This invention relates to pharmaceutical compositions comprising a methylthioribose (MTR) kinase inhibitor and an inhibitor of djg. novo methionine synthesis and their use as medicinal agents.

MTR-kinase is a microbial enzyme important in the recycling of methionine. US-A-4820692 discloses analogues o MTR as a new class of antimicrobial drugs because of their ability to perturb the growth of MTR kinase-containing microorganisms. These analogs may act through inhibition of methionine recycling from MTR, resulting in methionine depletion, or by conversion to toxic products. For example, it has been proposed that 5-trifluoromethylthioribose (TFMTR serves as a suicide substrate for the methionine salvage pathway through conversion to trifluoro ethionine or carbonothioic difluoride .Paras, oloςry Today, £ (10) , 1989, 330) .

Microbial biosynthesis of cysteine and methionine proceeds along a branched convergent pathway in one arm of which sulfate is reduced to sulfide while serine is acetylated to O-acetylserine. The next step consists of formation of cysteine from sulfide and O-acetylserine by the enzyme O-acetylserine sulf ydrylase. Methionine is then synthesised from cysteine via cystathionine in a sequence of reactions catalysed by cystathionine γ-synthase, cystathionine β-lyase, and methionine synthase. Thus, unlik mammalian cells which synthesize cysteine from methionine, enteric bacteria derive methionine from cysteine. Compound which inhibit O-acetylserine sulfhydrylase, cystathionine γ-synthase, cystathionine β-lyase or methionine synthase inhibit the synthesis of methionine and can be termed inhibitors of _Q. novo methionine synthesis. The synthesis o methionine and the methionine salvage pathway is summarised in the following scheme.

1, 2, 4-Trlazolo Propargylglycine

Θ

O- ^Θ Acety ilserine Cystathionine Cystathionine sulfhydrylase γ-synthase β-lyase

This invention is based upon the dis<tøfc&a_7y,.-fchat. inhibitors of uo. novo methionine synthesis act in synergy with MTR-kinase inhibitors to inhibit the growth of MTR- kinase containing microorganisms. Inhibition of ς__. novo methionine synthesis would appear to increase reliance on the methionine salvage pathway for maintenance of methionine levels, thereby leading to increased efficacy of MTR-kinase inhibitors against such microorganisms by disruption of the methionine salvage pathway.

Thus, in a first aspect the present invention provides a pharmaceutical composition comprising an MTR-kinase inhibitor, and an inhibitor of de. novo methionine synthesis and a pharmaceutically acceptable carrier.

Suitably an MTR-kinase inhibitor is a compound of the formula (1) :

wherein R is H, CI, F, Br, I or R ¹ S in which R ¹ is CI_I _Q linear or branched chain alkyl or halogenated linear or branched chain alkyl and R ² to R ⁴ are H or OH with the proviso that at least one of R ² to R ⁴ is OH.

A preferred MTR-kinase inhibitor is TFMTR.

Preferred inhibitors of __ novo methionine synthesis include inhibitors of O-acetylserine sulfhydrylase, cystathionine γ-synthase, cystathionine β-lyase, methionine synthase or mixtures thereof.

Examples of O-acetylserine sulfhydrylase inhibitors are 1,2, -triazole or azaserine (O-diazoacetylserine) .

An example of a cystathionine γ-synthase inhibitor is propargylglycine (2-amino-4-pentynoate) .

An example of a methionine synthase inhibitor is nitrous oxide (N2O) .

In a second aspect this invention provides a method of treating a mammal infected with an MTR-kinase containing. microorganism which comprises administering to said mammal an effective amount of an MTR-kinase inhibitor and an effective amount of an inhibitor of d≤. novo methionine synthesis.

Suitably the MTR-kinase inhibitor and inhibitor of de. novo methionine synthesis are administered concurrently either as a pharmaceutical composition as hereinbefore described or as separate pharmaceutical compositions.

Alternatively the MTR-kinase inhibitor and inhibitor of de. novo methionine synthesis are administered non-concurrently (for example more than 1 hour apart) as separate pharmaceutical compositions.

A pharmaceutical composition comprising both medicaments together and pharmaceutical compositions comprising the separate medicaments can be formulated in accordance with standard pharmaceutical practice.

They may be administered in standard manner, for example orally, sub-lingually, parenterally, transdermally, rectally, via inhalation, via buccal administration, or to the eye.

When given orally or via buccal administration they can be formulated as liquids, syrups, tablets, capsules and lozenges. An oral liquid formulation will generally consist of a suspension or solution of the medicament in a liquid carrier for example, ethanol, glycerine or water with a flavouring or colouring agent. Where the composition is in

the form of a tablet, any pharmaceutical carrier routinely used for preparing solid formulations may be used. Examples of such carriers include magnesium stearate, starch, celluloses, lactose and sucrose. Where the composition is i the form of a capsule, any routine encapsulation is suitable, for example using the aforementioned carriers in a hard gelatin capsule shell. Where the composition is in the form o a soft gelatin shell capsule any pharmaceutical carrier routinely used for preparing dispersions or suspensions may be considered, for example aqueous gums, celluloses, silicates or oils and are incorporated in a soft gelatin capsule shell.

Typical parenteral compositions consist of a solution or suspension of the medicament in a sterile aqueous or non- aqueous carrier optionally containing a parenterally acceptable oil or solublising agent, for example polyethylene glycol, polyvinylpyrrolidone, 2-pyrrolidone, cyclodextrin, lecithin, arachis oil, or sesame oil.

A typical suppository formulation comprises the medicament with a binding and/or lubricating agent, for example polymeric glycols, gelatins, cocoa-butter or other low melting vegetable waxes or fats or their synthetic analogues.

Typical transdermal formulations comprise a conventional aqueous or non-aqueous vehicle, for example a cream, ointment, lotion or paste or are in the form of a medicated plaster, patch or membrane.

Typical compositions for inhalation are in the form of a solution, suspension or emulsion that may be administered in the form of an aerosol using a conventional propellant such as dichlorodifluoromethane or trichlorofluoromethane, or are in the form of a powder for insufflation.

Formulations for administration to the eye include solutions, suspensions, ointments or creams as hereinbefore described.

Preferably the composition is in unit dosage form, for example a tablet, capsule or metered aerosol dose, so that the patient may administer to himself a single dose.

Each dosage unit contains suitably from 150-1500 mg, preferably 100 to 500 mg, of an MTR-kinase inhibitor and from 50 to 1500 mg of an inhibitor of da novo methionine synthesis preferably from 500 to 1000 mg of an O-acetylserine sulfhydrylase inhibitor or from 100 to 500 mg of a cystathionine γ-synthase inhibitor.

The daily dosage regimen is suitably about 5-30 mg/kg of an MTR-kinase inhibitor and about 5 to 100 mg/kg of an inhibitor of d≤. novo methionine synthesis, preferably about 10 to 50 mg/kg of an O-acetylserine sulfhydrylase inhibitor or about 5 to 30 mg/kg of cystathionine γ-synthase inhibitor.

Examples of MTR-kinase containing microorganisms whose growth is inhibited by the compositions and methods of the present invention include Klebsiella pnenmoniae _r Enterpbacter E. sakazaki and E. n1oar:af_ _r Serra ia marcescans _r Proteus v.ιlgaris _r YftrSJnϊa ssp, MQrganella ssp and Erwinia ssp.

MTR-kinase-containing parasitic protozoa do not synthesize methionine ds. novo but recycle methionine via the methionine salvage pathway previously described and also via methionine synthase. Inhibition of methionine synthase in such protozoa would increase reliance on the methionine salvage pathway for maintenance of methionine levels, thereby leading to increased efficacy of MTR-kinase inhibitors against such protozoa by disrupting the methionine salvage pathway. Examples of such protozoa include Plasmodiu falciparum (responsible for the most deadly form of malaria) , Giardia Iambiia and Qchr m n S malhamensis.

Thus, in a further aspect the present invention provides a method of treating a mammal infected with an MTR-kinase containing protozoan which comprises administering to said mammal an effective amount of an MTR-kinase inhibitor and an effective amount of a methionine synthase inhibitor. Examples of MTR-kinase inhibitors and methionine synthase inhibitors are as hereinbefore described.

MTR-kinase inhibitors are known from US-A-4820692 and can be prepared according to methods disclosed therein. TFMTR is suitably prepared according to the method disclosed in J. Biol. Chem. 265; 831, 1990.

Inhibitors of O-acetylserine sulfhydrylase such as 1,2,4-triazole or azaserine are commercially available and can be prepared as disclosed in Orςr. Syn. 40 : 99, 1960 and J. Am. hf.τn. Soc. 76 : 2878, 1954, respectively.

Inhibitors of cystathionine γ-synthase such as propargylglycine are commercially available and can be prepared as disclosed in J. Am. Chem. Soc. 95 : 6124, 1973

The following biological test method serves to illustrate this invention.

Inhibition of the Growth of Klebsiella pneumoniae

Materials: Azaserine (O-diazoacetylserine) , DL- propargylglycine and 1,2,4-triazole were purchased from Sigma Chemical Company (St. Louis, MO) . 5-Trifluoromethylthioribose (TFMTR) was synthesised as described previously.

Bacterial strains and culture conditions - A clinical isolate of Klebsiella pneumoniae, obtained from the Department of Veterans Affairs Medical Center, Portland, Oregon, was utilised in this study. &_. pneumoniae was maintained in a chemically-defined medium containing: 25mM NH4CI; 35mM glucose; 1.5mM KC1; 0.4mM MgSθ4,-

0.045mM NaCl; 0.025mM FeS04,- 0.025 μg/ml thia ine; and 66.6mM Na2HP04~NaH2P04. The following icronutrient solution was added with the indicated final concentrations : CaCl2 (5 x 10 ^_7 M), CoCl (5 x 10 ^-8 M), MnCl ₂ (10 ^"7 M) , HB0 ₃ (5 x 10~ ⁷ M), ZnCl ₂ (10 ^-8 M) , CuC0 ₃ (10 ^-8 M) , (NH ) 6M07O24 (5 x 10 ^_9 M) , and the pH was adjusted to 7.2.

Dose inhibition studies were conducted in 5ml cultures inoculated with ~10 ⁴ cells per ml and maintained in a rotary shaker incubator at 37°C. Growth was monitored by optical density at 470nm 12-15 hrs after incubation when control cultures had reached an optical density of 0.4. Isoboles representing the activity of drug mixtures were performed and analysed as described by Hewlett, Biometrics, 25: 477-87, 1969. Briefly, TFMTR and 1,2,4-triazole, azaserine, or propargylglycine were serially-diluted so that the organisms were simultaneously exposed to drug mixtures. The ability of the various drug combinations to inhibit growth by 50% (IC50) relative to control values was measured. When combined and plotted graphically, the results yield an isobologram. As described by Hewlett, isoboles for two separately active drugs resemble: 1) a straight line for additive action; 2) a convex line for subadditive action; and 3) a concave line for potentiation.

Results

Effect of methionine on the inhibitory action of TFMTR - In order to gain insight into the mechanism of action of TFMTR, the ability of methionine to reverse the inhibitory effect of TFMTR was tested. I . pneumoniae was cultured in defined medium containing iμM TFMTR and varying amounts of methionine. The organisms were inoculated at a density of ~10 ⁴ /ml and incubated for 15 hrs at 37°C. In the absence of added methionine, TFMTR totally inhibited cell growth. Increasing the concentration of methionine to 100μM restored growth to nearly 60% of control. The inhibitory action of TFMTR was completely abrogated by the addition of l,000μM methionine.

These results support the notion that TFMTR blocks cell growt by exploiting methionine recycling and suggest that blocking microbial methionine biosynthesis might potentiate the inhibitory action of TFMTR.

Potentiation of TFMTR bv 1.2.4-Triazole - 1,2,4-Triazole is an inhibitor of O-acetylserine sulfhydrylase the final step in microbial cysteine biosynthesis (J. Biol. Chem. 250 : 7324, 1975). We tested the ability of 1,2,4-triazole to potentiate the antimicrobial activity of TFMTR. The clinical strain of ___ pneumoniae used in our studies contains MTR kinase, actively salvages methionine from MTA in vivo, and is very sensitive to the effects of TFMTR, exhibiting an IC50 value in the submicromolar range. 1,2,4-Triazole is a weak but effective inhibitor of bacterial growth with an IC50 of

2.5 mM. The concave line of the isobologram drawn from the collected IC50 values indicates synergy between the two drugs. In combination, 0.1 μM TFMTR decreased the IC50 for 1,2,4- triazole by 10-fold to 0.25 mM. The degree of potentiation measured as the joint action ratio for the drug combination was calculated to be 2.5.

Potentiation of TFMTR bv azaserine - Azaserine is a substrate for O-acetylserine sulfhydrylase. Upon reaction of azaserine with this enzyme, diazoacetate, a highly-reactive and toxic product, is formed. Potentiation studies with TFMTR and azaserine demonstrated a striking synergy. The IC50 value for azaserine alone against _ _ pneumoniae was 2.0 μM. At all drug ratios, the amount of either drug required to produce 50 percent inhibition was less with the combination than with either drug alone, and became minimal when 0.05 μM TFMTR was combined with 0.25 μM azaserine. The degree of potentiation for TFMTR and azaserine measured as the joint action ratio was 3.6.

Potentiation of TFMTR by propargylglycine - Propargylglycine is an irreversible inhibitor of cystathionine γ-synthase, a pyridoxal phosphatedependent enzyme involved in microbial

methionine synthesis. The growth inhibitory effects of propargylglycine are reversed by methionine. In our studies, 375 μM propargylglycine was required to inhibit Klebsiella growth by 50 percent. Combining as little as O.lμM TFMTR with 20μM propargylglycine produced the same degree of growth inhibition. All points from the TFMTR-propargylglycine combinations used in the study fell well below the line of addition. The degree of potentiation between the two drugs was calculated to be 3.2.

Our results show that all three compounds act in synergy with TFMTR to inhibit the growth of Klebsiella pneumonia . Chemotherapy employing multiple drugs with different modes of action is important to prevent the emergence of drug-resistant pathogens. Ideally, such drugs should act synergistically toward the elimination of the invading parasite. From the results presented here, it is clear that compounds which block de novo methionine synthesis act synergistically with TFMTR. In summary, the combination of TFMTR with an antagonist of microbial methionine synthesis provides a novel approach towards the control of infections caused by MTR kinase- containing pathogens.