The present invention relates to the use of hydroxyguanidines in the prevention and treatment of a variety of diseases as well as to the protection of organs intended for transplantation. The present invention also relates to pharmaceutical compositions containing hydroxyguanidines intended for such use. Furthermore the invention relates to the manufacture of medicaments containing hydroxyguanidines useful for such prevention and treatment.
Xanthine dehydrogenase (EC 1.1.1.204) and xanthine oxidase (EC 1.1.3.22) are closely related molybdenum, iron-sulphur and flavin containing enzymes that catalyze the oxidation of purines such as hypoxanthine and xanthine leading to formation of, respectively, xanthine and uric acid (Pritsos and Gustafson 1994; Hille and Nishino, 1995). It is presumed that xanthine oxidase is formed from the dehydrogenase both in vivo and in vitro by reversible and irreversible postranslational processes. This conversion may occur reversibly by sulfhydryl oxidation, and irreversibly by proteolytic processes (Amaya et al., 1990; Nishino, 1994). The enzymes are composed of two identical subunits of about 1330 amino acids each, which amino acid sequences among mammals are highly conserved (Amaya et al., 1990; Wright et al., 1993; Ichida et al. 1993; Hille and Nishino, 1995). The oxidase and dehydrogenase forms of the enzyme exhibit different reactivities towards molecular oxygen and NAD+, the former oxidant is preferred by the oxidase and the latter by the dehydrogenase (Hille and Nishino, 1995). When oxygen is used as electron acceptor both superoxide radicals (i.e., superoxide anions) and hydrogen peroxide may be generated both of which are considered to be harmful and capable of causing tissue damage (Hille and Nishino, 1995). In particular the oxygen radical is considered to be harmful when present in a living organism, because of its reactivity and the fact that when a radical reacts with a non-radical matter of the organism a new potentially harmful radical is being produced (Kooij, 1994). The superoxide radical may react with H+ under formation of the perhydroxyl radical HO2. which is capable to react substantially faster than superoxide with tissue components (Kooji, 1994). Moreover, the perhydroxyl radical may form hydrogen peroxide which also may disintegrate to the extremely reactive hydroxyl radical .OH (Xia et al., 1996).
Both xanthine oxidase and xanthine dehydrogenase are capable of using oxygen as the oxidizing substrate whereby oxyradicals are formed. However, in the case of the dehydrogenase form NAD+ is the preferred substrate. In the presence of NAD+ the utilization of oxygen thus will be limited (Saugstad, 1996). Pathophysiological role of xanthine dehydrogenase/oxidase. Increased conversion of xanthine dehydrogenase to its oxidase form is implicated in pathological conditions of which diseases associated with hypoxia have attracted most attention. Conversion of the enzyme is assumed to contribute to tissue damage by generation of superoxide radicals produced from oxygen during the period of re-oxygenation of tissues after re-perfusion of an area previously deprived of blood flow (McCord, 1984; Nishino, 1994). According to this hypothesis the enzyme is converted to its oxidase form in conjunction with an ischemic period. Due to the inability of ATP-regeneration in the absence of adequate oxidative phosphorylation the cellular ATP pools will also become deprived and converted to hypoxanthine during ischemia. Upon eventual tissue reoxygenation (e.g. due to recovery of blood-circulation) xanthine oxidase will however start to utilize oxygen and then oxidize hypoxanthine to xanthine, and xanthine to uric acid, thus concomitantly generating toxic oxyradicals which may cause cell damage (Nishino, 1994; Saugstad, 1996). Hypoxia induced conversion of xanthine dehydrogenase to xanthine oxygenase has been shown experimentally in hepatocytes, Kupfer cells and endothelial cells (Wiezorek et al. 1994). The xanthine dehydrogenase/xanthine oxidase enzyme can be detected in a variety of tissues such as liver, kidney, heart, central nervous system, skeletal muscle, spleen, adrenal gland, intestine, skin, kidney, lung and placenta (Wajner and Harkness, 1989; Kooij, 1994). In particular interest has been focused on the fact that the enzyme may be present in endothelial cells (Moriwaki et al., 1993) and that damage to these cells may be involved in the pathology of reperfusion injuries by capillary leakage and formation of oedema. This sort of phenomenon could be involved in, for instance, induction of increased intracranial pressure in stroke and in high altitude sickness, and be the reason of the high mortality in these conditions.
Other abnormal conditions which have been associated with xanthine oxygenase induced oxyradical formation are complications seen in preterm infants such as periventricular leucomalacia (PVL), bronchopulmonary dysplasia (BPD), and retinopathy of prematurity (ROP) (Russell et al., 1995).
It has also been shown that increased conversion of xanthine dehydrogenase to xanthine oxidase can be induced in endothelial cells in vitro by activated neutrophils (Wakabayashi et. al. 1995) and experimental evidence indicates that xanthine dehydrogenase/xanthine oxidase may be an causative enzyme under influence of a variety of inducing factors such as TNF, interferon-gamma, IL-6, IL-1, and dexamethasone (Pfeffer et al., 1994). Moreover, the promoter region of the rat xanthine dehydrogenase/xanthine oxidase gene has been isolated; its sequence suggests several possible regulatory elements, including an NF-IL6 motif upstream of the transcriptional start site (Chow et al., 1994). Moreover, pre-treatment of mice and bacterial lipopolysaccharide or interferon-alpha increases xanthine oxidase activity (see Saugstad, 1996). The results quoted here thus indicate a role of xanthine dehydrogenase/xanthine oxygenase in inflammation.
In this context the term xe2x80x9ccausativexe2x80x9d and/or xe2x80x9cinducedxe2x80x9d and/or xe2x80x9cupregulatedxe2x80x9d is intended to mean that the content and/or activity of the xanthine oxidase/xanthine dehydrogenase enzyme becomes increased and/or is increased in a tissue compared to that of the normal level. A normal level is in this context intended to mean the level found in the corresponding tissue of a healthy individual.
Besides having the patophysiological roles described above it has been suggested that xanthine oxidase may be involved in a variety of other conditions such as xanthinuria, molybdenum-cofactor deficiency, gout, hyperuricemia, inflammation, airway obstruction, duodenal ulceration, arthritis, Parkinson""s disease, Alzheimer""s disease, paraquat intoxication, thermal skin injury, hyperthermia, pancreatitis, adult respiratory distress syndrome, nephrosis, adriamycin nephrosis, malaria, distant organ injury, cutaneous porphyrin photosensitation, inflammatory and autoimmune rheumatic diseases, rheumatoid arthritis, athereosclerosis, scleroderma and tumour promotion (see Kooji, 1994; Salim, 1994, Closa et al., 1994; Sakai et al., 1995; Misawa and Nakano, 1993; Misawa and Arai, 1993; Singh and Aggarwal, 1995; and references therein). Hepatotoxicity after viral infections (as well as after interferon treatment) was shown to be due to formation of xanthine oxidase and to oxyradical formation (see Saugstad, 1996).
Finally it should be mentioned that substantial amounts of xanthine oxidase may be released from the liver and intestine into the circulation during, e.g., hypoxia and/or shock, and that this circulating enzymatic activity may cause tissue damage at distant sites and organs of the body (Saugstad, 1996).
Known methods to prevent damage from xanthine dehydro-genase/xanthine oxidase derived free radicals. Due to the potential involvement of xanthine dehydrogenase/xanthine oxidase in the above mentioned conditions methods have been devised that are thought to interfere with xanthine dehydrogenase/xanthine oxidase or which may act on products(s) formed due to the action of these enzymes. One such method is to use an inhibitor of adenosine deaminase to limit tissue damage during reperfusion after a hypoxic episode (Xia et al., 1996). Thereby it is intended to prevent adenosine accumulated by the breakdown of cellular ATP during anoxia to form inosine which subsequently might be transformed to hypoxanthine, the very substrate for xanthine oxidase, thereby promoting the formation of superoxide radicals. Along this line the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) was tested experimentally in rabbits; results indicated that free radical formation was reduced and the contractile force increase in the hearts of EHNA treated animals compared to those of controls (Xia et al., 1996). One drawback with this approach is however that the adenosine deaminase inhibitor has little effect on the hypoxanthine and/or xanthine already formed during a hypoxic episode. EHNA is therefore expected to be effective only when applied prior and/or during a hypoxic period which in the clinical situation may often not be a realistic approach.
Another attempt was to use an inhibitor of xanthine oxidase to prevent breakdown of hypoxanthine/xanthine and, thereby, formation of superoxide. Xanthine oxidase inhibitors allopurinol and oxypurinol were applied experimentally by Das et al. (1987) in isolated pig hearts. The data generated by Das and co-workers did though not strongly support attenuation of the generation of free radicals by these compounds during reperfusion of the heart after a hypoxic period. In later studies it was however shown that oxypurinol reduced radical production during experimentally induced ischemia/reperfusion injury in the rat cerebral cortex (Phillis and Sen, 1993). In another attempt rats were pretreated with the xanthine oxidase inhibitor amflutizole prior to application of cerebral ischemia; this was found to strongly reduce the release of free radicals during brain reperfusion (Phillis et al., 1994). In vivo studies in experimental animals suggested that oxypurinol may afford protection against ischemic brain injury (Lin and Phillis, 1992). Recent clinical studies also suggest that allopurinol is useful in cardiac surgery when by-pass grafting is performed; it both improves postoperative recovery and reduces lipid perodixation (Coghlan et al., 1994; Castelli et al., 1995).
Free radicals have been implicated to have a role in duodenal ulceration. It was thus shown by Salim (1994) that cimetidine in combination with allopurinol, or cimetidine in combination with the radical scavenger DMSO (dimethylsulphoxide) gave remarkably better healing and reduced relapse rate in patients with refractory duodenal ulceration than did cimetidine alone.
In this context it should be stressed that there may be limitations to the use of blockers that prevent formation of uric acid from hypoxanthine and xanthine effected by administration of drugs such as EHNA, allopurinol, oxypurinol or amflutizole. This is because uric acid is one of the most important endogenous antioxidants and it may have great physiological importance by providing a tissue protective effect (see Saugstadt, 1996).
Another approach comprises the administration of a spin trap agent capable of reacting with free radicals to form a more stable species, such as N-tert-butyl-alpha-phenylnitrone (PBN) which reduced ischemic brain damage in experimental animals (Phillis and Clough-Helfman, 1990; Clough-Helfman and Phillis, 1991). The approach does not seem to have been widely applied and further evaluated. This method does not impede the formation of radicals but only traps them upon their formation, which is a clear disadvantage.
The administration of superoxide dismutase, superoxide dismutase derivatives or superoxide dismutase mimetics has also been attempted. (Closa et al., 1993; Hardy et al., 1994; Radak et al., 1995). However, superoxide dismutase may also be disadvantageous since it is known to increase ischemic re-perfusion injuries at high doses, an effect attributed to the capability of superoxide dismutase to enhance production of the highly toxic hydroxyl radical in the presence of Fe2+ (Mao et al., 1993). Moreover, administration of a protein macromolecule such as superoxide dismutase to a human may involve many complications of pharmaceutical, pharmacokinetical, toxicological and immunological nature.
Other agents well known in the art to be useful as free radical trapping or destroying agents are DCF (2xe2x80x2-deoxycoformycin), catalase, vitamin E (alpha-tocopherol), vitamin C (ascorbate; ascorbic acid), glutathione, uric acid, N-acetyl-cysteine (NAC), dimethylthiourea (DMU) and betacarotens.
Hydroxyurea, hydroxyguanidine and derivatives of hydroxyguanidine. Hydroxyurea is known to possess anticarcinogenic effect (Goodman and Gilman, 1970). The mechanism of action for that effect has been suggested to be due to the inhibition of DNA synthesis by inhibition of the enzymatic conversion of ribonucleotides to deoxyribonucleotides (Goodman and Gilman, 1970). In 1972 Adamson reported that hydroxyguanidine (N-hydroxyguanidine) also possesses antitumour activity. A number of hydroxyguanidine analogs were synthesised by Bailey et al. (1973) and found to have antihypertensive effect.
A pharmaceutical preparation of the hydroxyguanidine guanoxabenz is claimed to be particularly suitable for the treatment of diarrhoea and scours (EP 0 112 061 A2). More recently several other derivatives of N-hydroxyguanidine have been synthesised, some of which were reported to exhibit antiviral and/or antineoplastic activity (Tai et al., 1984; T""ang et al., 1985; Wang et al., 1990; Doubell and Oliver,1992; Koneru et al. 1993; Hui et al., 1994). These antiviral and antineoplastic effects have been associated with a possible inhibitory effect on ribonucleotide reductase (Weckbecker et al. 1987, 1988).
The metabolism of hydroxyguanidine derivatives is essentially unknown. Hydroxyguanidine itself was reported be metabolised to guanidine when injected intraperitonally into rats (Waler and Walker 1959). The mechanism underlying this metabolism is unknown.
It is an object of the invention to provide a treatment of conditions, including preventive treatment, related to the radical generating nature of the xanthine dehydrogenase/xanthine oxidase.
It is another object of the invention to provide means and methods to prevent or reduce the formation of oxygen radicals in the human body.
It is a further object of the invention to provide means for treatment of xanthine dehydrogenase/xanthine oxidase mediated disease. A xe2x80x9cxanthine dehydrogenase/xanthine oxidase mediated diseasexe2x80x9d is herein defined as a condition caused by the generation and/or accumulation of oxygen derived free radicals under catalysis of the xanthine dehydrogenase/xanthine oxidase enzyme. Specific examples of xanthine dehydrogenase/xanthine oxidase mediated disease are tissue damage, organ damage and inflammation caused by oxygen derived free radicals, the radicals being formed under catalysis by the xanthine dehydrogenase/xanthine oxidase enzyme. Throughout this specification the term xe2x80x9cxanthine oxidase/xanthine dehydrogenasexe2x80x9d has the same meaning as the term xe2x80x9cxanthine dehydrogenase/xanthine oxidasexe2x80x9d.
It is a still further object of the intention to provide means for treatment of xanthine oxidase/anthine dehydrogenase mediated ischemic disease or condition. By xe2x80x9cxanthine oxidase/xanthine dehydrogenase mediated ischemic disease or condition is intended a xanthine oxidase/xanthine dehydrogenase mediate disease or condition which is associated with ischemia of the body, an organ and/or tissue which is optionally followed by a period of reoxygenation.
Further objects of the invention are apparent from the following description of the invention including the appended claims.