WO1994027606A1

WO1994027606A1 - Method of treating helicobacter pylori infection

Info

Publication number: WO1994027606A1
Application number: PCT/AU1994/000290
Authority: WO
Inventors: George Louis Mendz; Stuart Lloyd Hazell
Original assignee: Unisearch Limited
Priority date: 1993-05-28
Filing date: 1994-05-30
Publication date: 1994-12-08

Abstract

The present invention relates to a method of treating Helicobacter pylori infection. The method involves the administration of a composition, the active ingredient of which inhibits or prevents the action of fumarate reductase.

Description

METHOD OF TREATING Helicobacter pylori INFECTION The present invention relates to a method for the treatment of Helicobacter pylori infection, and to a class of compounds that have been shown to have bactericidal activity against Helicobacter pylori .

Helicobacter pylori is the type species of a new genus of gastric colonising and related bacteria that constitute both autochthonous microbiota (normal flora) in animals and have a pathogenic role in upper gastrointestinal disease in humans. It has been established as the major aetiological agent of active chronic gastritis (Marshall & Warren, 1984; Morris & Nicholson, 1987), has been associated with peptic ulcers (Hornick, 1987), and more recently has been linked to the development of gastric cancer in humans (Parsonnet et al . , 1991; Nomura et al . , 1991).

The isolation of Helicobacter pylori represents a milestone in the elucidation of the mechanisms of gastritis, peptic ulcer disease and gastric carcinogenesis (Goodwin et al . , 1986). The accumulated knowledge of the last eleven years leads to the conclusion that eradication of this bacterium will result in the prevention or cure of significant upper gastrointestinal pathology. In a recent meeting at the National Institutes of Health (USA) the consensus decisions of the panel were (The Economist, 1994) :

(a) all patients who have or have had an ulcer should be tested for H. pylori ; and

(b) two weeks of antimicrobial treatment should be given to those whose test shows them to be infected.

The recent history of the treatment of peptic ulcer disease has seen the advent of agents designed to suppress acid such as histamine H2-receptor antagonists and proton pump inhibitors. These have helped the healing of ulcers, but not the cure of the disease, since ulcers often recur after treatment is stopped. On the other hand, the use of known antibacterial agents directed against H. pylori has been instrumental in the cure of peptic ulcer disease in many instances (Graham et al . , 1992). The current approaches, however, do not constitute ideal therapeutic regimens. Up to date, the eradication of H. pylori makes use of a combination of three antimicrobial agents: bismuth, a nitroi idazole, and amoxicillin or tetracycline. This triple therapy cannot be considered the final therapeutic protocol because of problems with resistance of some strains of the bacterium to nitroimidazoles, poor compliance, drug intolerance, and undesirable side effects. Newer drug combinations, such as proton pump inhibitors together with an antibiotic, are generally better tolerated than the triple therapy, but efficacy to a satisfactory level has not been demonstrated (Unge et al . , 1989) .

A major shortcoming with these treatments is the lack of a sound scientific foundation which limits their further development. The triple therapy was found by trial and error. The combinations of proton pump inhibitors with an antibiotic arose from a desire to broaden the indication profile of these products, rather than from an attempt to develop new and more effective anti-H. pylori drugs. The development of new therapeutic agents targeted against Helicobacter pylori will represent a significant advance in the treatment of upper gastrointestinal disease. The traditional approach to drug development has been to screen naturally occurring or synthetic products against a pathogen. Promising candidates serve as prototypes for the preparation of structural analogs that will show better cytotoxicity, selectivity, targeting, and pharmacological characteristics. This procedure has led to the appearance of most of the compounds presently in clinical use. However, this path is limited by the number of existing drugs for human studies, or off-the-shelf compounds, that can be employed in in vitro antimicrobial screenings and animal trials. The lack of understanding of how specific agents work against H. pylori limits substantially the possibility of improving drugs by design. The present inventors adopted an alternative approach which proceeds through an investigation of the biochemistry and microbiology of the organism to establish a scientific foundation for the rational selection of targets for antibacterial agents. The importance of this "enzyme-targeted chemotherapy" is highlighted by the fact that in 1992 little was known about the basic biological functions in H. pylori . Accordingly, the present inventors began to study the metabolism of the bacterium with the aim of understanding its physiology, and identifying areas where the therapeutic intervention may be feasible.

The present inventors have discovered that Helicobacter pylori can reduce fumarate to succinate via a fumarate reductase. As this enzyme is not present in humans the fumarate reductase enzyme constitutes a prime target for selective chemotherapeutic intervention against Helicobacter pylori .

The metabolism of fumarate by this bacterium was investigated employing one- and two-dimensional *H and l^C nuclear magnetic resonance spectroscopy (NMR) .

Metabolically competent cells generate malate and succinate from fumarate as the sole substrate indicating the presence of fumarase and fumarate reductase activities in H. pylori . The results indicated the existence of active fumarate catabolism in the bacterium. Employing ^H-NMR spectroscopy the inventors developed a method to study fumarate reductase activity in whole cells, lysates and membranes (Mendz & Hazell, 1993).

Similar observations were made in suspensions of H. pylori lysates or membranes, and the reaction rates measured depend on the conditions of the incubations. e.g., substrate concentration, type of bacterial preparation, etc. In these time-courses other metabolites besides succinate are observed to accumulate at later times. They have been identified as lactate, acetate, formate and alanine (Mendz & Hazell, 1993).

Fumarate reductase activities were observed in all the strains tested including wild types, urease-negative mutants and catalase-negative mutants, suggesting that it is a basic function of the bacterium. Additional evidence for the presence of fumarate reductase in H. pylori was obtained from incubations of bacterial lysates with citrate as the sole substrate. Time courses employing ^H-NMR spectroscopy showed that main products of citrate metabolism were succinate, formate and acetate, suggesting a partial or total reversal of the dicarboxylic acid segment of the Krebs' cycle which would include reduction of fumarate to succinate.

The inventors have found also that aspartate is a readily available source of fumarate for the bacterium. In incubations of bacterial lysates with L-aspartate as the only substrate, the amino acid was rapidly catabolized to fumarate indicating the presence of strong aspartase activity. Accordingly, the present invention consists in a method of treating Helicobacter pylori colonisation in a subject comprising administering to the subject a composition including as an active ingredient a compound which inhibits or prevents the action of fumarate reductase.

In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following examples. Figure 1 shows sequential spectra acquired using *H- NMR spectroscopy. The times at which the spectra were recorded are shown on the right-hand side of the figure. Addition of fumarate (F) to a suspension of H. pylori cells produces malate (M^, M2) in a rapid burst. After the concentrations of fumarate and malate reach equilibrium in the reaction catalysed by fumarase, the levels of fumarate and malate decline with time and succinate (S) accumulates, demonstrating the presence of fumarate reductase activity.

Effects of Inhibitors on Fumarate Reductase Activity The 1H-N R method developed by the present inventors, which allows direct measurements of fumarate reductase activity, was employed to investigate the effects on H. pylori fumarate reductase of three inhibitors used as antiparasitics. Morantel {1,4,5,6- tetrahydro-l-methyl-2-[2-(3-methyl-2- thienyl)ethenyl]pyrimidine, Merck Index 6175}, oxantel {(E)-3-[2-(l,4,5,6-tetrahydro-l-methyl-2- pyrimidinyl)ethenyl]phenol, Merck Index 6876}, and thiabendazole {2-(4-thiazolyl)-lH-benzimidazole, Merck Index 9217} are the active components of anthelminthics employed in animals and humans (Vanden Bossche, 1985).

This is the first time these inhibitors have been applied to study their effect on the fumarate reductase activity of bacteria. The presence of either of the three antimetabolites inhibited the catalysis of fumarate to succinate in H. pylori cell suspensions. Inhibition of fumarate reductase activity was also observed in preparations of bacterial lysates or membranes in the presence of either of the three compounds. The stronger inhibitory effect was observed for oxantel, and morantel caused the weaker inhibition.

Effects of Fumarate Reductase Inhibitors on Bacterial Growth and Viability The effects of morantel, oxantel and thiabendazole on cell growth and viability were studied in liquid cultures incubated at 37°C in 10% CO2, 95% humidity. Bacterial growth was monitored by the changes in the absorbance of the cell suspensions measured at 600 nm, and bacterial viability was measured by taking aliquots from the liquid cultures and using the method of Miles & Misra (1938).

All three inhibitors showed bactericidal effects; the strongest effect was observed for oxantel, and the weakest for morantel. Figure 2 shows growth and viability curves of H. pylori cultures with (■) and without (•) morantel.

Figure 3 shows the number of viable cells/ml in the same cultures as a function of time determined by the method of Miles & Misra (1938). (■) with morantel; (•) without morantel. Minimal Inhibitory Concentration (MIC) and Minimal Bacteridical Concentration (MBC) of Fumarate Reductase Inhibitors MIC is defined as the lowest concentration of drug that completely inhibits growth under conditions specified. MBC is defined as the lowest concentration of drug which results in a kill of >99.99% of organisms.

Sterile microtitre trays were used for the experiment. Isosensitest broth with 5% horse serum (60 μl) was added to each well; 50 μl of the inhibitor was then serially diluted from the 11th column to the 1st column of a row. Each well was then inoculated with 50 μl of the adjusted culture (5 x 10^ cfu) . The plates were incubated at 37°C in 10% CO2, 95% humidity. After 72 hours, the plates were inspected for growth which was determined by the formation of a 'button' or pellet at the bottom of the wells. The lowest concentration of the inhibitor that completely inhibited the growth of H. pylori was taken as the MIC.

After the determination of the MIC, the plates were shaken on a plate reader for 60 s. A culture from each well (10 μl) was sampled onto Columbia agar base (Oxoid) with 5% whole horse blood (Amadeus). The plates were incubated at 37°C in 10% CO2, 95% humidity for 10 days. Plates were inspected for growth on the 3rd, 5th, 7th and 10th days. MBC is that concentration of the inhibitor that kills >99.99% of the cells. In this protocol, less than 50 viable organisms should remain per well. Therefore 10 μl drops should contain less than 5 organisms.

MIC and MBC values were determined for morantel, oxantel and thiabendazole known to inhibit fumarate reductase activity in parasitic worms. The values obtained are given in the following table:

INHIBITOR MIC (mM) MBC (mM)

Morantel 3.13 3.13

Oxantel 0.94 0.94

Thiabendazole 1.88 1.88

Dose-Response Curves of Fumarate Reductase Inhibitors

The kinetics of the bactericidal effects of morantel, oxantel and thiabendazole was investigated by determining the cell growth and viability dose-response curves for of each inhibitor. Liquid cultures in Isosensitest broth with 0.5% BSA and 0.1% catalase inoculated with 10^-10^ H. pylori cells/ml were incubated for two hours at 37°C in 10% CQ2, 95% humidity in the presence of each inhibitor at concentrations between 0 and 1.2 mM. In Figure 4 the effects of the inhibitors on cell growth are presented as the absorbance of the suspensions measured at 600 nm, and the effects of cell viability as the change in the log number of viable cells/ml (♦ Morantel; • Thiabendazole; ■ Oxantel). Figure 5 shows the number of viable cells determined by the method of Miles & Misra (1938) (Legend as for Fig. 4). The dose response curves indicate that oxantel is the most effective inhibitor under the experimental conditions employed.

Effects of Fumarate on the Bactericidal Action of Oxantel The data on the inhibition of fumarate reductase activity, cell growth and cell viability by morantel, oxantel and thiabendazole, and the dose-response curves of the three antiparasitics suggest that oxantel is the most potent inhibitor for H. pylori . The selectivity of the target(s) affected by oxantel was investigated by comparing the bactericidal effects of the inhibitor in liquid Isosensitest with 5% horse serum cultures incubated at 37°C in 10% CO2, 95% humidity with or without added fumarate. The time-evolution of the number of viable cells was monitored in four cultures: (a) control (•), (b) with fumarate added (A) , (c) with oxantel added (♦), and (d) with oxantel and fumarate added (■) . The data on cell viability are shown in Figure 6.

The results show that the presence of fumarate did not have major effects on cell growth in the control cultures, but it reduced the rate of cell death caused by the inhibitor at the beginning of the incubations, suggesting that the bacterial fumarate reductase is a target for the inhibitor and that protection of this target by the presence of the natural substrate (fumarate) reduces significantly the bactericidal effects of oxantel.

The utilization of fumarate reductase inhibitors as antibacterial agents is not obvious because:

(i) many bacteria do not have this enzyme, and (ϋ) some pathogenic bacteria which have this enzyme, e.g., Eεcherichia coli, also possess alternative biochemical pathways which allow the organism to survive under conditions in which fumarate reductase is not utilized. The discoveries of the present inventors that: (a) Helicobacter pylori possesses a fumarate reductase enzyme;

(b) the bacterium has a readily available source of fumarate; (c) formate is an important metabolic product of the organism that could act as a donor substrate for fumarate reduction; and (d) bacterial growth and viability are inhibited in vitro by the presence of known fumarate reductase inhibitors such as morantel, oxantel and thiabendazole; demonstrate that under in vitro standard conditions fumarate reductase activity is an essential requirement for the growth and replication of the bacterium. Other Inhibitors of Fumarate Reductase

Based on the chemical structure of morantel, oxantel and thiabendazole and their relative potency as anti- H. pylori agents, and taking into consideration the chemical structures of other known active parasitic agents such as oxfendazole (Vanden Bossche, 1985), the present inventors screened a number of compounds for anti-fumarate reductase and bactericidal activity.

These compounds had not been considered previously as potential fumarate reductase inhibitors. Screening tests showed that a number of such compounds inhibited the fumarate reductase and had bactericidal activity (Table 1) .

TABLE I Activities of Different Inhibitors of Fumarate Reductase

INHIBITOR NMR Test* MIC (mM) MBC (mM)

1_I4,5,6-tetrahydro-1-methyl-2-[2-(3-methyl-2-thienyl)ethenyl]pyrimidine Medium 3.13 3.13

(E)-3-[2-(1,4,5,6-tetrahydro-1-methyl-2-pyrimidinyl)ethenyl]phenol Strong 0.94 0.94

2-(4-thiazolyl)-1H-benzimidazole Strong 1.88 1.88

(E)-1-(2-pyridyl)-2-(4-pyridyl)ethylene Weak ND ND ethyl (E)-cinnamate None ND ND

(Z)-1 ,2-bis(2-pyridyl)ethylene Medium 6.25 6.25

(E)-1 ,2-bis(2-pyridyl)ethylene Medium ND ND

1-vinylimidazole None ND ND

2-vinypyridine Medium 6.25 6.25

(E)-4-[4-(dimethylamino)styryl]-1 -methylpyridinium iodide Strong 0.78 0.78

(Z)-2-[4-(dimethylamino)styryl]-1 -methylpyridinium iodide Medium ND ND

2-vinylnapthalene None ND ND

The 1 H-NMR tests were carried out at inhibitor concentrations of 1 mM, observing the effects on the rates of conversion of fumarate to succinate. The results were scored in four grades: Strong, Medium, Weak or None. ND indicates that the test was not done.

In particular, (E)-4-[4-(dimethylamino)styryl]-l- methylpyridinium iodide strongly inhibited H. pylori fumarate reductase in the ¹H-NMR test, and scored MIC and MBC of 0.78 mM, the lowest values thus far determined for antimetabolites of H. pylori fumarate reductase.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

References

Goodwin, C.S., Armstrong, J.A. & Marshall, B.J. (1986) J. Clin . Path . 39, 353-365

Graham, D.Y., Lew, G.M., Malaty, H.M., Evans, D.G., Evans, D.J., Klein, P.D., Alpert, L.C. & Genta, R.M. (1992) Gastroenterology 102, 493-496.

Hornick, R.B. (1987) New Eng. J. Med. 316, 1598-1600.

Marshall, B.J. & Warren J.R. (1984) Lancet i, 1311-1315.

Mendz, G.L. & Hazell, S.L. (1993) Biochem. Mol . Biol . Int . 31, 325-332.

Miles, A.A. & Misra, S.S. (1938) J. Hyg. 38, 732-749.

Morris, A. & Nicholson, G. (1987) Am. J. Gastroent. 82, 192-199.

Nomura, A., Stemmermann G.N., Chyou, P.H., Kato, I., Perez-Perez, G.I. & Blaser, M.J. (1991) New Engl . J. Med. 325, 1132-1136.

Parsonnet, J., Friedman, G.D., Vandersteen, D.P., Chang, Y., Vogelman, J.H., Orentreicht, N.R. & Sibbley, R.K. (1991) New Eng. J. Med. 325, 1127-1131.

The Economist (1994) March 5, pp. 91-92, London.

Unge, P., Gad, A., Gnarpe, H. & Olsson, J. (1989) Scand. Gastroenterol . 167, 49-54.

Vanden Bossche, H. (1985) in Handbook of Experimental Pharmacology (Vanden Bossche, H., Thienpont, D. & Janssens, P.G., eds). Chapter 4, pp 153-157, Springer-Verlag, Berlin.

Claims

CLAIMS : -

1. A method of treating Helicobacter pylori colonisation in a subject comprising administering to the subject a composition including as an active ingredient a compound which inhibits or prevents the action of fumarate reductase.

2. A method as claimed in claim 1 in which the active ingredient is selected from the group consisting of 1,4,5,6-tetrahydro-l-methyl-2-[2-(3-methyl-2- thienyl)ethenyl]pyrimidine, (E)-3-[2-(1,4,5,6- tetrahydro-1-methyl-2-pyrimidinyl)ethenyl]phenol, 2-(4- thiazolyl)-l H-benzimidazole and (E)-4-[4- (dimethylamino)styryl]-1-methylpyridinum iodide.