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Publication number
WO1992018118A1
WO1992018118A1 PCT/US1992/003094 US9203094W WO9218118A1 WO 1992018118 A1 WO1992018118 A1 WO 1992018118A1 US 9203094 W US9203094 W US 9203094W WO 9218118 A1 WO9218118 A1 WO 9218118A1
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Prior art keywords
inhibitor
mtr
methionine
kinase
tfmtr
Prior art date
Application number
PCT/US1992/003094
Other languages
French (fr)
Inventor
Michael K. Riscoe
Paula A. Tower
John H. Fitchen
Adolph J. Ferro
Original Assignee
STATE OF OREGON, acting by and through THE OREGON STATE BOARD OF HIGHER EDUCATION, on behalf of THE OREGON HEALTH SCIENCES UNIVERSITY AND OREGON STATE UNIVERSITY
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Application filed by STATE OF OREGON, acting by and through THE OREGON STATE BOARD OF HIGHER EDUCATION, on behalf of THE OREGON HEALTH SCIENCES UNIVERSITY AND OREGON STATE UNIVERSITY filed Critical STATE OF OREGON, acting by and through THE OREGON STATE BOARD OF HIGHER EDUCATION, on behalf of THE OREGON HEALTH SCIENCES UNIVERSITY AND OREGON STATE UNIVERSITY
Publication of WO1992018118A1 publication Critical patent/WO1992018118A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to pharmaceutical compositions comprising a methylthioribose (MTR) kinase inhibitor and an inhibitor of de novo methionine synthesis and their use as medicinal agents.
  • MTR methylthioribose
  • 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.
  • TFMTR 5-trifluoromethylthioribose
  • 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
  • the next step consists of formation of cysteine from sulfide and O-acetylserine by the enzyme O-acetylserine sulfhydrylase.
  • Methionine is then synthesised from cysteine via cystathionine in a sequence of reactions catalysed by cystathionine ⁇ -synthase,
  • cystathionine ⁇ -lyase and methionine synthase.
  • enteric bacteria derive methionine from cysteine.
  • Compound which inhibit O-acetylserine sulfhydrylase, cystathionine ⁇ -synthase, cystathionine ⁇ -lyase or methionine synthase inhibit the synthesis of methionine and can be termed
  • This invention is based upon the discovery that
  • inhibitors of de novo methionine synthesis act in synergy with MTR-kinase inhibitors to inhibit the growth of MTR-kinase containing microorganisms. Inhibition of de 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.
  • the present invention provides a pharmaceutical composition comprising an MTR-kinase inhibitor, and an inhibitor of de novo methionine synthesis and a
  • an MTR-kinase inhibitor is a compound of the formula (1) :
  • R is H, CI, F, Br, I or R 1 S in which R 1 is C 1-10 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 de novo methionine synthesis include inhibitors of O-acetylserine sulfhydrylase,
  • cystathionine ⁇ -synthase cystathionine ⁇ -lyase, methionine synthase or mixtures thereof.
  • O-acetylserine sulfhydrylase inhibitors are 1,2,4-triazole or azaserine (O-diazoacetylserine) .
  • An example of a cystathionine ⁇ -synthase inhibitor is propargylglycine (2-amino-4-pentynoate).
  • An example of a methionine synthase inhibitor is nitrous oxide (N 2 O).
  • 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 de novo methionine synthesis.
  • 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.
  • MTR-kinase inhibitor and inhibitor of de novo methionine synthesis are administered non-concurrently (for example more than 1 hour apart) as separate
  • 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.
  • 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 liquid carrier for example, ethanol, glycerine or water with a liquid carrier for example, ethanol, glycerine or water with a liquid carrier for example, ethanol, glycerine or water with a liquid carrier for example, ethanol, glycerine or water with 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,
  • compositions celluloses, lactose and sucrose.
  • any routine encapsulation is suitable, for example using the aforementioned carriers in a hard gelatin capsule shell.
  • composition is in the form of 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
  • oil or solublising agent for example polyethylene glycol, polyvinylpyrrolidone, 2-pyrrolidone, cyclodextrin, lecithin, arachis oil, or sesame oil.
  • a typical suppository formulation comprises the
  • 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.
  • 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 de novo methionine synthesis preferably from 500 to 1000 mg of an O-acetylserine
  • sulfhydrylase inhibitor or from 100 to 500 mg of a
  • cystathionine ⁇ -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 MTR-kinase inhibitor
  • inhibitor of de 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 ⁇ -synthase inhibitor.
  • MTR-kinase containing microorganisms whose growth is inhibited by the compositions and methods of the present invention include Klebsiella pnenmoniae, Enterpbacter
  • 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.
  • 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
  • 1,2,4-triazole or azaserine are commercially available and can be prepared as disclosed in Org. Syn. 40 : 99, 1960 and
  • propargylglycine are commercially available and can be
  • K. pneumoniae was maintained in a chemically-defined medium containing: 25mM NH 4 CI; 35mM glucose; 1.5mM KCl; 0.4mM MgSO 4 ; 0.045mM NaCl; 0.025mM FeSO 4 ,- 0.025 ⁇ g/ml thiamine; and 66.6mM Na 2 HPO 4 -NaH 2 PO 4 .
  • the following micronutrient solution was added with the indicated final concentrations : CaCl 2 (5 x 10 -7 M), CoCl 2 (5 ⁇ 10 -8 M), MnCl 2 (10 -7 M), HBO 3 (5 ⁇ 10 -7 M), ZnCl 2 (10 -8 M), CuCO 3 (10 -8 M), (NH 4 ) 6 Mo 7 O 24 (5 ⁇ 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.
  • 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.
  • K. pneumoniae was cultured in defined medium containing i ⁇ M 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
  • TFMTR totally inhibited cell growth. Increasing the concentration of methionine to 100 ⁇ M restored growth to nearly 60% of control. The inhibitory action of TFMTR was completely abrogated by the addition of l,000 ⁇ M methionine.
  • 1,2,4-Triazole is a weak but
  • 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 IC 50 value for azaserine alone against K. pneumoniae was 2.0 ⁇ M. 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 ⁇ M TFMTR was combined with 0.25 ⁇ M azaserine.
  • the degree of potentiation for TFMTR and azaserine measured as the joint action ratio was 3.6.
  • TFMTR Potentiation of TFMTR by propargylglycine - Propargylglycine
  • Propargylglycine is an irreversible inhibitor of cystathionine ⁇ -synthase, a pyridoxal phosphatedependent enzyme involved in microbial methionine synthesis.
  • the growth inhibitory effects of propargylglycine are reversed by methionine.
  • 375 ⁇ M propargylglycine was required to inhibit Klebsiella growth by 50 percent.
  • Combining as little as 0.1 ⁇ M TFMTR with 20 ⁇ M 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.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
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Abstract

Pharmaceutical compositions are described comprising methylthioribose (MTR) kinase inhibitors (e.g. trifluoromethylthioribose) and inhibitors of de novo methionine synthesis (e.g. 1,2,4-triazole, azaserine or propargylglycine).

Description

MEDICAMENTS
This invention relates to pharmaceutical compositions comprising a methylthioribose (MTR) kinase inhibitor and an inhibitor of de 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 trifluoromethionine or
carbonothioic difluoride (Paras, tology Today, 5 (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 sulfhydrylase. 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, unlike 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 de novo methionine synthesis. The synthesis of methionine and the methionine salvage pathway is summarised in the following scheme.
Figure imgf000004_0001
This invention is based upon the discovery that
inhibitors of de novo methionine synthesis act in synergy with MTR-kinase inhibitors to inhibit the growth of MTR-kinase containing microorganisms. Inhibition of de 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) :
Figure imgf000005_0001
wherein R is H, CI, F, Br, I or R1S in which R1 is C1-10 linear or branched chain alkyl or halogenated linear or branched chain alkyl and R2 to R4 are H or OH with the proviso that at least one of R2 to R4 is OH.
A preferred MTR-kinase inhibitor is TFMTR.
Preferred inhibitors of de 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 1,2,4-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 de 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 in 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 of 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 de 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 de 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, Enterpbacter
aerogenes, E. sakazaki and E. cloacae, Serratia marcescans, Proteus vulgaris, Yersinia ssp, Morganella ssp and Erwini a ssp.
MTR-kinase-containing parasitic protozoa do not
synthesize methionine de 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 Plasmodium falciparum
(responsible for the most deadly form of malaria), Giardia Iambl i a and Ochromonas 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 Org. Syn. 40 : 99, 1960 and
J. Am. Chem. 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. K. pneumoniae was maintained in a chemically-defined medium containing: 25mM NH4CI; 35mM glucose; 1.5mM KCl; 0.4mM MgSO4; 0.045mM NaCl; 0.025mM FeSO4,- 0.025 μg/ml thiamine; and 66.6mM Na2HPO4-NaH2PO4. The following micronutrient solution was added with the indicated final concentrations : CaCl2 (5 x 10-7M), CoCl2 (5 × 10-8M), MnCl2 (10-7M), HBO3 (5 × 10-7M), ZnCl2 (10-8M), CuCO3 (10-8M), (NH4)6Mo7O24 (5 × 10_9M), and the pH was adjusted to 7.2.
Dose inhibition studies were conducted in 5ml cultures inoculated with ~104 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. K. pneumoniae was cultured in defined medium containing iμM TFMTR and varying amounts of methionine. The organisms were inoculated at a density of ~104/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 by 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 K. 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 K. 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 0.1μM 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 pneumoniae. 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.

Claims

Claims :
1. A pharmaceutical composition comprising an MTR-kinase inhibitor, an inhibitor of de novo methionine synthes and a pharmaceutically-acceptable carrier.
2. A composition according to claim 1 wherein the MT kinase inhibitor is a compound of the formula (1) :
Figure imgf000013_0001
wherein R is H, CI, F, Br, I or R1S in which R1 is C1-10 linear or branched chain alkyl or halogenated linear or branched chain alkyl and R2 to R4 are H or OH with the provis that at least one of R2 to R4 is OH.
3. A composition according to claim 2 wherein the MT kinase inhibitor is TFMTR.
4. A composition according to any one of claims 1 to wherein the inhibitor of de novo methionine synthesis is an O-acetylserine sulfhydrylase inhibitor.
5. A composition according to claim 4 wherein the O-acetylserine sulfhydrylase inhibitor is 1,2,4-triazole or azaserine.
6. A composition according to any one of claims 1 to wherein the inhibitor of de novo methionine synthesis is a cystathionine γ-synthase inhibitor.
7. A composition according to claim 6 wherein the cystathionine γ-synthase inhibitor is propargylglycine.
8. 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 de novo methionine synthesis.
9. A method according to claim 8 wherein the MTR- kinase inhibitor is a compound of the formula (1) :
Figure imgf000014_0001
wherein R is H, CI, F, Br, I or R1S in which R1 is C1-10 linear or branched chain alkyl or halogenated linear or branched chain alkyl and R2 to R4 are H or OH with the provis that at least one of R2 to R4 is OH.
10. A method according to claim 9 wherein the MTR- kinase inhibitor is TFMTR.
11. A method according to any one of claims 8 to 10 wherein the inhibitor of de novo methionine synthesis is an O-acetylserine sulfhydrylase inhibitor.
12. A method according to claim 11 wherein the O-acetylserine sulfhydrylase inhibitor is 1,2,4-triazole or azaserine.
13. A method according to any one of claims 8 to 10 wherein the inhibitor of de novo methionine synthesis is a cystathionine γ-synthase inhibitor.
14. A method according to claim 13 wherein the
cystathionine γ-synthase inhibitor is propargylglycine.
15. 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.
16. A method according to claim 15 wherein the MTR-kinase inhibitor is a compound of the formula (1) :
Figure imgf000015_0001
wherein R is H, CI, F, Br, I or R1S in which R1 is C1-10 linear or branched chain alkyl or halogenated linear or branched chain alkyl and R2 to R4 are H or OH with the proviso that at least one of R2 to R4 is OH.
17. A method according to claim 16 wherein the MTR-kinase inhibitor is TFMTR.
18. A method according to any one of claims 15 to 17 wherein the methionine synthase inhibitor is nitrous oxide.
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WO2010010383A1 (en) * 2008-07-21 2010-01-28 Isis Innovation Limited Treatment of obesity

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