GB2191693A - Anti-inflammatory agents - Google Patents

Abstract

Use of known histamine H2-receptor antagonists as anti-inflammatory agents by virtue of their ability to act as 5-lipoxygenase pathway inhibitors which leads to an inhibition of the production of leukotrienes. The histamine H2-receptor antagonists include cimetidine and ranitidine which can be formulated for both internal and topical administration, e.g. in suppository form.

Description

SPECIFICATION Anti-inflammatory agents This invention relates to the use of a class of known histamine H2-receptor antagonists in the treatment of inflammatory diseases, and, in particular, to the use of said histamine H2-receptor antagonists as lipoxygenase pathway inhibitors and as a consequence, general purpose anti-inflammatory agents.

Histamine is comprised of an imidazole ring with a side chain of two carbon atoms and a terminal amino group. At physiological pH it exists mainly in the positively charged ammonium form. The development of histamine H2-receptor antagonists was based on chemical alteration of histamine to provide analogues which had the capacity to inhibit gastric acid secretion in rats.Histamine and a number of histamine H2-receptor antagonists are represented by the following formula:

Compound R1 R2 Histamine H -(CH3) NH+3 Metiamide CH3 -CH2SCH2CH2NHCSN CH3 Burimamide H -(CH2)4 NH=C=SNHCH3 Cimetidine CH3 -CH2SCH2CH2NH=CNCNNHCH3 The ubiquitous nature of histamine and its role in many pathophysiological processes have prompted a variety of experimental and clinical studies which sought to explore the therapeutic potential of the histamine H2-receptor antagonists. In 1972 Black and his colleagues reported the synthesis and antagonist activity of N-methyl-N'-(4-(5)-imidazol-4-yl)butyl)-thiourea, or burimamide (Black, J.W. etal Nature, (1972) 236, 385). Burimamide was found not to be sufficiently potent for oral use, and substitution on the imidazole ring led to the synthesis of N-methyl-N'-(2-(5-methylimidazol-4-yl)methylthio)ethylthiourea or metiamide (Black, J. W. eft at Nature (Lond.) (1974)248,65).

Burimamide and metiamide appear to be well absorbed. Using radiolabelled material, oral, intraperitoneal or intravenous drug administration showed 7090% of the radioactivity in the urine within 24 hours. Recoveries after oral dosing were similar to parenteral administration. However, using orally dosed rats which were anaesthetized within 5 minutes, burimamide showed little absorption from the stomach after 2 hours. In experiments with pylorus-ligated cats, metiamide also did not appear two be absorbed from the stomach. Experiments in man have also shown no absorption from the stomach. It thus appears that neither burimamide or metiamide are absorbed from the acidic stomach.This is consistent with pKa's of the compounds around 7.0 in a medium of approximately pH 2.0, where the compounds would be almost entirely in the cationicform (Hessebo. T. In: International Symposium on histamine H2receptor antagonists, (1973), P. 29).

The minor adverse effects found with toxicity tests on metiamide were attributed to the thiourea group.

Modification led to the synthesis of N"-cyano-N-methyl-N'-[2-[[(5-methyl-1 H-imidazol-4 yl)methyl]thio]ethyl]guanidine, or cimetidine (Brimblecombe, R. W. etalj. Int. Med. Res. (1975)3,86.

The introduction of cimetidine has radically altered the management of duodenal and prepyloric ulcers.

The therapeutic success appears to be based firmly on the specificity and effectiveness of the histamine H2receptor antagonists inhibiting gastric acid secretion. Dosages of 1 g daily for 4 to 6 weeks produces healing in at least 70% of ulcers, with concomitant rapid relief of symptoms.

Bardhan (Bardhan, K. D. ln Cimetidine -The Westminster Hospital Symposium, Edinburgh and London, (1978) P. 31) summarized the results of six open trials of cimetidine in duodenal ulcer disease, a double-blind multicentre trial, and nineteen short term trials. Most of the trials confirmed the value of cimetidine treatment compared to placebo, although the difference was not significant in every case.

In a double blind study (Bodemar, G. and Walan, Al. Lancet(1976) No. 11, 161) 90% of or patients on cimetidine healed after six weeks at either 800 mg or 1200 mg daily dose. This compared with 36% healing rate in patients on placebo. Similar results were found using 1 g/day (Gray, G. R. eft at Lancet, (1977) No. I, 4). In a multicentretrial on 164 patients (Albano, O. etailtal. J. Gastroenterol (1978) 10, 247) 72% of patients on 1 g/day healed after 4 weeks compared to 34% on placebo. Other trials showing similar results have been reported (Domschke, W. etalActa Hepatogastrenterol. (1976)23,441; Moshal, M. G. petal S. Afr. Med.

J. (1977)52,760; Hetzel, D. J. eta/Gastroenterology, (1978) 74,389).

The treatment of gastric ulcers with cimetidine has also shown considerable success with symptomatic relief and healing rates significantly greater than placebo. A multicentre trial carried out by Bader and his colleagues (Bader, J. P. etalln: Proceedings of the 2nd International Symposium on Histamine H2-receptor antagonists. (1976) Excerpta Medica P. 287) showed a healing rate of 69% using 1 g/day for 28 days compared to 37% on placebo. In addition, cimetidine treatment causes more rapid and greater relief of pain.

Studies by Frost etal (Frost, F. petal. Brit. Med. J. (1977)2,795) showed a healing rate of 78% in patients on 1 glday for 6 weeks compared to 27% on placebo. Other trials showed similar success (Landecker, K. D. etal Med. J. Austr. (1979)2,43; Garbol, A. J. eft at Vgeskr. Laegar (1979)141,3301; Machado, L. P. eft at Gen.

(1978)32,461.

It has been observed that ulcer patients with psoriasis, a chronic inflammatory skin disease, showed clinical improvement after treatment with cimetidine at 1 g/day (Giacosa, A. etna!; Lancet (1978) ii, 1211; Wallack, D. etal Nouvelle Presse Medicale, (1979) 8,2981; Raffle, E. J. Lancet (1978) ii, 1314). The therapeutic implications of histamine H2-receptor antagonists in skin diseases was discussed recently (Davies, M. G. and Greaves, M. W. Brit. J. Dermatol. (1981) 104, 601. Other studies have demonstrated a reversal of anergy in Crohn's disease (Bicks, R. 0. and Rosenberg, E. Q. Lancet (1980) i, 552) indicating that cimetidine can enhance T cell immune competence in immunologically-comprised Crohn's disease.

Cimetidine has also been found to help alleviate joint pain in arthritic conditions (Sundar, S. Med. J. Aust.

(1978)1 98; Permin, H. etalAllergy(l981) 36,435). In addition, studies on rats showed that cimetidine reduced both the acute and chronic inflammatory response following administration of Freunds adjuvant to an extent comparable with the effect produced by indomethacin (Al-Haboubi and Zeitlin, Br. J. Pharmacol.

(1979) 67,446). Since cimetidine has been known primarily as an histamine H2-receptor antagonist, the antiinflammatory nature of the drugs which is proposed hereunder was not known, and expianation when offered on the action of cimetidine in psoriasis or arthritic conditions, was based on histamine H2-receptor antagonist activity.

Work by Taylor (In: International Symposium on histamine H2-receptor antagonists (1973) P. 43) showed that 4060% of administered metiamide is excreted unchanged in rats and dogs, with its sulphoxide as the major metabolite. As with metiamide, cimetidine is mainly excreted unchanged in urine, with the sulphoxide as principal metabolite (Brimblecombe, R. W. etaij. Int. Med. Res. (1975)3,86).

Metiamide was shown to be ten times more active than burimamide as a histamine H2-receptor antagonist, but although it was found to be a potent inhibitor of stimulated acid secretion in man, it was found to be associated with reversible granulocytopaenia in a few cases. It was believed that this was due to the thiourea group on the molecule, and this led to the development of cimetidine as a non-toxic alternative.

Cimetidine acts as a competitive antagonist of histamine-stimulated gastric acid secretion in animals and man, and its safety and efficacy have been established in treatment of acute peptic ulcers. It has been shown to relieve symptoms and promote healing in both gastric and duodenal ulcers. For many years the dictum "no acid - no ulcer" has dominated our understanding and management of peptic ulcer disease.

However, not all patients have enhanced acid secretion. It has been shown that only 30%40% of duodenal ulcer patients have increased secretion of gastric acid, and patients with gastric ulcers have either normal or reduced secretion. It is now believed that patients with gastric ulcers have decreased mucosal resistance and peptic ulcers may result from imbalance between acid/pepsin secretion and mucosal resistance.

Recently, the term "cytoprotection" has been introduced to encompass all the physiological processes which protect the gastric mucosa from acid/pepsin digestion. The cytoprotection mechanism comprises the following (1) a mucus layer, which maintains the pH gradient between the lumen and epithelial cell surface, and prevents back-diffusion of both hydrogen ions and pepsin; (2) bicarbonate secretion by the epithelial cells which maintains the pH of its surface at neutrality; (3) cellular regeneration, which provides a continual repair process for maintenance of the integrity of the mucosa; and (4) an adequate mucosal blood flow, which protects the mucosal cells by removal of locally accumulated acid. The cytoprotective processes are dependent at least partially on the production of prostaglandins, which play an important role in maintenance of mucosal integrity.Prostaglandin E2 (PgE2) can protect gastric mucosa by regulating mucosal blood flow (Gerber, J. G. and Nies, A. S. Dig. Dis. Sci. (1982)27,870), stimulate mucus production (Johansson, C. and Kollberg, B. Eur. J. Clin. Invest. (1979)9,229; Bolton, J. P. etalSurg. Forum (1976) 27, 402) and has an inhibitory effect on gastric acid production (Roberts, A. etaiGastroenterology (1976) 70, 359.

It is well known that cyclooxygenase inhibitors are detrimental to the mucosal cells and can lead to local inflammatory changes in the stomach and possible ulceration. It is not well understood why certain histamine H2-receptor antagonists e.g. cimetidine, can heal peptic ulcers, particularly when gastric acid secretion is not raised in many cases.

Leukotrienes are potent mediators of inflammation (Smith, M. J. H. etaij. Pharm. Pharmacol. (1980) 32,517). Leukotriene B4 (LTB4) is a potent chemotactic and chemokinetic agent for leukocytes (Ford Hutchinson A. W.,etaiNature(1980)286,264; Goetzl, E. J.andPickett,W. C. J. Immunol. (1980)125,1789).

It causes in vivo leukocyte accumulation (Bray, M. A. et al Brit. J. Pharmacol. (1981)22,483), modulates pain responses (Rackham, A. and Ford-Hutchinson, A. W. Prostaglandins (1983)25, 193) and causes changes in vascular permeability (Bray, M. A., et al, supra (1981); Wedmore, C. V. and Williams, T. J. Nature (1980)284, 646). The peptido-lipid conjugates (leukotriene C4, D4 and E4) collectively account for the biological activity known as "slow reacting substance of anaphylaxis" (Samuelsson, B. Science (1983) 568). They are potent smooth muscle contractile agents, and are therefore believed to be important mediators of asthma and other hypersensitivity reactions.The inhibition of the 5-lipoxygenase pathway is believed to play a central role in anti-inflammatory actions (Randall, R. Wet al Agents Actions (1980) 10, 553; Myers, R. F. and Seigel, M. I. Biochem. Biophys. Res. Commun. (1983)112, 586), and the development of specific antileukotriene agents are expected to play a major role in the search for potent anti-inflammatory drugs.

The cause of inflammatory diseases and peptic ulcer disease is unknown and there are no known cures at this time. The pathogenesis of these conditions is usually genetically related and precipitated by environmental factors. Treatment is normally by use of steroidal and non-steroidal anti-inflammatory drugs (NSAID's). Both classes of drug act on arachidonic acid metabolism by inhibition of the pathways leading to the formation of prostaglandins and leukotrienes. Most of these drugs have some attendant side effects with prolonged use and recently some NSAID's have been contraindicated-in certain inflammatory diseases (Rampton, D. S. and Hawkey, C. J. Gut (1984) 25,1399).

U.S. Patent Specification No. 3,950,333 and 4,128,658 respectively describe cimetidine and ranitidine as potent histamine H2-receptor antagonists.

It is an object of the present invention to demonstrate the anti-inflammatory nature of histamine H2receptor antagonists and propose a second medical use for these drugs as general purpose antiinflammatory compounds by virtue of the fact that they act as 5-lipoxygenase pathway inhibitors, i.e. inhibit the production of leukotrienes. It is a further object of this invention to provide a non-toxic alternative for treating inflammatory diseases.

The invention provides the use of at least one histamine H2-receptor antagonist for the preparation of a medicament for use as a 5-lipoxygenase pathway inhibitor and hence as an inhibitor of leukotriene production. Consequently, the invention provides the use of at least one histamine H2-receptor antagonist for the preparation of a medicamentfor use as a general purpose antiinflammatory agent.

The present invention also provides the use of at least one histamine H2-receptor antagonist for the preparation of a medicament for use as a stimulant of prostaglandin production.

Particularly suitable for general purpose anti-inflammatory agents according to the invention are N"cyano-N-methyl-N'-[2-[[(5-methyl-1 H-imidazol-4-yl]methyl]thio]ethyl]guanidine (cimetidine) and N-[2-(((5 [dimethylamino)methyli-2-furanyl)methylthio)ethyI)-N'-methyl-2-nitrn-1 -ethene-diami (ranitidine).

For the treatment of inflammatory skin diseases particularly preferred H2-receptor antagonists include N-methyl-N '-(4-(5)-imidazol-4-yl)butyl )-thiourea (burimamide), cimetidine, N-methyl-N'-(2-(5-methylimidazol-4-yl)methyl thio)ethylthiourea (metiamide) and ranitidine.

During the course of use in the present studies of the compositions of U.S. Patent Specification No.

3,950,333 and 4,128,658 supra for the treatment of duodenal ulcers, both compounds were observed to have anti-leukotiene properties i.e. they act as 5-lipoxygenase inhibitors as hereinafter described.

The general purpose 5-lipoxygenase pathway inhibitors for use in the present invention can be used in the treatment of a variety of inflammatory diseases. The compounds are suitable for internal administration. By internal administration is meant generally oral, enteral and parenteral administration.

Pharmaceutical compositions for internal administration according to the invention will comprise at least one histamine H2-receptor antagonist optionally in association with a pharmaceutically acceptable diluent or excipient therefor.

The histamine H2-receptor antagonists may also be formulated for topical application as indicated above.

According to the invention the aforementioned histamine H2-receptor antagonists can be used in the treatment of chronic inflammatory diseases, including rheumatoid arthritis and other inflammatory joint diseases, inflammatory skin disease (psoriasis, eczema, and dermatitis herpetiformis) chronic inflammatory bowel disease (Crohn's disease and ulcerative colitis) inflammatory liver disease (chronic active hepatitis and alcoholic hepatitis) and sarcoidosis of the lung, in addition to peptic ulcer disease.

Pharmaceutical compositions containing the histamine H2-receptor antagonists for use as a 5lipoxygenase pathway inhibitor according to the invention are preferably present in the composition in the range of 0.05 to 5% by weight.

The pharmaceutical compositions may be in a variety of forms. Forms suitable for oral administration include tablets, capsules (e.g. hard or soft gelatin capsules), syrups and liquids. As stated above, the compositions may also be formulated for enteral or parenteral administration and suitable forms for such administration include injectable solutions and perfusion solutions. Other forms of pharmaceutical compositions include rectal suppositories and vaginal pessaries. A particularly suitable formulation for the treatment of inflammatory bowel disease is an enema.

The invention will be further illustrated by the following examples: EXAMPLE 1 Tablets having the following composition are prepared: Cimetidine 200 mg Lactose 50 mg Avicel (Avicel is a Trade Mark) 40 mg Magnesium stearate 6 mg by mixing the active ingredient with the other constituents and compressing the product to form the tablets.

EXAMPLE 2 Capsules are prepared having the following composition: Cimetidine 200 mg Lactose 90 mg Magnesium stearate 7 mg by intimately mixing the above ingredients and pouring the mixture into hard gelatin capsules.

EXAMPLE 4 Suppositories are prepared, having the following composition: Cimetidine 200 mg Lactose 300 mg Witespol (Trade Mark) W45 q.s.p. 1.5 g The active ingredient is mixed with the lactose and then uniformly suspended in the Witespol W45 (suppository base) and heated to form a molten mass in conventional manner. The suspension is poured into cooled moulds to form suppositories weighing 1.5 g.

EXAMPLE 5 A vanishing cream is made up of the following: Oil Phase % w/w Liquid paraffin 30 Span 65 (Span is a Trade Mark) 1 Cetostearyl alcohol 5 EXAMPLES Aqueous Phase % wtw Carbopol 934 Gel 36 Chlorhexidine gluconate 1 Tween 85 (Tween is a Trade Mark) 2 Glycerol 20 Cimetidine 5 A 1% Carbopol 934 Gel is prepared by dispersing 2 g of Carbopol in 150 g water. The pH is adjusted to 7 with 1 N NaOH. The gel is then brought to 200 g with water. It is heated to 700C with the chlorhexidine gluconate, Tween 85 and glycerol. The cimetidine is then added. The oil phase is similarly heated and added to the water phase in a mortar and sheared for at least 5 minutes.

Synthesis of histamine H2-receptor antagonists 1) Burimamide 4(5)-(4-aminobutyl)imidazole is treated with methyl isothiocyanate in boiling ethanol. The product is purified by crystallization from acetonitrile or water and meits at 124-130". (Du rant G. J. eft at British Patent No. 1 307 539 (1973).

2) Metiamide 4-hydroxymethyl-5-methylimidazole is condensed with cysteamine in the presence of HCI or HBr. The resulting amine salt is collected and neutralized with aqueous potassium carbonate, and the liberated amine is treated directly with methyl isothiocyanate to give metiamide. Purification is effected by crystallization from water and the crystals have a melting point of 150152 (Durant G. J. et alBritish Patent No.1 338169(1973).

3) Cimetidine S,N-dimethyl-N'-cyano-isothiourea is treated with 4-methyl-5-imidazole-methylthio-ethylamine.

Purification is effected by crystallization from acetonitrile to give crystals with a melting point of 141142 (Durant, G. J. eft at British Patent No. 1 397436(1975).

4) Ranitidine Can be prepared according to the process described in Irish Patent No. 45456.

Pharmacological data for cimetidine The kinetics and metabolism of cimetidine are similar in rats, dogs and man so that preclinical toxicological and pharmacological studies may be considered to be directly relevant to man (Griffiths, Ret a/, In: Cimetidine, Exerpta Medica (1977) P. 38).

After administration of '4C-cimetidine, a 24-hour urinary recovery of up to 95% of the dose was found with up to 70% excreted as unchanged cimetidine, with the sulphoxide as main metabolite (Griffiths, R. et a!. supra). It is rapidly absorbed after oral administration with peak blood concentrations of 0.4 to 2.25 ,ug/ml in fasting subjects 1 to 2 hours after a single 200 mg dose. Following administration of 400 mg cimetidine, a peak serum concentration of 0.61 to 2.5 ,ug/ml was seen during same period. Administration of cimetidine with meals delays the peak serum concentration time, but does not significantly alter the proportion of the dose absorbed.The blood serum half-life was found to be 2 hours, and it was then rapidly excreted via the kidneys (Burland, W. L. et al Br. J. Pharmacol. (1975)2,481). A review of the pharmacologicai properties and clinical efficacy of cimetidine has been published (Finklestein, W. and Issel-Bacher, K. J. N. Engl. J. Med.

(1978)299,992).

The pharmacokinetics of burimamide and metiamide in rats and dogs demonstrated rapid absorption and elimination of both drugs in the unchanged form in amounts up to 60% of the administered dose, while fecal excretion (probably biliary metabolites) accounted for a further 10%. Dermal penetration was studied by Sutton (Sutton, T. J. Toxicol. Appl. Pharmacol. (1979) 50,459). Results from primary irritation and sensitization tests in rabbits and guinea pigs respectively showed that burimamide, metiamide and cimetidine penetrated the intact skin of the experimental animals only to a slight extent. In vitro studies with human skin showed similar slow absorption of each drug.

Toxicology of cimetidine Cimetidine has been demonstrated to have low acute toxicity (Leslie, G. B. and Walker, T. K. (In: Proceedings of 2nd International Symposium on histamine H2-receptorantagonists, (1976). Exerpta Medica (1977) P. 38). Acute toxicity is shown in table 1.

TABLE 1 Cimetidine-Acute LD60 Values Species Route LD50 (mglkg) Mouse i.v. 150 Mouse i.p. 470 Mouse p.o. 2600 Rat i.v. 106 Rat i.p. 650 Rat p.o. 5000 Hamster i.p. 880 Hamster p.o. 4000 Dog p.o. Ca2600 Repeated oral dosing up to 12 months in rats showed very few adverse effects, using doses up to 950 mg/kg. No significant difference between groups dosed with 150,378 and 950 mg/kg were observed in body weight, food consumption, blood chemistry, urinalysis or ophthalmoscopy. In males, there was a reduction in size of prostate glands in all groups and a reduction in size of testes and seminal vescicles in the rats at 950 mg/kg.

Repeated oral dosing up to 12 months studies was carried out on dogs. Doses ranged from 37 to 336 mg/kg. Prostate gland weights were reduced, but no other dose-related abnormalities were observed.

Haematology, blood chemistry, urinalysis, opthalmology, electrocardiography and histopathology (including gut) were normal.

Repeated intravenous dose studies in rats were carried out at 75 mg/kg for 14 days. Raised serum cholesterol levels were observed in the top dose group but all other parameters were within normal limits.

Intravenous infusion studies up to 126 mg/kg for 10 days causes no drug-related clinical effects.

Repeated intravenous infusion studies in dogs up to 84 mg/kg caused vomiting in 2 of 6 dogs on top doses. Weight loss also occurred in top dose dogs, but all other clinical parameters were normal.

Moderate tachycardia occurred in intravenous dose studies up to 41 mg/kg for 14 days, in the top dose dogs. Slight tachycardia occurred at lower doses but no other abnormalities were observed.

Teratological studies were carried out in rats, rabbits and mice at oral doses up to 950 mg/kg. Rats showed a few more embryos lost between ovulation and implantation compared to controls, but eventual litter size did not differ between dosed groups and controls.

Afew unexplained deaths occurred in higher dosed rabbit groups and embryo losses were higher after implantation. However, there were no adverse effect on foetal development.

No adverse effects were shown in mice studies, but high dosed dams grew less quickly than controls.

Fertility tests in male and female rats using daily doses up to 950 mg/kg for at least 70 days showed no dose related effects on mating performance and fertility in males or females and all offspring were normal.

The effect of cimetidine in retarding the development of both prostate glands and seminal vessicles is reversible. Cimetidine has been shown to have weak anti-androgenic activity (Eviatar, A. et al Arch. In.

Pharmacodyn. Ther. (1961)133,75) and could have caused the observed changes.

Interaction studies Acute interaction studies was carried out with cimetidine and a number of drugs. In rats studies using acute i.v. toxicity there was no significant interactions between cimetidine and atropine, trifluoperazine, chlordiazepoxide, propantheline, isopropamide, phenobarbitone sodium, diazepam, meprobamate, pethidine, succinylcholine, d-tubocurarine, thiopentone sodium, noradrenalin, adrenalin, lidnocane, propanolol, hydralazine, a-methyldopa, digoxin, hydrocortisone, frusemide, hydroxyzine, ampicillin, cephalotin, warfarin or heparin.

Mechanism of action The mechanism of action of histamine H2-receptor antagonists acting as general purpose antiinflammatory drugs according to the invention has been elucidated using cimetidine and ranitidine as preferred active ingredients, and duodenal ulcer disease as model for peptic ulcer disease. The biochemical effects of ranitidine were found to be similar to those of cimetidine on both PgE2 and LTB4 secretion. The two groups were not split and therefore the data presented hereunder is compiled from studies using both drugs, since cimetidine and ranitidine showed identical trends.

Twenty patients with endoscopically proven duodenal ulcer disease were selected for this study. All patients were either newly diagnosed or had chronic recurrent duodenal ulcers, and were off treatment for at least 2 months before commencement of this study. Fifteen patients were given cimetidine (1 glday) and 5 were given ranitidine (300 mg/day) for 4 weeks. Endoscopy was then repeated in all patients. It was demonstrated in all patients in this study that the ulcer was healed after 4 weeks therapy. Control subjects were chosen without history of duodenal ulcers or other gastrointestinal disease.

Blood samples were taken from all patients before and after treatment, and a single blood sample was obtained from the control subjects and processed alongside the patients' blood. Twenty ml of peripheral blood was drawn after overnight fast into sodium heparin vacutainers. Samples were also taken into vacutainers without anticoagulant for isolation of serum. Leucocyte-rich plasma obtained by dextran sedimentation was further fractionated using hypaque-ficoll gradient centrifugation. The peripheral blood mononuclear cells were harvested from the interface. Viability and purity were assessed by fluorescence microscopy, using ethidium bromide and acridine orange staining. Dual esterase staining was employed to determine the percentage of monocytes in each peripheral blood mononuclear cell fractions.Using these techniques it was found that there were no contaminating neutrophils in the isolated peripheral blood mononuclear cell fraction. In addition, freshly isolated cells were shown to have a viability of > 98%.

Opsonization of zymosan Awas carried out by incubating 1 ml samples (10 mg/ml) with 1 ml autologous serum for 30 min at 370C. The samples were centrifuged and the opsonized zymosan was resuspended in 5 ml phosphate buffered saline giving a final concentration of 2 mg/ml opsonized zymosan A. This method provided complement (C3b)-coated yeast cell-coat particles which acted as inflammatory stimulus to the monocytes.

Two samples of each peripheral blood mononuclear cell fraction (0.5 mi) were stimulated with 0.5 ml opsonized zymosan for 30 min (1) in the absence and (2) in the presence of 10 nmoles of arachidonic acid. In the absence of added arachidonic acid, the enzyme pathways are activated as phospholipase A2 (Scheme A). The phospholipase A2 releases membrane-bound arachidonic acid and the free acid then proceeds into the cyclooxygenase pathway to produce prostaglandins, and lipoxygenase pathway to produce leukotrienes. In the presence of added arachidonic acid, the requirement for endogenous substrate is bypassed and the added free arachidonic acid is metabolised in the cyclooxygenase and lipoxygenase pathways.

Scheme A Metabolism of arachidonic acid

MEMBRANE BOUND ARACHIDONIC ACID PHOSPHOLIPASE A2 FREE ARACHIDONIC ACID LIPOXYGENASE CYCL60XYGENASE LIPOXYGENASE LEUKOTRIENES PROSTAGLANDINS Following incubation, the cells and zymosan were removed by centrifugation and the supernatants assayed for secreted PgE2 and LTB4. Since lymphocytes do not produce any appreciable amounts of PgE2 or LTB4, secretion was assumed to be due entirely to the monocytes in each peripheral blood mononuclear cell fraction. Radioimmunoassay was employed to assess the secreted PgE2 and LTB4, and secretion was corrected for esterase-positive monocytes, and expressed as nag!1 06 monocytes.The data are presented as mean +SEM and Students "t" for unpaired data was used to analyse the results. "Significance" is indicated by p values < 0.05.

Determination of PgE2 and LTB4 production Locally produced prostaglandins are believed to play a role in the treatment and prevention of duodenal ulcer disease. Prostaglandin biosynthesis and secretion by gastric mucosal cells have been extensively studied particularly because PgE2 has important cytoprotective properties in the gastric mucosa.

Table 2 shows the optimal PgE2 secretion by peripheral blood monocytes from control subjects, and patients with duodenal ulcer disease before and 4 weeks after treatment with cimetidine or ranitidine. A significant reduction of PgE2 secretion by untreated patients was found compared to control subjects in the absence of added arachronic acid (p < 0.001). There was a small but non-significant increase in PgE2 secretion by the patients after 4 weeks treatment. This data would suggest that there is a block in either the phospholipase A2 step or cyclooxygenase pathway in the patient's monocytes.

TABLE 2 Optimal PgE2 secretion by monocytes from control subjects, and patients with duodenal ulcers (before and 4weeks after treatment), stimulated with opsonized zymosan.

PgE2 Secretion (n) (ng/106 monocytes) Control Subjects (20) 34.8 + 5.25 Untreated Patients (20) 18.1 + 1.70 p < 0.001 Treated Patients (20) 19.6 * 2.04 This reduction in PgE2 secretion observed in the patients monocytes correlates with other studies in which reduced prostaglandin levels were demonstrated in plasma and gastric juice in patients with untreated duodenal ulcers (Hinsdale, J. G. eft at Prostaglandins (1974) 6, 595).

Reduced PgE2 production by gastric mucosa was also reported in patients with duodenal ulcers (Sharon, P. etal Scand. J. Gastroenterol. (1983)18, 1045) and a later study by the same group showed that cimetidine treatment could significantly increase PgE2 production by gastric mucosal cells (Branski, D. etal.

Scand. J. Gastroenterol. (1984)19,457).

Table 3 shows the production of PgE2 when the monocytes were stimulated in the presence of added arachidonic acid. PgE2 secretion was marginally lower than control subjects in the untreated patients but did not reach statistical significance. However, after 4 weeks treatment the levels of PgE2 secretion was increased towards normal levels in the duodenal ulcer patients.

TABLE 3 Optimal PgE2 secretion by monocytes from control subjects, and patients with duodenal ulcers (before and 4weeks after treatment), stimulated with opsonized zyrgosan and 10 nmoles arachidonic acid.

PgE2 Secretion (n) (ng/106 monocytes) Control Subjects (20) 595 + 56.7 Untreated Patients (20) 392 + 53.0 Treated Patients (20) 508 + 66.7 This demonstrated that the cyclooxygenase pathway was not impaired in the monocytes from patients with duodenal ulcer disease. The reduced levels of PgE2 production shown in Table 2 could be due to either a block at phospholipase A2 (see Scheme A) or perhaps a deficiency of endogenous arachidonic acid in the cells. To test the latter possibility, endogenous levels of arachidonic acid were measured in the peripheral blood mononuclear cells of the control subjects and the patients before and after treatment. The results are presented in Table 4. The methods used for this procedure involved the transesterification of extracted lipids and measurement of methylated fatty acids using gas-liquid chromatography, compared to authentic standards.

TABLE 4 Endogenous arachidonic acid levels in peripheral blood mononuclear cells from control subjects, and patients with duodenal ulcers (before and 4 weeks after treatment).

Arachidonic Acid (n) Xug/mg DNA Control Subjects (20) 41.0 + 4.60 Untreated Patients (20) 38.5 * 5.16 Treated Patients (20) 38.1 +4.80 It can be seen that there is not a deficiency of endogenous arachidonic acid in the patients cells. It therefore seemed that the reduced levels of PgE2 secretion by the monocytes from patients with duodenal ulcers (Table 2) may have been due to a block at the phospholipase A2 step. To test this hypothesis, LTB4 was measured in the same subjects, using the same procedures. A block at phospholipase A2 would be expected to produce a similar reduction in LTB4 production using zymosan stimulation, with normal secretion using zymosan with added arachidonic acid.

TABLE 5 Optimal LTB4 secretion by monocytes from control subjects, and patients with duodenal ulcers (before and 4 weeks after treatment), stimulated with opsonized zymosan.

LTB4 Secretion (n) (ng/106 monocytes) Control Subjects (20) 1.8 + 0.25 Untreated Patients (20) 6.89 + 1.10 p < 0.001 Treated Patients (20) 3.39 + 0.53 p < 0.001 Table 5 shows the optimal LTB4 secretion by monocytes from control subjects, and duodenal ulcer patients, before and after 4 weeks treatment. A significant increase in LTB4 secretion by untreated patients compared to control subjects was observed using zymosan stimulation (p < 0.001). After 4 weeks treatment, a significant reduction of LTB4 secretion was observed in the patients (p < 0.001), but the levels did not reach normal values.This data demonstrated that there was not a block at the phospholipase A2 step, but would suggest a significant enhancement of the 5-lipoxygenase pathway in the monocytes of untreated patients (perhaps due to the disease process), with a significant reduction in this pathway after 4 weeks treatment with histamine H2-receptor antagonists.

Table 6 shows the production of LTB4 when the monocytes were stimulated in the presence of added arachidonic acid. LTB4 secretion was significantly higher than controls in the untreated patients (p < 0.001).

After 4 weeks treatment with histamine H2-receptor antagonists, LTB4 secretion was reduced towards normal values. Again, this demonstrated a significant enhancement in the 5-lipoxygenase pathway in monocytes of untreated patients, with a significant reduction in this pathway after 4 weeks treatment with cimetidine or ranitidine.

TABLE 6 Optimal LTB4 secretion by monocytes from control subjects, and patients with duodenal ulcers (before and after 4 weeks treatment), stimulated with opsonized zymosan and 10 nmoles arachidonic acid.

LTB4 Secretion (n) (ng/106 monocytes) Control Subjects (20) 8.50 + 1.50 Untreated Patients (20) 40.3 + 7.00 p < 0.001 Treated Patients (20) 12.0 + 1.86 The data presented herein would suggest that the 5-lipoxygenase pathway is enhanced in untreated patients with duodenal ulcers, and this would result in diversion of substrate into this pathway with a concomitant reduction in PgE2 production (Tables 2 and 3). Treatment with cimetidine and ranitidine inhibits LTB4 production (Tables 5 and 6) and may divert the substrate towards the cyclooxygenase pathway. This would account for the small increase in the PgE2 production in treated patients' monocytes as shown in Tables 2 and 3. Any drug which causes an increase in PgE2 production can contribute towards healing by cytoprotective properties, as outlined earlier.Reduction in LTB4 production is considered to be 3 potent anti-inflammatory action. It is therefore proposed that each of the histamine H2-receptor antagonists, cimetidine and ranitidine, may not only act by its known action of inhibition of acid production, but may also have general purpose anti-inflammatory drug action which promotes healing, due to inhibition of the 5lipoxygenase pathway.

Since inflammatory diseases are increasingly being associated with enhanced production of leukotrienes (Ford -- Hutchinson, A. W., J. Allergy Clin. Immunol. (1984) 74, 437), the results of Tables 5 and 6 demonstrated that the histamine H2-receptor antagonists, cimetidine and ranitidine, are potentially drugs which will inhibit the production of inflammatory leukotrienes and lead to improvement of inflammatory diseases.

As stated above it has been observed that ulcer patients with psoriasis, a chronic inflammatory skin disease, showed clinical improvement after treatment with cimetidine at 1 g/day. Other studies have demonstrated a reversal of anergy in Crohn's disease indicating that cimetidine can enhance T cell immune competence in immunologically-compromised Crohn's disease. Other indications of the anti-inflammatory properties of cimetidine are indicated in the prior art section of the present Specification.

According to the data presented herein, it is likely that the histamine H2-receptor antagonists, dmetidine and ranitidine, have the ability to inhibit leukotiene production, and hence they have the capacity to act as general purpose anti-inflammatory drugs. It has recently been reported that various non-steroidal anti-inflammatory drugs inhibit the lipoxygenase pathway as well as the cycloxygenase pathway (Randall, R. W. petal, Agents Actions (1980)10,553; Meyers, R. F. and Siegel, M. I. Biochem. Biophys. Res. Commun.

(1983)112,586). It has also been shown that these drugs inhibit the 11and 15-lipoxygenase pathways (Seigel, M. I. etal Biochem. Biophys. Res. Commun. (1980)92,688; Randall R. W. (1980) supra). I now believe that the true anti-inflammatory nature of various non-steroidal anti-inflammatory drugs (as opposed to the palliative analgesic and antipyretic action) is related to the inhibition of the highly potent products of the lipoxygenase pathways. The data presented herein would support the concept that healing of gastric ulcers by H2-receptor antagonists (where there is not usually hyperacidity) could be related to the inhibition of production of inflammatory LTB4. The data presented herein would also suggest that cimetidine and ranitidine are specific inhibitors of the 5-lipoxygenase pathway and would therefore be expected to have potent anti-inflammatory properties in the treatment of inflammatory diseases. A number of other lipoxygenase pathway inhibitors are currently under investigation, and it is now believed that development of specific 5-lipoxygenase pathway inhibitors will play a major role in treating inflammatory diseases.

Claims (11)

1. Use of at least one histamine H2-receptor antagonist for the preparation of a medicament for use as a 5-lipoxygenase pathway inhibitor.

2. Use of at least one histamine H2-receptor antagonist for the preparation of a medicament for use in the human treatment of chronic inflammatory diseases including rheumatoid arthritis and other inflammatory joint diseases, psoriasis and other inflammatory skin diseases, chronic inflammatory bowel disease such as Crohn's disease and ulcerative colitis, inflammatory liver disease such as chronic active hepatitis, and sarcoidosis of the lung.

3. Use of at least one histamine H2-receptor antagonist for the preparation of a medicament for use as a general purpose anti-inflammatory drug for human use.

4. Use of at least one histamine H2-receptor antagonist for the preparation of a medicament for use as a stimulant of prostaglandin production.

5. Use according to any one of the Claims 1 to 4, characterized in that the histamine H2-receptor antagonist is selected from N"-cyano-N-methyl-N '-[2-[[(S-methyl-1 H-imidazol-4-yl)methyl]thiojethyl]- guanidine (cimetidine) and N-[2-(((-5-[(dimethylamino)-methyl]-2-fu ranyl)methyl)th io)ethyl )-N '-methyl-2- nitro-1,1-ethenediamine (ranitidine).

6. Use according to Claim 5, characterized in that the histamine H2-receptor antagonist is N"-cyano methyl-N'-[2-[[(S-methyl-1 H-imidazol-4-yl)methyl]thiojethyl]guanidine (cimetidine).

7. Use according to any one of Claims 1 to 6 for the preparation of a medicament for topical application characterized in that the histamine H2-receptor antagonist is selected from N-methyl-N'-(4-(5)-imidazol-4- yl)butyl)-thiourea (burimamide), N"-cyano-N-methyl-N'-[2-[[(5-methyl-1 H-imidazol-4-yl )methyl]thio]ethyl]- guanidine (cimetidine), N-methyl-N'-(2-(5-methylimidazol-4-yl)methyi thio)ethylthiourea (metiamide), or N [2-(((-5-[(dimethylamino)-methyl]-2-furanyl)methyl )thio)ethyl )-N '-methyl-2-nitro-1 , 1 -ethene-diamine (ranitidine).

8. A suppository comprising at least one histamine H2-receptor antagonist and a suppository base.

9. A suppository as claimed in Claim 9, wherein the histamine H2-receptor antagonist is burimamide, cimetidine, metiamide or rantidine.

10. Atopical formulation comprising at least one histamine H2-receptor antagonist and a topical carrier.

11. A topical formulation as claimed in Claim 10, wherein the histamine H2-receptor antagonist is burimamide, cimetidine, metiamide or rantidine.