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  Pharmaceutical Patents  

 

Title:  Carbon monoxide as a biomarker and therapeutic agent
United States Patent: 
7,678,390
Issued: 
March 16, 2010

Inventors:
 Choi; Augustine M. K. (Pittsburgh, PA), Otterbein; Leo E. (New Kensington, PA)
Assignee:
  Yale University (New Haven, CT), John Hopkins University (Baltimore, MD)
Appl. No.:
 10/053,535
Filed:
 January 15, 2002


 

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Abstract

The present invention relates to the use of carbon monoxide (CO) as a biomarker and therapeutic agent of heart, lung, liver, spleen, brain, skin and kidney diseases and other conditions and disease states including, for example, asthma, emphysema, bronchitis, adult respiratory distress syndrome, sepsis, cystic fibrosis, pneumonia, interstitial lung diseases, idiopathic pulmonary diseases, other lung diseases including primary pulmonary hypertension, secondary pulmonary hypertension, cancers, including lung, larynx and throat cancer, arthritis, wound healing, Parkinson's disease, Alzheimer's disease, peripheral vascular disease and pulmonary vascular thrombotic diseases such as pulmonary embolism. CO may be used to provide anti-inflammatory relief in patients suffering from oxidative stress and other conditions especially including sepsis and septic shock. In addition, carbon monoxide may be used as a biomarker or therapeutic agent for reducing respiratory distress in lung transplant patients and to reduce or inhibit oxidative stress and inflammation in transplant patients.

Description of the Invention

FIELD OF THE INVENTION

The present invention relates to the use of carbon monoxide (CO) as a biomarker and therapeutic agent of heart, lung, liver, spleen, brain, skin and kidney diseases and other conditions and disease states including, for example, asthma, emphysema, bronchitis, adult respiratory distress syndrome, sepsis, cystic fibrosis, pneumonia, interstitial lung diseases, idiopathic pulmonary diseases, other lung diseases including primary pulmonary hypertension, secondary pulmonary hypertension, cancers, including lung, larynx and throat cancer, arthritis, wound healing, Parkinson's disease, Alzheimer's disease, peripheral vascular disease and pulmonary vascular thrombotic diseases such as pulmonary embolism. CO may be used to provide anti-inflammatory relief in patients suffering from oxidative stress and other conditions especially including sepsis and septic shock. In addition, CO may be used to store organs prior to transplantation. In addition, carbon monoxide may be used as a biomarker or therapeutic agent for reducing respiratory distress in lung transplant patients, to reduce or inhibit oxidative stress, inflammation or rejection of transplants in transplant patients.

BACKGROUND OF THE INVENTION

Heme oxygenase (HO) catalyzes the first and rate limiting step in the degradation of heme to yield equimolar quantities of biliverdin IXa, carbon monoxide (CO), and iron (Choi, et al., Am. J. Respir. Cell Mol. Biol. 15: 9-19; and Maines, Annu. Rev. Pharmacol. Toxicol. 37: 517-554). Three isoforms of HO exist; HO-1 is highly inducible while HO-2 and HO-3 are constitutively expressed (Choi, et al., supra, Maines, supra and McCoubrey, et al., E. J. Bioch. 247: 725-732). Although heme is the major substrate of HO-1, a variety of non-heme agents including heavy metals, cytokines, hormones, endotoxin and heat shock are also strong inducers of HO-1 expression (Choi, et al., supra, Maines, supra and Tenhunen, et al., J. Lab. Clin. Med. 75: 410-421). This diversity of HO-1 inducers has provided further support for the speculation that HO-1, besides its role in heme degradation, may also play a vital function in maintaining cellular homeostasis. Furthermore, HO-1 is highly induced by a variety of agents causing oxidative stress including hydrogen peroxide, glutathione depletors, UV irradiation, endotoxin and hyperoxia (Choi, et al., supra, Maines, supra and Keyse, et al., Proc. Natl. Acad. Sci. USA. 86: 99-103). One interpretation of this finding is that HO-1 can serve as a key biological molecule in the adaptation and/or defense against oxidative stress (Choi, et al., supra, Lee, et al., Proc Natl Acad Sci USA 93: 10393-10398; Otterbein, et al., Am. J. J. Respir. Cell Mol. Biol. 13: 595-601; Poss, et al., Proc. Natl. Acad. Sci. USA. 94: 10925-10930; Vile, et al., Proc. Natl. Acad. Sci. 91: 2607-2610; Abraham, et al., Proc. Natl. Acad. Sci. USA. 92: 6798-6802; and Vile and Tyrrell, J. Biol. Chem. 268: 14678-14681. Our laboratory and others have shown that induction of endogenous HO-1 provides protection both in vivo and in vitro against oxidative stress associated with hyperoxia and lipopolysaccharide-induced tissue injury (Lee, et al., supra, Otterbein, et al., supra and Taylor, et al., Am. J. Physiol. 18: L582-L591). We have also shown that exogenous administration of HO-1 via gene transfer can provide protection against oxidant tissue injury and elicit tolerance to hyperoxic stress (Otterbein, et al., Am. J. Resp. Crit. Care Med. 157: A565 (Abstr)).

Carbon monoxide (CO) is a gaseous molecule with known toxicity and lethality to living organisms (Haldane, Biochem. J. 21: 1068-1075; and Chance, et al., 1970, Ann. NY Acad Sci. 174: 193-204.). However, against this known paradigm of CO toxicity, there has been renewed interest in recent years in CO behaving as a regulatory molecule in cellular and biological processes based on several key observations. Mammalian cells have the ability to generate endogenous CO primarily through the catalysis of heme by the enzyme heme oxygenase (HO) (Choi, et al., supra and Maines, supra). The total cellular production of CO is generated primarily via heme degradation by HO (Marilena, Biochem. Mol. Med. 61: 136-142 and Verma, et al., 1993 Science 259: 381-384). Moreover, CO akin to the gaseous molecule nitric oxide, plays important roles in mediating neuronal transmission (Verma, et al., supra and Xhuo, et al., Science 260: 1946-1950) and in the regulation of vasomotor tone (Morita, and Kourembanas, 1995, J. Clin. Invest. 96: 2676-2682.; Morita, et al., 1995 Proc. Natl. Acad. Sci. USA 92:-1479; and Goda, et al., 1998, J. Clin. Inv. 101: 604-12). There is no data in the literature substantiating a protective role for CO in vivo against oxidative stress.

Septic shock and sepsis syndrome, resulting from excessive stimulation of immune cells, particularly monocytes and macrophages, remains one of the leading causes of death in hospitalized patients. Parillo, et al., Ann. Intern. Med. 113, 991-992 (1992). The pathophysiological alterations observed in sepsis are often not due to the infectious organism itself, but rather to the uncontrolled production of pro-inflammatory cytokines and chemokines including TNF-.alpha., IL-1, and MIP-1 that leads to leukocyte recruitment, capillary leak and ultimately participates in the lethality of sepsis. Beutler, et al., 232, 977-980 (1986); Netea, et al., Immunology 94, 340-344 (1998); and Wolpe, et al., J. Exp. Med. 167, 570-581 (1988). Lipopolysaccharide (LPS), a constituent of the gram negative bacterial cell wall, is the leading cause of sepsis, and when administered experimentally to macrophages or mice, mimics the same inflammatory response. Following LPS administration, there is a rapid but transient increase in these pro-inflammatory mediators which are subsequently down-modulated by a battery of anti-inflammatory cytokines including interleukin-10 (IL-10) and interleukin-4 (IL-4), which inhibit the synthesis of the pro-inflammatory cytokines and chemokines. J. Exp. Med. 177, 1205-1208 (1993). LPS initially binds to the CD14 and toll-like receptor (TLR) 2 (or 4) at the cell surface, [Yang, et al., Nature. 395: 284-288 (1998) and Chow, et al., J. Biol. Chem. 274: 10689-10692 (1999)] and has then been shown to activate the mitogen activated protein (MAP) kinase pathways including p38, p42/p44 ERK and JNK (MAP) kinases. Liu, et al., J. Immunol. 153, 2642-2652 (1994); Hambleton, et al., Proc. Natl. Acad. Sci. USA. 93, 2274-2778 (1996); Han, et al., J. Biol. Chem. 268, 25009-25014 (1993); Han, et al., Science 265, 808-811 (1994); Sanghera, et al., J. Immunol. 156, 4457-4465 (1996), and Raingeaud, et al., J. Biol. Chem. 270, 7420-7426 (1995). The relationship between the activation of these signaling molecules, downstream cytokine expression, and physiologic function represents an active line of investigation.

The United States Government has provided support for research which led to the present invention under one or more of NIH grant numbers HL60234, A142365 and HL55330. Consequently, the government retains certain rights in the invention.

OBJECTS OF THE INVENTION

It is an object of the present invention to provide novel gaseous mixtures containing low concentrations of carbon monoxide which may be used as therapeutic compositions.

It is another object of the present invention to provide a method for treating oxidative stress in a patient.

It is another object of the present invention to provide a method for treating a number of diseases and conditions in which oxidative stress occurs or is secondary.

It is yet another object of the present invention to provide a method for using carbon monoxide as a biomarker to determine that a patient producing carbon monoxide is suffering from oxidative stress or a condition or disease state in which oxidative stress is implicated.

At least one or more of these and/or other objects of the present invention may be readily gleaned from a review of the description of the invention which follows.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to novel pharmaceutical compositions for delivering to patients suffering from the effects of oxidative stress, the compositions comprising effective concentrations of carbon monoxide in a gaseous mixture comprising oxygen and optionally, nitrogen gas (as well as other minor optional gaseous components). An additional aspect of the present invention is directed to a method for delaying the onset of, inhibiting or alleviating the effects of oxidative stress, the method comprising delivering a therapeutic gas comprising carbon monoxide in an amount and for a time effective to delay the onset of, inhibit or alleviate the affects of oxidative stress in the patient. It has unexpectedly been discovered that the delivery of a therapeutic gas comprising low concentrations (i.e., concentrations ranging from about 1 ppb (part per billion) to about 3,000 ppm (preferably above about 0.1 ppm within this range) of the gas, preferably about 1 ppm to about 2,800 ppm, more preferably about 25 ppm to about 750 ppm, even more preferably about 50 ppm to about 500 ppm) of carbon monoxide is an extremely effective method for delaying the onset of, inhibiting or reversing the effects of oxidative stress in a patient. This is an unexpected result. It is noted here that in the method aspects of the present invention, an amount of carbon monoxide in the therapeutic gaseous composition which is in excess of 0.3% may sometimes be used, depending upon the condition or disease state to be treated.

Another aspect of the present invention is directed to the use of carbon monoxide as a biomarker for determining that a patient is suffering from oxidative stress and is at risk for or is suffering from a number of conditions or disease states which are secondary to or result in oxidative stress, for example, asthma, emphysema, bronchitis, adult respiratory distress syndrome, sepsis, cystic fibrosis, pneumonia, interstitial lung diseases, idiopathic pulmonary diseases, other lung diseases including primary pulmonary hypertension, secondary pulmonary hypertension, cancers, including lung, larynx and throat cancer, arthritis, wound healing, Parkinson's disease, Alzheimer's disease, peripheral vascular disease and pulmonary vascular thrombotic diseases such as pulmonary embolism, among others. The method comprises detecting carbon monoxide in a patient's breath to determine whether detectable levels of carbon monoxide occur in the breath. If detectable levels of carbon monoxide appear in the patient's breath, the patient may be diagnosed with oxidative stress or for being at risk for oxidative stress. The manifestations of oxidative stress may take the form of one or more of the above-referenced conditions or disease states. Appropriate therapeutic steps or other steps may be taken after such diagnosis to alleviate or treat the condition which is responsible for the oxidative stress in the patient.

Another aspect of the present invention relates to the finding that in certain patients, the administration of carbon monoxide in effective amounts to the patient may be used to induce HO-1 enzyme in the patient and prevent or limit oxidative stress in the patient, especially including oxidative stress caused by hyperoxia or sepsis. HO-1 enzyme is implicated in maintaining homeostasis in the cells of the patient.

Still another aspect of the present invention relates to the use of carbon monoxide to delay the onset of, inhibit or alleviate the effects of oxidative stress which occur in transplant patients, in particular, organ transplant patients, especially, but not exclusively lung transplant patients.

Another aspect of the present invention relates to a method for inhibiting the production of pro-inflammatory cytokines such as TNF-.alpha., IL-1.beta., IL-6, MIP-1.beta. and augmenting the production (expression) of the anti-inflammatory cytokine IL-10 and IL-4 in a patient comprising administering to the patient an effective amount of CO.

Still another aspect of the present invention relates to a method to preserve organs or tissue for transplants comprising adding to media in which the organs or tissue are stored a preservative effective amount or concentration of carbon monoxide. In this aspect of the present invention, the inclusion of carbon monoxide in effective amounts reduces, inhibits or alleviates the formation of reactive oxygen in the stored organ or tissue, thus extending the period in which organ transplants can be effectively stored without suffering oxidative damage.

Another aspect of the present invention relates to a method to prevent or reduce the likelihood of damage caused by oxidative stress associated with hyperoxia in a patient comprising administering an effective amount of carbon monoxide to a hyperoxic patient.

DETAILED DESCRIPTION OF THE INVENTION

Thus, according to an aspect of the present invention, a patient suspected of being in oxidative stress or at risk for oxidative stress is monitored to determine whether detectable levels of carbon monoxide may be measured in the exhaled breath of the patient. If detectable levels of carbon monoxide are seen (i.e., an amount of carbon monoxide of at least about 0.01 ppm in the patient's breath), then the attending physician or caregiver may then begin to administer therapeutic doses of carbon monoxide to treat oxidative stress or any one or more of the conditions or disease states which are secondary to or result in oxidative stress.

The following conditions or disease states may be treated using low dosages of CO in effective amounts pursuant to the teachings of the present invention. These include: asthma, emphysema, bronchitis, adult respiratory distress syndrome, sepsis, cystic fibrosis, pneumonia, interstitial lung diseases, idiopathic pulmonary diseases, other lung diseases including primary pulmonary hypertension, secondary pulmonary hypertension, cancers, including lung, larynx and throat cancer, arthritis, wound healing, Parkinson's disease, Alzheimer's disease, peripheral vascular disease and pulmonary vascular thrombotic diseases such as pulmonary embolism.

Low dosage CO may also be used in the present invention to induce HO-1 enzyme in patients and prevent or limit oxidative stress, especially oxidative stress caused by hyperoxia or sepsis. Induced HO-1 is implicated in maintaining homeostasis in the cells of the patient.

Low dosage CO may also be used to delay the onset of, or alleviate the effects of oxidative stress in transplant patients, in particular organ transplant patients, especially lung transplant patients. Low dosage CO may also be used to treat inflammatory conditions of the lungs or inflammation which occurs secondary to sepsis or rejection in transplant patients. While not being limited by way of theory, low dosage CO is believed to act as an anti-inflammatory agent by inhibiting the production and/or effect of pro-inflammatory cytokines such as TNF-.alpha., IL-1, IL-6, MIP-1 and induces or promotes the action of anti-inflammatory cytokines IL-4 and IL-10.

The present invention also relates to the use of CO as a preservative for storing organs to be used in transplants. It is an unexpected result that the inclusion of low dosage CO in the storage media in which organs to be transplanted are stored will substantially reduce the likelihood of oxidative damage to the organs during storage and substantially enhances the storage time that organs to be transplanted may be safely stored without suffering irreversible oxidative damage. Thus, in this aspect of the present invention, an effective amount of CO is bubbled into storage media either before or preferably when an organ is first placed in the media or shortly thereafter. CO may also be used to enhance the storage stability of organs which have been stored for some time in media, but in those instances, oxidative damage may have become irreversible, thus limiting the intended effect.

Administration of compounds according to the present invention is generally through the mouth or nasal passages to the throat and lungs, where the CO may exert its effect directly or be readily absorbed into the patient's blood stream. The concentration of active compound (CO) in the therapeutic gaseous composition will depend on absorption, distribution, inactivation, and excretion (generally, through respiration) rates of the drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. 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 composition. The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of time. Acute, sub-acute and chronic administration of CO are contemplated by the present invention, depending upon the condition or disease state to be treated.

In delivering CO to patients or in other applications at concentrations ranging from about 0.001 to about 3,000 ppm pursuant to the present invention, gaseous compositions according to the present invention may be prepared by mixing commercially available compressed air containing CO (generally about 1% CO) with compressed air or gas containing a higher percentage of oxygen (including pure oxygen), and then mixing the gasses in a ratio which will produce a gas containing a desired amount of CO therein. Alternatively, compositions according to the present invention may be purchased pre-prepared from commercial gas companies. In a preferred embodiment, patients are exposed to oxygen (O.sub.2 at varying doses) and CO at a flow rate of about 12 liters/minute in a 3.70 cubic foot glass exposure chamber. To make a gaseous composition containing a pre-determined amount of CO, CO at a concentration of 1% (10,000 ppm) in compressed air is mixed with >98% O.sub.2 in a stainless steel mixing cylinder, concentrations delivered to the exposure chamber or tubing will be controlled. Because the flow rate is primarily determined by the flow rate of the O.sub.2 gas, only the CO flow is changed to generate the different concentrations delivered to the exposure chamber or tubing. A carbon monoxide analyzer (available from Interscan Corporation, Chatsworth, Calif.) is used to measure CO levels continuously in the chamber or tubing. Gas samples are taken by the analyzer through a portion the top of the exposure chamber of tubing at a rate of 1 liter/minute and analyzed by electrochemical detection with a sensitivity of about 1 ppb to 600 ppm. CO levels in the chamber or tubing are recorded at hourly intervals and there are no changes in chamber CO concentration once the chamber or tubing has equilibrated. CO is then delivered to the patient for a time (including chronically) sufficient to treat the condition and exert the intended pharmacological or biological effect.

One of ordinary skill will readily recognize the symptoms of oxidative stress, inflammation, one or more of the conditions or disease states in which oxidative stress is implicated, sepsis or septic shock, and other conditions in which the delivery of CO represents a viable therapeutic option. All of these conditions or disease states are well known in the art.

In addition to using CO as a therapeutic agent, the measurement of CO may be a useful diagnostic tool to determine whether a patient is in oxidative stress or has a condition or a disease state where CO may be implicated. In this aspect of the present invention, a patient will have his or her exhaled breath analyzed for the presence of CO. CO content in a patient's breath is measured by a CO monitor (for example, using a Logan LR2000) which is sensitive to the detection of CO from 0 to about 1000 ppm (with a sensitivity as low as 1 ppb). In this method, the subjects exhale slowly from functional FVC into the breath analyzer with a constant flow (5-6 l/m) over a 20-30 second interval. Two successful recordings are made and mean values will be used for all calculations. Ambient CO levels are recorded before each breath in order to provide control or background values. While any elevation in CO levels from background numbers may implicate an actual or incipient state of oxidative stress, an amount of CO of at least about 1 ppm provides a clear indication that the patient is in or is about to suffer oxidative stress.


Claim 1 of 79 Claims

1. A method of treating emphysema secondary to or resulting in oxidative stress to a patient, comprising: identifying a patient suffering from emphysema secondary to or resulting in oxidative stress; and administering to the patient a therapeutically effective amount of a gaseous composition comprising carbon monoxide at a concentration of about 10 ppm to about 3000 ppm, wherein the patient inhales the gaseous composition.

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