the body's needs, especially when the person is overweight. Type 2 diabetes accounts for 90 to 95 percent of diabetes. Type 2 diabetes is nearing epidemic proportions due to an increasing prevalence of obesity and sedentary lifestyles. A combination of dietary measures, weight reduction and oral medication can keep the condition under control for a period of time, but most people with Type 2 diabetes ultimately require insulin injections.

Islet cell transplantation can be used as a treatment for the amelioration of type 1 diabetes (Lacy et al, Annu. Rev. Immunol., 2: 183-98, 1984; Weir e/ α/., J. Am. Optom. Assoc. 69:727-32, 2000; Berney et al, Langenbechs. Arch. Surg. 385: 378-8, 2000; Shapiro et al, N Engl. J. Med., 343:230-8, 2000). However, the processes of clinical islet transplantation are made difficult by a number of factors. One factor is primary nonfunction (PNF) of the graft. Another is the high number of donor islets needed for a successful reversal of diabetes (Shapiro et al, N Engl. J. Med., 343:230-8, 2000). Both situations reflect the same pathophysiology: the substantial cell loss in the graft within the first weeks after transplantation. After transplantation, islets suffer a variety of stress factors such as hypoxia before secondary vascularization (Carlsson et al, Diabetes 47: 1027-32, 1998) and exposure to pro-inflammatory cytokines and free radicals released from macrophages in the microenvironment of the transplant (Rabinovitch et al., Diabetes 48:1223-9, 1999; Kaufman et al, J Exp Med. 772:291-302, 1990; Corbett et al , Proc. Natl . Acad. Sci USA 90: 1731 -5, 1993) and from resident islet macrophages (Mandrup-Poulsen et al, J. Immunol. 739:4077-82, 1987; Arnush et al, J. Clin Invest. 702:516-26, 1998). The toxic effects of immunosuppressive drugs as well as rejection (Weir et al., Diabetes 46:1247-56, 1997) also contribute to islet cell loss. The existence of PNF after experimental syngeneic islet transplantation (Nagata et al, Transplant Proc. 22:855-6, 1990; Aήta et al., Transplantation 65: 1429-33, 1998) indicates that nonspecific inflammation plays a major role in this scenario. SUMMARY

The present invention is based, at least in part, on the discovery that administering HO-I and/or one or more degradation products of heme can treat and/or prevent diabetes. Accordingly, the present application features methods of treating, delaying the onset of, reducing the symptoms of, or preventing (reducing the likelihood of developing) diabetes (e.g., type 1 diabetes or autoimmune diabetes) in a patient. The methods include administering to the patient a first treatment that includes inducing HO- 1 in the patient; increasing the level of expression of HO-I in the patient; inducing apoferritin in the patient; increasing the level of expression of apoferritin in the patient; and/or administering a first pharmaceutical composition that includes carbon monoxide (CO), a CO-releasing compound, HO-I, hemin, bilirubin, biliverdin, ferritin, iron, desferoxamine (DFO), salicylaldehyde isonicotinoyl hydrazone (SIH), iron dextran, and/or apoferritin to the patient, in an amount sufficient to treat, delay the onset of, reduce the symptoms of, or prevent the diabetes. Optionally, the methods can include first identifying a patient suffering from or at risk for diabetes (e.g., type 1 diabetes or autoimmune diabetes). In certain embodiments, the methods include administering to the patient a combination of at least two of the above treatments. In some embodiments, the patient is newly diagnosed with diabetes (e.g , type 1 diabetes or autoimmune diabetes). In some embodiments, the diabetes is overt diabetes. In further embodiments, the diabetes is in the remission or honeymoon period. While the methods can be done in conjunction with a transplantation step (e.g., transplantation of pancreatic islets, 9- cells, or other cells that secrete insulin), in some embodiments, the method does not include a transplantation step. The methods can further include administering to the patient a second pharmaceutical composition that includes an agent described herein, e.g., an antiinflammatory agent, an immunosuppressive agent, insulin, an agent that mimics an effect of insulin, an anti-diabetic agent, and/or an agent effective to treat a symptom of diabetes. Further, the invention features methods of transplanting insulin secreting cells

(e.g., pancreatic islet cells) that include providing insulin secreting cells (e.g., from a donor); transplanting the insulin secreting cells into a patient suffering from diabetes or at risk for diabetes (e.g., type 1 diabetes or autoimmune diabetes); and before, during, or after the transplanting step, administering to the donor, cells, and/or patient a treatment including inducing HO-I or apoferritin in the donor, cells, and/or patient, increasing the level of expression HO-I or apoferritin in the donor, cells, and/or patient, inducing peroxisome-proliferator-activated receptor gamma (PPARK) in the donor, cells, and/or patient, increasing the level of expression of PPARK in the donor, cells, and/or patient, and/or administering a pharmaceutical composition that includes CO, a CO-releasing compound, HO-I, hemin, bilirubin, biliverdin, ferritin, desferoxamine, salicylaldehyde isonicotinoyl hydrazone, iron dextran, apoferritin, a PPARK agonist, and/or a toll-like receptor 4 (TLR4) antagonist to the donor, cells, and/or patient, wherein the treatment administered to the patient is sufficient to enhance survival or function of the insulin secreting cells after transplantation of the organ to the recipient. Optionally, the methods can include identifying a patient suffering from or at risk for diabetes (e.g., type 1 diabetes or autoimmune diabetes). In certain embodiments, the methods include administering to the patient a combination of the above treatments. In some embodiments, the donor is syngeneic (e.g., an identical twin) or the patient him- or herself (autologous transplantation). In some embodiments, the insulin secreting cells are syngeneic or autologous to the patient. In some embodiments, the insulin secreting cells (e.g., syngeneic or autologous insulin secreting cells) are derived from stem cells or another cell type. The insulin secreting cells can be treated (e.g., cultured, passaged, expanded, or administered a composition described herein) ex vivo prior to transplantation.

The methods can further include administering to the patient a second pharmaceutical composition that includes a pharmaceutical agent described herein, e.g., an anti-inflammatory agent, an immunosuppressive agent, insulin and/or an agent that mimics an effect of insulin.

Still further, the features methods of treating or reducing the symptoms of type 1 diabetes or autoimmune diabetes in a patient. The methods include administering to the patient a first treatment that includes one or more of inducing PPARK in the patient; increasing the level of expression of PPARK in the patient; and/or administering a PPARK agonist or TLR4 antagonist to the patient in an amount sufficient to treat or reduce the symptoms of the diabetes. Optionally, the methods can include first identifying a patient suffering from or at risk for diabetes. In some embodiments, the patient is newly diagnosed with diabetes. In some embodiments, the diabetes is overt diabetes. In further embodiments, the diabetes is in the remission or honeymoon period.

. Exemplary PPARK agonists include prostaglandins (e.g., prostaglandin J₂ and 15-deoxy-Δ^12>14-prostaglandin J₂) and thiazolidinediones (e.g., rosiglitazone, pioglitazone, troglitazone, ciglitazone, and MCC-555).

The methods can further include administering to the patient a second pharmaceutical composition that includes an agent described herein, e.g., an antiinflammatory agent, an immunosuppressive agent, insulin, an agent that mimics an effect of insulin, an anti-diabetic agent, and/or an agent effective to treat a symptom of diabetes.

A step of determining an HO-I parameter in the patient may be included in any of the treatment methods described herein. An "HO-I parameter" is HO-I activity, HO- 1 expression, level or presence of HO-I induction in response to a stimulus, or a polymorphism in the promoter of HO-I (e.g., a polymorphism associated with modified HO-I induction in response to a stimulus). The polymorphism can be a single nucleotide. polymorphism, restriction fragment length polymorphism or microsatellite polymorphism, e.g., referring to the number or length of repeats of a nucleotide sequence (e.g., (GT)_n in the promoter of HO-I).

In still further aspects, the application features compositions (e.g., pharmaceutical compositions) that include at least one of: an anti-inflammatory agent, an immunosuppressive agent, insulin, and an agent that mimics an effect of insulin; and at least one of CO, a CO-releasing compound, HO-I, hemin, bilirubin, biliverdin, ferritin, iron, DFO, SIH, iron dextran or apoferritin.

The term "pharmaceutical composition" is used throughout the specification to describe a gaseous, liquid, or solid composition containing an active ingredient described herein that can be administered to a patient. The invention contemplates use of any two, three, four, or five of these in combination or in sequence. The skilled practitioner will recognize which form of the pharmaceutical composition, e.g., gaseous, liquid, and/or solid, is preferred for a given application. Further, the skilled practitioner will recognize which active ingredient(s) should be included in the pharmaceutical composition for a given application.

The term "patient" is used throughout the specification to describe an animal, human or non-human, to whom treatment according to the methods of the present invention is provided. Veterinary applications are clearly anticipated by the present invention. The term includes but is not limited to mammals, e.g., humans, other primates, pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep and goats. The terms "effective amount" and "effective to treat," as used herein, refer to an amount or concentration that is effective within the context of its administration for causing an intended effect or physiological outcome. The terms "treat" or "treatment" are used herein to describe inhibiting, decreasing, or alleviating the effects of a disease or condition, e.g., a disease or condition described herein. When the terms "prevent," "preventing," "prevention," and "delaying onset" are used herein in connection with a given condition, they mean that the treated patient either does not develop a clinically observable level of the condition at all, or develops it more slowly and/or to a lesser degree than the patient would have absent the treatment. These terms are not limited to a situation in which the patient experiences no aspect of the condition whatsoever. For example, a treatment will be said to have "prevented" inflammation if it is given before, during, or after exposure to an otherwise inflammatory stimulus and results in a level of inflammation experienced by the patient that is lower than would have been expected without the treatment.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

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

The present invention includes providing, e.g., administering, inducing and/or expressing heme oxygenase 1 (HO-I) or at least one product of heme degradation in a patient to treat diabetes (e.g., type 1 diabetes or autoimmune diabetes), alone or in combination with one or more second treatments. Alternatively or in addition, HO-I can be provided to a patient (e.g., by administering HO-I protein, inducing HO-I in the patient or expressing HO-I in the patient) in conjunction with a product of heme degradation, e.g., carbon monoxide (CO), biliverdin, bilirubin, iron, and/or ferritin, to treat diabetes.

Use of Heme Oxygenase-1 and Products of Heme Degradation

Heme Oxygenase-1

HO-I can be provided to a patient by inducing or expressing HO-I in the patient, or by administering exogenous HO-I directly to the patient. As used herein, the term "induce(d)" means to cause increased production of a protein, e.g., HO-I or ferritin, in the body of a patient, using the patient's own endogenous (e.g., non-recombinant) gene that encodes the protein.

HO-I can be induced in a patient by any method known in the art. For example, production of HO-I can be induced by hemin (PANHEMATIN^, by iron protoporphyrin, or by cobalt protoporphyrin. A variety of non-heme agents including heavy metals, cytokines, hormones, nitric oxide, COCl₂, endotoxin and heat shock are also strong inducers of HO-I expression (Otterbein et al., Am. J. Physiol. Lung Cell

MoI. Physiol. 279:L1029-L1037, 2000; Choi et al, Am. J. Respir. Cell MoI. Biol. 15:9- 19, 1996; Maines, Annu. Rev. Pharmacol. Toxicol. 37:517-554, 1997; and Tenhunen et al, J. Lab. Clin. Med. 75:410-421, 1970). HO-I is also highly induced by a variety of agents and conditions that create oxidative stress, including hydrogen peroxide, glutathione depletors, UV irradiation and hyperopia (Choi et al, Am. J. Respir. Cell MoI. Biol. 15: 9-19, 1996; Maines, Annu. Rev. Pharmacol. Toxicol. 37:517-554, 1997; and Keyse et al, Proc. Natl. Acad. Sci. USA 86:99-103, 1989). A "pharmaceutical composition comprising an inducer of HO-I" means a pharmaceutical composition containing any agent capable of inducing HO-I in a patient, e.g., any of the agents described herein, e.g., hemin, iron protoporphyrin, and/or cobalt protoporphyrin.

The present application contemplates that HO-I can be expressed in a patient via gene transfer. As used herein, the term "expressed)" means to cause increased production of a protein, e.g., HO-I or ferritin, in the body of a patient using an exogenously administered gene (e.g., a recombinant gene). The HO-I or ferritin is preferably of the same species (e.g., human, mouse, rat, etc.) as the patient, in order to minimize any immune reaction. Expression could be driven by a constitutive promoter (e.g., cytomegalovirus promoters) or a tissue-specific promoter (e.g., milk whey promoter for mammary cells or albumin promoter for liver cells). An appropriate gene therapy vector (e.g., retroviruses, adenoviruses, adeno-associated viruses (AAV), pox (e.g., vaccinia) viruses, human immunodeficiency virus (HIV), the minute virus of mice, hepatitis B virus, influenza virus, Herpes Simplex Virus-1, and lentiviruses) encoding HO-I or ferritin would be administered to the patient orally, by inhalation, or by injection at a location appropriate for treatment of a condition described herein. Particularly preferred is local administration directly to the site of the condition. Similarly, plasmid vectors encoding HO-I or ferritin can be administered, e.g., as naked DNA, in liposomes, or in microparticles.

Further, exogenous HO-I protein can be directly administered to a patient by any method known in the art. Exogenous HO-I can be directly administered in addition to, or as an alternative to, the induction or expression of HO-I in the patient as described herein. The HO-I protein can be delivered to a patient, for example, in liposomes, and/or as a fusion protein, e.g., as a TAT-fusion protein (see, e.g., Becker-Hapak et al., Methods 24, 247—256, 2001). In the context of surgical procedures such as transplantation, it is contemplated that HO-I can be induced and/or expressed in, and/or administered to, donors, recipients, and/or the organ or cells to be transplanted.

In one embodiment, HO-I or an agent that increases the expression of HO-I is used as medicament (or in the preparation of a medicament) for treating diabetes in a patient or for administering to a patient at risk of developing diabetes.

Heme Degradation Products

Additionally or alternatively, product(s) of heme degradation can be administered to patients to treat the diseases or conditions described herein. "Heme degradation products" include CO, iron, biliverdin, bilirubin and (apo)ferritin. Any of the above or prodrugs thereof can be provided to patients, e.g., as an active ingredient in a pharmaceutical composition or by other methods as described herein.

Biliverdin and Bilirubin

The terms "biliverdin" and "bilirubin" refer to the linear tetrapyrrole compounds that are produced as a result of heme degradation.

Pharmaceutical compositions comprising biliverdin and/or bilirubin may be administered to patients in aqueous or solid forms. Biliverdin and bilirubin useful in the methods described herein can be obtained from any commercial source, e.g., any source that supplies biochemicals for medical or laboratory use. In the preparation, use, or storage of biliverdin and bilirubin, it is recommended that the compounds be exposed to as little light as possible.

The amount of biliverdin and/or bilirubin to be included in pharmaceutical compositions and to be administered to patients will depend on absorption, distribution, inactivation, and excretion rates of the bilirubin and/or biliverdin, as well as other factors known to those of skill in the art. Effective amounts of biliverdin and/or bilirubin are amounts that are effective for treating a particular disease or condition.

Effective amounts of biliverdin can fall within the range of about 0.1 to 1000 micromoles/kg/day, e.g., at least 5 μmol/kg/day, e.g., at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 micromoles/kg/day. Preferred ranges include 5 to 500 μmol/kg/day, 20 to 200 μmol/kg/day, and 25 to 100 μmol/kg/day. Because biliverdin is rapidly converted to bilirubin in the body (via biliverdin reductase), the present application contemplates that doses of biliverdin above 1000 micromoles/kg/day can be administered to patients. The entire dose of biliverdin can be administered as a single dose, in multiple doses, e.g., several doses per day, or by constant infusion.

Effective amounts of bilirubin can be administered to a patient to generate serum levels of bilirubin in a range of from about 0.1 to about 300 μmol/L, e.g., at least about 50 to about 200 μmol/L, or about 50 to about 100 μmol/L. To generate such serum levels, individual doses of bilirubin can be administered that can fall within the range of about 0.1 to 1000 mg/kg, e.g., at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 mg/kg. Preferred ranges include 5 to 500 mg/kg, 20 to 200 tag/kg, and 25 to 150 mg/kg. The entire dose of bilirubin can be administered as a single dose, in multiple doses, e.g., several doses per day, or by constant infusion.

A skilled practitioner will appreciate that amounts of bilirubin and/or biliverdin outside of these ranges can be used depending upon the application. Acute, sub-acute, and chronic administration of pharmaceutical compositions comprising biliverdin and/or bilirubin are contemplated by the present application, depending upon, e.g., the severity or persistence of the disease or condition in the patient. The compositions can be delivered to the patient for a time (including indefinitely) sufficient to treat the condition and exert the intended pharmacological or biological effect. In one embodiment, bilirubin or biliverdin is used as medicament (or in the preparation of a medicament) for treating diabetes in a patient or for administering to a patient at risk of developing diabetes.

The present invention contemplates that biliverdin and/or bilirubin can be bound to carriers. Such carriers include, for example, albumin or cyclodextrin. Binding of biliverdin and/or bilirubin to such a carriers could increase the solubility of biliverdin and/or bilirubin, thereby preventing deposition of biliverdin and/or bilirubin in the tissues. The present invention contemplates that it is possible to individually administer unbound biliverdin and/or bilirubin and albumin to the patient to produce the desired effect. Alternatively or in addition, it is contemplated that biliverdin reductase can be induced, expressed, and/or administered to a patient in situations where it is deemed desirable to increase bilirubin levels in the patient. The biliverdin reductase protein can be delivered to a patient, for example, in liposomes. Further, the present invention contemplates that increased levels of biliverdin reductase can be generated in a patient via gene transfer. An appropriate gene therapy vector (e.g., plasmid, adenovirus, adeno- associated virus (AAV), lentivirus, or any of the other gene therapy vectors mentioned herein) that encodes biliverdin reductase, with the coding sequence operably linked to an appropriate expression control sequence^ would be administered to the patient orally, via inhalation, or by injection at a location appropriate for treatment of a condition described herein. In one embodiment of the present invention, a vector that encodes biliverdin reductase is administered to an organ affected by a condition described herein, and biliverdin is subsequently or simultaneously administered to the organ, such that the biliverdin reductase breaks down the biliverdin to produce bilirubin in the organ. Iron and Ferritin

The release of free iron by the action of HO-I on heme stimulates the induction of apoferritin, which rapidly sequesters the iron to form ferritin. The present invention includes inducing or expressing apoferritin in a patient to treat inflammation or ischemia or cell proliferation associated with various diseases or conditions in the patient. Apoferritin can be induced in a patient by any method known in the art. For example, apoferritin can be induced by administering iron dextran to the patient. As another example, apoferritin levels in a patient can be increased by exposing the patient to ultraviolet radiation (Otterbein et ah, Am. J. Physiol. Lung Cell MoI. Physiol. 279:Ll029-L1037, 2000).

A "pharmaceutical composition comprising an inducer of apoferritin" means a pharmaceutical composition containing any agent capable of inducing apoferritin, e.g., heme, iron, and/or iron dextran, in a patient. Typically, a pharmaceutical composition comprising an inducer of apoferritin is administered to a patient in aqueous or solid form. Inducers of apoferritin useful in the methods of the invention, e.g., iron or iron dextran, can be obtained from any commercial source, e.g., a commercial source that supplies chemicals for medical or laboratory use.

An effective amount of an inducer of apoferritin, e.g., iron or iron dextran, is an amount that is effective for treating a disease or condition. Effective doses of iron dextran can be administered once or several times per day, and each dose can fall within the range of about 1 to 1000 mg/kg, e.g., at least 2, 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 800, or 900 mg/kg. Preferred ranges for iron dextran include 10 to 900 mg/kg, 100 to 800 mg/kg, 300 to 700 mg/kg, or 400 to 600 mg/kg. Free iron can be delivered to the patient, for example, as one or multiple doses of a commercially available iron supplement, e.g., a tablet containing iron.

Further, the present invention contemplates that increased levels of apoferritin and/or ferritin, e.g., H-chain apoferritin and/or H-chain ferritin, can be generated in a patient via gene transfer. An appropriate gene therapy vector (as described herein) would be administered to the patient orally or by injection or implantation at a location appropriate ferritin or apoferritin can be directly administered in addition to, or as an alternative to the induction or expression of apoferritin in the patient as described herein. The apoferritin protein can be delivered to a patient, for example, in liposomes, and/or as a fusion protein, e.g., as a TAT-fusion protein (see, e.g., Becker-Hapak et aL, Methods

24:247-256, 2001).

Alternatively or in addition, it is contemplated that other iron-binding molecules can be administered to the patient to create or enhance the desired effect, e.g., to reduce free iron levels. As one example, the present invention contemplates that apoferritin can be administered to a patient, as well as any type of iron chelator, e.g., desferoxamine

(DFO) or salicylaldehyde isonicotinoyl hydrazone (SIH) (see₇ e.g., Blaha et al, Blood

91(11):4368-4372, 1998), to create or enhance the desired effect.

Effective doses of DFO can be administered once or several times per day, and each dose can fall within the range of from about 0.1 to 1000 mg/kg, e.g., at least 2, 2.5.,

5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 800, or 900 mg/kg. Preferred ranges for DFO include 0.5 to 800 mg/kg, 1 to 600 mg/kg, 2 to 400 mg/kg, or 2.5 to 250 mg/kg.

Effective doses of SIH can be administered once or several times per day, and each dose can fall within the range of from about 0.02 to 100 mmol/kg, e.g., 0.02 to 50 mmol/kg, or 0.2 to 20 mmol/kg.

Effective doses of apoferritin can be administered once or several times per day, and each dose can fall within the range of about 1 to 1000 mg/kg, e.g., at least 2, 2.5, 5,

10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 800, or 900 mg/kg. Preferred ranges include 10 to 500 mg/kg, 20 to 200 mg/kg, and 25 to 150 mg/kg.

The skilled practitioner will recognize that any of the above can be administered as a single dose, in multiple doses, e.g., several doses per day, or by constant infusion.

Further, any of the above can be administered continuously, and for as long as necessary to produce the desired effect. Further, the skilled practitioner will recognize that any of the above can be administered in amounts outside the ranges given, depending upon the application.

In one embodiment, one or more of the above agents are used as medicament (or in the preparation of a medicament) for treating diabetes in a patient or for administering to a patient at risk of developing diabetes. Carbon Monoxide

The term "carbon monoxide" (or "CO") as used herein describes molecular carbon monoxide in its gaseous state, compressed into liquid form, dissolved in a liquefied gas (e.g., a propellant), or dissolved in aqueous solution. An effective amount of carbon monoxide for use in the present invention is an amount that is effective for treating a disease or condition. For gases, effective amounts of carbon monoxide generally have a concentration in a carrier gas within the range of about 0.0000001% to about 0.3% by weight, e.g., 0.0001% to about 0.25% by weight, preferably at least about 0.001%, e.g., 0.005%, 0.010%, 0.02%, 0.025%, 0.03%, 0.04%, 0.05%, 0.06%, 0.08%, 0.10%, 0.15%, 0.20%, 0.22%, or 0.24% by weight of carbon monoxide. For liquid solutions of CO, effective amounts generally fall within the range of about 0.0001 to about 0.0044 g CO/100 g liquid, e.g., 0.0001, 0.0002, 0.0004, 0.0006, 0.0008, 0.0010, 0.0013, 0.0014, 0.0015, 0.0016, 0.0018, 0.0020, 0.0021, 0.0022, 0.0024, 0.0026, 0.0028, 0.0030, 0.0032, 0.0035, 0.0037, 0.0040, or 0.0042 g CO/100 g aqueous solution. A skilled practitioner will appreciate that amounts outside of these ranges can be used depending upon the application.

A carbon monoxide composition can be a gaseous carbon monoxide composition. Compressed or pressurized gas useful in the methods of the invention can be obtained from any commercial source, and in any type of vessel appropriate for storing compressed gas. For example, compressed or pressurized gases can be obtained from any source that supplies compressed gases, such as oxygen, for medical use. The pressurized gas including carbon monoxide used in the methods of the present invention can be provided such that all gases of the desired final composition (e.g., CO, and any more or more of He, NO, CO₂, O₂, N₂, and/or air) are in the same vessel. Optionally, the methods of the present invention can be performed using multiple vessels containing individual gases. For example, a single vessel can be provided that contains carbon monoxide, with or without other gases, the contents of which can be optionally mixed with the contents of other vessels, e.g., vessels containing oxygen, nitrogen, carbon dioxide, compressed air, or any other suitable gas or mixtures thereof. Gaseous carbon monoxide compositions administered to a patient according to the present invention typically contain 0% to about 79% by weight nitrogen, about 21% to nearly 100% by weight oxygen and about 0.0000001% to about 0.3% by weight (corresponding to about 1 ppb or 0.001 ppm to about 3,000 ppm) carbon monoxide. Preferably, the amount of nitrogen in the gaseous composition is about 79% by weight, the amount of oxygen is about 21% by weight and the amount of carbon monoxide is about 0.0001% to about 0.25% by weight, preferably at least about 0.001%, e.g., 0.005%, 0.010%, 0.02%, 0.025%, 0.03%, 0,04%, 0.05%, 0.06%, 0.08%, 0.10%, 0.15%, 0.20%, 0.22%, or 0.24% by weight of carbon monoxide. It is noted that gaseous carbon monoxide compositions having concentrations of carbon monoxide greater than 0.3% (such as 1% or greater) can be used for short periods (e.g., one or a few breaths), depending upon the application.

A gaseous carbon monoxide composition can be used to create an atmosphere that comprises carbon monoxide gas. An atmosphere that includes an appropriate level of carbon monoxide gas can be created, for example, by providing a vessel containing a pressurized gas comprising carbon monoxide gas and releasing the pressurized gas from the vessel into a chamber or space to form an atmosphere that includes the carbon monoxide gas inside the chamber or space. Alternatively, the gases can be released into an apparatus that culminates in a breathing mask or breathing tube, thereby creating an atmosphere comprising carbon monoxide gas in the breathing mask or breathing tube, ensuring the patient is the only person in the room exposed to significant levels of carbon monoxide.

Carbon monoxide levels in an atmosphere can be measured or monitored using any method known in the art. Such methods include electrochemical detection, gas chromatography, radioisotope counting, infrared absorption, colorimetry, and electrochemical methods based on selective membranes (see, e g., Sunderman et al., Clin. Chem. 28:2026-2032, 1982; Ingi et al., Neuron 16:835-842, 1996). Sub-parts per million carbon monoxide levels can be detected by, e.g., gas chromatography and radioisotope counting. Further, it is known in the art that carbon monoxide levels in the sub-ppm range can be measured in biological tissue by a midinfrared gas sensor (see, e.g., Morimoto et al, Am. J. Physiol. Heart. Circ. Physiol 280.H482-H488, 2001). Carbon monoxide sensors and gas detection devices are widely available from many commercial sources. A pharmaceutical composition comprising carbon monoxide can also be a liquid composition. A liquid can be made into a pharmaceutical composition comprising carbon monoxide by any method known in the art for causing gases to become dissolved in liquids. For example, the liquid can be placed in a so-called "CO2 incubator" and exposed to a continuous flow of carbon monoxide, preferably balanced with carbon dioxide, until a desired concentration of carbon monoxide is reached in the liquid. As another example, carbon monoxide gas can be "bubbled" directly into the liquid until the desired concentration of carbon monoxide in the liquid is reached. The amount of carbon monoxide that can be dissolved in a given aqueous solution increases with decreasing temperature. As still another example, an appropriate liquid can be passed through tubing that allows gas diffusion, where the tubing runs through an atmosphere comprising carbon monoxide (e.g., utilizing a device such as an extracorporeal membrane oxygenator). The carbon monoxide diffuses into the liquid to create a liquid carbon monoxide composition.

The liquid can be any liquid known to those of skill in the art to be suitable for administration to patients (see, for example, Oxford Textbook of Surgery, Morris and Malt, Eds., Oxford University Press, 1994). In general, the liquid will be an aqueous solution. Examples of solutions include Phosphate Buffered Saline (PBS), Celsior™ solution, Perfadex™ solution, Collins solution, citrate solution, and University of

Wisconsin (UW) solution (Oxford Textbook of Surgery, Morris and Malt, Eds., Oxford University Press, 1994).

The present invention contemplates that compounds that release CO into the body after administration of the compound (e.g., CO-releasing compounds, e.g., photoactivatable CO-releasing compounds), e.g., dimanganese decacarbonyl, tricarbonyldichlororuthenium (II) dimer, and methylene chloride (e.g., at a dose of between 400 to 600 mg/kg, e.g., about 500mg/kg), can also be used in the methods of the present invention, as can carboxyhemoglobin and CO-donating hemoglobin substitutes. Agents capable of delivering doses of CO gas or liquid can also be utilized (e.g., CO releasing gums, creams, ointments or patches)

Any suitable liquid can be saturated to a set concentration of carbon monoxide via gas diffusers. Alternatively, pre-made solutions that have been quality controlled to contain set levels of carbon monoxide can be used. Accurate control of dose can be achieved via measurements with a gas permeable, liquid impermeable membrane connected to a carbon monoxide analyzer. Solutions can be saturated to desired effective concentrations and maintained at these levels.

A patient can be treated with a carbon monoxide composition by any method known in the art of administering gases and/or liquids to patients. The present invention contemplates the systemic administration of liquid or gaseous carbon monoxide compositions to patients (e.g., by inhalation, ingestion, or artificial lung), and the topical administration of the compositions to the patient's organs, e.g., the pancreas.

A patient's CO exposure can be monitored by assaying the percentage of blood hemoglobin that is bound by CO (i.e., carboxyhemoglobin (COHb)). The amount and timing of CO administration can be tailored to achieve a desired percentage of COHb (e.g., 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% 11%, 12%, 13%, 14%, or 15%). Alternatively, the amount and timing of CO administration can be tailored to achieved a desired increase in percentage of COHb over the initial CO level of the patient (e.g., an increase of 0.2%, 0.5%, 1.0%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%). The desired COHb level can be achieved as an equilibrium level for a period of time, or the desired level can be reached and then allowed to decrease over time.

Gaseous carbon monoxide compositions are typically administered by inhalation through the mouth or nasal passages to the lungs, where the carbon monoxide can exert its effect directly or be readily absorbed into the patient's bloodstream. The concentration of active compound (CO) utilized in the therapeutic gaseous composition will depend on absorption, distribution, inactivation, and excretion (generally, through respiration) rates of the carbon monoxide as well as other factors known to those of skill in the art. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed invention. Acute, sub- acute and chronic administration of carbon monoxide are contemplated by the present invention, depending upon, e.g., the severity or persistence of disease or condition in the patient. Carbon monoxide can be delivered to the patient for a time (including indefinitely) sufficient to treat the condition and exert the intended pharmacological or biological effect. Examples of methods and devices that can be utilized to administer gaseous pharmaceutical compositions comprising carbon monoxide to patients include ventilators, face masks and tents, portable inhalers, intravenous artificial lungs (see, e.g., Hattler e/ α/., Artif. Organs 18(l l):806-812, 1994; and Golόb et aL, ASAIO J., 47(5):432-437, 2001), and normobaric chambers, as described in further detail below. The present invention further contemplates that aqueous solutions comprising carbon monoxide can be created for systemic delivery to a patient, e.g., by oral delivery to a patient.

Alternatively or in addition, carbon monoxide compositions can be applied directly to the organs of a patient. For example, carbon monoxide compositions can be applied to the interior and/or exterior of the entire gastrointestinal tract, or to any portion thereof, by any method known in the art for insufflating gases into a patient. For example, gases, e.g., carbon dioxide, are often insufflated into the gastrointestinal tract and the abdominal cavity of patients to facilitate examination during endoscopic and laparoscopic procedures, respectively (see, e.g., Oxford Textbook of Surgery, Morris and Malt, Eds., Oxford University Press, 1994). The skilled practitioner will appreciate that similar procedures could be used to administer carbon monoxide compositions directly to the gastrointestinal tract of a patient.

Aqueous carbon monoxide compositions can also be administered directly to the organs of a patient. Aqueous forms of the compositions can be administered by any method known in the art for administering liquids to patients. For example, the aqueous form can be administered orally, e.g., by causing the patient to ingest an encapsulated or unencapsulated dose of the aqueous carbon monoxide composition. As another example, liquids, e.g., saline solutions, can be injected into the gastrointestinal tract and the abdominal cavity of patients during endoscopic and laparoscopic procedures, respectively. The skilled practitioner will appreciate that similar procedures could be used to administer liquid carbon monoxide compositions directly to the organs of a patient.

In one embodiment, carbon monoxide is used as medicament (or in the preparation of a medicament) for treating diabetes in a patient or for administering to a patient at risk of developing diabetes.

Combination Therapy

The present invention contemplates that any-of the treatments described herein, e.g., induction/expression/administration of HO-I and/or apoferritin, and the administration of CO, bilirubin, and/or biliverdin, can be used individually or in any combination to treat the disorders or conditions described herein. Further, the present invention contemplates that in any treatment regimen using any combination of the herein-described treatments, the treatments can be administered simultaneously on a single or multiple occasions, and/or individually at varying points in time, e.g., at different phases of a disease or condition. For example, a patient can receive both bilirubin and iron, or both of those phis CO, or bilirubin plus apoferritin, or two or more inducers of HO-I . Simultaneous administration can be effected either with two or more of the treatments combined into a single composition or with all treatments in separate compositions.

Treatment of Patients with Pharmaceutical Compositions of the Present Invention

A patient can be treated with pharmaceutical compositions described herein by any method known in the art of administering liquids, solids, and/or gases to a patient.

Systemic Delivery of Pharmaceutical Compositions

Aqueous and Solid Pharmaceutical Compositions

The present invention contemplates that aqueous pharmaceutical compositions can be created for systemic delivery to a patient by injection into the body, e.g., intravenously, intra-arterially, intraperitoneally, and/or subcutaneously. Aqueous pharmaceutical compositions can also be prepared for oral delivery, e.g., in encapsulated or unencapsulated form, to be absorbed in any portion of the gastrointestinal tract, e.g., the stomach or small intestine. Similarly, solid pharmaceutical compositions can be created for systemic delivery to a patient, e.g., in the form of a powder or an ingestible capsule.

Aqueous and solid pharmaceutical compositions typically include the active ingredient and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial. and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g.,

IS intravenous, intradermal, subcutaneous, oral and/or rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ solubilizer (BASF, Parsippany, NJ), or phosphate buffered saline

(PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents, e.g., sugars, polyalcohols such as mannitol or sorbitol, or sodium chloride can be included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. Microbeads, microspheres, or any other physiologically- acceptable methods, e.g., encapsulation, can be used to delay release or absorption of the active ingredients. Sterile injectable solutions can be prepared by incorporating the active ingredient in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying, and freeze-drying that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Oral compositions, which can be aqueous or solid, generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Pharmaceutically compatible binding agents and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel3 sodium carboxymethyl cellulose, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery. The active ingredients can be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycoHc acid, collagen, polyorthoesters, and polylactic acid.

Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies specific for viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD5O/ED5O.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

Gaseous pharmaceutical compositions Gaseous pharmaceutical compositions, e.g., pharmaceutical compositions containing carbon monoxide, can be delivered systemically to a patient by inhalation through the mouth or nasal passages to the lungs. The following methods and apparatus for administering carbon monoxide compositions are illustrative of useful systemic delivery methods for the gaseous pharmaceutical compositions described herein.

Ventilators

Medical grade carbon monoxide (concentrations can vary) can be purchased mixed with air or another oxygen-containing gas in a standard tank of compressed gas (e.g., 21% O2, 79% N2). It is non-reactive, and the concentrations that are required for the methods of the present invention are well below the combustible range (10% in air). In a hospital setting, the gas presumably will be delivered to the bedside where it will be mixed with house air in a blender to a desired concentration in ppm (parts per million). The patient will inhale the gas mixture through a ventilator, which will be set to a flow rate based on patient comfort and needs. This is determined by pulmonary graphics (i.e., respiratory rate, tidal volumes etc.). Fail-safe mechanism(s) to prevent the patient from unnecessarily receiving greater than desired amounts of carbon monoxide can be designed into the delivery system. The patient's carbon monoxide level can be monitored by studying (1) carboxyhemoglobin (COHb), which can be measured in venous blood, and (2) exhaled carbon monoxide collected from a side port of the ventilator. Carbon monoxide exposure can be adjusted based upon the patient's health status and on the basis of the markers. If necessary, carbon monoxide can be washed out of the patient by switching to 100% O2 inhalation. Carbon monoxide is not metabolized; thus, whatever is inhaled will ultimately be exhaled except for a very small percentage that is converted to CO2. Carbon monoxide can also be mixed with any level of O2 to provide therapeutic delivery of carbon monoxide without consequential hypoxic conditions. Face Mask and Tent

A carbon monoxide containing gas mixture is prepared as above to allow passive inhalation by the patient using a facemask or tent. The concentration inhaled can be changed and can be washed out by simply switching over to 100% O2. Monitoring of carbon monoxide levels would occur at or near the mask or tent with a fail-safe mechanism that would prevent too high of a concentration of carbon monoxide from being inhaled.

Portable inhaler Compressed carbon monoxide can be packaged into a portable inhaler device and inhaled in a metered dose, for example, to permit intermittent treatment of a recipient who is not in a hospital setting. Different concentrations of carbon monoxide could be packaged in the containers. The device could be as simple as a small tank (e.g., under 5 kg) of appropriately diluted CO with an on-off valve and a tube from which the patient takes a whiff of CO according to a standard regimen or as needed.

Intravenous Artificial Lung

An artificial lung (a catheter device for gas exchange in the blood) designed for O₂ delivery and CO2 removal can be used for carbon monoxide delivery. The catheter, when implanted, resides in one of the large veins and would be able to deliver carbon monoxide at given concentrations either for systemic delivery or at a local site. The delivery can be a local delivery of a high concentration of carbon monoxide for a short period of time at the site of the procedure, e.g., in proximity to the pancreas or implanted insulin-secreting cells (this high concentration would rapidly be diluted out in the bloodstream), or a relatively longer exposure to a lower concentration of carbon monoxide (see, e.g., Hattler et al, Artif. Organs 18(1 l):806-812, 1994; and Golob et al, ASAIO J., 47(5) :432-437, 2001).

Normobaric chamber In certain instances, it would be desirable to expose the whole patient to carbon monoxide. The patient would be inside an airtight chamber that would be flooded with carbon monoxide (at a level that does not endanger the patient, or at a level that poses an acceptable risk, or for non-human donors or brain-dead donors, at any desired level) without the risk of bystanders being exposed. Upon completion of the exposure, the chamber could be flushed with air (e.g., 21% O₂, 79% N2) and samples could be analyzed by carbon monoxide analyzers to ensure no carbon monoxide remains before allowing the patient to exit the exposure system.

Topical Delivery of Pharmaceutical Compositions

Alternatively or in addition, pharmaceutical compositions can be applied directly to an organ, tissue, or area of the patient's body to be treated.

A queous and Solid Pharmaceutical Compositions

Aqueous and solid pharmaceutical compositions can also be directly applied to an organ of a patient, or to an area of the patient targeted for treatment, by any method known in the art for administering liquids or solids to patients. For example, an aqueous or solid composition can be administered orally, e.g., by causing the patient to ingest an encapsulated or unencapsulated dose of the aqueous or solid pharmaceutical composition. Further, liquids, e.g., saline solutions, are often injected into the abdominal cavity of patients during endoscopic and laparoscopic procedures, respectively. The skilled practitioner will appreciate that similar procedures could be used to administer aqueous pharmaceutical compositions directly to an organ or, e.g., in the vicinity of an organ to be treated, to thereby expose the organ in situ to an aqueous pharmaceutical composition.

In the context of transplantation, in situ exposures can be performed by any method known in the art, e.g., by in situ flushing of the organ with a liquid pharmaceutical composition prior to removal from the donor (see Oxford Textbook of Surgery, Morris and Malt, Eds., Oxford University Press, 1994). Such exposures are described in further detail below.

Gaseous pharmaceutical compositions

A gaseous pharmaceutical composition can be directly applied to an organ of a patient, or to an area of the patient targeted for treatment, by any method known in the art for insufflating gases into a patient. For example, gases, e.g., carbon dioxide, can be insufflated into the abdominal cavity of patients to facilitate examination during endoscopic and laparoscopic procedures, respectively (see, e.g., Oxford Textbook of Surgery, Morris and Malt, Eds., Oxford University Press, 1994). Further, the skilled practitioner will appreciate that gaseous pharmaceutical compositions can be insufflated into the abdominal cavity of patients, e.g., in the vicinity of an organ to be treated, to thereby expose the organ in situ to a gaseous pharmaceutical composition.

Pharmaceutical Agents

Numerous anti-inflammatory and other pharmaceutical agents are useful in the methods described herein. Some pharmaceutical agents may at least partially depend on HO-I for their beneficial effect. For example, beneficial effects displayed by certain pharmaceutical agents may be replicated, in vivo or in vitro, by upregulation of HO-I , at least to some degree.

Useful agents include steroidal anti-inflammatory agents, non-steroidal antiinflammatory agents (NSAIDS), cyclooxygenase inhibitors (e.g., general cyclooxygenase inhibitors or specific COX-I or COX-2 inhibitors), lipoxygenase inhibitors (e.g., inhibitors of 5-lipoxygenase, 12/15 -lipoxygenase, or 15-lipoxygenase), statins, adenosine and adenosine A2a receptor (A2aR) agonists, probucol (and its derivatives and similar therapeutics), anti-inflammatory cytokines (e.g., IL-IO), prostaglandins, VEGF (and compounds that mimic VEGF)₅ immunosuppressants (e.g., rapamycin and cyclosporin), TNFI inhibitors (e.g., infliximab), interferons (e.g., alpha or gamma interferon), modulators of factors that regulate HO-I, compounds that release nitric oxide and/or modulate guanylate cyclase pathways. Also useful are agents that induce the expression or production of endogenous factors listed above.

Exemplary steroidal anti-inflammatory agents include corticosteroids, e.g., glucocorticoids, such as hydrocortisone, cortisone acetate, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclometasone, fludrocortisone acetate, deoxycorticosterone acetate, and aldosterone.

Exemplary non-steroidal anti-inflammatory agents (NSAIDS) include acetaminophen; salicylates, such as aspirin', methyl salicylate, and diflunisal; arylalkanoic acids, such as diclofenac, indomethacin, and sulindac; 2-arylpropionic acids, such as ibuprofen, ketoprofen, naproxen, carprofen, fenoprofen, and ketorolac; N- arylanthranilic acids, such as mefenamic acid, oxicams, such as piroxicam and meloxicam; sulfonanilides, such as nimesulide; and COX-2 inhibitors, such as celecoxib, rofecoxib, valdecoxib, parecoxib, etoricoxib. The methods described herein may be useful to reduce dosages of COX-2 inhibitors needed to cause a physiological effect, thus reducing the number, severity, and/or risk of unwanted side effects in the patient.

Exemplary statins include atrovastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pravastatin, rosuvastatin, and simvastatin. Exemplary A2a receptor agonists include 5'-(N-ethylcarboxamido)adenosine

(NECA), 2-(6-cyano-l-hexyn-l-yl)adenosine, regadenoson, 2-p- (2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680), ATL-146e, ATL-313, 2-cyclohexylmethylidene-hydrazinoadenosine. The methods described herein can also be used with non-adenosine analog A2a receptor agonists, such as ketamine.

An exemplary probucol derivative with anti-inflammatory effects is AGI- 1067 [butanedioic acid, mono[4-[[l-[[3,5-bis(l,l-dimethylethyl)-4-,hydroxyphenyl]thio]-l- methylethyl]thio]-2,6-bis (l,l-dimethylethyl)phenyl] ester].

Exemplary prostaglandins include prostaglandin J₂ and its derivatives and analogs such as 15-deoxy-Δ¹²'¹⁴-prostagIandin J2, Δ¹²-prostaglandin J₂, and 9,10- dihydro-15-deoxy-Δ¹²'¹⁴-prostaglandin J₂.

Exemplary immunosuppressants include alkylating agents, such as cyclophosphamide, nitrosourea, and platinum compounds; antimetabolites such as folic acid analogs (e.g., methotrexate), purine analogs (e.g., azathioprine and mercaptopurine), pyrimidine analogs, and protein synthesis inhibitors; cytostatics, such as dactinomycin, anthracyclines, mitomycin C, bleomycin, and mitramycin; immunosuppressive antibodies, such as antibodies against CD3 (e.g., visilizumab), CDl Ia (e.g., efalizumab), CD20 (e.g., rituximab or basiliximab), HL-2 receptor (e.g., daclizumab), and T-cell receptors; cyclosporin; tacrolimus; rapamycin; anti-TNFI agents; and mycophenolate mofetil.

Exemplary HO-I regulators include nrf2, HEF- Ia, STATs, SMADs and CEBP.

Exemplary NO/guanylate cyclase modulators include NO, nitroprusside, nitrite, nitroglycerin, sildenafil citrate, and other phosphodiesterase inhibitors.

Useful agents that mimic one or more effects of insulin include insulin analogs (e.g., insulin aspart, insulin lispro, insulin glargine, and insulin detemir), certain cytokines (e.g., visfatin), inorganic compounds (e.g., vanadium, pervanadate, and selenium), peroxisome-proliferator-activated receptor gamma (PPARK) agonists, stimulants of intracellular insulin signaling intermediates, inhibitors of substances that deactivate insulin receptor tyrosine kinase activity, thiazolidinediones (e.g., troglitazone, ciglitazone, pioglitazone, and rosiglitazone), and phytochemicals (e.g., polyphenols and/or other compounds from cinnamon, fungi, buckwheat, witch hazel, black and green teas, allspice, bay leaves, nutmeg, and cloves). Useful anti-diabetic agents include alpha-interferon, sulfonylurea agents (e.g., glyburide, glipizide, and glimerpiride), other beta cell stimulants (e.g., repaglinide and nateglinide), alpha-glucosidase inhibitors (e.g., acarbose and miglitol), biguanide agents (e.g., metformin), and thiazolidinediones (e.g., troglitazone, pioglitazone, rosiglitazone, ciglitazone, and MCC-555). Exemplary agents useful to treat symptoms associated with diabetes (e.g., sequelae) include agents for treating cardiovascular disease or disorders, such as, for example, antihypertensive agents, including, for example, angiotensin converting enzyme inhibitor (ACE-inhibitor), alpha-adrenergic agonists, beta-adrenergic agonists, alpha-adrenergic blockers, angiotensin II receptor antagonists; diuretics, including, for example, aldosterone antagonists, benzothiadiazine derivatives, organomercurials, purines, steroids (for example, canrenone, oleandrin, spironolactone), sulfonamide derivatives, or uracils; antianginal agents; antiarrhythmic agents; antiarteriosclerotic agents; antihyperlipoproteinemic agents; anicholelithogenic agents; anticholesteremic agents; antihypercholesterolemic agents; antihyperlipidemic agents; antihypertensive agents; antihypotensive agents; antilipidemic agents; calcium channel blockers; cardiac depressant agents; dopamine receptor agonists; dopamine receptor antagonists; HMG CoA reductase inhibitors; hypocholesteremic agents; hypolipidemic agents; hypotensive agents; monoamine oxidase inhibitors; muscle relaxants; potassium channel activators; pressor agents; serotonin uptake antagonists; thrombolytic agents; vasodilator agents; vasopressor agents; or vasoprotectant agents.

HO-I Parameters

An HO-I parameter, i.e., HO-I activity, HO-I expression, level or presence of HO-I induction in response to a stimulus, or a polymorphism in the promoter of HO-I (e.g., a polymorphism associated with modified HO-I induction in response to a stimulus) can be observed in the patient. A decision whether to provide a treatment such as inducing HO-I or apoferritin in the patient using a suitable inducer, expressing HO-I or apoferritin in the patient, or administering a pharmaceutical composition comprising HO-I, CO, bilirubin, biliverdin, ferritin, iron, desferoxamine, salicylaldehyde isonicotinoyl hydrazone, iron dextran, or apoferritin, can be made based on the measurement.

HO-I activity can be measured in a cell or tissue sample by measuring the release of products of heme degradation, such as biliverdin, CO, and iron. One or more heme degradation products can be measured in a labeled (e.g., isotopically labeled) form.

Expression of HO-I can be measured by standard means, e.g., by Western blot, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), radio immunoassay (RIA), immunofluorescence, protein micrσarray, Northern blot, reverse transcription-polymerase chain reaction (RT-PCR) (e.g., real-time RT-PCR), nuclease protection, primer extension, serial analysis of gene expression (SAGE), in situ hybridization and/or nucleotide microarray. Expression can be measured in a sample, e.g., a cell, tissue, or fluid sample, obtained from a patient. HO-I induction in response to stimulus can also be measured. HO-I expression can be induced in a patient by any method known in the art. For example, production of HO-I can be induced by hemin, by iron protoporphyrin, or by cobalt protoporphyrin. A variety of non-heme agents including heavy metals, cytokines, hormones, nitric oxide, COCl₂, endotoxin and heat shock are also strong inducers of HO-I expression (Otterbein et al., Am. J. Physiol. Lung Cell MoI. Physiol. 279:L1029-L1037, 2000; Choi et al, Am. J. Respir. Cell MoI. Biol. 15:9-19, 1996; Maines, Annu. Rev. Pharmacol. Toxicol. 37:517-554, 1997; and Tenhunen et al, J. Lab. Clin. Med 75:410-421, 1970). HO-I is also highly induced by a variety of agents and conditions that create oxidative stress, including hydrogen peroxide, glutathione depletors, UV irradiation and hyperoxia (Choi et al, Am. J. Respir. Cell MoI. Biol. 15: 9-19, 1996; Maines, Annu. Rev. Pharmacol. Toxicol. 37:517-554, 1997; and Keyse et al, Proc. Natl. Acad. Sci. USA 86:99-103, 1989). Induction can be measured by providing a sample, e.g., a cell, tissue, or fluid sample, from a patient, subjecting the sample to an HO-I inducing stimulus, and measuring expression of HO-I in response to the stimulus, e.g., as compared to HO-I expression in the absence of the stimulus or in response to a control stimulus that does not induce HO-I. Individuals with lower than average induction of HO-I, e.g., 80%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5% or less than average induction, can be administered a treatment described herein. The presence or absence of an HO-I polymorphism can be observed. An HO-I polymorphism can be, e.g., a single nucleotide polymorphism, restriction fragment length polymorphism, or microsatellite polymorphism, e.g., the number or length of repeats of a nucleotide sequence (e.g., (GT)_n in the promoter of HO-I). In some instances, the polymorphism can be correlated with the activity of HO-I, the expression of HO-I, or the level or strength of HO-I induction in response to a stimulus.

One particularly useful polymorphism correlated with HO-I induction is a microsatellite polymorphism of a (GT)_n repeat in the promoter region of HO-I. The length or number of microsatellite repeats can be measured, e.g., by PCR. Exemplary primer pairs for measuring repeat length are 5'-AGAGCCTGCAGCTTCTCAGA-3 '

(SEQ ID NO: 1) and 5'-ACAAAGTCTGGCCATAGGAC-S' (SEQ ID NO:2) (Kaneda et al., Arterioscler. Thromb. Vase. Biol., 22:1680-5, 2002) or 5'-ATC AC ACCC AGAGCCTGC AGC-3¹ (SEQ ID NO:3) and 5'-GGGGTGGAGAGGAGCAGTCAT-S' (SEQ ID NO:4) (Li et al, Chin. Med. J. (Engl)., 118: 1285-90, 2005). Shorter repeats are associated with greater up-regulation of HO-I in response to stimuli (Schillinger et al., J. Am. Coll. Cardiol., 43:950-7, 2004). Individuals with one or both alleles of HO-I having, e.g., at least 26 (e.g., 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more) GT repeats can be administered a treatment described herein.

Diabetes and Testing For Diabetes

Diabetes or diabetes mellitus is a metabolic disease that involves the presence of chronically elevated levels of blood glucose. Diabetes is caused by abnormal metabolism of glucose, protein and lipids, due to a deficiency or insufficiency of the actions of insulin. Typical signs of diabetes include an abnormal increase in serum glucose and excretion of glucose in the urine. Classic symptoms of diabetes mellitus in adults include polyuria, polydipsia, ketonuria, rapid weight loss, other acute manifestations of hyperglycemia, and elevated levels of plasma glucose. (See, e.g., Genuth, "Diabetes Mellitus," pp. 9.VI.1-35, in WebMD Scientific American Medicine, Dale and Federman, eds., WebMD Corp., 2004) .

Type 1 diabetes (also called insulin-dependent diabetes mellitus (IDDM), juvenile diabetes, brittle diabetes, or sugar diabetes) is accompanied by reduction of insulin producing cells, pancreatic beta cells. Symptoms of Type 1 diabetes include, e.g., high levels of sugar in the blood and/or urine, unusual thirst, frequent urination, extreme hunger but loss of body weight, blurred vision, nausea and vomiting, extreme weakness and tiredness, irritability, and mood changes. Complications associated with Type 1 include, e.g., hypoglycemia (low blood sugar in response to insulin administration), hyperglycemia (high blood sugar due to poor disease maintenance), and ketoacidosis (diabetic coma or loss of consciousness due to untreated or under-treated diabetes).

Type 2 diabetes (also called non-insulin-dependent diabetes mellitus (NIDDM)) is caused by a reduction in insulin sensitivity or insulin secretion. Type 2 diabetes is characterized by insulin resistance, i.e., a failure of the normal metabolic response of peripheral tissues to the action of insulin. Progression of Type 2 diabetes is generally characterized by increasing concentrations of blood glucose, coupled with a decrease in the rate of glucose-induced insulin secretion. The beta cells of Type 2 diabetics typically retain the ability to synthesize and secrete insulin. Type 2 diabetes is often accompanied by obesity.

Other subclasses of diabetes include maturity-onset diabetes of the young (MODY), hyperglycemia caused by continuous administration of steroids such as glucocorticoids (steroid diabetes), Cushing Syndrome, acromegaly and gestational diabetes. The methods described herein can be used to treat a patient with insulitis.

Insulitis is an inflammation caused by infiltration of lymphocytes into pancreatic islets of Langerhans. Insulitis can result in the destruction of beta cells in the islets, as seen in Type 1 diabetes. Upon complete onset of Type 1 diabetes, all or nearly all (e.g., 80%, 85%, 90%, 95% or more) of the pancreatic beta cells have been destroyed. A number of standard test have been developed to determine whether a patient has diabetes. These tests can also be used to track the patient's response to treatment.

One useful diagnostic test is the fasting glucose test. Typically, a blood sample of about 5 milliliters is taken from a patient after a ten to twelve hour fast. The patient repeats the fast another day and provides another blood sample. A patient without diabetes would have a fasting glucose level between about 80 mg/dl and about 120 mg/dl (or about 4 mmol/L to about 7 mmol/L). If a patient has two fasting glucose levels of 126 mg/dl (or 7 mmol/L) or greater, then the patient is typically diagnosed as having diabetes. Similar to the fasting glucose test is the oral glucose tolerance test (OGTT). The patient fasts for ten to twelve hours, then a blood sample is taken. A glucose dose of about 75 milligrams (or 100 milligrams if testing for gestational diabetes) is administered to the patient and blood samples are taken every thirty minutes for the next two hours. If the patient has a glucose level that is equal to or greater than 200 mg/dl (or 11.1 mmol/L), then the patient is typically diagnosed with diabetes.

An alternative to the fasting method is the two-hour postprandial plasma glucose (2 hr PPG) test. This test measures the amount of glucose in blood plasma after a person eats a meal loaded with a specific amount of sugar, typically about 75 milligrams. After two hours, the patient's glucose levels are ascertained by evaluating a blood sample. If a patient has a level of at least 200 mg/dl (or 11.1 mmol/L), then the patient is typically diagnosed as having diabetes.

A patient can also be diagnosed as diabetic if he or she exhibits a blood glucose level of greater than 200 mg/dl (or 11.1 mmol/L) and exhibits known symptoms of diabetes, as described above.

Insulin production in a patient suffering from diabetes or suspected of having diabetes can be measured indirectly by monitoring levels of C-peptide in the blood or urine of the patient. Patients that no longer produce insulin will have no detectable C-peptide. In adult women, gestational diabetes may be diagnosed when one or more of the following results are positive: (i) a fasting (for example, at least 8 hours) blood glucose level greater than about 95 mg/dl; (ii) a one-hour glucose level greater than about 180 mg/dl; (iii) a two-hour glucose level greater than about 155 mg/dl; or (iv) a three- hour glucose level greater than about 140 mg/dl. A subject can be confirmed to have diabetes when two or more diagnostic tests done on different days show blood glucose levels higher than normal for the particular subject. The particular test used to confirm the delay of onset of diabetes may vary and will generally be interpreted on a subject-by-subject basis determined by those skilled in the art. The methods disclosed herein can also be used to prevent or delay diabetes in a subject that is in the pre-insulitis stage of type 1 diabetes, or expresses a predisposition marker (e.g., autoantibodies against a beta cell or islet antigen such as insulin, glutamic acid decarboxylase (GAD), carboxypeptidase H, or the protein tyrosine phosphatase ICA512 (IA-2)), or expresses a predisposition marker but has not yet progressed to a hyperglycemic stage. In another embodiment, a subject of the methods herein is in the "pre-diabetic stage", e.g., exhibiting antibody positivity (presence of anti-islet cell or anti-beta cell antibodies) and/or abnormal first phase insulin test (insulin response to an i. v. glucose bolus).

The methods disclosed herein can also be used to treat diabetes in a subject with overt type 1 diabetes or autoimmune diabetes or a subject newly diagnosed with type 1 diabetes or autoimmune diabetes. In this stage, the subject can have clinical symptoms of diabetes but retains some capacity (albeit reduced) for insulin production (i.e., the patient still has some beta cells). Often, these subjects will have some detectable C-peptide in the blood and/or urine. In some instances, patients with overt type 1 diabetes will enter a remission or "honeymoon" period, in which blood sugar improves and beta cells partially recover.

The methods disclosed herein can also be used to treat diabetes in a subject with permanent type 1 diabetes or autoimmune diabetes. At this stage of the disease, all of the subject's beta cells have been destroyed and the subject is absolutely insulin deficient.

The non-obese diabetic ("NOD") mouse develops a spontaneous type 1 diabetes that shares many of the features associated with human IDDM, providing a well characterized and accepted animal model for this complex autoimmune disease (Makino et a/., Exp. Animal, 29:1-13, 1980; Sugihara el al., Histol. Histopathol., 4:397-404, 1989; Miyazaki ei al., Clin. Exp. Immunol., 60:622-630, 1985). The initial or pre- insulitis stage of disease begins around 3 weeks of age and involves immune cell infiltration in areas surrounding the pancreatic islets without apparent damage to the beta cells. The next phase of disease, known as insulitis, begins around the age of 6 weeks and involves a gradual increase in infiltration that ultimately overcomes the immunoregulatory mechanisms, leading to a progressive destruction of the beta cells. Loss of insulin production results in dysregulation of glucose metabolism; overt diabetes can manifest as early as 12 weeks of age. Progression from insulitis to diabetes correlates with a rise of ThI type cells specific for beta cell-associated antigens.

Cytokines such as IFNK and TNFI produced by these ThI cells stimulate recruitment of inflammatory cells capable of destroying beta cells. Transplantation

The present invention contemplates the use of the methods described herein to treat patients who undergo transplantation of an organ to treat diabetes. For convenience, the term "organ" is used throughout the specification as a general term to denote the tissue or cells being transplanted, whether that is most or all of an intact pancreas, or a portion thereof such as islets or beta cells isolated directly from a donor's pancreas, or islets or beta cells (or other insulin-secreting cells) cultured in vitro. Also included are artificial organs comprising beta cells or other insulin-secreting cells. The methods can be used to treat donors, recipients and/or the organ at any step of the organ harvesting, storage, and transplant process. For example, an organ can be harvested from a donor, treated with a pharmaceutical composition ex vivo in accordance with the present invention, and transplanted into a recipient. Islets or beta cells can be treated ex vivo during the process of isolating them from the donor's excised pancreas and during any in vitro culture steps. Alternatively or in addition, the organ can be treated in situ, while still in the donor (by treatment of the donor or by treating the organ). Optionally, a pharmaceutical composition can be administered to the recipient prior to, during, and/or after the surgery, e.g., after the organ is reperfused with the recipient's blood. The composition can be administered to the donor prior to or during the process of harvesting the organ from the donor. The terms "transplantation" is used throughout the specification as a general term to describe the process of transferring an organ to a patient. The term "transplantation" is defined in the art as the transfer of living tissues or cells from a donor to a recipient, with the intention of maintaining the functional integrity of the transplanted tissue or cells in the recipient (see, e.g., The Merck Manual, Berkow, Fletcher, and Beers, Eds., Merck Research Laboratories, Rahway, N. J., 1992). Transplants are categorized by site and genetic relationship between donor and recipient. The term includes, e.g., autotransplantation (removal and transfer of cells or tissue from one location on a patient to the same or another location on the same patient), allotransplantation (transplantation between members of the same species), and xenotransplantation (transplantations between members of different species).

The term "donor" or "donor patient" as used herein refers to an animal (human or non-human) from whom an organ, tissue, islets, or cells can be obtained for the purposes of storage and/or transplantation to a recipient patient. The term "recipient" or "recipient patient" refers to an animal (human or non-human) into which an organ, tissue, islets, or cells are transferred.

The terms "organ rejection," "transplant rejection," or "rejection" are art- recognized and are used throughout the specification as a general term to describe the process of rejection of an organ in a recipient. Included within the definition are, for example, three main patterns of rejection that are commonly identified in clinical practice: hyperacute rejection, acute rejection, and chronic rejection (see, e.g., Oxford- Textbook of Surgery, Morris and Malt, Eds., Oxford University Press, 1994).

Ex vivo exposure of an organ to a pharmaceutical composition can occur by exposing the organ to an atmosphere comprising a gaseous pharmaceutical composition, to a liquid pharmaceutical composition, e.g., a liquid perfusate, storage solution, or wash solution containing the pharmaceutical composition, or to both.

For example, in the context of exposing an organ to a gaseous pharmaceutical composition comprising carbon monoxide, the exposure can be performed in any chamber or area suitable for creating an atmosphere that includes appropriate levels of carbon monoxide gas. Such chambers include, for example, incubators and chambers built for the purpose of accommodating an organ in a preservation solution. An appropriate chamber can be a chamber wherein only the gases fed into the chamber are present in the internal atmosphere, such that the concentration of carbon monoxide can be established and maintained at a given concentration and purity, e.g., where the chamber is airtight. For example, a CO₂ incubator can be used to expose an organ to a carbon monoxide composition, wherein carbon monoxide gas is supplied in a continuous flow from a vessel that contains the gas.

As another example, in the context of exposing an organ to an aqueous pharmaceutical composition, the exposure can be performed in any chamber or space having sufficient volume for submerging the organ, completely or partially, in an aqueous pharmaceutical composition. As yet another example, the organ can be exposed by placing the organ in any suitable container, and causing a liquid pharmaceutical composition to "wash over" the organ, such that the organ is exposed to a continuous flow of the composition.

As yet another example, in the context of pharmaceutical compositions comprising carbon monoxide, the organ can be placed, e.g., submerged, in a medium or solution that does not include carbon monoxide, and placed in a chamber such that the medium or solution can be made into a carbon monoxide composition via exposure to a carbon monoxide-containing atmosphere as described herein. As still another example, the organ can be submerged in a liquid that does not include carbon monoxide, and carbon monoxide can be "bubbled" into the liquid. An organ can be harvested from a donor and transplanted by any methods known to those of skill in the art (see, for example, Oxford Textbook of Surgery, Morris and Malt, Eds., Oxford University Press, 1994). The skilled practitioner will recognize that methods for transplanting and/or harvesting organs for transplantation can vary depending upon many circumstances, such as the age of the donor/recipient or the nature of the organ being transplanted.

Methods of culturing and preparing insulin-secreting cells (e.g., syngeneic or autologous insulin secreting cells) for transplantation are known to those skilled in the art. See, e.g., U.S. Patents No. 7,022,520; 6,967,019; 6,815,203; 6,703,017; 6,670,180; 6,642,003; 6,562,620; 6,326,201 ; 5,874,306; and 5,821,121 ; and U.S. Patent Applications 2006/0040387; 2006/0040385; 2005/0276794; 2005/0069529; 2005/0048032; 2004/0142901; 2004/01 10287; 2003/0228287; 2003/0138948; 2003/0109036; 2002/0172665; and 2002/00721 15. The methods can be used to produce syngeneic or autologous insulin-secreting cells, e.g., from a twin or the patient intended to receive the cells. The present invention contemplates that any or all of the above methods for exposing an organ to a pharmaceutical composition can be used in a given procedure, e.g., used in a single transplantation procedure.

EXAMPLES The invention is illustrated in part by the following examples, which are not to be taken as limiting the invention in any way.

Example 1. Induction of HO-I by Cobalt Protoporphyrin Alleviates Diabetes

Seven NOD mice that had developed overt diabetes, based on a determination that they were hyperglycemic (blood glucose >300 mg/dl) for three successive days, were treated for two weeks with 10 mg/kg Cobalt Protoporphyrin IX (CoPP) intraperitoneal^ (i.p.) three times per week. CoPP is an inducer of heme oxygenase-1 (HO-I). AU seven animals returned to normoglycemia by the end of the treatment and maintained the normal status for more than 200 days. This example demonstrates that induction of HO-I is effective to treat diabetes in an accepted animal model.

Example 2. Induction of HO-I Promotes Survival of Syngeneic Islet Grafts NOD mice with autoimmune diabetes reject syngeneic islet transplants, presumably as a consequence of autoimmunity (Kbulmanda et al., Xenotransplantation, 10: 178-184, 2002). NOD mice that had developed permanent diabetes, based on a determination that they were hyperglycemic (blood glucose >300 mg/dl) for two weeks, were transplanted with syngeneic islets from NOD-scid mice. The transplanted mice were treated three times per week with 10 mg/kg CoPP (i.p.) for two weeks starting at the time of the transplant. The animals became normoglycemic and remained normoglycemic for >62, >127, >127 days, respectively. Control NOD mice were transplanted with syngeneic islets from NOD-scid mice and administered a depleting anti-CD4 antibody or no treatment. Untreated mice remained normoglycemic due to the survival of the islet transplants for 7-21 days, whereas mice treated with an anti-CD4 antibody remained normoglycemic for 14-21 days. This example demonstrates that induction of HO-I is effective to prolong survival of syngeneic grafts of insulin- producing cells in an accepted model of diabetes.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with a detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the foil owing claims

Claims

WHAT IS CLAIMED IS:

1. A method of treating diabetes in a patient, comprising: administering to a patient suffering from or at risk for diabetes at least one treatment selected from the group consisting of: inducing heme oxygenase 1 (HO-I) in the patient; increasing the expression of HO-I in the patient; inducing apoferritin in the patient; increasing the expression of apoferritin in the patient; and administering to the patient a pharmaceutical composition comprising at least one of carbon monoxide, HO- 1, hemin, bilirubin, biliverdin, ferritin, iron, desferoxamine, salicylaldehyde isonicotinoyl hydrazone, iron dextran, and apoferritin; in an amount sufficient to treat diabetes, wherein the method does not include a transplantation step.

2. The method of claim 1, wherein the treatment comprises administering to the patient a pharmaceutical composition comprising carbon monoxide.

3. The method of claim 1, wherein the treatment comprises inducing HO-I in the patient.

4. The method of claim 3, wherein inducing HO-I in the patient comprises administering to the patient a pharmaceutical composition comprising cobalt protoporphyrin.

5. The method of claim 1, wherein the treatment comprises administering to the patient a pharmaceutical composition comprising HO-L

6. The method of claim I, wherein the treatment comprises administering to the patient a pharmaceutical composition comprising biliverdin.

7. The method of claim 1, wherein the treatment comprises administering to the patient a pharmaceutical composition comprising bilirubin.

8. The method of claim I₃ wherein the diabetes is type 1 diabetes.

9. The method of claim 1, further comprising administering to the patient a second treatment comprising an agent selected from the group consisting of an antiinflammatory agent, an immunosuppressive agent, insulin and an agent that mimics an effect of insulin.

10. The method of any one of claims 1 to 9, further comprising determining the level of heme oxygenase 1 (HO-I) activity, expression, or induction in the patient, in response to a stimulus.

1 1. The method of any one of claims 1 to 9, further comprising determining whether a heme oxygenase 1 (HO-I) promoter in the patient comprises a polymorphism.

12. A pharmaceutical composition comprising insulin and at least one compound selected from the group consisting of: cobalt protoporphyrin, carbon monoxide, a carbon monoxide-releasing compound, HO-I, hemin, bilirubin, biliverdin, ferritin, iron, desferoxamine, salicylaldehyde isonicotinoyl hydrazone, iron dextran and apoferritin.

13. The method of claim 1, wherein at least two treatments from the group are administered to the patient.

14. A method of transplanting insulin secreting cells to a diabetic patient comprising:

(a) identifying a patient suffering from or at risk for autoimmune diabetes;

(b) providing syngeneic or autologous insulin secreting cells;

(c) transplanting the cells into the patient; and

(d) before, during, or after step (c), administering to the patient at least one treatment selected from the group consisting of: inducing heme oxygenase 1 (HO-I) in the patient; expressing HO-I in the patient; inducing apoferritin in the patient; expressing apoferritin in the patient; and administering to the patient a pharmaceutical composition comprising at least one of carbon monoxide, a carbon monoxide-releasing compound, HO-I₃ hemin, bilirubin, biliverdin, ferritin, iron, desferoxamine, salicylaldehyde isonicotinoyl hydrazσne, iron dextran, and apoferritin, wherein the treatment administered to the patient in step (d) is sufficient to enhance survival or function of the insulin secreting cells after transplantation of the cells into the patient.

15. The method of claim 14, wherein the treatment comprises administering to the patient a pharmaceutical composition comprising carbon monoxide.

16. The method of claim 14, wherein the treatment comprises inducing HO-I in the patient.

17. The method of claim 16, wherein inducing HO-I in the patient comprises administering to the patient a pharmaceutical composition comprising cobalt protoporphyrin.

18. The method of claim 14, wherein the treatment comprises administering to the patient a pharmaceutical composition comprising HO-I .

19. The method of claim 14, wherein the treatment comprises administering to the patient a pharmaceutical composition comprising biliverdin.

20. The method of claim 14, wherein the treatment comprises administering to the patient a pharmaceutical composition comprising bilirubin.

21. The method of claim 14, wherein the insulin secreting cells are pancreatic islet cells.

22. The method of claim 14, further comprising administering to the patient a second treatment different from the first treatment, the second treatment comprising an anti-inflammatory agent or an immunosuppressive agent.

23. The use of an agent that induces peroxisome-proliferator-activated receptor gamma (PPARK) in a patient, increases the level of expression of PPAJRK in a patient, or acts as a PPARK agonist or toll-like receptor 4 (TLR4) antagonist in the patient, as a medicament for the treatment of prevention of diabetes.

24. The use of an agent that induces heme oxygenase 1 (HO-I) in the patient, increases the expression of HO-I in a patient; induces apoferritin in a patient; increases the expression of apoferritin in a patient as a medicament for use in combination with a pharmaceutical composition comprising at least one of carbon monoxide, HO-I, hemin, bilirubin, biliverdin, ferritin, iron, desferoxamine, salicylaldehyde isonicotinoyl hydrazone, iron dextran, and apoferritin; wherein the medicament is useful for the treatment or prevention of diabetes in a subject that will not undergo transplantation.

25. A pharmaceutical composition comprising insulin and at least one compound selected from the group consisting of: cobalt protoporphyrin, carbon monoxide, a carbon monoxide-releasing compound, HO-I, hemin, bilirubin, biliverdin, ferritin, iron, desferoxamine, salicylaldehyde isonicotinoyl hydrazone, iron dextran and apoferritin.

.

26. The use of an agent that increases heme oxygenase 1 (HO-I) in a patient or increases apoferritin in the patient in combination with at least one of: carbon monoxide, a carbon monoxide-releasing compound, HO-I, hemin, bilirubin, biliverdin, ferritin, iron, desferoxamine, salicylaldehyde isonicotinoyl hydrazone, iron dextran, and apoferritin as a medicament for the treatment of diabetes, wherein the medicament is administered to enhance survival or function of the insulin secreting cells after transplantation of the cells into the patient.