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10.1517/13543770902858824 © 2009 Informa UK Ltd ISSN 1354-3776 663All rights reserved: reproduction in whole or in part not permitted

Therapeutic applications of the gaseous mediators carbon monoxide and hydrogen sulfi de Gerard L Bannenberg † & Helena LA Vieira † Department of Plant Molecular Genetics, Centro Nacional de Biotecnología / CSIC, Campus de la Universidad Autónoma, Calle Darwin 3, Cantoblanco, 28049 Madrid, Spain

Background : Hydrogen sulfide (H 2 S) and carbon monoxide (CO) are endogenously produced gaseous autacoids that regulate a number of physiological processes, including the inflammatory response, cell death and proliferation, neural transmission and smooth muscle tone. Objective/methods : The current review aims to provide a comprehensive overview of all recent patent applications that address the potential therapeutic applications of CO and H 2 S. Results/conclusion : Beyond the direct administration of CO and H 2 S, this review highlights the therapeutic applications of a variety of gas-releasing molecules that are being developed to deliver CO and H 2 S to diseased tissues at thera-peutic doses. The term autacoid, which, in addition to its pharmacological use to describe a locally-acting hormone, literally translates from Greek as ‘self-drug’, seems to particularly well describe the current approach to capture the potential therapeutic use of these two gasotransmitters. In summary, we can conclude that there is a markedly growing interest in harnessing the tissue-protective actions of CO and H 2 S.

Keywords: carbon monoxide , carbon monoxide releasing molecules , gasotransmitters , hydrogen sulfide , hydrogen sulfide releasing molecules

Expert Opin. Ther. Patents (2009) 19 (5):663-682

1. Introduction

The human body uses locally acting hormones, or autacoids, for the instantaneous regulation of tissue function. Many autacoids have been identified and include amine-containing compounds, fatty acid-derived eicosanoids and docosanoids, and a range of regulatory peptides. Over the past 30 years, the physicochemical nature of the autacoids has expanded with the identification of several endogenously produced gases. These include nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H 2 S) [1-4] . Among these three gasotransmitters, CO may have been the first identified endogenously produced gas, although its role as an endogenous regulator was not recognized at that time [5] . It was not until the 1990s that CO was described as cell-signaling molecule, acting as a neurotransmitter [6] . That the body is able to use gaseous autacoids for regulating physiology was first recognized and established for NO. The paradigmatic shift in our understanding of the distinct physicochemical nature and mechanism of action of NO soon led to experimental confirmation that other gases can also act as endogenous mediators [7] . Thus, CO and H 2 S were demonstrated to be produced endogenously and to regulate specific physiological functions. Several other gases, including acetaldehyde, sulfur dioxide and dinitrogen oxide, are now also being investigated with the aim of appreciating their putative endogenous functions in the body.

The potent biological actions identified for NO, CO and H 2 S are now actively being explored for their therapeutic potential. A range of studies has shown that administration of these gasotransmitters can stimulate a number of specific

1. Introduction

2. Patent applications on CO

3. Patent applications on

H2S-releasing molecules and

H2S liquid dosage forms

4. Exert opinion

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664 Expert Opin. Ther. Patents (2009) 19(5)

pharmacologic actions. The applicability of gases as medicine has limitations that relate to their storage, precision of dosage and targeting of specific sites of the body. Although the direct administration of biologically active gases can be useful in specific applications, current research towards developing pharmacological applications of the gasotransmitters focuses on finding suitable gas-releasing molecules. Such releasing molecules afford the administration of water-soluble and rela-tively stable forms of the gas, with enhanced pharmacokinetic properties, and which can release the gas under, in the best case, defined conditions at specific sites in the body.

Several mechanisms whereby the gas can be released can be predicted: i) spontaneous release at defined rates; ii) transfer of the gas from the releasing scaffold directly to a target molecule; iii) induced release; and iv) metabolism of a com-pound with concomitant formation of the gas. In the next sections of this review, we describe the formation and actions of CO and H 2 S in relation to recent patents and patent applications that seek to harness therapeutic applications based on the newly gathered knowledge on the physiological functions of CO and H 2 S. These patents will furthermore illustrate the different approaches taken for administration of gaseous autacoids.

1.1 Carbon monoxide CO is an endogenous product of heme degradation by heme-oxygenase (HO), which also generates free iron and biliverdin (a potent antioxidant) [8] . The isoform HO-1 is highly inducible in response to oxidative stress, ultraviolet (UV) light, heavy metals and inflammation, and plays an important role in cell redox state, acting as an antioxidant and cytoprotective enzyme [8] . The HO-2 isoform is constitutively expressed in neurons and is controlled by post-translational modifications [9] . More recently, HO-1 has also been described as a transcription factor activator important in the oxidative stress response [10,11] .

Administration of CO at low concentrations produces several beneficial actions in distinct tissues, such as a reduction in inflammation, an anti-proliferative response, vasodilation or inhibition of apoptosis. CO can be directly administrated as gas or as CO-releasing molecules (CO-RMs), which are small organic molecules that were first identified and characterized by Motterlini et al. [12-14] , representing a new class of compounds that preserve the physiological properties related to CO.

One of the best explored physiological functions of CO is its anti-inflammatory properties in several tissues and cell types. Both in vivo and in vitro , exogenous CO inhibits the expression of lipopolysaccharide (LPS)-induced pro-inflammatory cytok-ines, TNF- α , IL-1 β and macrophage inflammatory protein-1 β . The anti-inflammatory property of CO is mediated by p38 MAPK and its upstream MAPK kinase (MKK3) in RAW264.7 macrophages [15] . In experimental autoimmune encephalomyelitis, a model of multiple sclerosis, CO admin-istration suppresses neuroinflammation [16] . Inhalation of CO after hepatic ischemia and reperfusion reduces microcirculatory

deficits and inflammation [17] . In a rat lung model of ischemic tissue injury, CO mediates tissue protection and reduces thrombosis by inhibiting the transcription of early growth response 1, a potent transcription activator of inflammatory cascades [18] . In macrophages, CO-RMs reduce activation by LPS [19] , and CO gas treatment promotes activation and stabilization of hypoxia inducible factor 1, which regulates expression of genes involved in inflammation and apoptosis [20] .

CO imparts cell protective actions and controls processes that modulate cell death. CO generated by HO-1 prevents TNF- α -mediated apoptosis in endothelial cells, through p38 MAPK and NF- k B activation [21,22] . CO exposure also pro-tects endothelial cells against apoptosis induced by lung ischemia-reperfusion [23,24] . In neuronal primary cultures, CO exposure provides cytoprotective preconditioning and increases resistance against apoptosis induced by excitotoxicity and oxidative stress [25] . Exogenous administration of CO, furthermore, inhibits cytokine-mediated apoptosis in vascular smooth cells [26] and protects hepatocytes against apoptosis triggered by endotoxin shock [27] .

The maintenance of tissue homeostasis demands regulating a delicate balance between cell death and cell proliferation. In addition to its anti-apoptotic properties, CO is also able to inhibit cell proliferation of distinct cell types. It was first found by Morita et al. that endogenous CO (produced by HO-1 after an hypoxic insult) mediates an anti-proliferative action in smooth muscle cells [28] . More recently, it was demonstrated that atherosclerotic lesions, occurring after aorta transplantation, can be prevented by CO. Inhibition of leukocyte infiltration/activation and of smooth muscle cells proliferation contribute to this protective action of CO [29] . The anti-proliferative action of CO is mediated through activation of guanylate cyclase and p38 MAPK, and expression of the cell cycle inhibitor p21 [29] .

A reduction of pulmonary arterial hypertension by CO has been shown to occur through growth arrest and apoptosis of hyper-proliferating arterial smooth cells by affecting the G 0 /G 1 phase of the cell cycle [30] . CO has also been shown to reduce airway hyper-responsiveness towards methacholine in ovalbumin-sensitized mice [31] . In addition, CO can regulate CD3-activated T-lymphocyte activation and proliferation, a process involved in many patho-physiological processes, such as transplantation, asthma and other immunological diseases. This anti-proliferative effect is independent of MAPK or cGMP signaling, and requires inhibition of caspase-3 and -8 expression and activation [32] .

Vasodilatation is another well-studied property of CO, although at the systemic level CO is considered a relatively weak vasodilator in comparison to NO. The soluble heme-dependent guanylate cyclase (sGC)/cGMP pathway is implicated in the vasodilatory action of CO in aorta [33,34] and in vascular smooth muscle cells [28,35] . Activation of the sGC/cGMP pathway in airway smooth muscle induces relaxation and bronchodilatation [36] . Recently, cGMP-independent mechanisms

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of vasoregulation by CO have been demonstrated. In renal arterial vessels and in cerebral arterioles, CO modulates blood vessel perfusion by directly acting on calcium-dependent potassium channels [37-39] . CO generated by astrocytes can, thus, activate cerebral arteriolar dilation by regulating smooth muscle cell calcium-dependent potassium channels [40] .

Because of the enormous therapeutic potential of CO, large efforts have been initiated in the past years to develop new ways of delivering this gas to specific tissues and organs. Pharmaceutical CO administration by inhalation requires CO doses that are sufficiently high to allow diffusion of therapeutically effective amounts of CO from blood to tissues. Direct administration of CO by inhalation of the gas is limited by the high affinity of hemoglobin for CO, leading to potentially rapid intoxication owing to the facile formation of high levels of carboxy-hemoglobin. In addition, the direct use of CO gas has several disadvantages: special equipment is required (ventilators, masks, inhalers, etc.) and establishment of appropriate doses is a complex process, thus, limiting its utilization to specialized hospital settings. The administration of CO by inhalation can be considered more suitable for treat-ment of respiratory tract and lung disease or for the treatment of a transplant donor and/or ex vivo treatment of an organ to be transplanted.

To surpass the above limitations for effective systemic CO delivery, alternative pharmaceutical approaches have been designed that encompass: i) the use of pro-drugs that are metabolically converted into CO; and ii) the use of CO-RMs, compounds which are able to bind, stabilize, release and deliver CO. However, in many cases, the administration of CO-RMs can also cause an increase in carboxy-hemoglobin level, and efforts are needed to develop new molecules that are able to release CO in a tissue-specific manner.

The use of pro-drugs, such as methylene dichloride and organic aldehydes and esters (discussed below), find their use in the selective formation of CO in tissues that possess a specific enzymatic activity, which converts the compound with concomitant release of CO. Provided that a suitable combination of pro-drug and metabolic activity is used, this approach is potentially useful for the tissue-selective targeting of CO release. Thus far, cytochrome P450- and glutathione-S-transferase-mediated decarbonylation reactions have been implicated in the release of CO from methylene chloride.

The second strategy, based on CO-RMs, is regarded as more promising for therapeutic applications. Three main classes of CO-RMs with potential pharmacological actions have been described up-to-date: transition metal carbonyls, boranocarbonates and aldehydes. When at the end of the 1990s CO had emerged as a potential therapeutic molecule, it became necessary to discover solutions to deliver and store CO in a controlled manner, as well as to target it to diseased tissues. This needed to be achieved without interfering with hemoglobin-mediated oxygen transport in the systemic circulation. Developing ways of providing CO in a chemically stable form was imperative. Transition metal carbonyls, which

are compounds well described in the organometallic chemistry field for catalysis and purification processes, were found to be promising compounds for releasing CO. These complexes are constituted by a transition metal (such as manganese, cobalt or iron) surrounded by a certain number of carbonyl groups as coordinated ligands. CORM-1, with the formula (Mn 2 (CO) 10 ), was the first compound to present biological activity related to CO [12,13] . CO was released from CORM-1 after photo-excitation, which was measured by conversion of deoxymyoglobin into carbonmonoxymyoglobin (MbCO). Additionally, in isolated heart, CORM-1 produced attenua-tion of coronary vasoconstriction induced by an inhibitor of NO-synthase [41] . Following these first encouraging results, other CO-RMs have been developed. CORM-2 and CORM-3 are ruthenium-based compounds with formulae [Ru(CO) 3 Cl 2 ] 2 and [Ru(CO) 3 Cl(glycinate)], respectively [12,13] . CORM-2 has been shown to induce vasodilatation in iso-lated rat aorta. CORM-3 is water-soluble (unlike CORM-2) and has become the most studied CO-RM with many described actions; e.g. it can induce vasodilatation, has anti-inflamma-tory properties in macrophages and neuronal cells, and pro-tects the kidney from cold ischemia/reperfusion injury.

CORM-A1 (Na 2 H 3 BCO 2 ), unlike CORM-3, does not contain a transition metal but is a boronate compound, which releases CO when it comes in contact with water [39] . Under physiological conditions, it releases CO at a much slower rate than many transition metal CO complexes. In the presence of myoglobin, CORM-3 transfers CO forming MbCO with a very fast rate (t 1/2 = ∼ 1 min), whereas CORM-A1 releases CO much slower (t 1/2 = ∼ 21 min). Therefore, it is thought that CORM-3 would be more applicable for therapeutic applications in which CO acts as a prompt signaling mediator, whereas CORM-A1 would be more suitable for chronic diseases, mimicking better a sustained HO-mediated CO formation. Several recent reviews are available that address these topics [14,42,43] . New compounds are being developed that deliver CO in a more controlled manner with respect to tissue and pathology-specificity, minimization of carboxy-hemoglobin formation and velocity of CO release.

1.2 Hydrogen sulfi de H 2 S is a gas that is produced endogenously through specific cysteine desulfhydration pathways, which are widely found in the body [2] . It is thought that the two major enzymatic pathways that function under physiological conditions for cata-lyzing the desulfhydration of cysteine are cystathionine- β -synthase or cystathione- γ -lyase (cystathionase) [44] . In the brain, a principal route of H 2 S synthesis is thought to occur through cystathionine- β -synthase [45] . Cystathione- γ -lyase is a major route of H 2 S formation in liver, is expressed in ileum, portal vein and thoracic aorta, and is present in vascular smooth muscle cells [46,47] . A third enzymatic route contributing to H 2 S production from cysteine has recently been identified, namely, 3-mercaptopyruvate sulfurtransferase in combination

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666 Expert Opin. Ther. Patents (2009) 19(5)

with cysteine aminotransferase [48] . The mobilization of bound sulfide in astrocytes by intracellular alkalinization in response to depolarization has been shown to constitute another physiologically relevant source of H 2 S [49] . This latter route may constitute a rapidly responsive H 2 S-generating system, which responds to neuronal activity. In addition, H 2 S-producing intestinal bacteria may provide unknown quantities of H 2 S to the circulation.

Local tissue H 2 S concentrations are not only determined by the rate of formation but also by degradation of H 2 S. Dissolved gaseous H 2 S is in a pH-dependent equilibrium with the sulfide anion (HS - ), which can oxidize to sulfite, sulfate, thiosulfate, sulfur (elemental), polysulfides, dithionate and polythionates. The major routes of degradation are through non-enzymatic oxidation of sulfide to sequentially thiosulfate, sulfite, and sulfate; the last is excreted in urine [44] . The relative roles of the different enzymatic and chemical routes of H 2 S/H 2 S anion homeostasis in vivo are only partially understood. Concentration ranges of H 2 S/H 2 S anion in blood and tissues have been measured in the 10 – 200 µM range, which are surprisingly high for a locally acting hormone. Bound forms of sulfide are suspected to contribute to these values in assays used at present. Direct gas phase measurements of blood–alveolar gas concentrations point to endogenous H 2 S concentrations, which are in the low nanomolar range [50] . Local formation and rapid degradation of H 2 S are likely to regulate H 2 S signaling while maintaining overall tissue concentrations in the low nanomolar range.

Like NO and CO, H 2 S can bind to specific metal groups in metalloenzymes, such as the coordinated iron in carbonic anhydrase and cytochrome c oxidase. In contrast to CO, H 2 S can directly react with several free radicals, such as superoxide anion radical, NO and peroxynitrite, to form hydrogen disulfide. Sulfide can also react with disulfide groups, or with sulfhydryl groups forming persulfides. Another important reaction is the electrophilic addition of H 2 S to proteins, a reaction that determines its action on ATP-sensitive potassium channels, myoglobin and hemoglobin.

Potent biological actions have been uncovered for H 2 S in just the past 10 years, which include roles in the regulation of blood pressure, inflammation, cell viability, cellular oxygen consumption, pain processing and insulin secretion. Exoge-nously administered H 2 S, at micromolar concentrations, can induce vasorelaxation in vitro and in vivo [47,51] . H 2 S is pro-duced in smooth muscle of various vascular beds [47] . The blood pressure lowering action is mediated by activation of ATP-sensitive potassium channels in vascular smooth muscle and seems to be independent from the activation of guany-late cyclase. H 2 S can facilitate the vasorelaxant action of NO [46] . On the other hand, H 2 S reduces the biosynthetic pathway for NO formation [52,53] and may interact directly with NO [54] , indicating that the vascular role of H 2 S is not a simple one. Exogenous H 2 S can also cause vasoconstriction and antagonize the actions of vasodilators [54,55] . It has to be borne in mind that oxygen tension determines the vasoactivity of H 2 S [56] ,

and it is likely that at high oxygen tension more sulfide-derived oxidation products may be formed.

Endogenous vascular formation of H 2 S may make a significant contribution to maintaining vascular tone under normal conditions [57,58] . In addition, H 2 S is likely to be of substantial importance during cardiovascular disorders. This includes a contribution of H 2 S during hypotension and shock [59] , and a decreased H 2 S formation has been measured in spontaneous hypertensive rats [60] . A cardioprotective role for H 2 S has been established, and H 2 S offers protection against myocardial ischemia-reperfusion injury. A deficient H 2 S formation seems to play a role in neo-intima formation after catheter-induced carotid artery injury. In addition, a more central regulatory role of H 2 S as an oxygen sensor in oxygen receptive cells, such as carotid bodies and neuroepithelial bodies, has been proposed [61] . In such a model, oxygen tension determines the rate of oxidation of H 2 S, which controls the concentration of active H 2 S in these specific tissues.

H 2 S also has relaxing actions of smooth muscle cells from the airways, stomach, intestines and urinary bladder [62] . H 2 S has, furthermore, been identified as a neuromodulator; it is formed in response to glutamate-mediated neuronal excitation, facilitates NMDA-receptor-activated neurotransmission and is implicated in memory [63] .

The molecular mechanisms whereby H 2 S exerts its biological actions are far from being completely understood. Guanylate cyclase-independent actions of H 2 S have been demonstrated, which may be mediated by extracellular calcium-influx [64] . Activation of ATP-activated K + -channels and T-type Ca 2+ -channels can also mediate H 2 S-induced for acti-vation of cellular responses [47,65] . Several intracellular protein kinases are activated by H 2 S, but it is not yet known whether these are activated directly or secondarily to upstream targets for H 2 S. H 2 S also directly regulates endothelial NO synthase activity [53] .

Changes in H 2 S have also been associated with different pathologies. In Alzheimer’s disease, reduced levels of H 2 S have been measured, whereas the opposite seems to occur in Down syndrome [66,67] . During injury of the gastric mucosa by NSAIDs, endogenous H 2 S formation may be compromised [68] . An interesting regulatory role of H 2 S has been revealed in acute inflammation. Importantly, H 2 S can reduce the adherence of leukocytes to vascular endothelium of mesenteric venules. Inhibition of endogenous H 2 S formation induces both normal and aspirin-induced injury-mediated leukocyte adherence, indicating that H 2 S has a constitutive modulatory function in controlling leukocyte-mediated inflam-mation [69] . It, furthermore, can control fluid extravasation during experimentally-induced acute inflammation [70] . These roles have provided an important new means for modulating inflammation and form the basis for several patent applications, as discussed below. In sepsis, an apparent opposite role for H 2 S has been determined, in this case a pro-inflammatory role [71] .

Apparently opposing physiological actions of H 2 S have been described at several occasions in the literature. For

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example, both pro- and anti-inflammatory activities have been reported, H 2 S can both induce apoptosis and protect from apoptosis, and both vasorelaxation and vasoconstriction can be induced by H 2 S. There are several considerations to make in understanding the variety of actions ascribed to H 2 S. Tissue specific actions are an important factor to consider; for example, H 2 S has pro-nociceptive actions in rat hind paw, but lowers pain perception in its gastrointestinal tract [72,73] . Secondly, the interaction of H 2 S with pathways activated by other gasotransmitters and autacoids is, further-more, likely to dictate the actions induced by H 2 S in the intact tissue or organism [74] . Additionally, it is possible that some observations on the actions of H 2 S have been made in vitro or in isolated cell systems that were not studied in their full physiological context. For example, the control of oxygen tension is critical for correct extrapolation of results observed in vitro to the in vivo situation [56] .

Given the overall beneficial properties of H 2 S, it is not surprising that several attempts are being made to deliver H 2 S to the body. There are several disadvantages in the use of H 2 S gas itself. H 2 S is unstable in the presence of oxygen, and formation of oxidation products can occur during pro-duction, transport and storage. Inhalation of H 2 S has tradi-tionally been considered toxic and can cause nausea, irritation of the mucosa of the eyes, shortness of breath, hypotension and loss of consciousness, and is lethal at high ppm concen-trations. The toxicity of H 2 S after inhalation depends on dose, duration of exposure, and intrinsic distribution kinetics and sensitivity of the exposed organism. The narrow thera-peutic window of inhaled H 2 S combined with lack of control over the targeting to specific tissues makes administration of H 2 S gas by inhalation a route that is not optimal, except possibly for direct delivery to the airways. Consequently, the development of liquid dosage forms of sulfides with reduced rates of oxidation, or local delivery of H 2 S by release from donor molecules, are at present being explored actively.

2. Patent applications on CO

Three different approaches have been proposed for harnessing the therapeutic potential of CO: i) direct administration of CO gas; ii) use of pro-drugs; and iii) transport and delivery of CO by means of specific CO carriers. Several patent families that use these approaches are described below.

2.1 Therapeutic use of CO gas and/or CO solutions Otterbein and colleagues have filed nine patent families describing CO as a therapeutic agent, as well as for prevention of a variety of diseases. The first patent (WO03094932) [75] presents two main directives: i) measurement of CO in exhaled air for diagnosis of diseases associated with HO-1 induction; and ii) the use of CO inhalation for treatment of several diseases. These include vascular diseases (vascular thrombotic disease, pulmonary hypertension, peripheral vascular disease); inflammatory diseases (sepsis, asthma, bronchitis);

neurodegenerative diseases (Parkinson’s disease, Alzheimer’s disease); lung diseases (emphysema, adult respiratory distress syndrome, cystic fibrosis, pneumonia, interstitial lung diseases, idiopathic pulmonary diseases); wound healing and cancer.

The second patent (WO03000114) [76] concerns the use of CO for organ and tissue transplantation. CO is prepared and administered in gaseous compositions or liquid solutions; the treatment can also contemplate HO-1 inducers, HO-1 gene transfer, HO-1 protein delivery, as well as NO mixed in the gaseous compositions, with or without phosphodiesterase inhibitor. CO is claimed to be used for treatment of the trans-plant donor, and/or ex vivo treatment of the transplanted organ, tissue or cells, and/or treatment of the recipient. The target organs and tissues disclosed in the patent are: liver, kidney, heart, skin, small intestines, pancreas as well as pancre-atic islets or cells (pancreatic β cells, liver cells, fibroblasts, bone marrow cells, neuronal cells, myocytes and stem cells).

Vascular diseases are the CO target of patent WO03072024 [77] with indications for intimal hyperplasia associated with angioplasty, atherosclerosis and transplant atherosclerosis. Applications can be systemic or topical, gas-eous or liquid compositions of CO, as well as CO releasing gums, creams, patches and intraperitoneal insufflation. Administration of HO-1 inducers, HO-1 gene or protein transfer, biliverdin, iron or ferritin, is also contemplated.

Patent WO03088923 [78] claims CO administration for the treatment of ileus of the stomach, small intestine or colon by systemic or topical delivery of gaseous or liquid compositions. The administration can be done before, during and/or after surgical procedures for preventing and/or treating ileus.

WO03088981 [79] describes the use of CO in patients (newborns and/or premature infants) suffering from or at risk of necrotizing enterocolitis. The pharmaceutical composition can be administrated by gaseous, liquid or solid compositions through inhalation, insufflation, infusion, injection and/or ingestion. Treatment with CO can be in conjunction with: i) inducing HO-1, expressing recombinant HO-1 or ferritin in the patient; and ii) administration of a composition com-prising HO-1, bilirubin, biliverdin, ferritin, apoferritin, iron desferoxamine or iron dextran.

Methods for treating hepatitis with CO are disclosed in WO03096977 [80] . The indications are for hepatitis caused by virus (hepatitis viruses A, B, C, D, E and G), by drugs (iso-niazid, methyldopa, acetaminophen, amiodarone, nitrofurantoin) or by alcoholic liver disease. The use of CO to improve liver transplantation (treatment of donor, of transplant ex vivo and/or of the recipient) is also contemplated but not claimed.

The anti-proliferative property of CO is claimed in WO03103585 [81] for the treatment of cancer and several diseases related to unwanted angiogenesis, by preventing VEGF production. CO-related treatments are similar to the previous patents of Otterbein and colleagues, alone or in combination with chemotherapy, radiotherapy, immunotherapy, gene therapy and/or surgery. Indications are for p21-expressing cancer cells and unwanted angiogenesis associated with, for

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668 Expert Opin. Ther. Patents (2009) 19(5)

example, rheumatoid arthritis, lupus, psoriasis, diabetic retinopathy and myocardial angiogenesis. This patent application also provides results showing inhibition of VEGF production and decrease of tumor volume by CO, as well as results demon-strating that CO increases survival of mice administrated with a lethal number of mesothelioma.

Hemorrhagic shock can be treated by CO (WO2004043341) [82] by preventing the systemic inflammation and/or systemic tissue injury. At therapeutically effective doses, CO does not have an effect on blood pressure nor does it seem to aggravate the shock-mediated hypotension.

Otterbein and colleagues have filed an application (WO2008008513) [83] for the treatment of inflammatory disorders with a combination of various pharmaceutical agents with: i) an inducer of HO-1 and/or apoferritin in the patient or ii) administration of compositions containing HO-1, apoferritin, hemin, CO, CO-releasing compounds, bilirubin, biliverdin, ferritin, iron, desferoxamine, salicylaldehyde ison-icotinoyl hydrazone and/or iron dextran. This combination treatment potentiates the activity of anti-inflammatory agents. In other aspects, this combination treatment can be used for organ transplants, for patients undergoing angioplasty or surgery (other than transplant surgery), for cancer, unwanted angiogenesis, hepatitis, restenosis or intimal hyperplasia. Patent WO2004000368 [84] is very similar to the previous one, but in this case NO is the key anti-inflammatory factor, which is administrated in combination with the agents described in WO2008008513 [83] for the treatment of inflammatory disorders.

Based on the finding that binding of CO or NO to some of the four heme groups of hemoglobin facilitates cooperative binding of oxygen to the other heme groups, patent US5885621 [85] claims the use of these gases for the treatment of patients with a hemoglobinopathy. These pathologies are characterized by a reduced affinity of hemoglobin for oxygen. In the case of sickle cell anemia, a further advantage for the proposed treatment is found, namely that CO and NO pre-vent the tendency of hemoglobin to polymerise. The gases can be administrated by inhalation. The ex vivo treatment of erythrocytes is considered as well.

In patent FR2816212 [86] filed by Air Liquide, the use of CO gas is claimed for the treatment or prevention of cardiovascular inflammation, such as ischemia, reperfusion, stenosis, restenosis and platelet aggregation. CO administration can be combined with anti-inflammatory and anti-thrombotic drugs, as well as with NO donors. In a second patent filed by Air Liquide (WO0209731) [87] , CO is proposed for the treatment of inflammation of the upper airway and bronchial tree, occurring in diseases such as asthma, cystic fibrosis, pneumopathy and bronchial pneumopathy. CO gas can be combined with other gases such as NO, carbon dioxide, helium, oxygen or nitrogen, as well as with anti-inflamma-tory drugs. In both patents, CO- and NO-donating drugs are also claimed, but no therapeutic applications or examples of CO/NO-donor compounds are disclosed.

The use of a CO/xenon mixture is claimed by Petzelt and Jakobsson for the protection of cells, tissues and organs from apoptosis or necrosis, or from aberrant programming of cell division, occurring after ischemia or hypoxia of the heart, brain, kidney, other peripheral organs as well as in tumor cells (WO2005067945) [88] . The application is based on the follow-ing findings: i) xenon at high concentrations provides cell protection against excess neurotransmitter release; ii) xenon reduces heart infarct size during reperfusion; and iii) xenon is an NMDA-receptor antagonist. At effective doses, xenon also presents anesthetic actions. The objective of this invention is to improve and extend the therapeutic applications of xenon by combining it with CO, allowing the effective con-centrations of xenon to be decreased. Different gas mixture compositions are discussed in the patent, although the mode of gas administration is not disclosed.

2.2 Therapeutic use of CO-RMs The use of the metal carbonyl complexes iron pentacarbonyl ( 1 ) and iron enneacarbonyl ( 2 ) is claimed by the company Lohmann Therapy Systems for the transdermal delivery of CO ( Figure 1 ) (WO9535105) [89] . The compounds can be administrated through different pharmaceutical formulations: creams, ointments, gel colloids or skin patches. The indications are for hypertension, platelet aggregation, inflammation and neurotransmission disorders in the peripheral and central nervous systems.

The invention described by Kao and colleagues in US5670664 uses norbornadienone derivatives to release CO on irradiation with UV or near UV light [90] . After photo-activation, norbornadienone ( 3 ) decomposes to yield the highly stable molecule benzene and CO ( Figure 2 ). Deriva-tives of norbornadienone are inert precursor molecules that can be taken up by cells and remain stable inside the cell until irradiated with UV light. Several norbornadienone derivatives are described in the patent, and examples of their chemical synthesis are disclosed. Experimental applications in cultured cells or tissues are claimed, but no therapeutic applications are disclosed.

A patent application filed by the biotechnology company Sangstat (WO02078684) [91] is based on the in vivo metabolic conversion of methylene chloride (dichloromethane) into CO and CO 2 by cytochrome P450 and glutathione S-trans-ferase ( 4 ; Figure 3 ). The claims provide methods and composi-tions to treat vascular, inflammatory and immunological disorders. Several therapeutic applications for methylene chloride are provided, namely protection against ischemia/reperfusion injury in the liver on transplantation, protection from apoptosis and liver allograft survival, protection against allograft arteriosclerosis due to intima hyperplasia, inhibition of neointimal growth after wire injury and therapeutic actions in collagen-induced arthritis. Inflammatory diseases mentioned in the claims are septic shock, rheumatoid arthritis, Crohn’s disease and colitis. Cytochrome P450-mediated formation of CO from methylene chloride occurs mostly in the liver. For delivery of hepatocyte-generated CO to other tissues, further

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FeOC

CO

CO

CO

CO

1 2

Fe

C

FeCO

CO

CO

OC

OC

OCCO

CO

O

Figure 1. Metal carbonyl complexes iron pentacarbonyl (1) and iron enneacarbonyl (2).

O

CO +

3

Figure 2 . Formation of CO via UV light-induced decomposition of norbornadienone (3).

arteriosclerosis, post-ischemic organ damage, myocardial infarc-tion, angina, hemorrhagic shock, sepsis, penile erectile dysfunc-tion and adult respiratory distress syndrome. One of the disclosed compounds is the water-soluble metal carbonyl compound CORM-3 (Ru(CO) 3 Cl-glycinate) ( 5 ; Figure 4 ), which can liberate CO in in vitro , ex vivo and in vivo biological models. CORM-3 is one of the best studied CO-RMs and many examples of its actions are described in the literature, for example, cardioprotection, protection against ischemia-reperfusion injury and allograft rejection [43] .

Patent WO200404558 is also based on metal carbonyl compounds, but in this case the delivered CO is used for the treatment of post-ischemia damaged organs [93] . The organ can be treated extracorporeally, for instance, during transplantation procedures or can be an isolated organ inside or attached to the body but isolated from the blood flow. Examples are described for CORM-3 [43,94] . The invention claimed by WO2004045598 is based on metal carbonyl administration in combination with a guanylate cyclase stimulant or stabilizer [93] . Vascular relaxation by NO and CO is mediated in part by increased intracellular cGMP levels through activation of sGC. Because CO is a much weaker stimulator of sGC than NO ( in vitro ), this patent application claims to improve the therapeutic actions of CO by co-administration of a stabilizer or stimulant of sGC. The benzyl-indazole derivative YC-1 is disclosed as one example of an activator of sGC. YC-1 can be administrated concommitantly with, or before, CO. The indications are the same as described in WO02092075 [92] .

Boranocarbonates, another class of CO-releasing compounds, are disclosed in US2007/0065485 by Motterlini and col-leagues [96] . Typically, boranocarbonates present the minimal structure ( 6 ) described in Figure 5 . It is believed that the attachment of three hydrogen atoms to boron facilitates CO release. Structures in which a carboxylate group is attached to boron, that is, -COO - , -COOH and -COOX (with X being any pharmaceutically suitable sterifying group) can also be considered. All examples described in this patent concern CORM-A1 ( 7 ; disodium boranocarbonate; Na 2 (H 3 BCO 2 )).

The main difference between CORM-A1 and CORM-3 is the rate of CO release. CORM-A1 is considered to be a ‘slow releaser’, presenting a half-life of around 21 min in the presence of myoglobin, whereas CORM-3 has a half-life of around 1 – 2 min. Slow releasers, such as CORM-A1, allow the design of pharmaceuticals that could be more versatile for the treatment of certain chronic diseases (i.e., arthritis, inflammation, cancer, organ preservation, septic shock, chronic hypertension, prevention of restenosis after balloon angioplasty, post-operative ileus) in which a continuous release and longer lasting action of CO may be required [14] .

Patent WO2007/085806 [97] provides a pharmaceutical composition for delivery of CO comprising carbonyl ligands and a cyclopentadienyl, indenyl or fluorenyl ligand covalently bonded to the transition metal center. Some examples of the core structures ( 8 – 10 ) and two water-soluble

H

HCl

Cl

4

Figure 3 . Methylene chloride.

transport through the systemic circulation would be required, which involves binding to hemoglobin. A potential disad-vantage of cytochrome P450-mediated biotransformation of pro-drugs to form CO is the inhibition of the same enzyme by the formed CO, which limits the efficacy of the process.

Motterlini and colleagues (HemoCORM Ltd) have filed six patent applications related to CO-RMs. Two main classes of CO-RMs are described in their patents: i) metal carbonyl compounds, typically with a transition metal of low molecular mass (Ru, Fe, Co and Mn) and ii) boranocarbonates. Patent application WO02092075 [92] discloses two classes of CO-RMs: organometallic compounds with the general formula M(CO) x A y B z (M, a metal preferably Fe, Ru or Co; A, an atom or group bonded to M by ionic, covalent or coordination bonds; and B, various groups) and organic compounds, such as formates, oxalates and derivatives. The release of CO from the metal carbonyl complexes is measured spectrophotometrically by assessing the conversion of deoxymyoglobin to MbCO. The applications claimed in this patent are: treatment of hypertension (such as acute pulmonary and chronic hypertension), radiation damage, endotoxic shock, inflammation, inflammation-related diseases such as asthma and rheumatoid arthritis, hyperoxia-induced injury, cancer, transplant rejection,

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670 Expert Opin. Ther. Patents (2009) 19(5)

CO-RMs (CORM-337; 11 and CORM-351; 12 ) are given in Figure 6 . Attaching substituents to the cyclopentadienyl ( 8 ), indenyl ( 9 ) or fluorenyl ( 10 ) ring of a transition metal complex subtly alters the electronic properties of the mole-cule, and allows modulation of the CO release rate to a physiological target. In addition, it may increase the solubil-ity of the CO-RM in aqueous physiological fluid. It is believed that the presence of specific substituents R 1 on the cyclopentadienyl ring (such as shown in 11 and 12 ) increases the solubility and/or stabilizes the resulting CO-RM by-prod-uct, effectively preventing formation of an insoluble species. The patent provides descriptions of the synthesis of these novel compounds. The biological activities of this class of CO-RMs are demonstrated by in vitro approaches with RAW264.7 macrophages and are based on cytotoxicity and anti-inflammatory assays (e.g., reduction in NO production after LPS challenge). The same therapeutic applications are claimed in WO02092075 [92] .

The most recent patent filed by Motterlini is WO2008/003953 [98] that describes manganese carbonyl compounds for the therapeutic delivery of CO. This patent application is an extension of earlier work [92,93] , and the claimed therapeutic applications, the method used to measure CO release, as well as the biological actions of these CO-RMs, are the same as described in these previous patents. The general structures of CO-RMs described in this proposal are given in Figure 7 ( 13 , 14 ). Ligands X, Y and Z can be a wide variety of chemical groups. All described compounds were tested for cytotoxicity and anti-inflammatory actions using in vitro assays with RAW264.7 cells. Two water-soluble compounds, CORM-371 ( 15 ) [(Me 4 N)(Mn(CO) 4 (thioacetate) 2 )] and

B

O

OX

B

H

H

H

O

O

2Na+

2-

6 7

Figure 5 . Chemical structure of boranocarbonates.

CORM-376 ( 16 ), with CO release half-lifes of 32 and 9 min, respectively, are shown to induce relaxation of pre-contracted rat aorta at concentrations ≥ 50 µM.

The first patent filed by Haas and colleagues from Alfama Co. (WO2003/066067) [99] claims the in vivo use of CO-releasing compounds for the treatment and/or prevention of chronic inflammation (such as rheumatoid arthritis) and of diseases with a strong inflammatory component, such as atherosclerosis, stroke, coronary vascular disease and Alzheimer’s disease. This application emphasizes the need for CO to be delivered to specific sites in the body that are affected by a disease. Seven different classes of CO-RMs are described as candidates. Class 1 consists of simple 18 electron organometallic carbonyl complexes, or modifications thereof, to improve solubility in physiological media ( 17 – 20 ; Figure 8 ). In compounds 17 – 19 , the metal group M is chosen from manganese or rhenium, whereas in compound 20 the metal is molybdenum or wolfram. Substituent X can be a halide, an alkyl or aryl group, a weakly coordinating anion, or an ether or thioether. Class 2 compounds are class 1 organometallic complexes linked to another molecule, such as a carrier or a drug ( 21, 22 ; Figure 8 ). Class 3 consists of supramolecular aggregates made of CO-containing organometallic complexes (from class 1 and 2) encapsulated in, for instance, cyclodextrins, liposomes or mesoporous materials. Class 4 drug candidates are inor-ganic complexes bearing ligands containing nitrogen and/or sulfur donors that function as reversible CO carriers ( 23 – 25 ; Figure 8 ). The complexes disclosed are macrocycles bearing bidentate N donors (diamines, diglyoximes) or bidentate nitrogen and sulfur-containing donors (aminothiols, cysteine) and nitrogen donors (imidazole, histidine). Class 5 candi-dates are compounds from class 4 that are linked through a variety of molecular spacers to other pharmacologically important molecules ( 26 ; Figure 8 ). The introduction of a spacer between the organometallic complex and a carrier or drug can potentially aid in preserving the functionality of both entities. Class 6 compounds are organic substances that release CO either by an enzymatic process or spontaneous decarbonylation, and are soluble in physiologically compa-tible supports ( 27 – 29 ; Figure 8 ). The substituent R can be hydrogen, or an alkyl or aryl group. Finally, class 7 compounds are class 6 compounds that are encapsulated in a cyclodex-trin host, in liposomes and/or in a suitable inorganic or organic support.

The present invention does not provide results in direct support of these compounds as being CO-RMs. In addition, there are no biological in vitro or in vivo results presented showing the efficacy of these compounds. The novelty of this application is the emphasis on selective CO delivery to diseased tissues by conjugating CO-RMs with other compounds or drugs said to accumulate in particular tissues. For example, CO-RMs can be conjugated with aspirin or other organic acids that are enriched in inflamed tissues; with biphosphonates that are targeted to inflamed joints of arthritis patients; or with α -lipoic acid that accumulates in the vasculature.

Figure 4 . CORM-3 (Ru(CO) 3 Cl-glycinate).

Ru

HN

O

CO

Cl

OC

OCO

5

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Expert Opin. Ther. Patents (2009) 19(5) 671

In US2007/0207993, US2007/0207217 and WO2007073225 filed by Haas et al. (Alfama Co.), molybdenum carbonyl complexes are disclosed as CO-RMs useful for inhibiting TNF production and for treating inflammatory diseases [100] . The general formula of the compounds is (Mo(CO) 5 Y)Q, where Y is bromide, chloride or iodide. Q is (NR 4 )

+ and is complexed with one cyclic polyether molecule or one or more acyclic polyether molecules, or NH 4

+ Na + , K + , Mg 2+ , Ca 2+ or Zn 2+ , wherein each is free or in complex with one cyclic polyether molecule or one or more acyclic polyether

molecules. Each R is independently an alkyl group. Some examples ( 30 – 32 ) are given in Figure 9 . Results are provided that demonstrate the spontaneous CO release from the com-pounds, as well as CO release in vivo . In addition, results show the ability of compound 30 in preventing TNF- α production in mice, and in treating adjuvant arthritis in rats. Examples of the synthesis of the compounds are provided as well. The disclosed indications for CO-RMs herein are arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, osteoarthritis, asthma, chronic obstructive

R1

(R2)r

R1

(R2)r

(R3)s

(R1)

(R3)t

8 9 10

11 12

(R3)s

PF6-

Fe+

OCCO

CO

OOH

O

BF4-

O

OFe+

OCCO

CO

Figure 6 . Examples of substituted CO-releasing organometallic carbonyl complexes claimed by Motterlini and colleagues.

-

Mn

CO

CO

OC

OC

S

C

C

O

S

O

-

CO

Mn

CO

OC Y

OC X

O

O

O

Mn

CO

Mn

OC

OC

Ac

Ac CO

13 14

15 16

OC CO

Ac

K+

Y

X

Z

Mn

CO

Mn

OC

OC CO

OC CO

NMe4+

Figure 7 . Examples of CO-releasing manganese carbonyl complexes claimed by Motterlini and colleagues.

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672 Expert Opin. Ther. Patents (2009) 19(5)

M

CO

OC

XOC

CO

CO

17

M

CO

OC

XOC

CO

CO

+

X-

18

M

X

M

X CO

CO

CO

CO

CO

OC

OC

CO

19

M

X

M

X CO

CO

CO2-

[N(alkyl)4]+

2

CO

CO

OC

OC

Co

20

Fe

CO

N

CO

S

N

S

=

N

S

Aminothiole or cysteine

Class 4

Class 2

Class 1

Fe

N

N

CO

L

N

N

=

N

N NN

ary l ary l

HO OH

=Fe

CO

CO

CO

CO

LL L

L

Diimines, glyoximes, amino-alcohols,aminothiols, aminoacids

23

21

24

25

Mn

CO

(spacer)-aspirin

CO

OC

22

(spacer)-aspirin

COOC CO

CCl

Cl

Cl

O

ONa

C

28

O

SCCl

Cl

Cl

OR

29

O

OR

C CCl

Cl

Cl

27

26

Fe

CO

N

CO

S

N

S

= Aminothiole or cysteine

(spacer)-aspirin

N

S

Class 5

Class 6

Figure 8 . Organometallic carbonyl complexes and organic CO precursors claimed by Haas and colleagues.

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Expert Opin. Ther. Patents (2009) 19(5) 673

pulmonary disease, inflammatory lung disease, ulcerative colitis, Crohn’s disease, inflammatory disease associated with ischemia/reperfusion injury, atherosclerosis, Alzheimer’s disease, myocardial infarction, stroke or organ transplantation. These indications largely overlap with those in other CO patent applications, but are somewhat more committed to rheumatoid arthritis and more specifically directed towards the inhibition of TNF- α production.

De Matos and colleagues (Alfama Co.) have filed a patent application for treating inflammatory diseases by administrating aldehydes and derivatives thereof (US2008/0026984 A1) [101] . The general formula of these aldehydes ( 33 ) is given in Figure 10 . The compounds exhibit anti-inflammatory properties at least in part by delivering CO in normal and inflamed tissues. CO is generated from the aldehydes by decarbonylation activated by the presence of reactive oxygen species, a change in pH, hydrolysis, metabolic activation or any other biological change that affects the stability of the aldehyde. Some aldehydes (or derivatives thereof) generate CO exclusively or preferentially in the presence of reactive oxygen species, and are expected to have beneficial effects in diseases associated with oxidative stress. The application demonstrates the anti-inflammatory actions of compounds 34 (trimethylacetaldehyde) and 35 (2-methyl-2-phenylpropionaldehyde) in rats after induction of adjuvant arthritis ( Figure 10 ). Treatment was initiated 10 days after adjuvant arthritis induction using daily injec-tion for 30 consecutive days at a dose of 25 or 100 mg/kg for both molecules. The indications claimed in this patent application are very similar to previous therapeutic applica-tions sought after by Alfama: arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, psoriasis arthritis, osteoar

thritis, inflammatory lung disease, Alzheimer’s disease, Par-kinson’s disease, amyotrophic lateral sclerosis or multiple sclerosis, inflammatory bowel disease, inflammatory skin disease, myocardial infarction, stroke or transplant rejection, Gram-positive or Gram-negative shock, sepsis, septic shock, hemorrhagic or anaphylactic shock, and systemic inflammatory response syndrome.

3. Patent applications on H 2 S-releasing molecules and H 2 S liquid dosage forms

Patent applications that address the potential therapeutic applications of H 2 S liquid dosage forms and H 2 S-releasing molecules are all very recent. Several applications reflect the strong confidence that H 2 S can add to the therapeutic value of known drugs, reduce their side effects or that H 2 S by itself has beneficial therapeutic actions.

Wallace and colleagues have disclosed several patent applications that illustrate a powerful approach to use H 2 S-releasing compounds for adding to the known thera-peutic action of other drugs and treatments, or which reduce their side effects. In one application, one of several H 2 S-releasing compounds is used as a specific compound that exerts an anti-inflammatory action through modulation of nuclear tran-scription factor activity (WO2006085127) [102] . These com-pounds include: 5-( p -hydroxyphenyl)-3H-1,2-dithiol-3-thione ( Figure 11 , compound 36 ), 1,3-dithiol-2-thione-5-carboxylic acid ( 37 ), 3-thioxo-3H-l,2-dithiole-5-carboxylic acid ( 38 ) and 3-thioxo-3H-l,2-dithiole-4-carboxylic acid ( 39 ). The H 2 S-releasing compounds are included in a topical formulation of Sangre do Grado, an extract obtained from a resin with

Mo

CO

CO

Br

CO

OC

CO

N+(Et)4

-

30

Mo

CO

CO

Cl

CO

OC

CO

N+(Et)4

-

31

Mo

CO

I

CO

CO

OC

CO

N+(Et)4

-

32

Figure 9 . Examples of CO-releasing molybdenum carbonyl complexes claimed by Haas and colleagues.

O

H

R3

R2

R1

O

H

O

H

33 3534

Figure 10 . Examples of aldehydes which can generate CO, claimed by De Matos and colleagues.

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674 Expert Opin. Ther. Patents (2009) 19(5)

known wound healing properties of specific species of Croton , a tree found in tropical and sub-tropical regions. The thera-peutic application is the topical treatment of pathological disorders caused by a variety of insect bites, jelly fish stings, as well as anorectal disorders, including hemorrhoids and anal fissures. A number of other compounds, such as cysta-thione, methionine, N-acetyl-penicillamine and other sulfur-containing compounds that can potentially be transformed to form H 2 S after metabolism can also be included in the formulation. The overall aim of the application is to find a way to treat the inflammatory sequelae of tissue damage by com-bining the wound healing properties of Sangre do Grado with the H 2 S-releasing compounds that will lower inflammation through reduced NF- k B activity. Furthermore, the combination aims to lower the required concentration of Sangre do Grado (which owing to its ardent red color is not comfortably used for topical applications).

Whereas in this patent application the mentioned H 2 S-releasing compounds are used as such, in subsequent patent applications these compounds, structurally-related H 2 S-releasing compounds, and other H 2 S-releasing compounds, are coupled covalently to several therapeutically useful, often off-patent, drugs. Overall, the authors aim to combine the pharmacodynamic activities of the scaffolding drug and a H 2 S-releasing group. Thus, in one patent application (WO2008009118 A1), Wallace and colleagues disclosed the 4-hydroxy-thiobenzamide chemical moiety ( 40 ), which when covalently coupled to a suitable drug, forms a thiocarbamoyl phenyl ester compound ( 41 ) that can release H 2 S ( Figure 12 ) [103] . Alternatively, the 4-hydroxy-thiobenzamide as such may be used as a salt together with a second compound. The application discloses 4-hydroxy-thiobenzamide-derivatives of drugs encompassing all major areas of pharmacotherapy, but more specifically claims compounds found in the classes of NSAIDs, anticolitic drugs, analgesics and anti-hyperlipidemic drugs, in particular statins [103] . The aim of the patent application is the devel-opment of drug derivatives with reduced side effects and/or enhanced pharmacological activity compared to the drug alone. This may be a useful development strategy for drugs with known side effects on the gastrointestinal tract, which may be further enhanced under conditions of oxidative stress and inflammation such as occurring during gastrointestinal

damage and colitis. The application mentions redox-cycling antitumoral drugs, anti-thrombotic drugs, NSAIDs, mucolytics, bone-resorption inhibitors and antibiotics as examples of drugs that can display side effects on the gastrointestinal tract, such as local tissue injury and inflammation.

Results are provided that show a markedly improved reduction of experimental colitis by using 4-hydroxy-thiobenzamide-coupled mesalamine, a reduction in pain perception and a reduction in neutrophil adherence in aspirin-induced tissue-injury. Another example is 4-hydroxy-thiobenzamide-naproxen, which is shown to completely lack the gastric toxicity of naproxen itself. The inhibition of cyclooxygenase-mediated generation of thromboxane is maintained by the derivatization of naproxen with 4-hydroxy-thiobenzamide, indicating that the cyclooxygenase-inhibitory activity of naproxen is preserved. The results indicate that by including H 2 S release from con-ventional drugs, it is possible to counteract some known side effects although maintaining the principal pharmacodynamic properties of the drugs. Several compounds, such as thiocar-bamoylbenzoate-naproxen, were demonstrated to induce a significantly smaller increase in systolic blood pressure when administered to hypertensive rats, compared to when the drug itself was administered. A reduced propensity to elevate blood pressure may reduce the cardiovascular side effects frequently seen with the use of some of the drugs. An indica-tion to enhanced drug potency was further provided by the demonstration that the 4-hydroxy-thiobenzamide-derivative of naproxen displayed an increased reduction in prostaglandin E 2 formation in inflammatory exudates. It is furthermore shown that the 4-hydroxy-thiobenzamide-naproxen conju-gate can release significantly more H 2 S than the 4-hydroxy-thiobenzamide moiety alone, either in buffer or in a diluted liver homogenate.

The most specific claims of this application disclose the beneficial actions of 4-hydroxy-thiobenzamide-coupled statins, such as enhanced pharmacological activity by coupling of a H 2 S-releasing moiety to simvastatin (succinic acid 2-(2-[8-(2,2-dimethyl-butyryloxy)-2,6-dimethyl-1,2,6,7,8,8a-hexa-hydro-naphthalen-1-yl]-ethyl)-6-oxo-tetrahydro-pyran-4-yl ester 4-thiocarbamoyl-phenyl ester). When compared to the corresponding statin alone, the compound had a higher efficacy in reducing platelet aggregation, accompanied by an increase

S

SS

COOH S

S

S

COOH

S

S

S

COOH

36 37 38 39

OH

S

S

S

Figure 11 . Examples of H 2 S-releasing compounds claimed by Wallace and colleagues.

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Expert Opin. Ther. Patents (2009) 19(5) 675

in platelet cAMP. It is not known and not addressed how modification of the drugs by the 4-hydroxy-thiobenzamide moiety affects the pharmacological activity of the statin on other parameters that determine its pharmacological profile, such as uptake, transport, clearance and inhibitory action of HMG-CoA reductase. The same considerations may apply to all of the other proposed drug/H 2 S-releasing molecule combinations.

In separate patent applications, the H 2 S-releasing versions of the anticolitic drugs 4-aminosalicylic acid and 5-aminosalicylic acid (mesalamine) (WO2007140611) [104] as well as the spasmolytic drug trimebutine (WO2008/009127) [105] , are claimed to afford a superior safety profile in the gastrointestinal tract compared to the unmodified drugs alone. In these appli-cations, the parent drugs are modified with an H 2 S-releasing moiety through an azo-, ester-, anhydride-, thioester- or amide-linkage, which can be broken down enzymatically within the GI tract. A wide range of structurally different H 2 S-releasing moieties is contemplated. Three preferred compounds are the H 2 S-releasing aminosalicylates shown in Figure 13 ; 5-amino-2-hydroxy-benzoic acid 4-(5-thioxo-5H-3dithiol-3-yl)-phenyl ester ( 42 ), 4-thiocarbamoylphenyl-4-hydroxybenzoate or 5-amino-2-hydroxybenzoate ( 43 ) and 4- or 5-amino-2-hydroxy-benzoic acid anhydride with N-acetylcysteine ( 44 ). The modification of the aminosalicylates may provide the extra advantage of converting the drugs into pro-drugs, which are protected from too rapid absorption in the upper gastrointestinal tract, permitting more effective delivery to the lower GI tract (colon and rectum). The released H 2 S may additionally lower inflammation, reduce pain resulting from colon distension and relax intestinal smooth muscle.

Kawabata disclosed the use of the sodium, potassium, ammonium and calcium sulfide salts for the prevention or treatment of diseases of the digestive system (JP2007063167) [106] . In particular, the application encompasses a wide range of formulations for the treatment of ulcers of the stomach and duodenum, gastritis, diarrhoea and enteritis. H 2 S plays an endogenous role in limiting neutrophil attachment to the

40 41

NH2

S

OH

NH2

S

OO

R′

Figure 12 . The 4-hydroxy-thiobenzamide chemical moiety (40) and H 2 S-releasing thiocarbamoyl phenyl ester conjugate (41).

vascular endothelium of the GI tract, and inhibits inflammation when the mucosa is damaged, for example, in a small per-centage of people by NSAID use [68] . Furthermore, H 2 S has been shown to promote healing of gastric ulcers in the rat [107] . Wallace and colleagues disclosed an application that makes use of these protective actions of H 2 S. By coupling one of several different H 2 S-releasing groups ( 36 , 40 ; and 45 – 47 ; Figure 14 ) to a variety of common NSAIDs, they aim to reduce the side effects of NSAID use [108] . One example of these so-called HS-NSAIDs, ATB-337, is diclofenac linked to an H 2 S-releasing moiety. The compound has been shown to possess a markedly reduced inflammatory profile on the stomach mucosa and vasculature, yet preserves its capacity to inhibit cyclooxygenase activity and eicosanoid formation [109] . Administration of these compounds leads to an increased plasma H 2 S level, and also inhibits platelet cyclooxygenase. In fact, the cyclooxygenase inhibitory activity seems to be increased when 4-hydroxy-thiobenzamide-derivatized NSAIDs were compared with the parent NSAIDs. The patent applica-tion also provides routes of synthesis. The novel compounds are claimed to be useful in a range of inflammatory disor-ders, not only in humans, but also in cattle and pets. A large set of results is provided to support the anti-inflammatory activity of the various HS-NSAIDs.

Another example of the modification of an existing class of drugs with H 2 S-releasing molecules is disclosed by Sparatore and colleagues (EP1630164 A1) [110] . They prepared chemically-modified inhibitors of phosphodiesterase to obtain H 2 S-releasing compounds for the treatment of impotence and disorders of the cardiovascular system (angina, hypertension, heart failure and atherosclerosis) and the digestive system (alterations in gut motility, irritable bowel syndrome, gastropathy, and ulcer healing and prevention). In particular, a series of pyrazolo-[4,3-d]pyrimidin-7-ones (such as sildenafil, vardenafil and tadalafil), selective inhibitors of type-V cyclic GMP phosphodiesterases (PDE5), are modified on the carboxylic acid functional group with one of several possible H 2 S-releasing agents. The compounds are mentioned to be particularly useful in patients with mild to severe endothelial dysfunction who respond relatively poorly to PDE5 inhibitors, and can poten-tially also be used as an alternative to organic nitrates in patients who experience side effects from excessive NO production. The authors mention that two analogues, 5-(p-hydroxyphenyl)-3H-1,2-dithiol-3-thione-modified sildenafil and cysteine-modified sildenafil, have K i values of 6.8 – 7.2 nM for PDE5 inhibition (compared to K i of sildenafil of ∼ 1 – 2 nM [111] ), indicating that the inhibitory action of sildenafil is largely preserved by the proposed structural modifications.

Scherrer and Sparatore assessed the usefulness of a series of H 2 S-releasing benzoic acid-derivatives to inhibit inflammation, which underlies metabolic syndrome, diabetes, obesity, dys-lipidemia and insulin resistance (WO 2006066894 A1) [112] . The compounds were reported to preserve blood pressure, endothelium-dependent vasodilation and glutathione levels when rats were exposed to the glutathione biosynthesis

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676 Expert Opin. Ther. Patents (2009) 19(5)

inhibiting agent buthionine sulfoxime to activate hypertension through the induction of oxidative stress. Tested compounds are 2-acetoxy-benzoic acid 4-(thioxo-5H-[1,2]dithiol-3-yl)-phenyl ester, 2-hydroxybenzoic acid 4-(thioxo-5H-[1,2]dithiol-3-yl)-phenyl ester, and 2-(5-[1,2]dithiolan-3-yl-pentanoyloxy)benzoic acid ethyl ester.

Zhu disclosed the use of H 2 S and the H 2 S-donor sodium bisulfide for the treatment of cardiovascular disease, such as myocardial infarction (CN101011413A) [113] . Pharmacological properties were reduction of formation of active oxygen species, acceleration of vessel growth, negative inotropic action or as calcium channel antagonist.

Exposure to H 2 S and other sulfide compounds has been shown to reduce metabolism, reversibly lower body temperature and induce bradycardia [114] . These actions can confer protection

from tissue damage resulting from hypoxia and ischemia/reperfusion [115] . Low concentrations of H 2 S may allow tighter coupling of mitochondrial respiration and ATP pro-duction and create a more efficient use of energy substrates. Two patent applications include the use of H 2 S or a H 2 S-donor to make use of these hypo-metabolic and tissue-preserving properties of H 2 S, and hence improve the use of another therapeutic compound. Wintner disclosed thiomorpholine and thiomorpholine analogues with the aim of reversibly inhibiting metabolism in cells, tissue or organs of a mamma-lian organism exposed to hypoxia (WO2008/070741A1) [116] . The thiomorpholine compounds are mentioned to be used in conjunction with H 2 S or related chalcogenide compounds. Results are provided that show the lowering of body temperature on administration of thiomorpholine in mice

SS

S

OO

C

OH

H2N

O

O

OH

H2N

S

NH2

O

O

SH

NH

O

O

OH

H2N

42 43

44

Figure 13 . H 2 S-releasing aminosalicylates.

SS

O

OH

OH

SS

N

OH

N OHCS

45 4746

Figure 14 . Examples of H 2 S-releasing groups which can be coupled to existing pharmacologically active compounds, claimed by Wallace and colleagues.

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Expert Opin. Ther. Patents (2009) 19(5) 677

exposed to hypoxia (3.5 – 4% O 2 ). The potential use of therapeutic hypothermia induced by thiomorpholine is dem-onstrated by results in which thiomorpholine infusion delayed death of mice when given before a high dose of LPS. In particular, the application is aimed at the treatment of bleeding, hemorrhagic shock, surgery, and tissue protection during chemotherapy and radiation therapy. Other applications relate to the preservation of dead animals, plant tissue, and for impeding spoilage of food and beverages. The mechanism whereby thiomorpholine compounds induce the reported hypometabolism is, however, not explained in the application.

Another example in which co-administration of H 2 S is reported to add to the actions of another drug is provided in an application by Dobson (WO2008011670A1) [117] . Here, potassium channel openers, adenosine receptor agonists and local anesthetic drugs (such as lignocaine) are used to reduce injury of cells and tissues during trauma. The compounds can be administered once, intermittently or continuously. The approach is based on aiming to maintain the cell mem-brane potential during ischemia, lower tissue energy demand, and inhibit inflammatory and coagulation responses. The co-administration of H 2 S is said to contribute to these three tissue-preserving actions.

The potential protective actions of H 2 S are further highlighted by a patent application presented by Szabo and colleagues (WO2008079993) [118] . The application aims for the use of H 2 S (sulfides) to lower the cellular toxicity and side effects associated with therapeutic treatment with NO or NO-donors, and vice versa. The sulfide and NO compounds can both be administered by inhalation and/or as a solution. Peroxynitrite is one potentially noxious product of NO. It is formed through the reaction of NO with superoxide anion radical and is a strong oxidant. When it is formed, it can mediate oxidative damage through nitrosation reaction with proteins and lipids, and is a mediator of tissue damage in some clinical disease settings. H 2 S may potentially lower the nitrosative stress mediated by peroxynitrite. Overall, the application predicts the improvement of the treatment of respiratory, cardiovascular, pulmonary and blood disease, the reduction of tissue injury resulting from hypoxia and ischemia-reperfusion events, as well as the treatment of cancer (or other hyperpro-liferative disease), inflammation, infections, shock sepsis stroke and recovery from extreme hypothermia and hyperthermia. The application also provides details to a device that can administer the two therapeutic gases, as well as to the administration of stabilized solutions of sulfide through other routes of administration (as described in [119] ). Results are presented which demonstrate that pretreatment of J744 murine macrophages with H 2 S (60 µM to 1 mM sulfide solutions) for 30 min or 24 h reduces the cytotoxicity induced by per-oxynitrite or the NO-donor S-nitroso-glutathione (1 – 10 mM). The administration of sulfide also lowers pro-inflammatory cytokine release in a murine LPS-stimulated peritonitis model. Of interest, the action of H 2 S on IL-1 β release seems to be mediated through HO, as the sulfide action was

fully reversed by co-administration of tin-protoporphyrin IX. The authors propose that H 2 S may exert both a short-term antioxidant effect and a long-term ‘pre-conditioning’ action.

Faikovich disclosed the use of H 2 S for the treatment of Helicobacter pylori infections in humans [120] (RU2304967C2). A 10 day p.o. treatment with 450 ml H 2 S solution daily (containing 10 or 25 mg/l water), in combination with omepra-zole and furazolidone, led to eradication of the infection and significant healing of stomach tissue. Another example of substantial pharmacological activity in humans using treatment directly with H 2 S-containing solutions is given in a patent by Botvineva (RU2321386) [121] . Here, topical applications of sulfide-containing mud during 10 – 15 min, alternated with H 2 S/carbonic acid warm water baths, are demonstrated to be beneficial for diabetic patients. The actions of this so called peloidotherapy are measurable in all tested patients and include a lowering of blood sugar levels and improved peripheral circulation, two actions that can reduce the angiopathy associated with diabetes.

H 2 S-releasing drugs have also found their way in an application for the prevention or reduction of tumoral disease. Sparatore and colleagues disclosed the use of the 5-(p-hydroxyphenyl)-3H-1,2-dihiol-3-thione ester of valproic acid for the treatment of a wide variety of cancer types (EP1886681A2) [122] . The compound can be used in com-bination with existing chemotherapeutic agents. Valproic acid has recently been recognized as a histone deacetylase inhib-itor with selective antitumor activities, and possibly targets epigenetic alterations in cancerous cells [123] . Additionally, the application claims the use of the compound for the reduction of oxidative stress associated with diseases of the cardiovascular, respiratory, nervous, gastrointestinal and uro-genital systems, in tumors, with diseases of the skin and connective tissue, and with infectious disease. The applica-tion shows the reduction in inflammatory cytokine release from the cancer cell line HeLa by the compound acetyl thiosalicylic acid 4-(nitroxymethyl)-phenyl ester, and a 75% reduction in basal and TNF- α -stimulated NF- k B activity in a reporter cell system. Furthermore, this compound is able to inhibit the expression of the inducible form of NO synthase via stimulating the expression of HO-1 in a zymosan-stimulated murine macrophage cell line. The application, thus, discloses the development of modulators of nuclear transcription factors that not only inhibit inflammation, but may also stimulate production of endogenous anti-inflammatory enzymes.

Tomaselli and colleagues disclosed the use of liquid dosage forms of H 2 S (sulfide) salts and other chalcogenide salts (of which only selenium is considered further) (WO2008043081) [119] . The pharmaceutical aspects of this application are well defined. A variety of salts is mentioned in the application, such as sodium, potassium, rubidium, ammonium and calcium salts. The sulfide salts make up at least 80% (w/v) of the solution, in the concentration range of 95 – 150 mM, pH 6.5 – 8.5 and an osmolarity of 250 – 330 mOsmol/l. Different ways for

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678 Expert Opin. Ther. Patents (2009) 19(5)

stabilizing the solutions are provided: by keeping oxygen concentration below 5 µM, addition of a metal chelator (such as DTPA), a reducing agent (dithiothreitol), a free radical scavenging agent or antioxidant (e.g., Trolox or TCEP), or other preservatives such as benzyl alcohol or methyl paraben. In addition, the solution can be stored in light-impermeable containers, or under an inert atmosphere. The invention provides many routes of administration (e.g., orally, intravenously, topically and subcutaneously). The solution can be used for the prevention or treatment of injured biological material, such as platelets, transplant tissue or tissue at risk for reperfusion injury or bleeding (such as occurring during surgery). The application contains a large set of experimental results that support the stability of the solutions, as well the release in vivo of H 2 S (by detection of the main metabolite thiosulfate in urine in rat) after i.v. administration. Furthermore, infusion of Na 2 S is shown to prevent hypoxia-induced death in mice and reduces ischemia-reperfusion injury.

Although most patent applications related to therapeutic aspects of H 2 S provide almost irresistible support for the beneficial actions of H 2 S, several patent applications specifically address the lowering of endogenous H 2 S production. Yokawa and colleagues disclose a method for lowering H 2 S in the intestines (JP2007099672A) [124] . The authors describe that lowering H 2 S prevents dysfunction of large intestine and affords a general improvement in health. In addition, they predict that the functional damage of intestines induced by H 2 S can lead to dystrophy, acne, rash and paralysis of limbs. Increased H 2 S formation is known to be associated with ulcerative colitis and is reported to be caused by an altered microbial composition or metabolism in ulcerative colitis feces [125] . The lowering of intestinal H 2 S production is pro-posed to be achieved by the administration of water-soluble but poorly digestible polysaccharides, which include galacto-mannan, low molecular mass alginate, non-digestibility dextrin, polydextrose, chicory fiber, arabinogalactan and various oli-gosaccharides. The addition of polyphenolic compounds, such as catechin, epicatechin, and gallocatechin, is said to further improve the reduction in H 2 S production.

Kawabata and colleagues aim to lower endogenous H 2 S production with the objective of reducing hyperalgesia mediated by H 2 S (JP2007112735) [126] . Several inhibitors of endogenous enzymatic pathways for H 2 S-formation, namely the cystathionine- γ -lyase inhibitors propargylglycine and β -cyanoalanine, are claimed as novel analgesic drugs. In rats, the H 2 S-lowering compounds are shown to increase the threshold for mechanical hyperalgesia in hind paws induced by administration of LPS or by cysteine, a precursor for H 2 S biosynthesis.

4. Exert opinion

The patent applications on therapeutic applications of CO and H 2 S submitted thus far are illustrative for one increasingly desirable avenue for small molecule therapy, namely the

controlled release of endogenous autacoids with useful phar-macological actions. This approach aims to make use of the already in place mechanisms for tuning down specific physi-ological reactions, such as vasoconstriction and inflamma-tion. Such agonists for counter-regulation are expected to be inherently safer than inhibitors, as they may bring back dis-turbed physiological systems into endogenously acceptable margins.

Several of the proposed strategies for the delivery of CO from CO-RMs present interesting and advantageous concepts. Compounds can be designed to release CO at the target tissue by conjugation with other drugs or compounds that can accumulate in specific tissues. Targeted CO release in a specific tissue circumvents the need for systemic transport of CO through binding to hemoglobin after inhalation (with the potential risk for substantial saturation of hemoglobin). Effectively, the role of hemoglobin becomes one of elimination of small quantities of CO targeted to that tissue. CO-RMs can also be designed to deliver CO in response to a specific stimulus, such as the presence of increased levels of reactive oxygen species, which may occur in diseases with a marked component of oxidative stress. Further pharmaceutical improvements can be made such as encapsulation of CO-RMs in supramolecular structures that protect metal carbonyl complexes for prolonged time periods and mask their reactivity, or manipulation of compound solubility to alter tissue dis-tribution (for instance, hydrophobic molecules can be more effective for targeting a compound to the CNS).

The availability of several different chemical structures that release CO and H 2 S may help in designing and optimizing the right combinations of bearing scaffold and gasotransmitters. We are still some time away from understanding how to control the release of CO and H 2 S at rates that are thera-peutically effective at the right place in the body. Optimization of gasotransmitter-carrier combinations with respect to pharmacokinetics and pharmacodynamics will, therefore, be an interesting drug developmental challenge for the near future. In particular, this issue stands out given the different chemical mechanisms for release and donation of CO and H 2 S, as well as the tremendous variety of ligands that can be used. In the specific case in which the gasotransmitter-releasing scaffold is a known drug, the balance between maintaining the rate of formation of the parent biologically active drug versus the release of gas, as well as the action of the gasotransmitter on the pharmacological profile of the carrier drug, may require a substantial research effort. The coupling of biologically active compounds and introduction of novel chemical moieties that inadvertently create chemically reactive and potentially toxic groups is also an important point to consider in this respect [127] . Given that small modifications in the structure of a drug can create large changes in pharmacological properties, it is important to determine in greater detail how modification of known drugs with, for example, the used H 2 S-releasing compounds affects their pharmacokinetic and pharmacodynamic

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properties. The use of spacers to separate a CO-releasing pharmacophore from another chemical moiety (carrier or drug) has been indicated for CO-RMs [99] , but this strat-egy has not yet been proposed in patent applications for H 2 S-releasing drugs.

It is possible that a number of patent applications for therapeutic actions of CO-RMs and H 2 S-releasing molecules will be evaluated based on the still limited knowledge on the mechanisms of action of these gasotransmitters. Notably, in some cases, apparently opposite actions may make it difficult to claim the therapeutic applicability of a given action in a more generalized way, as the actions of CO and H 2 S can differ between tissues and situations. Many patent applica-tions claimed for a large number of therapeutic indications; however, few biological data related to these indications have generally been provided, especially concerning in vivo data. Up-to-date, in May 2008, Ikaria concluded a Phase I clinical trial for i.v. sodium sulfide (commercial name IK1001) as treatment for several hypoxic/ischemic conditions, including myocardial infarction, cardiopulmonary bypass surgery and acute lung injury. The same company is at present also develop-ing Covox(R) (CO by inhalation) in Phase II trials for kidney transplantation.

Taken together, it is to be expected that the development of pharmaceutical applications based on CO and H 2 S will provide us with novel ways to treat disease, especially because the organism is likely to accept well such endogenously produced gasotransmitters once they are released. However, we are still early in the development of the field to tell which approaches are going to be the most successful.

Acknowledgements

The authors would like to thank Y Nakagawa and F Yarovinsky for help with translations of patents written in Japanese and Russian, and C Romão and J Clària for help in the revision of the manuscript and comments.

Declaration of interest

G Bannenberg is a Ramón y Cajal fellow supported by the Spanish Ministry of Education and Science, and the Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Madrid, Spain. HLA Vieira is supported by the Portuguese Fundação para a Ciência e Tecnologia, SFRH/BPD/27125/2006 fellow.

Bibliography Papers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers.

1. Wu L, Wang R. Carbon monoxide: endogenous production, physiological functions, and pharmacological applications. Pharmacol Rev 2005 ; 57 : 585 -630

2. Kamoun P. Endogenous production of hydrogen sulfi de in mammals. Amino acids 2004 ; 26 : 243 -5

3. Ignarro L. Endothelium-derived nitric oxide: actions and properties. FASEB J 1989 ; 3 : 31 -6

4. Moncada S, Higgs A. The L-arginine-nitric oxide pathway. NEJM 1993 ; 329 : 2002 -12

5. Sjöstrand T. Endogenous formation of carbon monoxide: the CO concentration in the inspired and expired air of hospital patients. Acta Physiol. Scand 1951 ; 22 (2-3):137-141

6. Verma A, Hirsch DJ, Glatt CE, et al. Carbon monoxide: a putative neural messenger. Science 1993 ; 259 : 381 -4

7. Li L, Moore PK. An overview of the biological signifi cance of endogenous gases: new roles for old molecules. Biochem Soc Trans 2007 ; 35 : 1138 -41

8. Ryter SW, Alam J, Choi AM. Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications. Physiol Rev 2006 ; 86 (2): 583 -650

• Broad and complete review on heme oxygenase and carbon monoxide.

9. Dore S. Decreased activity of the antioxidant heme oxygenase enzyme: implications in ischemia and in Alzheimer’s disease. Free Radic Biol Med 2002 ; 32 (12): 1276 -82

10. Lin Q, Weis S, Yang G, et al. Heme oxygenase-1 protein localizes to the nucleus and activates transcription factors important in oxidative stress. J. Biol. Chem 2007 ; 282 (28): 20621 -33

11. Lin QS, Weis S, Yang G, et al. Catalytic inactive heme oxygenase-1 protein regulates its own expression in oxidative stress. Free Radic Biol Med 2008 ; 44 (5): 847 -55

12. Motterlini R, Clark JE, Foresti R, et al. Carbon monoxide-releasing molecules: characterization of biochemical and vascular activities. Circ Res 2002 ; 90 (2): E17 -24

13. Clark JE, Naughton P, Shurey S, et al. Cardioprotective actions by a water-soluble carbon monoxide-releasing molecule. Circ Res 2003 ; 93 (2): e2 -8

14. Motterlini R, Sawle P, Hammad J, et al. CORM-A1: a new pharmacologically active carbon monoxide-releasing molecule. FASEB J 2005 ; 19 : 284 -6

15. Otterbein LE, Bach FH, Alam J, et al. Carbon monoxide has anti-infl ammatory effects involving the mitogen-activated protein kinase pathway. Nat Med 2000 ; 6 (4): 422 -8

16. Chora AA, Fontoura P, Cunha A, et al. Heme oxygenase-1 and carbon monoxide suppress autoimmune neuroinfl ammation. J Clin Invest 2007 ; 117 (2): 438 -47

17. Ott MC, Scott JR, Bihari A, et al. Inhalation of carbon monoxide prevents liver injury and infl ammation following hind limb ischemia/reperfusion. FASEB J 2005 ; 19 (1): 106 -8

18. Mishra S, Fujita T, Lama VN, et al. Carbon monoxide rescues ischemic lungs by interrupting MAPK-driven expression of early growth response 1 gene and its downstream target genes. Proc Natl Acad Sci USA 2006 ; 103 (13): 5191 -6

19. Sawle P, Foresti R, Mann BE, et al. Carbon monoxide-releasing molecules (CO-RMs) attenuate the infl ammatory response elicited by lipopolysaccharide in RAW264.7 murine macrophages. Br J Pharmacol 2005 ; 145 (6): 800 -10

Therapeutic applications of the gaseous mediators carbon monoxide and hydrogen sulfi de

680 Expert Opin. Ther. Patents (2009) 19(5)

20. Chin BY, Jiang G, Wegiel B, et al. Hypoxia-inducible factor 1 α stabilization by carbon monoxide results in cytoprotective preconditioning. Proc Natl Acad Sci USA 2007 ; 104 (12): 5109 -14

21. Brouard S, Otterbein LE, Anrather J, et al. Carbon monoxide generated by heme oxygenase 1 suppresses endothelial cell apoptosis. J Exp Med 2000 ; 192 (7): 1015 -26

22. Brouard S, Berberat PO, Tobiasch E, et al. Heme oxygenase-1-derived carbon monoxide requires the activation of transcription factor NF- κ B to protect endothelial cells from tumor necrosis factor-alpha-mediated apoptosis. J Biol Chem 2002 ; 277 (20): 17950 -61

23. Zhang X, Shan P, Otterbein LE, et al. Carbon monoxide inhibition of apoptosis during ischemia-reperfusion lung injury is dependent on the p38 mitogen-activated protein kinase pathway and involves caspase 3. J Biol Chem 2003 ; 278 (2): 1248 -58

24. Zhang X, Shan P, Alam J, et al. Carbon monoxide modulates Fas/Fas ligand, caspases, and Bcl-2 family proteins via the p38alpha mitogen-activated protein kinase pathway during ischemia-reperfusion lung injury. J Biol Chem 2003 ; 278 (24): 22061 -70

25. Vieira HLA, Queiroga CSF, Alves PM. Preconditioning induced by carbon monoxide provides neuronal protection against apoptosis. J Neurochem 2008 ; 107 : 375 -84

26. Liu XM, Chapman GB, Peyton KJ, et al. Antiapoptotic action of carbon monoxide on cultured vascular smooth muscle cells. Exp Biol Med 2003 ; 228 (5): 572 -5

27. Sarady JK, Zuckerbraun BS, Bilban M, et al. Carbon monoxide protection against endotoxic shock involves reciprocal effects on iNOS in the lung and liver. FASEB J 2004 ; 18 (7): 854 -6

28. Morita T, Perrella MA, Lee ME, Kourembanas S. Smooth muscle cell-derived carbon monoxide is a regulator of vascular cGMP. Proc Natl Acad Sci USA 1995 ; 92 (5): 1475 -9

29. Otterbein LE, Zuckerbraun BS, Haga M, et al. Carbon monoxide suppresses arteriosclerotic lesions associated with chronic graft rejection and with balloon injury. Nat Med 2003 ; 9 (2): 183 -90

30. Song R, Mahidhara RS, Liu F, et al. Carbon monoxide inhibits human airway smooth muscle cell proliferation via

mitogen-activated protein kinase pathway. Am J Respir Cell Mol Biol 2002 ; 27 (5): 603 -10

31. Ameredes BT, Otterbein LE, Kohut LK, et al. Low-dose carbon monoxide reduces airway hyperresponsiveness in mice. Am J Physiol Lung Cell Mol Physiol 2003 ; 285 (6): L1270 -6

32. Song R, Mahidhara RS, Zhou Z, et al. Carbon monoxide inhibits T lymphocyte proliferation via caspase-dependent pathway. J Immunol 2004 ; 172 (2): 1220 -12266

33. Graser T, Vedernikov YP, Li DS. Study on the mechanism of carbon monoxide induced endothelium-independent relaxation in porcine coronary artery and vein. Biomed Biochim Acta 1990 ; 49 (4): 293 -6

34. Hussain AS, Marks GS, Brien JF, Nakatsu K. The soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) inhibits relaxation of rabbit aortic rings induced by carbon monoxide, nitric oxide, and glyceryl trinitrate. Can J Physiol Pharmacol 1997 ; 75 : 1034 -7

35. Christodoulides N, Durante W, Kroll MH, Schafer AI. Vascular smooth muscle cell heme oxygenases generate guanylyl cyclase-stimulatory carbon monoxide. Circulation 1995 ; 91 (9): 2306 -9

36. Cardell LO, Ueki IF, Stjärne P, et al. Bronchodilatation in vivo by carbon monoxide, a cyclic GMP related messenger. Br J Pharmacol 1998 ; 124 (6): 1065 -8

37. Kaide JI, Zhang F, Wei Y, et al. Carbon monoxide of vascular origin attenuates the sensitivity of renal arterial vessels to vasoconstrictors. J Clin Invest 2001 ; 107 (9): 1163 -71

38. Jaggar JH, Leffl er CW, Cheranov SY, et al. Carbon monoxide dilates cerebral arterioles by enhancing the coupling of Ca2+ sparks to Ca2+-activated K+ channels. Circ Res 2002 ; 91 (7): 610 -7

39. Zhang F, Kaide J, Wei Y, et al. Carbon monoxide produced by isolated arterioles attenuates pressure-induced vasoconstriction. Am J Physiol Heart Circ Physiol 2001 ; 281 (1): H350 -8

40. Li A, Xi Q, Umstot ES, et al. Astrocyte-derived CO is a diffusible messenger that mediates glutamate-induced cerebral arteriolar dilation by activating

smooth muscle cell KCa channels. Circ Res 2008 ; 102 (2): 234 -41

41. Motterlini R, Foresti R, Grenn CJ. Studies on the development of carbon monoxide releasing molecules: potential applications for the treatment of cardiovascular dysfunction. In: Wang R, editor, CRC Press, Boca Raton, Florida, USA, 2002 . p. 149 -271

42. Mann BE, Motterlini R. CO and NO in medicine. Chem Commun 2007 ;(41): 4197 -208

43. Motterlini R, Mann BE, Foresti R. Therapeutic applications of carbon monoxide-releasing molecules. Expert Opin Investig Drugs 2005 ; 14 : 1305 -18

44. Dominy JE, Stipanuk MH. New roles for cysteine and transsulfuration enzymes: production of H2S, a neuromodulator and smooth muscle relaxant. Nutr Rev 2004 ; 62 : 348 -53

45. Abe K, Kimura H. The possible role of hydrogen sulfi de as an endogenous neuromodulator. J Neurosci 1996 ; 16 : 1066 -71

46. Hosoki R, Matsuki N, Kimura H. The possible role of hydrogen sulfi de as an endogenous smooth muscle relaxant in synergy with nitric oxide. Biochem Biophys Res Commun 1997 ; 237 : 527 -31

47. Zhao W, Zhang J, Lu Y, Wang R. The vasorelaxant effect of H2S as a novel endogenous gaseous K(ATP) channel opener. EMBO J 2001 ; 20 : 6008 -16

48. Shibuya N, Tanaka M,Yoshida M, et al. 3-Mercaptopyruvate sulfurtransferase produces hydrogen sulfi de and bound sulfane sulfur in the brain. Antioxid Redox Signal 2008 In press

49. Ishikami M, Hiraki K, Umemura K, et al. A source of hydrogen sulfi de and a mechanism of release in brain. Antioxid Redox Signal 2009 ; 11 : 1 -10

50. Furne J, Saeed A, Levitt MD. Whole tissue hydrogen sulfi de concentrations are orders of magnitude lower than presently accepted values. Am J Physiol Regul Integr Comp Physiol 2008 ; 295 : R1479 -85

51. Cheng Y, Ndisang JF, Tang G, et al. Hydrogen sulfi de-induced relaxation of resistance mesenteric artery beds of rats. Am J Physiol Heart Circ Physiol 2004 ; 287 : H2316 -23

52. Geng B, Cui Y, Zhao J, et al. Hydrogen sulfi de downregulates the aortic

Bannenberg & Vieira

Expert Opin. Ther. Patents (2009) 19(5) 681

L-arginine/nitric oxide pathway in rats. Am J Physiol Regul Integr Comp Physiol 2007 ; 293 : R1608 -11618

53. Kubo S, Doe I, Kurokawa Y, et al. Direct inhibition of endothelial nitric oxide synthase by hydrogen sulfi de: contribution to dual modulation of vascular tension. Toxicol 2007 ; 232 : 138 -46

54. Ali MY, Ping CY, Mok YY, et al. Regulation of vascular nitric oxide in vitro and in vivo; a new role for endogenous hydrogen sulphide? Br J Pharmacol 2006 ; 149 : 625 -34

55. Lim JJ, Liu Y, Khin ES, Bian JS. Vasoconstrictive effect of hydrogen sulfi de involves downregulation of cAMP in vascular smooth muscle cells. Am J Physiol Cell Physiol 2008 ; 295 : C1261 -70

56. Koenitzer JR, Isbell TS, Patel HD, et al. Hydrogen sulfi de mediates vasoactivity in an O2-dependent manner. Am J Physiol Heart Circ Physiol 2007 ; 292 : H1953 -60

57. Yang G, Wu L, Jiang B, et al. H2S as a physiological vasorelaxant: hypertension of mice with deletion of cystathionine- γ -lyase. Science 2008 ; 322 : 587 -90

58. Distrutti E, Mencarelli A, Santucci L, et al. The methionine connection: homocysteine and hydrogen sulfi de exert opposite effects on hepatic microcirculation in rats. Hepatol 2008 ; 47 : 659 -67

59. Mok YY, Atan MS, Yoke Ping C, et al. Role of hydrogen sulphide in haemorrhagic shock in the rat: protective effect of inhibitors of hydrogen sulphide biosynthesis. Br J Pharmacol 2004 ; 143 : 881 -9

60. Du J, Yan H, Tang C. Endogenous H2S is involved in the development of spontaneous hypertension (article in chinese). Beijing Da Xue Xue Bao 2003 ; 35 : 102

61. Olson KR. Hydrogen sulfi de and oxygen sensing: implications in cardiorespiratory control. J Exp Biol 2008 ; 211 : 2727 -34

62. Teague B, Asiedu S, Moore PK. The smooth muscle relaxant effect of hydrogen sulphide in vitro: evidence for a physiological role to control intestinal contractility. Br J Pharmacol 2002 ; 137 : 139 -45

63. Kimura H. Hydrogen sulfi de as a neuromodulator. Mol Neurobiol 2002 ; 26 : 13 -9

64. Zhao WWR. H2S-induced vasorelaxation and underlying cellular and molecular mechanisms. Am J Physiol Heart Circ Physiol 2002 ; 283 : 474 -80

65. Matsunami M, Tarui T, Mitani K, et al. Luminal hydrogen sulfi de plays a pro-nociceptive role in mouse colon. Gut 2008 In press

66. Eto K, Asada T, Arima K, et al. Brain hydrogen sulfi de is severely decreased in Alzheimer’s disease. Biochem Biophys Res Commun 2002 ; 293 (5): 1485 -8

67. Kamoun P. Endogenous hydrogen sulfi de overproduction in Down syndrome. Am J Med Genet A 2003 ; 116A (3): 310 -1

68. Fiorucci S, Antonelli E, Distrutti E, et al. Inhibition of hydrogen sulfi de generation contributes to gastric injury caused by anti-infl ammatory nonsteroidal drugs. Gastroenterol 2005 ; 129 : 1210 -24

69. Zanardo RC, Brancaleone V, Distrutti E, et al. Hydrogen sulfi de is an endogenous modulator of leukocyte-mediated infl ammation. FASEB J 2006 ; 20 : 2118 -20

70. Sidhapuriwala J, Li L, Sparatore A, et al. Effect of S-diclofenac, a novel hydrogen sulfi de releasing derivative, on carrageenan-induced hindpaw oedema formation in the rat. Eur J Pharmacol 2007 ; 569 : 149 -54

71. Zhang H, Zhi L, Moochhala SM, et al. Endogenous hydrogen sulfi de regulates leukocyte traffi cking in cecal ligation and puncture-induced sepsis. J Leukoc Biol 2007 ; 82 : 894 -905

72. Kawabata A. Hydrogen sulfi de as a novel nociceptive messenger. Pain 2007 ; 132 : 74 -81

73. Distrutti E, Sediari L, Mencarelli A, et al. 5-Amino-2-hydroxybenzoic acid 4-(5-thioxo-5H-[1,2]dithiol-3yl)-phenyl ester (ATB-429), a hydrogen sulfi de-releasing derivative of mesalamine, exerts antinociceptive effects in a model of postinfl ammatory hypersensitivity. J Pharmacol Exp Ther 2006 ; 316 : 325 -35

74. Li L, Moore PK. Putative biological roles of hydrogen sulfi de in health and disease: a breath of not so fresh air? Trends Pharmacol Sci 2008 ; 29 : 84 -90

75. Yale University. WO03094932A1; 2003

• First patent application fi led by Otterbein and colleagues.

76. Beth Israel Hospital. WO03000114; 2003

77. Beth Israel Deaconess Medical Center Inc. & University of Pittsburgh. WO03072024A2; 2003

78. University of Pittsburgh. WO03088923A2; 2003

79. University of Pittsburgh. WO03088981A1; 2003

80. Yale University & University of Pittsburgh. WO0309677; 2003

81. Yale University & University of Pittsburgh. WO03103585A2; 2003

82. University of Pittsburgh & Yale University. WO2004043341; 2004

83. Beth Israel Deaconess Medical Center Inc. WO2008008513; 2008

84. University of Pittsburgh. WO2004000368; 2004

85. The General Hospital Corporation. US5885621; 1997

86. Air Liquide santé International. FR2816212; 2002

87. Air Liquide santé International. WO0209731; 2002

88. Aga AB. WO2005067945; 2005

89. Lohmann Therapie Systems lts. WO9535105; 1995

90. University Maryland Biotechnology Institute. US5670664; 1997

91. Sangstat Medical Corporation. WO02078684; 2002

92. Northwick Park Institute for Medical Research & University of Sheffi eld. WO02092075A3; 2002

• First patent applied by Motterlini and colleagues (HemoCORM Ltd).

93. Northwick Park Institute for Medical Research & University of Sheffi eld. WO20040405598; 2004

94. Northwick Park Institute for Medical Research & University of Sheffi eld. WO20040405599; 2004

95. Foresti R, Hammad J, Clark JE, et al. Vasoactive properties of CORM-3, a novel water-soluble carbon monoxide-releasing molecule. Br J Pharmacol 2004 ; 142 : 453 -60

96. Hemocorm Ltd. WO20070065485; 2007

97. Hemocorm Ltd. WO2007085806A2; 2007

98. Hemocorm Ltd. WO2008003953A2; 2008

99. Alfama Inc. WO2003066067A3; 2003

• First patent application fi led by Alfama Inc.

100. Alfama Inc. WO2007073225; 2007

Therapeutic applications of the gaseous mediators carbon monoxide and hydrogen sulfi de

682 Expert Opin. Ther. Patents (2009) 19(5)

101. Alfama Inc. US20080026984A1; 2008

102. Antibe Therapeutics Inc. WO2006085127A1; 2006

103. Antibe Therapeutics Inc. WO2008009118A1; 2008

104. Antibe Therapeutics Inc. WO2006125295; 2006

105. Antibe Therapeutics Inc. WO2007140611A1; 2007

106. Fuso Pharmaceutical Ind. & University Kinki. JP2007063167; 2007

107. Wallace JL, Dicay M, McKnight W, Martin GR. Hydrogen sulfi de enhances ulcer healing in rats. FASEB J 2007 ; 21 : 4070 -6

108. Antibe Therapeutics Inc. WO 2008009127A1; 2008

109. Wallace JLCG, Santagada V, Cirino G, Fiorucci S. Gastrointestinal safety and anti-infl ammatory effects of a hydrogen sulfi de-releasing diclofenac derivative in the rat. Gastroenterol 2007 ; 132 : 261 -71

110. Expotrend Company S.A. EP1630164A1; 2006

111. Turko IVS, Ballard SA, Francis SH, Corbin JD. Inhibition of cyclic GMP-binding cyclic GMP-specifi c phosphodiesterase (type 5) by sildenafi l

and related compounds. Mol Pharmacol 1999 ; 56 : 124 -30

112. Metabono S.A. WO2006066894A1; 2006

113. Fudan University, China. CN101011413A; 2007

114. Volpato GP, Searles R, Yu B, et al. Inhaled hydrogen sulfi de: a rapidly reversible inhibitor of cardiac and metabolic function in the mouse. Anesthesiol 2008 ; 108 : 659 -68

115. Szabo C. Hydrogen sulphide and its therapeutic potential. Nat Rev Drug Discov 2007 ; 6 : 917 -35

116. Ikaria Inc. 2008

117. Hybernation Therapeutics Limited. WO2008011670A1; 2008

118. Ikaria Inc. WO2008079993A2; 2008

119. Ikaria Inc. WO2008043081A2; 2008

120. Gosudartvennoe ObschcheobrazovatelL’noe uchrezhdenie vysshego professional’nogo obrazovanija Kazanskij gosudarstvennij meditsinkij universitet (RU). RU2304967; 2007

121. Federal’noe G, Uchrezhdenie PJA. Pjatigorskij GNIK Rosdrava, Russia. RU2321386; 2008

122. CTG Pharma S.R.L. EP1886681A2; 2008

123. Dueñas-González A, Candelaria M, Pérez-Plasencia C, et al. Valproic acid as epigenetic cancer drug: preclinical, clinical and transcriptional effects on solid tumors. Cancer Treat Rev 2008 ; 34 : 206 -22

124. Taiyo Kagaku KK. JP2007099672A; 2007

125. Levine J, Ellis CJ, Furne JK, et al. Fecal hydrogen sulfi de production in ulcerative colitis. Am J Gastroenterol 1998 ; 93 : 83 -7

126. Fuso Pharmaceutical Ind. & University Kinki. JP2007112735; 2007

127. Hulsman N, Medema JP, Bos C, et al. Chemical insights in the concept of hybrid drugs: the antitumor effect of nitric oxide-donating aspirin involves a quinone methide but not nitric oxide nor aspirin. J Med Chem 2007 ; 50 : 2424 -31

Affi liation Gerard L Bannenberg † 1 Helena LA Vieira 2 † Author for correspondence 1 Campus de la Universidad Autónoma, Centro Nacional de Biotecnología / CSIC, Department of Plant Molecular Genetics, Calle Darwin 3, Cantoblanco, 28049 Madrid, SpainTel: +34 91 585 4531 ; Fax: +34 91 585 4506 ; E-mail: gbannenberg@cnb.csic.es 2 Animal Cell Technology Laboratory, ITQB-UNL/IBET, Apartado 12, 2780-901, Oeiras, Portugal