ZA200601854B - Use of boranocarbonates for the therapeutic delivery of carbon monoxide - Google Patents

Description

Therapeutic Delivery of Carborm Monoxide

FIELD OF THE XNVENTION

The pres ent invention relates to pha—rmaceutical compositions and compounds for the therapesutic delivery of carbon monoxicle to humans and other mammal.s. Another use of the composition and compounds is in organ perfusion.

BACKGROUND OF THE INVENTION

Mammalia cells constantly generate ecarbon monoxide (CO) gas via the eradogenous degradation of heme= by a family of - constitutive «HO-2) and inducible (HO-1) heme oxygenase enzymes “?. First described as a putative neural messenger °,

CO is now regarded as a versatile signalirmg molecule having essential regulatory roles in a variety off physiological and pathophysioclocgical processes that take plamce within the cardiovascular-, nervous and immune systemss. Indeed, CO produced in tlie vessel wall by heme oxygeraase enzymes possesses vasorelaxing properties and has been shown to prevent vasoconstriction and both acute arad chronic hypertension through stimulation of solubl_e guanylate cyclase 4-19 Endogenous CO appears to modulate sinusoidal tone in the hepatic circulation !!, control the proliferation of vascular smooth muscle cells !* and suppress the re—jection of transplanted hearts '*. The biological action of heme oxygenase-der3aved CO is substantiated by t=he pharmacological effects observed when this gas is applied exogenously to in vitro and in wivo gystems. At concentraticons ranging from 10 to 500 p.p.m. , CO gas has been reported tc mediate potent anti-inflammat-ory effects '*, prevent endothelial cell apoptosis *®*, dinhibit human airway smooth rmuscle cell proliferation and promote protection against hyperoxic as : well as ischermic lung injury '"*®*. In view of the pivotal role exerted by the heme oxygenase pathway in t=he control of cellular homeostasis *° and the emerging pleiotropic properties attributed to CO ?°, it is conceivable that this diatomic gas could be wsed as a therapeutic tool for the treatment of vascular dysfunction and immuno-related disease states.

At pmesent, three different approaches have been proposed for examining the therapeutic potential of CO: 1) direct administra tion of CO gas ?°; 2) use of pro-drugs (i.e. methylene chloride) which are catabolized by hepatic enzymes to generat e CO ?*; and 3) transport and delivery of CO by means of specifi ¢ CO carriers *. Some investigators have concentrat ed their efforts on the last strategic approach as it has been recently reported that certain transition met-al carbonyls possess the ability to liberate CO under approp-—rxiate conditions and function as CO-releasing molecules (CO-RMs ) in biological systems. In particular, it was shown that CO-REMs induce ves sel relaxation in isolated aortic tissue and prevent coronary vasoconstriction as well as acute hypertension ian vivo through specific mechanisms that can be simulated by activation of the HO-1/CO pathway ?’. Interestingly, the versatile chemistry of transition metals allows them to be effectively modified by coordinating biological ligands to the metal center in order to render the molecule less toxic, tmnore water soluble and to modulate the release of CO. It has besen recently reported that tricarbonyldchloro (glycinato) ruthenium(II) (here called CORM- 3), a newly synthesized water-soluble form of metal carbomyl that liberates CO in vitro, ex-vivo and in vivo biological models, protects myocardial cells and tissues against ischemia-reperfusion injury as well as cardiac allograft rejection 2%?°. Some of this work is published in

Internatiomal Patent Application WO 02/092075 (ref. 25).

In tlhe case of CORM-3, the chloride and glycinate li gands are labile and their substitution with higher affinity licgands present in the cellular or plasma environment (i.e. glutathione) would appear to accelerate dissociation of CO from the metal center 2’. When added to a solution contain ing myoglobin (Mb), the release of CO from CORM-3 is accelerated as 1 mole of CO per mole of compound is liberated within 1-2 min **. CORM-3 would, therefore, fall into a category of compounds that release CO very rapidly (“fast zreleasers”) which can be ideal for several clinical applications in which

CO acts as a signalling mediator (i.e. neurotr-ansmission, acute hyperterision, angina, ischemia-reperfusi_on); however, identifying compounds that release CO with a s low kinetics ("slow releasers”) would implement the design of

Pharmaceuticals that could be more versatile i n the treatment of certain chr-onic diseases (i.e. arthritis, imflammation, cancer, organ preservation; chronic hypertensi-on; septic shock prevention of restenosis after balloon angioplasty, post- operative ilewms) where the continuous and long -lasting effect of CO may be required.

An interesting example in the development. of transition metal carbonyl s that are used for medical appl-ications not related to the therapeutic use of CO is represented by carbonyls spec ifically designed for radio-imagfng technology.

The recently described technetium(I) complex [®Tc(OH,);- (CO)s]* has attracted mmuch interest as a precursor for technetium-99m radiopharmaceuticals *®. A number of biomolecule=s, for example, peptides, scFv , and CNS receptor ligands, have already been labeled with technetium by this approach, demorastrating the potential of [®*Tc(OH;),-(C0);]* for radiopharmacceutical application ?*. This compound can be prepared ira a single-step procedure from aqueous [’*"TcO4] in the presence of CO and BH, - as a reducing agent *°.However, the published pr—eparation of ("Tc (OH;) 3- (COD ,]* relying on gaseous carbon morioxide, is unsuitable for use in commercial radiopharmaceu.tical “kits”.

A recent study has reported the first commercia lly feasible preparation of [**™Tc(OH,);-(CO);]1* in physiologic al media using a boron-based carbonylating agent, potassium bo-ranocarbonate (K, [H;BCO,]), wirich acts as a CO source and a recducing agent at the same time 37%.

Wa 2005/013691 PCT/GB2004/003365

Boranocarbonates have been disclosed or suggested for physiological effects in the prior art. EKF-A-34238 and EP-A- 181721 describes anti-tumour and anti-hypem-lipidemic activities of amine-carboxboranes. US-A-43312989 discloses use

S of amine boranes to inhibit the inflammation process. US-A- 5254706 describes phosphite-borane compounds for anti-tumour, anti-inflammatory and hypolipidemic activity.

WO93/05795 discusses use of organic b oron compounds effective against osteoporosis and suggests also anti- inflammatory, anti-hyperlipidemic and antireoplastic activity.

The compounds disclosed are primarily of the amino-borane class, but Na,BH;COO is also tested. Hall et al., "Metal Based

Drugs", Vol. 2, No. 1, 1995, describes anti -inflammatory activity of acyclic amine-carboxyboranes ira rodents.

These documents reveal interest in thee boron compounds either because of the possible effect of boron itself or because the amino-boranes are analogous to the natural «o-amino acids.

SUMMARY OF THE INVENTION

As exemplified by the experimental data detailed below, the present inventors have found that boraraocarbonate compounds can be used to deliver CO to a playsiological target so as to provide physiological effect.

Accordingly the present invention provides a pharmaceutical composition, intended for alministration to a human or other mammal for delivery of carbon monoxide, comprising a boranocarbonate compound or ion adapted to make

CO available for physiological effect and at least one pharmaceutically acceptable carrier.

Boranocarbonates are a group of compowmds which can loosely be described as carboxylate adducts of borane and derivatives of borane. Boranocarbonates generally contain a group of the form -COO” or COOR (where R is HK or another group) attached to the boron atom, so that they may be called boranocarbo><cylates or carboxyboranes, but tlhe term boranocarborate seems to be preferred. The compound X, (H;BCOO) and the related K(H;BCOOH) are described in reference 31, where the compouncl K,(H,BCO0) is used for producineg Tc carbonyls. 5 Thus typically a boranocarbonate has t he molecular structure imcluding the moiety

Te 0

Prefex—red is the structure above with three hydrogen atoms attacs=hed to the boron (BH;-CO-), since this is believed to facilitate CO release.

Also poreferred are structures where a carboxylate group is attached to boron, i.e. -CO0™, -COOH-, -«00X where X may be any suitabl_e esterifying group acceptable pharmaceutically.

Prefe xably the boranocarbonate compoumid in the pharmaceut Mi. cal composition has an anion of the formula:

BH, (C200) ,Z, wher ein:- x is 1, 2 or 3 y is 1, 2 or 3

Zz is 0, 1 or 2

X + Y +z = 4, eaclna Q is 07, representing a carboxylate anionic form, or &s OH, OR, NH,, NHR, NR;, SR or halogen, where the or each R is alkyl (preferably of 1 to 4 carbon atoms) , eackn Z is halogen, NH;, NHR', NR',, &SR' or OR' where the or each R' is alkyl (preferably of 1 to 4 carbon atoms) .

Since thiss formula is analogous to the boxrranc anion

BH,", the structure generally is an anion. It may be a divalent &anion when one (COQ) is present as (C007). If the structure is an anion, a cation is require=d. Any physiolog ically suitable cation may be employed, particularly

WED 2005/013691 PCT/GB2004/003365 , a metal cation such as an alkali metal ion e.g. K' or Na' or an alkaline earth metal cation such as Ca™* or Mg**. Alternatively non-metal cations might be employed, suach as NR, where each R is H or alkyl (preferably of 1 to 4 cambon atoms) or PR,* where

R is alkyl (preferably of 1 to 4 carbor atoms). The cation may be selected in order to achieve a clesired solubility of the compound.

Preferably y is 1. Preferably x is 3.

Preferably the boranocarbonate i= soluble and is present in solution in a suitable solvent, e.g . an aqueous solvent, in the composition. Other possible solvents are ethanol, DMSO,

DMF and other physiologically compatilbwle solvents.

The boranocarbonates employed in the present invention vary in their ability to provide CO. The release of CO may be

PH and temperature dependent. Lower pH causes more or faster release. Thus a range of compounds is available, for choice of a suitable release rate for a particular application. Slow release over a long period, of hours or days, can be achieved.

Solutions can be provided containing clissolved CO, already released by the boranocarbonate. Alte=rnatively, release of CO may be triggered by change of conditieon (e.g. pH or temperature) or by contact with anoth er material, e.g. another solvent or agueous physiological flui d such as blood or lymph, or even at a physiological delivery site.

Typically the pharmaceutical cormpositions of the present invention release CO such as to make it available to a therapeutic target in dissolved form. However, in some circumstances CO may be released direxctly to a non-solvent acceptor molecule.

It will be apparent that pharma_ceutical compositions according to the present invention maay be capable of delivering CO therapeutically through one or more of the above= described modes of action.

The boranocarbonate compound meEay further comprise a targeting moiety, to facilitate rele ase of CO at an

A appropriate si. te. The targeting moiety is typically capable of binding a receptor on a particular target cell surface, in order to promo=te release of CO at the require=d site. The targeting moie=ty may be a part of a modulating ligand capable of binding to a receptor found on the surface. of the target cells, or may be derived from another molecul e, such as an antibody direc:ted against a particular recept or, joined to the boranocarbonat e molecule by a suitable linker.

The pharrmaceutical compositions of the poresent invention typically comprise a pharmaceutically accepta_ble excipient, carrier, buffe-r, stabiliser or other material s well known to those skilled in the art. Such materials should be non-toxic and should not. interfere unduly with the effi cacy of the active ingredi ent. The precise nature of the: carrier or other material may d.epend on the route of administr—ation, e.g. oral, intravenous, s ubcutaneous, nasal, intramuscul ar, intraperitonea 1, transdermal, transmucosal or— suppository routes.

Pharmacewatical compositions for oral adrministration may be in tablet, capsule, powder or liquid form. A tablet may include a soli. d carrier such as gelatin or ame adjuvant or a slow-release pmolymer. Liquid pharmaceutical compositions generally incl. ude a liquid carrier such as wa_ter, petroleun, animal or vege=table oils, mineral oil or synt_hetic oil.

Physiological saline solution, dextrose or ot-—her saccharide solution or gl.ycols such as ethylene glycol, propylene glycol or polyethylerme glycol may be included. Phar-maceutically acceptable amounts of other solvents may also be included, in particular whesre they are required for dissol ving the particular cowpound contained in the composit=ion.

For intravenous, cutaneous or subcutaneecous injection, or injection at tthe site of affliction, the acti ve ingredient will typically be in the form of a parenteral ly acceptable solution whicla is pyrogen-free and has suital>le pH, isotonicity armd stability. Those of relevant= skill in the art are well able to prepazxre suitable solutions using, for— example, isotonic vehiecles such as Sodium Chloride Inj ection,

Ringer's Injection, Laectated Ringer's Injection.

Preservatives, stabilisers, buffers, antioxidants and/” or other additives may be inclu-ded, as required. Delivery syst—ems for needle-free injection are also known, and compositionss for use with such systems may be prepared accordingly.

In pharmaceutica® compositions intended for deli—very by any route including bu.t not limited to oral, nasal, miacosal, intravenous, cutaneouss, subcutaneous and rectal the active substance may be micro encapsulated within polymeric sspheres such that exposure to body fluids and subsequent CO remlease is delayed in time.

Administration iss preferably in a prophylactical ly effective amount or a therapeutically effective amoun t (as the case may be, although prophylaxis may be considered therapy), - this being sufficient to show benefit to the individu_al. The actual amount administoered, and rate and time-course of administration, will clepend on the nature and severity of what is being treated. Prescription of treatment, e.g. de=cisions on dosage etc, is within the responsibility of generamal practitioners and otheer medical doctors, and typicall_y takes account of the disord er to be treated, the condition of the individual patient, tThe site of delivery, the method of administration and ot her factors known to practitione=rs.

Examples of the techn _iques and protocols mentioned akoove can be found in Remington._’s Pharmaceutical Sciences, 16tEa edition,

Osol, A. (ed), 1980.

When formulatincg pharmaceutical compositions according to the present inventiora, the toxicity of the active inegredient and/or the solvent mast be considered. The balance Tbhetween medical benefit and t—oxicity should be taken into ac count.

The dosages and formulations of the compositions wil 1 typically be determirmed so that the medical benefit provided outweighs any risks due to the toxicity of the const-ituents.

There is fur-ther provided a method of intro&ucing CO to a mammal comprising the step of administering a pha—rmaceutical composition according to the present invention. ®C0 is thought to act at least im part through stimulation or ac-tivation of guanylate cyclase. CO is thought to have functioms as, inter alia, a neurotransmitter and a vasodilating agent .

Accordingly there is provided a method of deliver—ing CO to a mammal for stimula tion of guanylate cyclase activzity. There is further provide d a method of delivering CO to = mammal for stimulating neurot ransmission or vasodilation. However the present applicants do not wish to be bound by theory and do not exclude the po ssibility that CO operates by otcher mechanisms.

The heme oxycyenase 1 (HO-1) pathway is thoug ht to represent a pivota 1 endogenous inducible defensive system against stressful stimuli including UVA radiationss, carcinogens, ischa emia-reperfusion damage, endotox<ic shock and several other cond itions characterised by production of oxygen free radicals (32, 19,2). The protective effect of HO-1 is attributed to the «generation of the powerful antioxidants biliverdin and bil irubin and the vasocactive gas CO.

Expression of HO-1 has been linked with cardiac Xenograft survival (33), supzoression of transplant arteriosclerosis (34) and amelioration oz=f post-ischemic myocardial dysfuanction (35).

HO-1 has also been directly implicated in the resolution phase of acute inflammatzion in rats (36). Other pathological situations, such ass haemorrhagic shock in brain amd liver as well as sepsis (37 —39), are characterized by induction of the

HO-1 gene, which seems to play a crucial role in counteracting the vascular dysfumiction caused by these pathophyssiological states. Increased generation of CO as a consequerace of HO-1 induction markedly affects vessel contractility arad diminishes acute hypertension in the whole organism (10,9). Exposure of animals to ambient air containing low concentrations of CO or transfection of thes HO-1 gene results in protection against hyperoxia-induced lung injury in vivo, a mechanism medi_ated by attenuation of both neu trophil inflammation and lung apooptosis (cell death) (17,40). Exogenous CO gas also has the abi lity to suppress pro-inflammato ry cytokines and modulate the expression of the anti- inflammatory molecule, IL-10, booth in vitro and in vivo (14). Therefore administration of CO in accordance with the inv-ention may be used for treatment= of any of these conditions, fo r modulation of inflammatory stz=ates and regression of other pat hophysiological conditions including cancer.

Accordingly there is provided a method of introdu cing CO to a mammal comprising the step of administering a pharmaceutical composit ion according to the present inzxrention, for treatment of hypert ension, such as acute, pulmonary.” and chronic hypertension, r-adiation damage, endotoxic shocl<, inflammation, inflammat ory-related diseases such as ast=hma, rheumatoid arthritis and small bowel disease, hyperoxi=a- induced injury, apoptosis, cancer, transplant rejectior, post- operative ileus, arteri_osclerosis, post-ischemic organ damage, myocardial infarction, angina, haemorrhagic shock, sepssis, penile erectile dysfunce=tion and adult respiratory distmress syndrome, and in procedBures such as balloon angioplastwsy (to treat restenosis followring balloon angioplasty) and ao=xtic transplantation. For e=xample, in balloon angioplasty =it may be advantageous to makes a local delivery of CO-releasimng compound before and/or after the angioplasty. Alterna‘tively, a stent may have a coatzing containing CO- releasing commpounds.

The present inven-tion also provides the use of a boranocarbonate compournd or ion as herein described in the manufacture of a medicament for delivering CO to a physiological target, poarticularly a mammal, to provid e a physiological effect, e.g. for stimulating neurotransm ission or vasodilation, or forxr treatment of any of hypertensi on, such as acute, pulmonary anci chronic hypertension, radiation damage, endotoxic shock, inflammation, inflammatory-re lated digeases suchr. as asthma, rheumatoid arthritis a.nd small bowel disease, hype roxia-induced injury, apoptosis, c ancer, transplant re jection, post-operative ileus, art eriosclerosis, sickle cell amemia or sickle cell disease, post -ischemic organ damage, myocardial infarction, angina, haemorrhaagic shock, sepsis, penile erectile dysfunction and adult resspiratory distress synd®-ome, and in procedures such as baR. loon angioplasty ard aortic transplantation. Such me™icaments may be adapted for administration by an oral, intravenous, subcutaneous, nasal, inhalatory, intramuscular, intraperitoneal, transdermal, transmucosal or sumppository route.

In a further aspect, the invention providess a method of treatment of a mammal comprising stimulation of neurotransmiss ion, vasodilation or smooth muscle relaxation by

CO as a physiologically effective agent, or the treatment of any of hypertemsion, radiation damage, endotoxic shock, inflammation, =inflammatory-related diseases, hyperoxia-induced injury, apoptosis, cancer, transplant rejection, post- operative ileus, arteriosclerosis, post-ischemic organ damage, myocardial infarction, angina, haemorrhagic shocks, sepsis, penile erectile dysfunction, adult respiratory di_stress syndrome, vascumlar restenosis, hepatic cirrhosis, cardiac hypertrophy, he=art failure and ulcerative colitiess, or treatment in ba.lloon angioplasty, aortic transpla_ntation or survival of a t ransplanted organ, by administrati on of a boranocarbonate compound or ion adapted to make C O available for physiologic-al effect. These are treatments a ssociated with the action of CO.

Preferably~, the method of treatment is stimmlation of neurotransmissicon, vasodilation or smooth muscle mrelaxation by

CO as a physiologically effective agent, or treatrment of any of acute or chronic systemic hypertension, radiation damage, endotoxic shock. hyperoxia-induced injury, apoptossis, cancer, transplant rejection, post-operative ileus, arteriosclerosis,

post-ischeamic organ damage, angina, haemorrhagic shock, sepsis, peanile erectile dysfunction, vascrilar restenosis, hepatic ci.rrhosis, cardiac hypertrophy, hesart failure and ulcerative= colitis, or treatment in balloon angioplasty, aortic tra.nmsplantation or survival of a trransplanted organ.

More preferably, the method of treatmment is stimulation of neurotr-ansmigsion, vasodilation or smooth muscle relaxation by CO as a physiologically effective agent=, or treatment of any of acu te or chronic systemic hypertenssion, hyperoxia- induced in jury, cancer by the pro-apoptotic effect of CO, transplant rejection, post-operative ileus , post-ischemic organ damacje, angina, haemorrhagic shock, penile erectile dysfunctiom, hepatic cirrhosis, cardiac hypertrophy, heart failure ancl ulcerative colitis, or treatmemt in balloon angioplasty or aortic transplantation.

Parti cularly, the method may be treat—ment of any of hyperoxia-i# nduced injury, cancer by the pres-apoptotic effect of CO, trarasplant rejection, post-operative ileus, post- ischemic oxrgan damage, angina, haemorrhagicc shock, penile erectile dysfunction, hepatic cirrhosis, cardiac hypertrophy, heart failuare and ulcerative colitis, or tmreatment in balloon angioplasty= or aortic transplantation.

By "srmooth muscle relaxation" is mean t treatment of conditions other than by vasodilation, sucka as chronic anal fissure, immternal anal sphincter disease arad anorectal disease.

More sspecific treatments to which the invention may be applied are the suppression of atherosclerotic legions following a ortic transplantation, ischemic lung injury, prevention of reperfusion induced myccardia.l damage, and also to achieve the pro-apoptotic effects of CO (e.g. in cancer treatments) .

The imvention further provides use of the boranocarbonate compounds o=xr ions here described in treatme mnt, e.g. by perfusion, of a viable mammalian organ extr acorporeally, e.g.

during storage and/or transport of an organ for transplant surgery. For this purpose, the boranocarb onate is in dissolved form, preferably in an aqueous s-olution. The viable organ may be any tissue containing living cells, such as a heart, a kidney, a liver, a skin or muscle flap, etc.

For example, isolated organs e.g. ext—racorporeal organs or in situ organs isolated from the blood Supply can be treated. TThe organ may be, for example, a scirculatory organ, respiratory organ, urinary organ, digestivee organ, reproducti ve organ, neurological organ, mussScle or skin flap or an artificial organ containing viable cellas. In particular, the organ way be a heart, lung, kidney or liver. However, the body tissiaie which is treatable are not lim=—ited and may be any human or mnammal body tissue whether extraceorporeal or in-situ in the body. It is further believed that #che compositions of the invention here described are useful to deliver CO to an extracorporeal or isolated organ so as to —xreduce ischaemic damage of the organ tissue.

With in the present invention, the bor—anocarbonates here described can be used in combination with == guanylate cyclase stimulant or stabilizer to deliver CO to a physioclogical target so as to provide an improved physioZlogical effect.

The —pharmaceutical preparation may contain the boranocarloonate and the guanylate cyclase sstimulant/stabilizer in a singMe composition or the two componemts may be formulated separately for simultaneous or s=sequential administration.

Thus the present invention provides aa method of introducirmg CO to a mammal as a therapeutiec agent comprising: a) administering a boranocarbonate which makes available CO suitable for physiological effect; and b) administering a guanylate cyclasse stimulant or gtabilisem.

In t his aspect, the method is particularly applicable to treatment of acute or chronic systemic hypeertension, pulmonary hypertension, transplant reject ion, post-operative ileus , ar-teriosclerosis, post-ischemic organ damage, myocardial imfarction, penile erectile dysfunction, vascular restenosis, hepatic cirrhosis, cardiac hypertrophy, heart failure, c=hronic arnal fissure, internal anal sphincter disease, anorectal. d#.sease, and ulcerative colitis or for treatment in ball_oon argioplasty or aortic transplantation.

Preferably, the stabilizer/stimulant is administer ed foirst followed by the boranocaxbonate but this order max.” be re=versed.

The guanylate cyclase stabilizer/stimulant compourmd may b e any compound which stimulat es production of guanylatee cyclase or which stabilizes guanylate cyclase, in parti-cular t he active form of guanylate cyclase. A single compound can be wm sed or a combination of compounds can be used either f or ssimultaneous or sequential administration, i.e. the var—ious aaspects include/use at least one guanylate cyclase stimulant/stabilizer.

Examples include 3-(5'-hydroxymethyl-2'-furyl)-1-¥oenzyl-

ZJindazole (YC-1), 4 pyrimidinarnine-5-cyclopropyl-2-{1-[ «(2- fluorophenyl) methyl] -1H-pyrazolo[3, 4-blpyridin-3-yl] (E23AY 41- 272), BAY 50-6038 (ortho-PALi), BAY 51-9491 (meta PAL) _, and

BAY 50-8364 (para PAL). The structures of ortho-, met=a- and —para- PAL are shown in Figure 9 attached. These comporunds have been found to bind to an activation site on the guanylate cyclase and any other compourads that similarly bind to the site may be useful as the guanylate cyclase stabilizexr—/ stimulant. Also useful are NO donors and l-benzyl-3- ( 3!- ethoxycarbonyl)phenyl-indazole, 1l-benzyl-3-(3'- hydroxymethyl)phenyl-indazole, 1-benzyl-3-(5'- diethylaminomethyl) -furyl-indazole, 1l-benzyl-3-(5'- methoxymethyl) furyl-indazole , 1l-benzyl-3-(5'- hydroxymethyl) furyl-6-methyl —indazole, 1-benzyl-3-(5'— hydroxymethyl) -furyl-indazol -benzyl-3- (5'-hydroxymethyr1) ~-furyl- indazole, l-benzyl-3- (5!'-hydxoxymethyl)-furyl-6-fluorc-

I inda_zole, 1-benzyl-3- (5*-hydroxymethyl )-furyl-6-methoxy- inda:=zole, and 1-benzyl-3- (5'-hydroxyme thyl) -furyl-5,6- methylenedioxoindazole or rharmaceutic=ally acceptable salts therseof .

For reasons relating to prior pafcent filings and for proprietary reasons, the present appli_cants may wish to exclvude use of the following two compounds from the protection givem to the present invention in any of its aspects as claiwmmed: -

I. K, (H,BCO0)

II. "Ne 2°

RN—B—¢

TN where R, R' = H, alkyl, perflumoroalkyl.

Ther efore this exclusion is now optioraally and provisionally made .

Throughout this application, ref erences to medical trea tment are intended to include botlm human and veterinary trea tment, and references to pharmaceutical compositions are acco-xrdingly intended to encompass compoositions for use in huma_n or veterinary treatment.

Experimental data illustrating t-he present invention will now be described.

In the accompanying drawings, Fi.gs 1 to 8 are graphs showwing results of the experiments of Examples 1 to 8 below.

Fig. 9 is chemical formulae mentioned above. Figs 10 and 11 are graphs showing results of Exampless 9 and 10 below.

EXAMPLES: 1 TO 8

Reagents

Tricarbo.nylchloro(glycinato) ruthen ium (II) ({Ru(CO) -53C1 (glycinate)] or CORM-3) was synthesized as previously described by Clark and ecollaborators 2¢. Disoditam boranoca=rbonate (Na,[H,BC0O,], indic=ated here as “CORM-Al1") was synthesi=zed as previously describecd by Alberto and collaborators *!'. Sodium borohydrid-e (NaBH,) and all other reagents were from Sigma Chemicals (Poole, Dorset).

Preparation of inactive CORM-Al and its use as negative control

The chemistry of boranocarbonate irm aqueous solution has be=en previously described *'. This comporand is relatively stable in distilled water at basic pH. The cocompound starts to release CO ag the pHI moves towards more physioslogical conditions (pH="7.4) and the r-ate of CO release is great.ly accelerated at acidic

PH. Base=d on this evidence, we gen.erated an inactive form of

CORM-Al1l ( iCORM-Al) by reaction of t he compound with acid.

Specifica lly, a small aliquot (10 ux 1) of concentrated hydrochlo ric acid (10 M) was added to 1 ml of CORM-Al in water (100 mM f£ inal concentration). The reeaction resulted in a rapid evolution of a gas (presumably CO); the solution was then bubbled with a stream of nitrogen imn order to remove the residual &C0 gas eventually dissolvecd. Aliquots of this solution wwere used as a negative comtrol of CORM-21 in the experiments conducted to quantify tlkae release of CO (i.e. MD assay) as well as the biological efficacy (i.e. vessel relaxatior). Since boron is a compornent of CORM-Al and because borohydricie could be formed during t—he liberation of CO froam

CORM-Al ira aqueous solution, sodium borohydride (NaBH,) was also utilized as a negative control in some experiments.

Detection of CO release

The release of CO £rom CORM-Al was assessed spectrophotometricaally by measuring the conversion of deoxymyoglobin (Mb) to carbonmonoxy myoglobin (MbCO) by a method previously described 2°. The amount of MbCO formed waes quantified by measwring the absorbance at 540 nm (extinctiora coefficient = 15.4 M*' cm) over time at 37 °C. Myoglobin solutions (approximmately 50 umol/L final concentration) were= prepared fresh by dissolving the protein in 0.04 M phosphates buffer (pH=7.4). So dium dithionite (0.1 %) was added to convert the oxidize d myoglobin to its reduced form prior to each reading. Some «experiments were also conducted using Mb at

PH=5.5 or at room teemperature (RT) in order to examine the kinetic of CO releasse from CORM-Al under different chemical and physical condit=ions.

Isolated aortic ring preparation: studies on vessel relaxatieon

Transverse ring sect-ions of thoracic aorta were isolated from male Lewis rats and suspended under a 2 g tension in an orgama bath containing oxycyenated Krebs-Henseleit buffer at 37 °C im a manner previously described '°. The relaxation response to

CORM-Al (40, 80 and 160 uM) was assessed in aortic rings pre— contracted with phermylephrine (3 uM}. Control rings were similarly treated by” adding equal doses of the inactive compound (iCORM-Al) or sodium borohydride (NaBH,) to the orga.n bath. Experiments wesre also conducted by comparing the effects of CORM-A1 and CORM—3 on vessel relaxation over time.

Example 1. Conversiosn of myoglobin (Mb) to carbon monoxide myoglobin (MbCO) by CO gas.

Myoglobin (Mb) in it s reduced state displays a characteristics spectrum with a maximal absorption peak at 555 nm (see Figure= 1, dotted line). Whem a solution of Mb (50 uM) is bubbled for-

1 min with CO gas (1%), a rapid convemrsion to carbon monoxide myoglobin (MbCO) is observed. As showr in Figure 1, MbCO displays a characteristic spectrum witzh two maximal absorption peaks at 540 and 576 nm, respectively (solid line). This method has been previously developed t—o monitor and determin. e the amount of CO released from CO-RMs % and can be used to examirne how various conditions such ass different pHs and temper-atures can affect the kinetics of C0 release (see

Exampl.es 4).

Example 2. Conversion of myoglobin (Ml>) to carbon monoxide myoglosbin (MbCO) by CORM-AL.

Additi on of CORM-Al (60 pM) to a solut=ion containing reduced

Mb (pH=7.4, temp. = 37 °C) resulted im a gradual formation o-=f

MbCO over time. As shown in Figure 2, a spectrum typical of reduced Mb (filled square) is converte=d to a spectrum charac teristic of MbCO after 210 min i.ncubation (inverted opesn triang le). The trace with asterisks sh.ows the spectrum of MbCZ0 when MX is saturated with CO gas (posi tive control) as descri ded in Materials and Methods.

Exampl € 3. Kinetics of CO release from- CORM-Al at room temper ature.

The amount of MbCO formed after additi on of CORM-Al to the Mio solution can be quantified by measurin_.g the absorbance at 540 nm knowing the extinction coefficient for MbCO {(¢ = 15.4 M? cm 1). CORM-Al at three different concentmrations was added to a solution containing Mb at room tempera ture and the formed MbCTO was ca lculated over time. Non-linear r egression analysis usirg one ph.ase exponential association (Gra—phPad Prism) resulted dn the be st fitting of the three curves (:x?>0.99). As shown in

Figure 3, the amount of MbCO formed fr.om CORM-Al increases with a defined kinetic in a concentrat ion-dependent manner.

The calculated Y,.x. for each plot (16.7—=1.2, 33.1%1.4 and 48,242 .5) was in very good agreement w_ith the three concentratio-ns of CORM-Al used (15.86, 31.1 and 46.7 uM, respectively). This indicates that the reaction leading to the formation of CO from CORM-Al in agueous solution goe=s to completion o ver time and that one mole of CO per mol.e of compound is liberated. From the fitted curves the awerage half-life of CORM-Al at room temperature is 112+3 mi_n.

Example 4. E ffects of temperature and PH on the rate of CO - release from CORM-Al.

The rate of CO release from CORM-Al was examined at different

PHs and temp eratures. CORM-Al (60 uM) wae added to t—he Mb solution und er three different conditions: 1) at room temperature (RT) and pH=7.4; 2) at 37 °C and PH=7.4; and 3) at 37 °C and pH = 5.5. The concentration of MbCO was ca lculated at different ti=me points and non-linear regression anal ysis was used to obta in the best fitting of the three curves as described in example 3. As shown in Figure 4, the ramte of CO release from CORM-Al is significantly accelerated by= increasing the temperature as well as by decreasing the pH.

Specifically , it can be calculated that the half-life of CORM-

Al is 104 min at RT/pH=7.4 (triangles), 18.5 min at 37 °C/pH=7.4 (diamonds) and 1.2 min at 37 °C/pH=5.5 (squares).

Example 5. C-omparison between CORM-Al and its inacti_ve form (iCORM-A1l) om their ability to liberate CO.

As described in the Materials and Methods section, C0 is rapidly lost when CORM-Al ig added to acidic soluticons. This step allows the generation of an inactive compound CC iCORM-A1l) that could b e used as an ideal negative control for testing the biologic al activity of these molecules. To verify that iCORM-Al has effectively lost its full ability to re=lease CO, the compound. (60 uM) was added to a solution contairaing Mb (50 pM) at pH=7. 4/RT and the MbCO formed over time was czalculated,

As shown in Figure 5, iCORM~Al (circles) is incapabl_e of generating a my detectable MbCO suggesting that the compound has been fully -inactivated. The effect of CORM-Al (squares) on

MbCO formation -is shown for comparison.

Example 6. Comparison between CORM-Al and CORM-3 =in their ability to elici.t vasorelaxation.

CORM-3 ([Ru(CO), Cl (glycinate)]) has been shown to promote a rapid and significant relaxation in isolated vesse=ls and this effect has been demonstrated to be mediated by co *. It is also known from recent works that the liberation of CO from

CORM-3 to Mb or in biological systems occurs very rapidly (approximately 5 min) 2%2?, which ie in agreement wwith the prompt pharmacological effects observed in isolate=d vessels.

In the case of CORM-Al, the release of CO at physi_ ological pH is slower (18.4 min) as shown in example 5. Thus, it is expected that thee pharmacological action of CORM-MA1 would reflect its biochemical behaviour. Indeed, as showsn in Figure 6, CORM-Al (80 uM) caused a much slower effect on relaxation compared to CORM -3 (80 uM). Specifically, CORM-3 ( solid line) added to isolated aortic rings pre-contracted witli phenylephrine (Phe) promoted a 75% relaxation with in 4-5 min whereas CORM-Al (dashed line) caused a gradual vas orelaxation which was maxima 1 (96%) 33 min following addition of the compound to the organ bath.

Example 7. Concemtration-dependent effect of CORM-_Al on vasorelaxation

Pre-contracted aortic rings were treated with incr easing concentrations of CORM-Al (40, 80 and 160 uM) and the percentage of vasorelaxation was calculated at dif ferent time points. As shown in Figure 7, CORMA-1 caused a sigmificant relaxation over time in a concentration-dependent wmanner. For instance, it can be seen from the graph that after 10 min, the percentage of relaxation elicited by the different concentrations ox CORM-Al compared to control was =as follows: 21.042.3% with 40© uM CORM-Al, 40.213.4% with 80 uM CORM-Al and

74.9%1.8% wvith 160 uM CORM-Al. The data are re presented as the meantS.E.M . of 6 independent experiments for e ach group.

Example 8. The vasorelaxant properties of CORM -Al are mediated by Co

Pre-contrac-ted aortic rings were treated with 80 uM CORM-Al, iCORM-Al (®:he inactive compound) or NaBH,, which was used as an additional negative control (see Materials and Methods for details) . Ms shown in Figure 8, only CORM-Al promoted a gradual ancd profound vasorelaxation whereas bo-th iCORM-Al and

NaBH, were totally ineffective. These results clearly suggest that CO likoerated from CORM-Al is directly responsible for the observed phiarmacological effect. The data are —represented as the mean+S .E.M. of 6 independent experiments fer each group.

Examples 9 and 10.

Stock solut—ions of sodium boranocarbonate (COREM-Al, 100 mM) were prepamed by solubilizing the compound in «distilled water prior to tlie experiment. 3-(5’-hydroxymethyl-2 *-furyl)-1- benzyl -ind=azole (YC-1)} was purchased from Sigma-Aldrich (Poole, Domrset) and prepared in dimethyl sulfo=xide (DMSO). All data axe empressed as mean t+ s.e.m., Differencess between the groups analilysed were assessed by the Student’s two-tailed t- test, and =an analysis of variance (ANOVA) was pperformed where more than t—wo treatments were compared. Resultss were considered statistically significant at P<0.05 .

Isolated acortic ring preparation: studies on veassel relaxation

Transverse ring sections of thoracic aorta were isolated from male Lewis rats and suspended under a 2 g tens-ion in an organ bath containing oxygenated Krebs-Henseleit buffer at 37 °C in a manner prewriously described [10]. The relaxatieon response to

CORM-Al (20 uM) in the presence or absence of ¥C-1 (1 uM final concentration) was assessed over time in aortiec rings pre- contracted with phenylephrine (1 mol/L). YC-1 was added to the is olated rings 30 min prior to contraction with phenyl ephrine. hi Animal studies: effect of CORM-Al and Y<'-1 on blood pressure

Lewis —xats (280-350 g) were anaesthetise=d by intramuscular inject-ion of 1 ml/kg Hypnorm. Specially designed femoral artery and versous catheters were then surgicall=y implanted and mean arteri=al pressure (MAP) monitored contiruously using a polygraph recorder in a manner previousl y described [23]. The effect of CORM-AL on mean arterial presssure (MAP) over time was asssessed following an intravenous (#® .v.) injection of 50 pmol key. Similar experiments were condwicted by administering

YC-1 (1.2 pmol kg™, i.v.) to animals 5 ruin prior to the bolus additicon of CORM-Al. Control experimentss using YC-1 alone were also performed.

Example= 9. Effect of CORM-Al and YC-1 orm aortic vasorelaxation

Pre-cortracted aortic rings were treated with CORM-Al and the percentcage of vasorelaxation was calculaated at different time points _ As shown in Figure 10, 20 pM COR®MA-1 caused 13+4.9% relaxat=—ion after 20 min; interestingly, a more pronounced and signifi cant relaxation response (61+6.2%) was detected after pre-treatment of vessels with ¥YC-1 (1 gM). Note that in control. vessels pre-treated with YC-1 al one and contracted with plaenylephrine there was only a wminosr relaxation response over time (2.8+1.1% after 20 min). The r-elaxation response of vesselss pre-treated with YC-1 was also very significant at 1 uM and 10 pM CORM-Al (35+9.8% and 5113.3 %, respectively). The data arce represented as the meants.e.m. of 6 independent experiments for each group. *P<0.05 vs. CORM-Al alone or YC-1 alone.

Examples 10. Effect of CORM-Al and CORM-3 on mean arterial pressur-e. Femoral artery and venous cathesters were surgically implanted into anesthetized Lew-is rats and blood pressures continuously monitored as previ ously described by us [23 1. The effect of CORM-Al and YC-1 on mean arterial pressure (MA.P) in vivo is represented in Figure 11. The compounds were inj ected intravenously as a bolus at a final concentration of 50 pmoles/kg for CORM-Al and 1.2 pmol kg? for YC-1. When tlae two compounds were given in combination, YC-1 was administer—ed 10 min prior to CORM-Al injection. As shown, CORM~-Al produc ed a gradual and sustained decrease in MAP over time; for ins tance, 60 min after CORM-Al injection MAP decreased by 6.3+1.5 -mmHg from the initial baseline value . Injection with YC-1 alo me also produced an effect on blood pressure; however, the decrease in MAP was only transient, reaching a maximum o £ 5.541.0 mmHg after 10 min and returning to basal levels 50 min after injection. Interestingly , the combination of CORM -Al and ¥C-1 produced a synergistic effect resulting in a rapid and profound hypotension. In fact, MAP significantly dec xeased by 16.1+5.6 mmHg after 10 min amd remained at this level for the rest of the experiment. The data are represented as the meanzs.e.m. of 5 independent experiments for each group. ‘P<0.05 vs. baseline (-10 min); ' P<0.05 vs. CORM-Al alome or

YC-1 alone.

The present invention ther-efore provides water-solimble compounds which are useful as CO carriers which can have selectable chemical properties, enabling novel therapeut ic approaches based on CO delivery . This offers significant advantages over inhalation of CO as it may circumvent th e problems related to the systemic effects of CO gas on oxygen transport and delivery. Moreover, the design of stable compounds with "fast" or "slow" kinetics of CO release t hat could target selective organs amd affect only a restrict ed area of the body is highly feasible. One application fo x the use of water-soluble compounds is in conditions where Co needs to be applied locally. For instance, in order to protec t vascular tissues during balloon angioplasty and prevent “blood

VO 2005/013691 PCT/GB2004/003365 vessel restenosis, CO-provid ing compounds may be applied to vessels prior to the angiopl asty procedure. Alternatively, vascular stents may be cover ed with specific boranocarbonate compounds that have the abil ity to release CO slowly to the injured vessels and inhibit smooth muscle cell proliferation.

Compounds whose kinetic of CO release is affected by temperature could also be used ex-vivo as an adjuvant to

Preservation solutions that are commonly employed to store organs prior to transplantat-ion. The protective role of HO-1 against organ rejection has loeen extensively reported and the concept of treating the orgari(s) rather than the recipient (8) will have much benefit in the clinical setting of transplantation.

ww 0 2005/013691 PCT/GB2004-/003365

References 1. Tenhunen R, Marver HS, Schmid R. Microsomal heme oxygenase. Characterization of the enzyme. J Biol CChem. 1969;244:6388-6394 . 2. Maines MD. The heme oxygenase gystem: a regulator of second messenger gases. Annu Rev Pharmacol Toxicol. 1997;37:517-554. 3. Verma A, Hirsch DJ, Glatt CE, Ronnett GV, Snyder SH .

Carbon monoxide: a putative neural messenger. Scien ce. 1993;259:381-384. 4. Sacerdoti D, Escalante B, Abraham NG, McGiff JC, Le-vere

RD, Schwartzman ML. Treatment with tin prevents the development of hypertension in spontaneously hyperteensive rats. Science. 1989;243:388-390. 5. Marks GS, Brien JF, Nakatsu XK, McLaughlin BE. Does «carbon monoxide have a physiological function? Trends Pharomacol

Sci. 1991;12:185-1838. 6. Coceani F, Kelsey I., Seidlitz E, Marks GS, McLaughl din BE,

Vreman HJ, Stevenson DK, Rabinovitch M, Ackerley C.

Carbon monoxide formation in the ductus arteriosus Hin the lamb: implications for the regulation of muscle ton-e. Br

J Pharmacecl. 1997;1.20:599-608. 7. Johnson RA, Colombari E, Colombari DSA, Lavesa M, T alman

WT, Nasjletti A. Role of endogenous carbon monoxide in . central regulation of arterial pressure. Hypertensi on. 1997;30:962-967. 8. Wang R, Wang ZZ, Wu LY. Carbon monoxide-induced vasorelaxation and the underlying mechanisms. Br J

Pharmacol. 1997;1271.:927-934.

/0138691 PCT/GB2004/003365 9. Mlotterlini R, Gonzales A, Foresti R, C lark JE, Green CJ,

Winslow RM. Heme oxygenase-l-derived c-arbon monoxide contributes to the suppression of acute-e hypertensive r—esponses in vivo. Circ Res. 1998;83:5 68-577. 10. Sammut IA, Foresti R, Clark JE, Exon DaJ, Vesely MJJ,

S-arathchandra P, Green CJ, Motterlini TR. Carbon monoxide i 8 a major contributor to the regulatieon of vascular tone i n aortas expressing high levels of haeeme oxygenase-1. Br

J~ Pharmacol. 1998;125:1437-1444. 11. 8S uematsu M, Goda N, Sano T, Kashiwagi .S, Egawa T, Shinoda

Y , Ishimura ¥. Carbon monoxide: an endecgenous modulator of sinusoidal tone in the perfused rat liver. J Clin

I"nvest. 1995;96:2431-2437. 12. Miorita T, Mitsialis SA, Koike H, Liu YX, Kourembanas S.

Carbon monoxide controls the prolifera tion of hypoxic vascular smooth muscle cells. J Biol CZhem. 1 997;272:32804-32809. 13. Sato K, Balla J, Otterbein L, Smith RN , Brouard S, Lin Y,

Cosizmadia E, Sevigny J, Robson SC, Ver cellotti G, Choi

AaM, Bach FH, Soares MP. Carbon monoxid.e generated by heme omxydgenase-1 suppresses the rejection o f mouse-to-rat c=ardiac transplants. J Immunol. 2001;1 66:4185-41%4. 14. Otterbein LE, Bach FH, Alam J, Soares M, Tao Lu H, Wysk

VMI, Davis RJ, Flavell RA, Choi AM. Carb-on monoxide has amnti-inflammatory effects involving th_e mitogen-activated rorotein kinase pathway. Nat Med. 2000; 6:422-8. 15. BE3rouard S, Otterbein LE, Anrather J, T—obiasch E, Bach FH,

Choi AM, Soares MP. Carbon monoxide ge=merated by heme oxygenase 1 suppresses endothelial cel.l apoptosis. J Exp

Med. 2000;192:1015-1026.

16. Song R, Mahidhazxa RS, Liu F, Ning W, Otterbein. LE, Choi

AM. Carbon monoxide inhibits human airway smoo th muscle cell proliferat-ion via mitogen-activated prote in kinase pathway. Am J Respir Cell Mol Biol. 2002;27:60.3-610. 17. Otterbein LE, Mantell LL, Choi AMK. Carbon moneoxide provides protection against hyperoxic lung injwury. Am J

Physiol. 1999;276:L.688-1694. 18. Fujita T, Toda KK, Karimova A, Yan SF, Naka Y, “et SF,

Pinsky DJ. Paradoxical rescue from ischemic lurg injury by inhaled carbon monoxide driven by derepression of fibrinolysis. Nat Med. 2001;7:598-604. 19. Foresti R, Motterrlini R. The heme oxygenase pat-—hway and its interaction with nitric oxide in the control of cellular homeost asis. Free Rad Res. 1999;31:459-475. 20. Otterbein LE. Ca rbon monoxide: innovative anti— inflammatory properties of an age-old gas moleccule.

Antioxid Redox Signal. 2002;4:309-319. 21. Chauveau C, Bouchet D, Roussel JC, Mathieu P, BBraudeau C,

Renaudin K, Tesson L, Soulillou JP, Iyer S, Bue=low R,

Briegon I. Gene transfer of heme oxygenase-1 andE carbon monoxide deliver~y inhibit chronic rejection. Ana J

Transplant. 2002 ;2:581-592. 22. Motterlini R, Foxesti R, Green CJ. Studies on t he development of carbon monoxide-releasing molecu. les: potential applications for the treatment of cardiovascular dwsfunction. In: Carbon Monoxide and

Cardiovascular Functions. Wang R, ed. 2002. CRC Press,

Boca Raton, Flordda. 23. Motterlini R, Claark JE, Foresti R, Sarathchandra P, Mann

BE, Green CJ. Caxbon monoxide-releasing molecules:

Ww 0 20051013691 PCT/GB2004/003365 characterization o f£ biochemical and vascular activities.

Circ Res. 2002;90:E17-E24. 24. Clark JE, Naughton P, Shurey S, Green CJ, Johnson TR,

Mann BE, Foresti R _, Motterlini R. Cardioprotective actions by a water —soluble carbon monoxide-releasing molecule. Circ Res - 2003;93:e2-e8. 25. Mann BE, Motterlini R. Therapeutic delivery of carbon monoxide. PCT. 200=;WO 02092075. 26. Johnson TR, Mann BE, Clark JE, Foresti R, Green CJ,

Motterlini R. Metal. carbonyls: a new class of pharmaceuticals? Aragew Chem Int Ed Engl. 2003;In press. 27. Motterlini R, Mann BE, Johnson TR, Clark JE, Foresti R,

Green CJ. Biocactivi ty and pharmacological actions of carbon monoxide-rel easing molecules. Curr Pharm Des. 2003;In press. ’ 28. Waibel R, Alberto R , Willuda J, Finnern R, Schibli R,

Stichelberger A, Eg li A, Abram U, Mach JP, Pluckthun A,

Schubiger PA. Stable one-step technetium-929m labeling of

His-tagged recombinant proteins with a novel Te({I)- carbonyl complex. Naat Biotechnol. 1999;17:897-901. ) 29. Egli A, Alberto R, Tannahill L, Schibli R, Abram U,

Schaffland A, Waibed R, Tourwe D, Jeannin L, Iterbeke K,

Schubiger PA. Organometallic 99mTc-aquaion labels peptide to an unprecedented high specific activity. J Nucl Med. 1999;40:1913-1917. 30. Alberto R, Schibli RR, Egli A, Schubiger AP, Abram U,

Kaden TA. A novel orrganometallic aqua complex of technetium for the Labeling of biomolecules: Synthesis of (Tc-99m(0OH2) (3) (CO) €3)1 (+) from ([(TcO4)-Tc-99m] (-) in aqueous solution and its reaction with a bifunctional ligand. J Am Chem Scoc¢. 1998;120:7987-7988.

31.. Alberto R, Ortner K, Wheatleey N, Schibli R, Schubiger AP.

Synthesis and properties of boranocarbonate: a conven dient in situ CO source for the acgueous preparation of ' ((99m) Tc(OH(2))3 (CO) 31+. J Am Chem Soc. 2001;123:3135—~ 3136. 32. Abraham NG, Drummond GS, Lut=ton JD, Kappas A. The biological significance and physiological role of heme oxygenase. Cell Physiol Biochem 1996;6:129-68. 33. Soares MP, Lin Y, Anrather JV, Cgizmadia E, Takigami K,

Sato K, Grey ST, Colvin RP, Choi AM, Poss KD, et al.

Expression of heme oxygenase:-1 can determine cardiac xenograft survival. Nature Med 1998;4:1073-7. 34. Hancock WW, Buelow R, Sayegh MH, Turka LA. Antibody- induced transplant arteriosc lerosis is prevented by graft expression of anti-oxidant amd anti-apoptotic genes.

Nature Med 1998;4:1392-5. 35. Clark JE, Foresti R, SarathcEiandra P, Kaur H, Green CJ,

Motterlini R. Heme oxygenase—l-derived bilirubin ameliorates post-ischemic mycocardial dysfunction. Am J

Physiol Heart Cire Physiol 2000;278:H643-51. 36. Willie D, Moore AR, Fredericl<= R, Willoughby DA. Heme oxygenase: a novel target forxr the modulation of inflammatory response. Natures Med 1996;2:87-90. 37. Bauer M, Pannen BHJ, Bauer I, Herzog C, Wanner GA,

Hanselmann R, Zhang JX, Cleme=ns MG, Larsen R. Evidence for a functional-link betweerm stress-response and vascular control in hepatic poortal circulation. Am J

Physiol 1996;271:G929-35.

38. Fukuda K, Panter SS, Sharp NFR, Noble LJ. Inducticn o f heme oxygenase-1 (HO-1) aftesr traumatic brain injury in the rat. Neurosci Lett 1995 ,.5199:127-30. 39. Yet SF, Pellacani A, Pattersson C, Tan L, Folta SC, F-oster

L, Lee WS, Hsieh CM, Perrella MA. Induction of heme oxygenase-1 expression in vascular smooth muscle cells. A link to endotoxic shock. J E3iol Chem 1997;272:4295-3=01. 190 40. Otterbein LE, Kolls JK, Mant=ell LL, Cook JL, Alam J, Choi

AMK. Exogenous administration of heme oxygenase-1 by gene transfer provides protectiorn against hyperoxia-induce=d lung injury. J Clin Invest 1.999;103:1047-54.

Ld

Claims (1)

1. Use of a boranocarbormnate compound or ion in the manufacture of a medicament, for the stimulation of neurotransmission, vasodilation or smooth muscle rela—xation by CO as a physiologically effective agent, or for the t-reatment of any of hypertension, radiation damage, endotoxic shock, inflammation, inflammators/-related diseases, hyperoxi-a-induced injury, apoptosis, cancer. transplant rejection, post - operative ileus, arteriosclerosis, post-ischemic organ damage, myocardial infarction, angina, haemorrhagic shock, sepsis, penile erectile dysfunction, adult respiratory distre.ss syndrome, vascular restenosis, hepatic cirrhosis, carediac hypertrophy, heart failure and ulcerative colitis or for treatment in balloon angioplasty, aortic transplantat._don or survival of a transplanted organ.

2. Use according to claim 1 wherein the medicament is for the stimulation of neurotwxansmission, vasodilation or smooth muscle relaxation by CO as a physiologically effective.e agent, or for the treatment of amy of acute or chronic systemmatic hypertension, radiation damage, endotoxic shock, hype—roxia- induced injury, apoptosis, cancer, transplant rejecti-on, post- operative ileus, arteriosclerosis, post-ischemic orga—n damage, angina, haemorrhagic shock, sepsis, penile erectile dysfunction, vascular restenosis, hepatic cirrhosis, cardiac hypertrophy, heart failure and ulcerative colitis or for treatment in balloon angioplasty, aortic transplantat ion or survival of a transplanted organ. ’

3. Use according to claim 1 wherein the medicament is for the stimulation of neurotwxansmission, vasodilation or smooth muscle relaxation by CO as a physiologically effectiv—e agent, or for the treatment of amy of acute or chronic systematic hypertension, hyperoxia-imaduced injury, cancer by the pro- apoptotic effect of CO, transplant rejection, post-op erative ileus, post-ischemic orgam damage, angina, haemorrhag-ic shock,

Penile erectile dysfunction, hepatic cirrhosDis, cardiac hypertrophy, heart failure and ulcerative colitis or for treatment in balloon angioplasty or aortic tmansplantation. 4 Use according to any one of claims 1, 2 and 3 wherein the me=dicament is suitable for administration by an oral, imtravenous, subcutaneous, nasal, inhalatory ~ intramuscular, imtraperitoneal, transdermal, transmucosal or— suppository route. 5a Use according to any one of claims 1 to 4 wherein the molecular structure of the boranocarbonate ccompound or ion irmcludes the moiety r= 0)

6. Use according to claim 5 wherein the bor—anocarbonate compound or ion includes the moiety BH,-CO-.

7. Use according to claim 5 or 6 wherein time boranocarbonate is a compound or anion of the formula: BH, (COQ) ,Z, wherein: - Xx is 1, 2 or 3 y is 1, 2 or 3 Zz is 0, 1 or 2 X+Y +2 =4, each Q is O°, representing a carboxylates anionic form, or is OH, OR, NH,, NHR, NR,, SR or halogeen, where the or each R is alkyl (preferably of 1 to <1 carbon atoms) , each Z is halogen, NH,, NHR', NR',, SR' or OR' where the or each R' is alkyl (preferably of 1. to 4 carbon atoms) .

8. Use according to claim 7 wherein z is 0.

9g. Use according to claim 8 or 9 where y is 1.

10. Use according to claim 7 where x is 3.

11. Use according to any one= of claims 7 to 10 where the boranocarbonate is an anion, with at least one Q in the fo—rnm of 0” or OR, and the composition includes at least one meta==al cation.

12. Use according to claim 11 wherein the or each metal cation is an alkali metal cat-ion or an alkaline earth metal cation.

13. Use according to claim 1 2 wherein the boranocarbonate= is Na, (H3;BCO,) .

14. Use according to any one of claims 1 to 13 wherein thme medicament further includes a guanylate cyclase stimulant cor stabilizer.

15. Use according to claim 1 4 wherein the guanylate cycla_se stimulant or stabilizer is a molecule or ion uncombined with the boranocarbonate compound or ion.

16. Use according to claim 1 4 or 15 wherein the guanylate= cyclase stimulant or gtabilizer is YC-1.

17. Use according to any one of claims 14 to 16 wherein t_he medicament is adapted for one of simultaneous and sequentiz=al administration of the boranocarbonate compound or ion and ®&he guanylate cyclase stimulant ox stabilizer..

18. Use according to any one of claims 1 to 17 wherein thue boranocarbonate compound or ion is other than

I. K, (H,BCOO)

II. _° R;N—B——¢C wl Sor where R, R' = H, alkyl, perfluorocalkyl.

19. Method of treatment of a mamma 1 comprising stimulation of neurotransmission, vasodilation or smooth muscle relaxation by CO as a Physiologically effective agent, or the treatment of any of hypertension, radiation damage, endotoxic shock, inflammation, inflammatory-related diseases, hyperoxia-induced injury, apoptosis, cancer, transplamt rejection, post- operative ileus, arteriosclerosis, peost-ischemic organ damage, myocardial infarction, angina, haemoesrrhagic shock, sepsis, penile erectile dysfunction, adult r-espiratory distress syndrome, vascular restenosis, hepat ic cirrhosis, cardiac hypertrophy, heart failure and ulcer ative colitis, or treatment in balloon angioplasty, ao rtic transplantation or survival of a transplanted organ, by administration of a boranocarbonate compound or ion adapted to make CO available for physiological effect.

20. Method according to claim 19 comprising stimulation of neurotransmission, vasodilation or semooth muscle relaxation by CO as a physiologically effective age=nt, or treatment of any of acute or chronic systemic hypertemision, radiation damage, endotoxic shock, hyperoxia-induced imjury, apoptosis, cancer, transplant rejection, post-operative ileus, arteriosclerosis, post-ischemic organ damage, angina, haemorrhagic shock, sepsis, penile erectile dysfunction, vascular restenosis, hepatic cirrhosis, cardiac hypertroplay, heart failure and ulcerative colitis, or treatment in balloon angioplasty, aortic transplantation or survival of a transplanted organ.

21. Method according to claim 19 cromprising stimulation of neurotransmission, vasodilation or smooth muscle relaxation by CO as a physiologically effective agent, or treatment of any of acute or chronic systemic hyperterasion, hyperoxia-induced injury, cancer by the pro-apoptotic effect of CO, transplant rejection, post-operative ileus, post=-ischemic organ damage, angina, haemorrhagic shock, penile er—ectile dysfunction, hepatic cirrhosis, cardiac hypertroplmy, heart failure and ulcerative colitis, or treatment in balloon angioplasty or aortic tranasplantation.

22. Method according to any one of clairms 19, 20 or 21 wherein including administration by an or-al, intravenous, subcutaneouss, nasal, inhalatory, intramus cular, intraperitorieal, transdermal, transmucosal or suppository route.

23. Method according to any one of claims 19 to 22 wherein the molecular structure of the boranocarboonate compound or jon includes the= moiety Fe 0]

24. Method according to claim 23 wherein the boranocarbonate compound or on includes the moiety BH;-CO—.

25. Method according to claim 23 or 24 wlhaerein the boranocarbonaate is a compound or anion of the formula: BH, (COQD),2Z, whereir:- X is 14 2 or 3 vy is 1,. 2 or 3 z is 0, 1 or 2 X+y + 2 =4, each Q is 0°, representing a carboxyl ate anionic form, or is (OH, OR, NH,, NHR, NR;, SR or haTlogen, where the or eactlm R is alkyl (preferably of 1 t—o 4 carbon atoms) , each Z is halogen, NH,, NHR', NR',, SERR' or OR' where the or each R' is alkyl (preferably of 1 to 4 carbon atoms) .

26. Method according to claim 25 wherein = is 0. :

27. Method according to claim 25 or 26 wh_ere y is 1.

28. Method according to claim 25 where x 4s 3.

29. Method according to any one of claims 25 to 28 where the boraanocarbonate is an anion, with at least one Q in the form of OO" or OR, and the composition includes a t least one metal cation.

30. Method according to claim 29 wherein ®-he or each metal cati on is an alkali metal cation or an alka line earth metal cati on.

31. Method according to claim 29 wherein t—he boranocarbonate is Na, {H;BCO,) .

32. Method according to any one of claims 19 to 31 wherein the wmedicament further includes a guanylate cyclase stimulant or s®&abilizer.

33. Method according to claim 32 wherein t—he guanylate cyclase stimulant or stabilizer is a molecule or ion uncorunbined with the boranocarbonate compounci or ion. 34, Method according to claim 32 or 33 whe-rein the guanylate cycl=ase stimulant or stabilizer is YC-1.

35. Method according to any one of claims 32 to 34 comprising simul taneous or sequential administration off the boraraiocarbonate compound or ion and the guaraylate cyclase stimulant or stabilizer,

36. Use according to any one of claims 19 —to 35 wherein the boraraocarbonate compound or ion is other than

I. Ka (H,BCOO)

IT. NG _° R;N—™—B——C ON, where R, R' = H, alkyl, perfluoroalk-—yl.

37. A method of treating a viable mammaliara organ extra corporeally or an isolated mammalian or-gan, compriging contacting t-he organ with a pharmaceutical composition comprising a. boranocarbonate compound or icen adapted to make CO available: for physiological effect.

38. A methc>d according to claim 37 whereim the boranocarbon .ate compound or ion is as defin ed in any one of claims 5 to 13.

39. Method according to 38 or 39 wherein t—he composition further incl—udes a guanylate cyclase stimulant or stabilizer.

40. Method according to claim 39 wherein the guanylate cyclase stimmulant or stabilizer is a molecule or icn uncombined w—ith the boranocarbonate compound or ion.

41. Method according to claim 39 or 40 whe=rein the guanylate cyclase stimualant or stabilizer is YC-1.

42. A medic:al or veterinary implant carryi ng, in a form releasable at= the implant site, a boranocarloonate compound or ion adapted t=o make CO available for physiological effect.

43. An impl ant according to claim 38 where in the boranocarbon=ate compound or ion is as defined in any one of claims 5 to M3.

44. An implaant according to 42 or 43 where _in the medicament further inclinades a guanylate cyclase stimul=ant or stabilizer.

45. An implant according to claim 44 wherezin the guanylate cyclase stimulant or stabilizer is a molecul.e or ion uncombined wi_th the boranocarbonate compounc or ion.

46. An impl=ant according to claim 44 or 45 wherein the guanylate cyczlase stimulant or stabilizer iss YC-1.

47. A methocd of introducing CO to a mammal as a therapeutic agent comprissing: a) ad ministering a boranocarbonate w-hich makes available CO suitable for physiological effe=ct; and b) ad ministering a guanylate cyclase stimulant or stabiliser.

48. A method according to claim 47, which ds for the stimulation o-f neurotransmission, vasodilati on or smooth muscle relaxa.tion by CO as a physiologically effective agent,

or for the treatment of any of hypertension, radiati_on damage, endotoxic shock, inflammation, inflammatory-related diseases, hyperoxia-indiaced injury, apoptosis, cancer, transpl.ant rejection, pPosst-operative ileus, arteriosclerosis, oost- ischemic orgarm damage, myocardial infarction, anginam., haemorrhagic s=hock, sepsis, penile erectile dysfunction, adult respiratory di stress syndrome, vascular restenosis, hepatic cirrhosis, cardiac hypertrophy, heart failure and ul cerative colitis or for- treatment in balloon angioplasty, aortic transplantatio-n or survival of a transplanted organ.

49. A method according to claim 47, which is for the stimulation of neurotransmission, vasodilation or sm_ooth muscle relaxat ion by CO as a physiologically effecti—ve agent, or for the tre atment of any of acute or chronic syst ematic hypertension, radiation damage, endotoxic shock, hyp-eroxia- induced injury , apoptosis, cancer, transplant reject ion, post- operative ileu s, arteriosclerosis, post-ischemic org<an damage, angina, haemorrhagic shock, sepsis, penile erectile dysfunction, v-ascular restenosis, hepatic cirrhosis, cardiac hypertrophy, h-eart failure and ulcerative colitis or for treatment in balloon angioplasty, aortic transplantation or survival of a -transplanted organ.

50. A method according to claim 47, which for the stimulation of neurotransm-oission, vasodilation or smooth muscle —relaxation by CO as a physsiologically effective agent, or for the treatment of amy of acute or chronic systematic hype-xtension, hyperoxia-indueced injury, cancer by the pro-apoptotic effect of CO, transplant rejection, post-operative ileus, pcoost- ischemic organ damage, angina, haemorrhagic shock, pe=nile erectile dysfumction, hepatic cirrhosis, cardiac hype=rtrophy, heart failure and ulcerative colitis or for treatmen®: in balloon angiopZlasty or aortic transplantation.

51. A method according to claim 47, which is for treatment of any of acute om chronic systemic hypertension, pulmornary hypertension, ®ransplant rejection, post-operative ileus,

WVV0 2005013691 PCT/GB24004/003365 arteriosclerosis, post-iscIemic organ damage, myocardi al infarction, penile erectiles dysfunction, vascular rest nosis, hepatic cirrhosis, cardiac hypertrophy, heart failure, chronic anal fissure, internal anal sphincter disease, anorectal disease, and ulcerative colitis or for treatment in balloon angioplasty or aortic transsplantation.

52. A method according to any one of claims 47 to 51 wherein the boranocarbonate compourad or ion is as defined in ary one of claims 5 to 13.

53. A method according to any one of claim 47 to 52 wherein the guanylate cyclase stimu. lant or stabilizer is a mole=cule or ion uncombined with the bor-anocarbonate compound or iomm.

54. A method according to any one of claims 47 to 53 wwherein the guanylate cyclase stimu lant or stabilizer is yYC-1.

55. A pharmaceutical composition comprising: a) a boranocarbonate= compound or ion which make s available CO suitable for piysiological effect; and b) a guanylate cycl=ase stimulant or stabiliser.

56. A composition accordimg to claim 55 wherein the boranocarbonate compound or ion is as defined in any onee of claims 5 to 13.

$7. A composition according to claim 55 or 56 wherein the guanylate cyclase stimulant or stabilizer is a molecule or ion uncombined with the boranocaarbonate compound or ion.

58. A composition accordineg to any one of claims 55 to 57 wherein the guanylate cyclasse stimulant or stabilizer iss vC-1.

59. A composition accordiney to any one of claims 55 to 58, adapted for one of simultarmeous and sequential administ—ration of the boranocarbonate compcound or ion and the guanylate cyclase stimulant or stabili. zer.