WO2008008513A2 - Combination treatments - Google Patents

Abstract

The present invention relates to the treatment of disorders using pharmaceutical agents in combination with heme oxygenase- 1 and/or heme degradation products, such as biliverdin or carbon monoxide.

Description

COMBINATION TREATMENTS

RELATED APPLICATIONS

This application claims priority to U.S. Application Serial No. 60/830480, entitled "Combination Treatments, filed on July 13, 2006, the contents of which are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to the treatment of conditions such as inflammatory disorders and to the potentiation of the effects of various pharmaceutical agents.

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. The release OfFe²⁺ 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.

SUMMARY

The present application is based, in part, on the discovery that HO-I and products of heme degradation can be used to potentiate the activity of various pharmaceutical agents, including anti-inflammatory agents.

Accordingly, in one aspect, the application features a method of treating, e.g., reducing, inflammation in a patient. The method includes administering to a patient diagnosed as sufFering from or at risk for inflammation: (i) a first pharmaceutical composition comprising an anti-inflammatory agent, and (ii) a second treatment selected from: inducing HO-I and/or apoferritin in the patient using a suitable inducer other than the anti- inflammatory agent; increasing the expression of HO-I and/or apoferritin in the patient; and administering a second pharmaceutical composition comprising HO-I, hemin, CO, a CO-releasing compound, bilirubin, biliverdin, ferritin, iron, desferoxamine, salicylaldehyde isonicotinoyl hydrazone, iron dextran, and/or apoferritin, in amounts sufficient to reduce inflammation.

In any of the methods described herein, the inflammation can be associated with asthma, adult respiratory distress syndrome, interstitial pulmonary fibrosis, pulmonary emboli, chronic obstructive pulmonary disease, primary pulmonary hypertension, chronic pulmonary emphysema, congestive heart failure, peripheral vascular disease, stroke, atherosclerosis, ischemia-reperfusion injury, heart attacks, glomerulonephritis, conditions involving inflammation of the kidney, infection of the genitourinary tract, viral and toxic hepatitis, cirrhosis, ileus, necrotizing enterocolitis, specific and nonspecific inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, cancer, deficient wound healing, infection, sepsis, Alzheimer's disease, Parkinson's disease, graft versus host disease, and/or hemorrhagic, septic, or anaphylactic shock. The inflammation can be inflammation of the heart, lung, liver, pancreas, joints, eye, bronchi, spleen, brain, skin, and/or kidney. The inflammation can also be an inflammatory condition localized in the respiratory tract or the gastrointestinal tract, e.g., asthma, bronchitis, sinusitis, pneumonia, amoebic dysentery, bacillary dysentery, schistosomiasis, Campylobacter enterocolitis, yersinia enterocolitis, enterobius vermicularis, radiation enterocolitis, ischaemic colitis, eosinophilic gastroenteritis, ulcerative colitis, indeterminate colitis, and Crohn's disease. Alternatively, it can be a systemic inflammation.

In another aspect, the application features a method of transplanting an organ, tissue, or cells, which includes administering to a donor (or to an organ of the donor in si(u) a first pharmaceutical composition comprising an anti-inflammatory agent and/or an immunosuppressive agent, in combination with administering at least one treatment selected from: inducing HO-I and/or apoferritin in the donor using a suitable inducer other than the first pharmaceutical composition; increasing the expression of HO-I and/or apoferritin in the donor; and administering a second pharmaceutical composition comprising CO, a CO-releasing compound, HO-I, hemin, bilirubin, biliverdin, ferritin, iron, DFO, SIH, iron dextran, and/or apoferritin to the donor; and transplanting an organ tissue or cells of the donor into a recipient, wherein the anti-inflammatory agent and/or immunosuppressive agent and treatment administered are sufficient to enhance survival or function of the transplant after transplantation into the recipient.

In still another aspect, the application features a method of transplanting an organ, tissue, or cells, which includes (a) providing an organ, tissue, or cells of a donor; (b) administering to the organ, tissue, or cells ex vivo a first pharmaceutical composition comprising an anti-inflammatory agent and/or an immunosuppressive agent, in combination with administering at least one treatment selected from: inducing HO-I and/or apoferritin in the organ, tissue, or cells using a suitable inducer other than the first pharmaceutical composition; expressing HO-I and/or apoferritin in the organ, tissue, or cells, and administering a second pharmaceutical composition comprising CO, a CO- releasing compound, HO-I, hemin, bilirubin, biliverdin, ferritin, DFO, SEH, iron dextran, and/or apoferritin; and (c) transplanting the organ, tissue, or cells into a recipient, wherein the anti-inflammatory /immunosuppressive agent and the treatment administered to the organ are sufficient to enhance survival or function of the transplant after transpl antati on .

In yet another aspect, the application features a method of transplanting an organ, tissue, or cells, which includes providing an organ, tissue or cells from a donor, transplanting the organ, tissue or cells into a recipient, and before, during, or after the transplanting step, administering to the recipient a first pharmaceutical composition comprising an anti-inflammatory agent and/or an immunosuppressive agent, in combination with administering at least one treatment selected from: inducing HO-I and/or apoferritin in the recipient using a suitable inducer other than the first pharmaceutical composition; increasing the expression of HO-I and/or apoferritin in the recipient; and administering a second pharmaceutical composition comprising CO, a CO-releasing compound, HO-I, hemin, bilirubin, biliverdin, ferritin, DFO, SIH, iron dextran, and/or apoferritin; wherein the anti-inflammatory agent and/or immunosuppressive agent and the treatment administered to the recipient are, in combination, sufficient to enhance survival or function of the organ after transplantation of the organ to the recipient. In transplantation methods, the first pharmaceutical composition can be administered in any one, two, or three of the following manners: (1) treatment of the donor prior to and/or during removal of the organ; (2) treatment of the organ ex vivo; and (3) treatment of the recipient prior to, during, or after transplant of the organ. The second treatment described herein (e.g., induction of HO-I, administration of CO, etc.) can be administered at the same time as, before, or after the first pharmaceutical composition. For example, the first pharmaceutical composition and CO could be administered to the donor, followed by bathing the organ in a biliverdin or bilirubin solution, followed by administration of the first pharmaceutical composition and apoferritin to the recipient. All other specific combinations and permutations of this method are contemplated, though not specifically listed herein.

In another aspect, the application provides a method of performing angioplasty on a patient, which includes performing angioplasty on the patient; and before, during, or after the performing step, administering to the patient a first pharmaceutical composition comprising an anti-inflammatory agent, in combination with administration of a second treatment selected from: inducing HO-I and/or apoferritin in the recipient using a suitable inducer other than the first pharmaceutical composition; increasing the expression of HO-I and/or apoferritin in the patient; and administering a second pharmaceutical composition comprising CO, a CO-releasing compound, HO-I, hemin, bilirubin, biliverdin, ferritin, DFO, SIH, iron dextran and/or apoferritin. The first pharmaceutical composition and second treatment are administered in an amount sufficient to reduce (e.g., prevent, at least in part) intimal hyperplasia in the patient. The angioplasty can be any angioplasty procedure, e.g., balloon angioplasty; laser angioplasty; atherectomy, e.g., directional atherectomy, rotational atherectomy, or extraction atherectomy; and/or any angioplasty procedure using a stent, or any combination of such procedures. One or both of the first pharmaceutical composition and second treatment can be administered via an implantable device, such as a stent. In yet another aspect, the application provides a method of treating (e.g., preventing or decreasing) restenosis or intimal hyperplasia in a patient. The method includes administering to a patient diagnosed as suffering from or at risk for restenosis or intimal hyperplasia: (i) a first pharmaceutical composition comprising an antiinflammatory agent, and (ii) a second treatment selected from inducing HO-I and/or apoferritin in the patient using a suitable inducer other than an anti-inflammatory agent; increasing the expression of HO-I and/or apoferritin in the patient; and administering a second pharmaceutical composition comprising HO-I, hemin, CO, a CO-releasing compound, bilirubin, biliverdin, ferritin, iron, DFO, SIH, iron dextran, and/or apoferritin, in amounts sufficient to treat restenosis or intimal hyperplasia. The intimal hyperplasia or restenosis can arise from any cause, e.g., balloon angioplasty; laser angioplasty; atherectomy, e.g., directional atherectomy, rotational atherectomy, or extraction atherectomy; and/or an angioplasty procedure using a stent, or any combination of such procedures. In still another aspect, the application features a method of performing surgery

(e.g., other than transplant surgery) e.g., vascular and/or abdominal surgery, on a patient, which includes performing surgery on the patient; and before, during, and/or after performing the surgery, administering to the patient a first pharmaceutical composition comprising an anti-inflammatory agent, in combination with administering at least one treatment selected from: inducing HO-I and/or apoferritin in the recipient using a suitable inducer other than the first pharmaceutical composition; increasing the expression of HO-I and/or apoferritin in the patient; and administering a second pharmaceutical composition comprising CO, a CO-releasing compound, HO-I, hemin, bilirubin, biliverdin, ferritin, DFO, SIH, iron dextran, and/or apoferritin. In other aspects, the application features a method of treating a cellular proliferative and/or differentiative disorder (e.g., naturally arising cancer) in a patient, which includes identifying a patient suffering from or at risk for a cellular proliferative and/or differentiative disorder (e.g., naturally arising cancer); and administering to the patient a first pharmaceutical composition comprising an anti-inflammatory agent in combination with administering at least one treatment selected from: inducing HO-I and/or apoferritin in the recipient using a suitable inducer other than the first pharmaceutical composition; increasing the expression of HO-I and/or apoferritin in the patient, and administering a second pharmaceutical composition comprising CO, a CO- releasing compound, HO-I, hemin, bilirubin, biliverdin, ferritin, DFO, SIH, iron dextran, and/or apoferritin to the patient, in amounts sufficient to treat the cellular proliferative and/or differentiative disorder.

In cancer treatment methods described herein, the cancer can be any type of cancer. The cancer can be cancer found in any part(s) of the patent's body, e.g., cancer of the stomach, small intestine, colon, rectum, mouth/pharynx, esophagus, larynx, liver, pancreas, lung, breast, cervix uteri, corpus uteri, ovary, prostate, testis, bladder, skin, kidney, brain/central nervous system, head, neck, throat, bone, or any combination thereof. It can be a hematopoietic disorder, such as a leukemia or lymphoma. For cancer treatments, the methods can be used alone or in combination with other methods for treating cancer in patients. Accordingly, in another embodiment, the methods described herein can include treating the patient using surgery (e.g., to remove a tumor or portion thereof), chemotherapy, immunotherapy, gene therapy, and/or radiation therapy. Treatments described herein can be administered to a patient at any point, e.g., before, during, and/or after the surgery, chemotherapy, immunotherapy, gene therapy, and/or radiation therapy.

In another aspect, the application features a method of treating unwanted angiogenesis in a patient. The method includes administering to a patient diagnosed as suffering from or at risk for unwanted angiogenesis: (i) a first pharmaceutical composition comprising an anti-inflammatory agent and/or angiogenesis inhibitor, and (ii) a second treatment selected from inducing HO-I and/or apoferritin in the patient using a suitable inducer other than the anti-inflammatory agent, increasing the expression of HO-I and/or apoferritin in the patient, and administering a second pharmaceutical composition comprising HO-I, hemin, CO, a CO-releasing compound, bilirubin, biliverdin, ferritin, iron, DFO, SIH₃ iron dextran, and/or apoferritin, in amounts sufficient to treat unwanted angiogenesis.

In still another aspect, the application features a method of treating hepatitis in a patient. The method includes administering to a patient diagnosed as suffering from or at risk for hepatitis: (i) a first pharmaceutical composition comprising an anti- inflammatory agent, and (ii) a second treatment selected from inducing HO-I and/or apoferritin in the patient using a suitable inducer other than the anti-inflammatory agent, increasing the expression of HO-I and/or apoferritin in the patient, and administering a second pharmaceutical composition comprising HO-I, hemin, CO, a CO-releasing compound, bilirubin, biliverdin, ferritin, iron, DFO, SIH, iron dextran, and/or apoferritin, in amounts sufficient to treat hepatitis.

The hepatitis can be the result of, or a person may be considered at risk for hepatitis because of, any of a number of factors, e.g., infections, e.g., viral infections, e.g., infection with hepatitis A, B, C₅ D, E and/or G virus; alcohol use (e.g., alcoholism); drug use (e.g., one or more drugs described herein, e.g., acetaminophen, anesthetics, anti-tuberculosis drugs, antifungal agents, antidiabetic drugs, neuroleptic agents, and drugs used to treat HTV infection and AIDS); autoimmune conditions (e.g., autoimmune hepatitis); and/or surgical procedures. In yet another aspect, the application features a method of reducing the effects of ischemia in a patient, which includes identifying a patient suffering from or at risk for ischemia; and administering to the patient a first pharmaceutical composition comprising an anti-inflammatory agent in combination with administering at least one second treatment selected from: inducing HO-I and/or apoferritin in the recipient, expressing HO-I and/or apoferritin in the patient, and administering a second pharmaceutical composition comprising CO, a CO-rel easing compound, HO-I, hemin, bilirubin, biliverdin, ferritin, DFO, SIH, iron dextran, and/or apoferritin to the patient, in amounts sufficient to reduce the effects of ischemia. In another aspect, the application features a method of potentiating the response of a patient to a first pharmaceutical agent (e.g., a pharmaceutical agent disclosed herein). The method includes administering to a patient: (i) a first pharmaceutical composition comprising a pharmaceutical agent, and (iϊ) a second treatment selected from inducing HO-I and/or apoferritin in the patient using a suitable inducer, expressing HO-I and/or apoferritin in the patient, and administering a second pharmaceutical composition comprising HO-I, hemin, CO, a CO-releasing compound, bilirubin, biliverdin, ferritin, iron, desferoxamine, salicylaldehyde isonicotinoyl hydrazone, iron dextran, and/or apoferritin, in amounts sufficient to potentiate the response of the patient to the pharmaceutical agent. The method can permit a response in the patient greater than that seen without the second treatment, e.g., in a patient that responds poorly to the pharmaceutical agent. Alternatively or in addition, the method may permit administering the pharmaceutical composition at a dosage level lower than would be effective in the absence of the second treatment, e.g., a dosage level that provides reduced number or extent of unwanted side effects. For example, the method may permit administering the pharmaceutical agent at a dosage level at least 2-fold, e.g., 5- fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or 2000-fold lower than would be effective in the absence of the second treatment.

In yet another aspect, the application features a method of potentiating the efficacy of a vaccine composition. The method includes administering to a patient (i) a vaccine composition comprising an immunogenic agent, and (ii) a second treatment selected from inducing HO-I and/or apoferritin in the patient using a. suitable inducer, expressing HO-I and/or apoferritin in the patient, and administering a second pharmaceutical composition comprising HO-I, hemin, CO, a CO-releasing compound, bilirubin, biliverdin, ferritin, iron, desferoxamine, salicylaldehyde isonicotinoyl hydrazone, iron dextran, and/or apoferritin, in amounts sufficient to potentiate the response of the patient to the vaccine composition.

In any of the methods described herein, the pharmaceutical agent or anti- inflammatory agent can be a statin, adenosine or an adenosine A2a receptor (A2aR) agonist, a cyclooxygenase inhibitor (e.g., a general cyclooxygenase inhibitor or a specific inhibitor of COX-I or COX-2), probucol, a lipoxin, a steroid, or a prostaglandin.

Any of the methods described herein can further include determining an HO- ϊ parameter of the subject. The HO-I parameter can be HO-I activity, HO-I expression, level or presence of HO-I induction in response to a stimulus, or an allele of a polymorphism in the promoter of HO-I (e.g., an allele associated with modified (e.g., increased or reduced) 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., the number or length of repeats of a nucleotide sequence (e.g., (GT)_n in the promoter of HO-I).

Pharmaceutical compositions can be in gaseous, liquid, or solid form, and can be administered to the patient by any method known in the art for administering gases and liquids to patients, e.g., via inhalation, insufflation, infusion, injection, and/or ingestion. The pharmaceutical composition can be in gaseous, powder, or liquid form (e.g., in the form of a mist or spray), and administered to the patient by inhalation. If in liquid or solid form, the pharmaceutical composition can be administered to the patient orally. The pharmaceutical composition can be in gaseous, solid, and/or liquid form, and administered topically to an organ of the patient and/or directly to the abdominal cavity of the patient. The pharmaceutical composition can be administered to the patient using an extracorporeal membrane gas exchange device or artificial lung.

Also described herein is the use of an anti-inflammatory and/or immunosuppressive agent along with CO, a CO-releasing compound, HO-I, hemin, bilirubin, biliverdin, ferritin, DFO, SIH, iron dextran, apoferritin, and/or an inducer of HO-I and/or apoferritin in the manufacture of a medicament for treatment or prevention of a condition described herein. The medicament can be in any form described herein, e.g., a liquid, gaseous, or solid composition. The term "pharmaceutical composition" is used throughout the specification to describe a gaseous, liquid, or solid composition containing an active ingredient, e.g., an agent described herein, that can be administered to a patient and/or an organ. The invention contemplates use of any two, three, four, five, six, seven or eight 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, rodent or non-rodent, to whom treatment according to the methods of the present invention is provided. Veterinary applications are clearly contemplated by the present invention. The term includes but is not limited to birds, reptiles, amphibians, and mammals, e.g., humans, other primates, pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep and goats. Preferred subjects are humans, farm animals, and domestic pets such as cats and dogs. The term "treat(ment)" is used herein to describe delaying the onset of, inhibiting, or alleviating the effects of a disease or condition, e.g., a disease or condition described herein. Skilled practitioners will appreciate that a patient can be diagnosed by a physician (or veterinarian, as appropriate for the patient being diagnosed) as suffering from or at risk for a condition described herein by any method known in the art, e.g., by assessing a patient's medical history, performing diagnostic tests, and/or by employing imaging techniques. The compositions described herein can be administered (and/or administration can be supervised) by any person, e.g., a health-care professional, veterinarian, or caretaker (e.g., an animal (e.g., dog or cat) owner), depending upon the patient to be treated, and/or by the patient him/herself, if the patient is capable of self- administration.

The terms "effective amount" and "effective to treat," as used herein, refer to an amount or concentration of active ingredients (e.g., an anti-inflammatory agent and at least one of: CO, a CO-releasing compound, HO-I, hemin, apoferritin (or an inducer of HO-I or apoferritin), bilirubin, and biliverdin) utilized for a period of time (including acute or chronic administration and periodic or continuous administration) that is effective within the context of its administration for causing an intended effect or physiological outcome. For example, an effective amount of a gaseous composition comprising CO is, e.g., an amount capable of reducing inflammation.

As used herein, the terms "inflammatory agent," "immunosuppressive agent," and "pharmaceutical agent" do not encompass HO-I, products of heme degradation (e.g., CO, biliverdin, bilirubin, iron, and ferritin), CO-releasing compounds, hemin, nitric oxide, hydrogen peroxide, COCb, desferoxamine, salicylaldehyde isonicotinoyl hydrazone, iron protoporphyrin, cobalt protoporphyrin, iron dextran, and apoferritin.

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.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

DESCRIPTION OF DRAWINGS FIG. l is a chart depicting TNFI inhibition by adenosine and 5'-(N- ethylcarboxamido)adenosine (NECA) in cells overexpressing HO-I (HO-I RAW264.7) or a vector control (NEO RAW264.7). Data are presented as mean ± SEM; *: p < 0.01.

FIG. 2 is a chart depicting TNFI inhibition by NECA at 10 TM and 10 nM in cells infected with adenovirus overexpressing HO-I (Ad HO-I), control adenovirus expressing EGFP (Ad EGFP), and wild-type cells. Data are presented as mean ± SEM; **: p < 0.01 ; *: p < 0.05.

FIG. 3 is a chart depicting fold induction of adenosine receptors A2a, A2b, and A3 in cells overexpressing HO-I (HO-I RAW264.7) or a vector control (NEO RAW264.7). Data are presented as mean ± SEM; *: p < 0.05 FIG. 4 is a line graph depicting percent survival of mice over time for four treatments: Iipopolysaccharide and D-galactosamine (LPS/GalN), LPS/GalN + NECA₃ LPS/GalN + cobalt protoporphyrin (CoPP), and LPS/GalN + NECA + CoPP. DETAILED DESCRIPTION

Use of Heme Oxygenase- 1 and Products of Heme Degradation

The present application includes providing to a patient heme oxygenase- 1 (HO- 1), in conjunction with administering an agent described herein, e.g., an anti- inflammatory agent, by administering exogenously-produced HO-I protein to the patient, by inducing HO-I expression in the patient, and/or by expressing an exogenously-introduced gene encoding HO-I in the patient, to treat various diseases or conditions, and/or to improve the outcome of various surgical procedures, e.g., transplantation procedures. 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 the treatment or prevention of various diseases or conditions, and/or to improve the outcome of various surgical procedures, e.g., transplantation procedures.

Optionally, HO-I can be provided to a patient in conjunction with administration of an agent described herein, e.g., an anti-inflammatory agent, along with any or all of the products of heme degradation, e.g., carbon monoxide (CO), biliverdin, bilirubin, iron, and ferritin. Alternatively, any or all of the products of heme degradation can be provided to the patient, along with an agent described herein, e.g., an anti-inflammatory agent, without providing HO-I to the patient. 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 apoferritin, 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, 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). 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 invention contemplates that HO-I can be expressed in a patient via gene transfer. As used herein, the term "express(ed)" means to cause increased production of a protein, e.g., HO-I or apoferritin, in the body of a patient using an exogenously administered gene (e.g., a recombinant gene). The HO-I or apoferritin 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 apoferritin 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 apoferritin can be administered, e.g., as naked DNA, in liposomes, or in microparticles.

Further, exogenous HO-] 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 to be transplanted. 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 carbon monoxide, iron, biliverdin, bilirubin and (apo)ferritin. Any of the above 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 can be administered to patients in aqueous or solid forms. Biliverdin and bilirubin useful in the methods of the invention 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 Tmols/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 μmols/kg/day, 20 to 200 μmols/kg/day, and 25 to 100 μmols/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 μmols/L, e.g., at least about 50 to about 200 μmols/L, or about 50 to about 100 μmols/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 mg/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 trie application. Acute, sub-acute, and chronic administration of pharmaceutical compositions comprising biliverdin and/or bilirubin are contemplated by the present invention, 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 the treatment or prevention of various diseases or disorders described herein. In another embodiment, bilirubin or biliverdin is used as medicament (or in the preparation of a medicament) for improving the outcome of a surgical procedure.

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 al., Am. J. Physiol. Lung Cell MoI. Physiol. 279:L1029-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, e.g., iron or iron dextran, useful in the methods of the invention 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 or ferritin, e.g., H-chain apoferritin 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 for treatment of a condition described herein. Further, exogenous ferritin or apoferritin can be directly administered to a patient by any method known in the art. Exogenous 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-fusϊon protein (see, e.g., Becker-Hapak el 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) (see, e.g., Blaha et aL, Blood 91(1 1):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. 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 treatment or prevention of various diseases or disorders or for improving the outcome of a surgical procedure.

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, He, NO, CO2, O2, N2, 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. 5 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 carbon0 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 appropriate levels of5 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 an0 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, gas5 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 ef 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 theQ 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, trtcarbonyldichlororuthenium (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 gastrointestinal tract. 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, subacute 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 et al, Artif. Organs 18(l l):806-812, 1994; and Golob et ai, 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 laproscopic 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 the treatment or prevention of various diseases or disorders or for improving the outcome of a surgical procedure. 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 in surgical procedures, to treat the disorders or conditions described herein, and or as a medicament to treat or prevent the disorders or conditions described herein, and/or as a medicament to improve the outcome of a surgical procedure. 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 plus CO, or bilirubin plus apoferritinj or two or more inducers of HO-I , in combination with a pharmaceutical agent.

Treatment of Patients with Pharmaceutical Compositions A patient can be treated with pharmaceutical compositions described herein or a medicament may be formulated 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., 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™ emulsifier (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 physiologicially- 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, PrimogeB, 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, polyglycolic 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 systemicaϊly 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% Na)- 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 small intestine (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 1):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. Aqueous 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, to treat the interior of the gastrointestinal tract or any portion thereof. Further, liquids, e.g., saline solutions, are often 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 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, 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 gaseous pharmaceutical compositions directly to the interior of the gastrointestinal tract, or any portion thereof. 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.

Anti-Inflammatory. Immunosuppressant, and Other Pharmaceutical Agents

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

Agents useful in the methods described herein include steroidal antiinflammatory agents, non-steroidal anti-inflammatory agents (NSAIDS), cyclooxygenase inhibitors (e.g., general cyclooxygenase inhibitors such as aspirin or specific inhibitors of COX-I or COX-2), statins, adenosine and adenosine A2a receptor (A2aR) agonists, probucol (and its derivatives and similar therapeutics), antiinflammatory cytokines ( e.g., BL-IO), prostaglandins, VEGF (and compounds that mimic VEGF), alcohol, tannins, heat shock proteins, immunosuppressants (e.g., rapamycin and cyclosporin), interferons ( e.g., interferon gamma), antihypertensives (e.g., bosentan, sildenafil and epoprostenol), estrogen and estrogen derivatives, antifungals (e.g., fluconozole and nystatin), TNFI inhibitors (e.g., infliximab), antiviral agents, chloroquine, isonϊazid, antibiotics, modulators of factors that regulate HO-I, compounds that release nitric oxide and/or modulate guanylate cyclase pathways and vaccine formulations. 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, budesonide, fluticasone, dexamethasone, betamethasone, triamcinolone, beclometasorte, fludrocortisone acetate, deoxycorticosterone acetate and aldosterone.

Exemplary NSADDS include acetaminophen; salicylates, e.g., aspirin, methyl salicylate and diflunisal; arylalkanoic acids, e.g., diclofenac, indomethacin, and sulindac; 2-arylpropionic acids, e.g., ibuprofen, ketoprofen, naproxen, carprofen, fenoprofen, and ketorolac; N-arylanthranilic acids, e.g., mefenamic acid; oxicams, such as piroxicam and meloxicam; sulfonanilides, e.g., nimesulide; and COX-2 inhibitors, e.g., celecoxib, rofecoxib, valdecoxib, parecoxib and etoricoxib. The methods described herein can be used to reduce the effective dose of COX-2 inhibitors, thereby reducing the number, severity, or risk of unwanted side effects. 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-y])adenosine, regadenoson, 2-p- (2-carboxyethyl)phenethylamino-5'-N-ethyicarboxamidoadenosine (CGS-21680), ATL-146e, ATL-313 and 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 (l₃l-dimethylethyl)phenyl] ester]. .

Exemplary prostaglandins include prostaglandin J2 and its derivatives and analogs such as 15-deoxy-Δ^12>14-prostaglandin J2, Δ¹ ^prostaglandin h and 9,10-dihydro- ] 5-deoxy-Δ¹²'¹⁴-prostaglandin J2.

Exemplary immunosuppressants include alkylating agents (e.g., cyclophosphamide, nitrosourea and platinum compounds); antimetabolites (e.g., folic acid analogs (e.g., methotrexate), purine analogs (e.g., azathioprine and mercaptopurine), pyrimidine analogs, and protein synthesis inhibitors); cytostatics, (e.g., dactinomycin, anthracycHnes, mitomycin C, bleomycin, and mitramycin); immunosuppressive antibodies (e.g., antibodies against EL-2 receptor and T-cell receptors); cyclosporin; tacrolimus (FK-506); rapamycin; anti-TNFI agents; and mycophenolate mofetil. Exemplary antiviral agents include amantadine, rimantadine, zanamavir, oseJtamivir and azidothymidine (AZT).

Exemplary HO-I regulators include nrf2, HIF- lα, STATs, SMADs, and CEBP. Exemplary NO/guanylate cyclase modulators include NO, nitroprusside, nitrite, nitroglycerin, sildenafil citrate and other phosphodiesterase inhibitors. Exemplary antibiotics include aminoglycosides, such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, and tobramycin; carbacephems, such as loracarbef; carbapenems, such as ertapenem, imipenem, cilastatin, and meropenem; first, second, third, and fourth generation cephalosporins, such as cefadroxil, cefazolin, cephalexin, cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperzone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, and cefepime; glycopeptides, such as teicoplanin and vancomycin; macrolides, such as azithromycin, clarithromycin, dirithromycin, erythromycin, and ^oleandomycin; monobactams, such as aztreonam; penicillins, such as amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, fiucl oxacillin, mezlocillin, nafcillin, penicillin, piperacillin, and ticarcillin; polypeptides, such as bacitracin, colistin, and polymyxin B; quinolones, such as ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, and trovafloxacin; sulfonamides, such as mafenide, sulfacetamide, sulfamethizole, sulfasalazine, sulfisoxazole, and trimethoprim; tetracyclines, such as demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline; and others such as chloramphenicol, clindamycin, ethambutol, fosfomycin, furazolidone, isonazid, linezolid, metronidazole, nitrofurantoin, pyrazinamide, quinupristin, dalfopristin, rifampin and spectinomycin.

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 an allele of a polymorphism in the promoter of HO-I (e.g., an allele associated with modified HO-I induction in response to a stimulus) can be observed in the patient. A decision whether to provide a second 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)₅ immunofluorescence, protein microarray, 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. Individuals with lower than average expression of HO-I, e.g., 80%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5% or less than average expression, can be administered a second treatment described herein. 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, COCI₂, 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. CHn. 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 second treatment described herein.

The presence or absence of an allele 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- 1). In some instances, the allele can be associated with the activity of HO-I₃ the expression of HO-I, or the level or strength of HO-I induction in response to a stimulus. An individual with one or more alleles associated with a reduced level of activity of HO-I , expression of HO-I, or induction of HO-I in response to at stimulus, e.g., 80%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5% or less than average, can be administered a second treatment described herein.

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^I-AGAGCCTGCAGCTTCTCAGA-3^I (SEQ ID NO: 1) and 5^I-ACAAAGTCTGGCCATAGGAC-3^I (SEQ ID NO:2) (Kaneda et αl., Arterioscler. Thromb. Vase. Biol., 22:1680-5, 2002) or 5'- ATCACACCCAGAGCCTGC AGC-3' (SEQ ID NO:3) and 5^I-GGGGTGGAGAGGAGCAGTCAT-3^I (SEQ ID NO:4) (Li et αl., Chin. Med. J. (Engl)., 1 18:1285-90, 2005). Shorter repeats are associated with greater up-regulation of HO-I in response to stimuli (Schillinger et ai, 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 second treatment described herein.

Disorders and Conditions

The methods and/or medicaments of the present invention can be used to treat one or more of the following inflammatory, respiratory, cardiovascular, renal, hepatobiliary, reproductive or gastrointestinal disorders; shock; or cellular proliferative and/or differentiative disorders; to reduce the effects of ischemia; and to aid in wound healing.

When the terms "prevent," "preventing," and "prevention" 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 Jesser 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 during 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.

Inflammatory Disorders

The methods of the present invention can be used to prevent or treat inflammatory disorders. The terms "inflammatory disorder(s)" and "inflammation" are used to describe the fundamental pathological process consisting of a dynamic complex of reactions (which can be recognized based on cytologic and histologic studies) that occur in the affected blood vessels and adjacent tissues in response to an injury or abnormal stimulation caused by a physical, chemical or biologic agent, including the local reactions and resulting morphologic changes, the destruction or removal of the injurious material, and the responses that lead to repair and healing. Inflammation is characterized in some instances by the infiltration of immune cells (e.g., monocytes/macrophages, natural killer cells, lymphocytes (e.g., B and T lymphocytes)) and/or glial cells. In addition, inflamed tissue may contain cytokines and chemokines that are produced by the cells that have infiltrated into the area. Often, inflammation is accompanied by thrombosis, including both coagulation and platelet aggregation. The term inflammation includes various types of inflammation such as acute, chronic, allergic (including conditions involving mast cells), alterative (degenerative), atrophic, catarrhal (most frequently in the respiratory tract), croupous, fibrinopurulent, fibrinous, immune, hyperplastic or proliferative, subacute, serous and serofibrinous inflammation. Inflammation localized in the gastrointestinal tract, or any portion thereof, liver, heart, skin, spleen, brain, kidney, pancreas, bladder, or the respiratory tract can be treated with the methods of the present invention. Inflammation associated with shock, e.g., septic shock, hemorrhagic shock caused by any type of trauma, and anaphylactic shock can also be treated. Further, it is contemplated that the methods of the present invention could be used to treat rheumatoid arthritis, lupus, and other inflammatory and/or autoimmune diseases; heightened inflammatory states due to immunodeficiency, e.g., due to infection with HIV; and hypersensitivities. Wound Healing

Based on the anti-inflammatory properties of HO-I and heme degradation products, the present invention contemplates that the methods described herein can be used to promote wound healing (e.g., in transplanted, lacerated (e.g., due to surgery), or burned skin). Although these methods typically involve administering the treatments described herein directly to the wound (e.g., as a wound dressing, lotion, or ointment), , they can alternatively or in addition be delivered systemically, before, during, or after the wounding event.

Respiratory Disorders

Examples of respiratory conditions include, but are not limited to asthma; Acute Respiratory Distress Syndrome (ARDS), e.g., ARDS caused by peritonitis, pneumonia (bacterial or viral), or trauma; idiopathic pulmonary diseases; interstitial lung diseases, e.g., Interstitial Pulmonary Fibrosis (IPF); pulmonary emboli; Chronic Obstructive Pulmonary Disease (COPD); emphysema; bronchitis; cystic fibrosis; lung cancer of any type; lung injury, e.g., hyperoxic lung injury; Primary Pulmonary Hypertension (PPH); secondary pulmonary hypertension; and sleep-related respiratory disorders, e.g., sleep apnea. Cardiovascular Disorders

Cardiovascular disorders include disorders involving the cardiovascular system, e.g., the heart, the blood vessels, and/or the blood. A cardiovascular disorder can be caused, for example, by an imbalance in arterial pressure, a malfunction of the heart, or an occlusion of a blood vessel, e.g., by a thrombus. Examples of such disorders include congestive heart failure, hypertension, peripheral vascular disease, pulmonary vascular thrombotic diseases such as pulmonary embolism, stroke, ischemia-reperfusion (I/R) injury to the heart, atherosclerosis, and heart attacks. Renal Disorders Disorders involving the kidney include but are not limited to pathologies of glomerular injury such as in situ immune complex deposition and cell-mediated immunity in glomerulonephritis, damage caused by activation of alternative complement pathway, epithelial cell injury, and pathologies involving mediators of glomerular injury including cellular and soluble mediators, acute glomerulonephritis, such as acute proliferative (poststreptococcal, postinfectious) glomerulonephritis, e.g., poststreptococcal glomerulonephritis and nonstreptococcal acute glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, membranous glomerulonephritis (membranous nephropathy), minimal change disease (lipoid nephrosis), focal segmental glomerulosclerosis, membranoproliferative glomerulonephritis, IgA nephropathy (Berger disease), focal proliferative and necrotizing glomerulonephritis (focal glomerulonephritis) and chronic glomerulonephritis. Disorders of the kidney also include infections of the genitourinary tract.

Hepatobiliary Disorders

Disorders involving the liver include but are not limited to hepatitis, cirrhosis and infectious disorders. Causative agents of hepatitis include, for example, infections, e.g., infection with specific hepatitis viruses, e.g., hepatitis A, B, C, D, E, and G viruses; or hepatotoxic agents, e.g., hepatotoxic drugs (e.g., isoniazid, methyldopa, acetaminophen, amiodarone, and nitrofurantoin), and toxins (e.g., endotoxin or environmental toxins). Hepatitis may occur postoperatively in liver transplantation patients. Further examples of drugs and toxins that may cause hepatitis (i.e., hepatotoxic agents) are described in Feldman: Sleisenger & Fordtran's Gastrointestinal and Liver Disease, 7th ed., Chapter 17 (Liver Disease Caused by Drugs, Anesthetics, and Toxins), the contents of which are expressly incorporated herein by reference in their entirety. Such examples include, but are not limited to, methyldopa and phenytoin, barbiturates, e.g., phenobarbital; sulfonamides (e.g., in combination drugs such as co-trim oxazole (sulfamethoxazole and trimethoprim); sulfasalazine; salicylates; disulfiram; β-adrenergic blocking agents e.g., acebutolol, labetalol, and metoprolol); calcium channel blockers, e.g., nifedipine, verapamil, and diltiazem; synthetic retinoids, e.g., etretinate; gastric acid suppression drugs e.g., oxmetidine, ebrotidine, cimetidine, ranitidine, omeprazole and famotidine; leukotriene receptor antagonists, e.g., zafirlukast; anti-tuberculosis drugs, e.g., rifampicin and pyrazinamide; antifungal agents, e.g., ketoconazole, terbinafϊne, fluconazole, and itraconazole; antidiabetic drugs, e.g., thiazolidinediones, e.g., troglitazone and rosiglitazone; drugs used in neurologic disorders, e.g., neuroleptic agents, antidepressants (e.g., fluoxetine, paroxetine, venlafaxine, trazodone, tolcapone, and nefazodone), hypnotics (e.g., alpidem, Zolpidem, and bentazepam), and other drugs, e.g., tacrine, dantrolene, riluzole, tizanidine, and alverine; nonsteroidal antiinflammatory drugs, e.g., bromfenac; COX-2 inhibitors; cyproterone acetate; leflunomide; antiviral agents, e.g., fialuridine, didanosine, zalcitabine, stavudine, lamivudine, zidovudine, abacavir; anticancer drugs, e.g., tamoxifen and methotrexate; recreational drugs, e.g., cocaine, phencyclidine, and 5-methoxy-3,4~ methylenedioxymethamphetamine; L-asparaginase; amodiaquine; hycanthone; anesthetic agents; e.g., halothane, enflurane, and isoflurane; vitamins e.g., vitamin A; and dietary and/or environmental toxins, e.g., pyrrolizidine alkaloids, toxin from

Amanita phalloides or other toxic mushrooms, aflatoxin, arsenic, Bordeaux mixture (copper salts and lime), vinyl chloride monomer; carbon tetrachloride, beryllium, dimethylformamide, dimethylnitrosamine, methylenedianiline, phosphorus, chlordecone (Kepone), 2,3,7, 8-tetrachloro-dibenzo/?~dioxin (TCDD), tetrachloroethane, tetrachloroethylene, 2,4,5-trinitrotoluene, 1,1,1-trichloroethane, toluene, and xylene, and known "herbal remedies," e.g., ephedrine and eugenol.

Symptoms of hepatitis can include fatigue, loss of appetite, stomach discomfort, and/or jaundice (yellowing of the skin and/or eyes). More detailed descriptions of hepatitis are provided, for example, in the The Merck Manual of Diagnosis and Therapy, 17^th Edition, Section 4, Chapter 42, Section 4, Chapter 44, and Section 4, Chapter 40, the contents of which are expressly incorporated herein by reference in their entirety.

Skilled practitioners will appreciate that a patient can be diagnosed by a physician as suffering from hepatitis by any method known in the art, e.g., by assessing liver function, e.g., using blood tests for serum alanine aminotransferase (ALT) levels, alkaline phosphatase (AP), or bilirubin levels.

Individuals considered at risk for developing hepatitis may benefit particularly from the invention, primarily because prophylactic treatment can begin before there is any evidence of hepatitis. Individuals "at risk" include, e.g., patients infected with hepatitis viruses, or individuals suffering from any of the conditions or having the risk factors described herein (e.g., patients exposed to hepatotoxic agents, alcoholics). The skilled practitioner will appreciate that a patient can be determined to be at risk for hepatitis by a physician's diagnosis. Gastrointestinal Disorders

Gastrointestinal disorders include but are not limited to ileus (of any portion of the gastrointestinal tract, e.g., the large or small intestine), inflammatory bowel disease, e.g., specific inflammatory bowel disease, e.g., infective specific inflammatory bowel disease, e.g., amoebic or bacϋlary dysentery, schistosomiasis, Campylobacter enterocolitis, yersinia enterocolitis, or enterobius vermicularis; non-infective specific inflammatory boweJ disease, e.g., radiation enterocolitis, ischaemic colitis, or eosinophilic gastroenteritis; and non-specific bowel disease, e.g., ulcerative colitis, indeterminate colitis, and Crohn's disease; necrotizing enterocolitis (NEC), and pancreatitis. Cellular Proliferative and/or Differentiate Disorders and Angiogenesis

Examples of cellular proliferative and/or differentiative disorders include, but are not limited to, carcinoma, sarcoma, metastatic disorders, and hematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin.

The term "cancer" refers to cells having the capacity for autonomous growth. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include cancerous growths, e.g., tumors; oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. Also included are malignancies of the various organ systems, such as respiratory, cardiovascular, renal, reproductive, hematological, neurological, hepatic, gastrointestinal, and endocrine systems; as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine, and cancer of the esophagus. Cancer that is "naturally arising" is any cancer that is not experimentally induced by implantation of cancer cells into a subject, and includes, for example, spontaneously arising cancer, cancer caused by exposure of a patient to a carcinogen(s), cancer resulting from insertion of a transgenic oncogene or knockout of a tumor suppressor gene, and cancer caused by infections, e.g., viral infections. The term "carcinoma" is art recognized and refers to malignancies of epithelial or endocrine tissues. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues. An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

The lerm "sarcoma" is art recognized and refers to malignant tumors of mesenchymal derivation. The term "hematopoietic neoplastic disorders" includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin. A hematopoietic neoplastic disorder can arise from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.

Cancers that may be treated using the methods and compositions of the present invention include, for example, cancers of the stomach, colon, rectum, mouth/pharynx, esophagus, larynx, liver, pancreas, lung, breast, cervix uteri, corpus uteri, ovary, prostate, testis, bladder, skin, kidney, brain/central nervous system, head, neck and throat; Hodgkins disease, non-Hodgkins leukemia, sarcomas, choriocarcinoma, and lymphoma, among others.

The methods of the present invention can also be used to inhibit unwanted (e.g., detrimental) angiogenesis in a patient and to treat angiogenesis dependent/associated conditions associated therewith. As used herein, the term "angiogenesis" means the generation of new blood vessels in a tissue or organ. An "angiogenesis dependent/associated condition" includes any process or condition that is dependent upon or associated with angiogenesis. The term includes conditions that involve cancer, as well as some that do not. Angiogenesis dependent/associated conditions can be associated with (e.g., arise from) unwanted angiogenesis, as well as with wanted (e.g., beneficial) angiogenesis. The term includes, e.g., solid tumors; tumor metastasis; benign tumors, e.g., hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis, lupus, and other connective tissue disorders; psoriasis; rosacea; ocular angiogenic diseases, e.g., diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osier- Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; and wound granulation. Other processes in which angiogenesis is involved include reproduction and wound healing. By inhibiting VEGF, the methods disclosed herein can also be useful in the treatment of diseases of excessive or abnormal stimulation of endothelial cells. Such diseases include, e g., intestinal adhesions, atherosclerosis, scleroderma, and hypertrophic scars, e.g., keloids, as well as endothelial cell cancers that are sensitive to VEGF stimulation.

Individuals considered at risk for developing cancer may benefit particularly from the invention, primarily because prophylactic treatment can begin before there is any evidence of the disorder. Individuals "at risk" include, e.g , individuals exposed to carcinogens, e.g., by consumption, e.g., by inhalation and/or ingestion, at levels that have been shown statistically to promote cancer in susceptible individuals. Also included are individuals at risk due to exposure to ultraviolet radiation, or their environment, occupation, and/or heredity, as well as those who show signs of a precancerous condition such as polyps Similarly, individuals in very early stages of cancer or development of metastases (i.e., only one or a few aberrant cells are present in the individual's body or at a particular site in an individual's tissue)) may benefit from such prophylactic treatment. The skilled practitioner will appreciate that a patient can be determined to be at risk for cancer by any method known in the art, e.g., by a physician's diagnosis. Skilled practitioners will also appreciate that chemotherapy, radiation therapy, immunotherapy, gene therapy, and/or surgery can be administered in combination with the treatments described herein, for example, to treat cancer. Neurological Disorders

The methods of the present invention can also be used to treat neurological disorders Neurological disorders include, but are not limited to disorders involving the brain, e.g., degenerative diseases affecting the cerebral cortex, including Alzheimer's disease, and degenerative diseases of basal ganglia and brain stem, including Parkinsonism and idiopathic Parkinson's disease (paralysis agitans). Further, the methods may be used to treat pain disorders. Examples of pain disorders include, but are not limited to, pain response elicited during various forms of tissue injury, e.g., inflammation, infection, and ischemia, usually referred to as hyperalgesia (described in, for example, Fields, H.L. (1987) Pain, New York:McGraw-Hill); pain associated with musculoskeletal disorders, e.g., joint pain; tooth pain; headaches; pain associated with surgery; pain related to irritable bowel syndrome; or chest pain. Also included in this category are seizure disorders, e.g., epilepsy.

Reproductive Disorders

The methods described herein can be used to treat or prevent certain reproductive disorders, e.g., impotence and/or inflammation associated with sexually transmitted diseases. Further, the methods of the present invention can be used to prevent premature uterine contractions, and may be used to prevent premature deliveries and menstrual cramps.

EXAMPLES Example 1. HO-I Overexpression Augments TNFI Inhibition

This example demonstrates that overexpression of HO-I augments the effect of the anti-inflammatory agents adenosine and 5'-(N-ethylcarboxamido) adenosine (NECA) on TNFα production by lipopolysaccharide (LPS) activated macrophages. RAW 264.7 mouse macrophage cell lines were stably transfected with either heme oxygenase- 1 (HO-I RAW264.7) or empty plasmid (NEO RAW264.7). The resulting cell lines were treated with 100 μM adenosine or 10 μM NECA 30 minutes prior to stimulation with 1 ng/ml LPSBS_5:05. After 4 hours, the supernatant of each group was analyzed for TNFα secretion by ELISA (R&D Systems). Overexpression of HO-I gave an approximately 1.5 to 2-fold greater inhibition of TNFα secretion compared to the vector control (Fig. 1).

Example 2. HO-I Overexpression Potentiates Anti-Inflammatory Effects

This example demonstrates that HO-I overexpression can potentiate the effects of low doses of anti-inflammatory agents. Cells of the mouse macrophage cell line RAW264.7 were infected with adenovirus (at 30 MOI) expressing either HO-I or EGFP (negative control). Cells were treated with 10 nM or 10 μM NECA, and TNFα secretion was measured by ELISA following stimulation by 1 ng/ml LPS for 4 hours. Whereas overexpression of HO-I caused a nearly 2-fold increase in inhibition of TNFα secretion in response to 10 μM NECA, the results were most striking in the cells treated at a 1000- fold lower concentration (Fig. 2). Treatment with 10 nM NECA had no observable effect on TNFα secretion in wild-type cells or EGFP expressing cells. However, in cells that overexpressed HO-I, 10 nMNECA inhibited TNFα secretion by approximately

20%, approximately the same inhibition seen by 10 TM NECA treatment of cells that do not overexpress HO-I. This demonstrates that overexpression of HO-I was able to stimulate in cells a response to the anti-inflammatory agent NECA at a level 1000-fold less than that required to produce an equivalent response in wild-type cells. Example 3. HO-I Overexpression Affects Expression of Adenosine Receptor

This example demonstrates that overexpression of HO-I increased the expression of one receptor specific for adenosine and adenosine analogs. Cells of the mouse macrophage cell line RAW 264.7 were stably transfected with either HO-I (RAW264.7) or empty plasmid (NEO RAW264.7) as described in Example 1, and real-time PCR was used to measure mRNA expression of the adenosine receptors A2a, A2b, and A3. Predesigned TaqMan3 probes specific for the genes of interest and the control transcript GAPDH were used to quantify expression levels in the cells. Overexpression of HO-I increased expression of A2a nearly five-fold (Fig. 3). Expression of A2b and A3 was not significantly affected. Example 4. HO-I Induction Potentiates Anti-inflammatory Effects In Vivo

This example demonstrates that induction of HO-I potentiated the antiinflammatory effects of NECA in vivo. C57BL/6 male mice were treated with 5 mg/kg LPS and 700 mg/kg D-galactosamine (GaIN) and one of four treatments: 1 mg/kg NECA (n = 3), 5 mg/kg cobalt protoporphyrin (CoPP) (n = 3), 1 mg/kg NECA + 5 rng/kg CoPP (n = 4), or a control saline injection (n = 3). The untreated mice selectively developed hepatic failure and died within 6-10 hrs. Administration of NECA alone or CoPP alone provided a modest increase in survival. However, all mice treated with the combination of NECA and CoPP survived for the entire twelve-hour course of the experiment. These results suggest that induction of HO-I can potentiate anti- inflammatory effects in vivo.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of treating inflammation in a patient, the method comprising: administering to a patient diagnosed as suffering from or at risk for inflammation:

(i) a first pharmaceutical composition comprising an anti-inflammatory agent; and

(ii) a second treatment selected from the group consisting of: inducing heme oxygenase 1 (HO-I) 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 administering to the patient a second pharmaceutical composition comprising one or more of carbon monoxide (CO), a CO-releasing compound, HO-I, bilirubin, biliverdin, ferritin, iron, desferoxamine, salicylaldehyde isonicotinoyl hydrazone, iron dextran, and apoferritin; wherein the first pharmaceutical composition and the second treatment are administered in an amount sufficient to treat inflammation.

2. A method of treating inflammation in a patient, the method comprising: determining the patient's level of heme oxygenase 1 (HO-I) activity, expression, or induction in response to a stimulus, and, if HO-I activity, expression or induction in response to a stimulus is determined to be reduced compared to a reference standard, administering to the patient in an amount sufficient to treat inflammation:

(i) a first pharmaceutical composition comprising an anti-inflammatory agent; and

(ii) a second treatment selected from the group consisting of: inducing HO-I 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 administering to the patient a second pharmaceutical composition comprising one or more of carbon monoxide (CO), a CO-releasing compound, HO-I, bilirubin, biliverdin, ferritin, iron, desferoxamine, salicylaldehyde isonicotinoyl hydrazone, iron dextran, and apoferritin.

3. The method of claim 2, wherein the level of HO-I activity is determined.

4. The method of claim 2, wherein the level of HO-I expression is determined.

5. The method of claim 2, wherein the level of HO-I induction in response to a stimulus is determined.

6. A method of treating inflammation in a patient, comprising: determining an allele of a polymorphism in heme oxygenase 1 (HO-I) promoter in the patient, and, if the HO-I promoter comprises a specified allele, administering to the patient in an amount sufficient to treat inflammation:

(i) a first pharmaceutical composition comprising an anti-inflammatory agent; and

(ii) a second treatment selected from the group consisting of: inducing HO-I 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 administering to the patient a second pharmaceutical composition comprising one or more of carbon monoxide (CO), a CO-rel easing compound, HO-I, bilirubin, biliverdin, ferritin, iron, desferoxamine, salicylaldehyde isonicotinoyl hydrazone, iron dextran, and apoferritin.

7. The method of any one of claims 1 to 6, wherein the anti-inflammatory agent is selected from the group consisting of statins, adenosine, cyclooxygenase inhibitors, probucol, steroids, and prostaglandins.

8. The method of any one of claims 1 to 6, wherein the second treatment is administering a pharmaceutical composition comprising carbon monoxide.

9. The method of any one of claims 1 to 6, wherein the second treatment is inducing HO-I in the patient.

10. The method of any one of claims 1 to 6, wherein the second treatment is administering a pharmaceutical composition comprising HO-I to the patient.

1 1. The method of any one of claims 1 to 6, wherein the second treatment is administering a pharmaceutical composition comprising biliverdin to the patient.

12. The method of any one of claims 1 to 6, wherein the second treatment is administering a pharmaceutical composition comprising bilirubin to the patient.

13. The method of any one of claims 1 to 6, wherein the second treatment is administering a pharmaceutical composition comprising ferritin to the patient.

14. The method of any one of claims 1 to 6, wherein the second treatment is administering a pharmaceutical composition comprising desferoxamine (DFO) or salicylaldehyde isonicotinoyl hydrazone (SIH) to the patient.

15. The method of any one of claims 1 to 6, wherein the second treatment is administering a pharmaceutical composition comprising iron dextran to the patient.

16. The method of any one of claims 1 to 6, wherein the second treatment is administering a pharmaceutical composition comprising apoferritin to the patient.

17. The method of any one of claims 1 to 6, wherein the second treatment is inducing apoferritin expression in the patient.

18. The method of any one of claims 1 to 6, wherein the inflammation is associated with a condition selected from the group consisting of: asthma, adult respiratory distress syndrome, interstitial pulmonary fibrosis, pulmonary emboli, chronic obstructive pulmonary disease, primary pulmonary hypertension, chronic pulmonary emphysema, congestive heart failure, peripheral vascular disease, stroke, atherosclerosis, ischemia- reperfusion injury, heart attack, glomerulonephritis, conditions involving inflammation of the kidney, infection of the genitourinary tract, viral hepatitis, toxic hepatitis, cirrhosis, ileus, necrotizing enterocolitis, specific and non-specific inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, cancer, wounds, Alzheimer's disease, Parkinson's disease, graft versus host disease, and hemorrhagic, septic, or anaphylactic shock.

19. The method of any one of claims 1 to 6, wherein the inflammation is inflammation of the heart, respiratory tract, liver, spleen, brain, joint, skin, gastrointestinal tract and/or kidney.

20. A method of potentiating the response of a patient to a pharmaceutical agent, the method comprising: identifying a first pharmaceutical agent as being one that can be potentiated by a second treatment selected from inducing HO-I or apoferritin in the patient, increasing the level of expression of HO-I or apoferritin in the patient, and administering a second pharmaceutical composition comprising one or more of HO-I, CO, a CO-releasing compound, bilirubin, biliverdin, ferritin, iron, desferoxamine, salicylaldehyde isonicotinoyl hydrazone, iron dextran, and apoferritin; administering to a patient the first pharmaceutical agent; and administering to the patient the second treatment in an amount sufficient to potentiate the patient's response to the first pharmaceutical agent.

21. The method of claim 20, wherein the first pharmaceutical agent is an i mmunosuppressant.

22. The use of a first agent that: induces heme oxygenase 1 (HO-I) in a patient, increases the level of expression of HO-I in a patient, induces apoferritin in a patient, increases the level of expression of apoferritin in a patient, or that comprises one or more of carbon monoxide (CO), a CO-releasing compound, HO-I, bilirubin, biliverdin, ferritin, iron, desferoxamine, salicylaldehyde isonicotinoyl hydrazone, iron dextran, and apoferritin in the preparation of a medicament for υse in combination with an anti- inflammatory agent, wherein the first agent and the anti-inflammatory agent are administered in an amount sufficient to treat inflammation.