HK1036222B - Enhancement of oxazolidinone antibacterial agents activity by using arginine derivatives - Google Patents

Description

Use of arginine derivatives to enhance oxazolidinone antibacterial agents

Technical Field

The present invention relates to methods and compositions for enhancing the anti-gram-negative biological efficacy of oxazolidinone antibacterial agents using an arginine derivative.

Background

Oxazolidinone antibacterial agents are a novel class of synthetic antimicrobials with potent activity against a variety of human and animal pathogens, including gram positive aerobic bacteria such as multiply tolerant staphylococci and streptococci, anaerobic organisms such as bacteroides and clostridia species, and acid resistant organisms such as mycobacterium tuberculosis and mycobacterium avium. In particular, oxazolidinone compounds of structures I-V have been found to be particularly effective.

However, certain oxazolidinones are generally poorly active against aerobic gram-negative organisms, such as Escherichia coli, Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, or Klebsiella pneumoniae, at useful concentrations. Thus, the use of these oxazolidinone antibacterial agents alone is limited to the state of infection caused by gram-positive bacteria. It is therefore an object of the present invention to provide a method for enhancing the bacteriostatic profile of oxazolidinones. We have now found that when an oxazolidinone antibacterial agent is administered with an arginine derivative, there is a significant synergistic effect against gram negative organisms. The effective amount of oxazolidinone antibacterial agent that is fully effective against aerobic gram-negative organisms is much less than the amount required when the oxazolidinone is not administered with these arginine derivatives.

Disclosure of information

International patent publication WO96/33285 discloses methods for screening microbial efflux pump inhibitors, including those that export antibiotics. The screening method is based on the intracellular concentration of a compound, such as an antibiotic or dye, when the bacterial cell is contacted with an efflux pump inhibitor. In addition, the invention provides pharmaceutical compositions containing such outward flux pump inhibitors, including certain arginine derivatives, and methods for treating microbial infections and enhancing the antimicrobial activity of certain antimicrobial agents.

The 36 th ICAAC abstract proposed by Pharmacia and Upjohn, inc. discloses that mutation of the AcrAB antibiotic efflux pump in escherichia coli brings sensitivity to oxazolidinone antibacterial agents.

Summary of The Invention

The present invention provides a method and composition for treating gram-negative biological infections in mammals comprising administering an effective amount of an oxazolidinone antibacterial agent and an arginine derivative of formula A or a pharmaceutically acceptable salt,

wherein

R₁Is that

a) Aryl, optionally substituted by C_1-4Alkyl radical, C_1-4Alkoxy radical, C_1-4Alkylthio, halogen or-NH₂The substitution is carried out by the following steps,

b)-(CH₂)_iaryl, wherein aryl is substituted by C_1-4Alkyl radical, C_1-4Alkoxy radical, C_1-4Alkylthio, halogen or-NH₂The substitution is carried out by the following steps,

c) thienyl, furyl, pyridyl, benzofuryl or benzothienyl;

z is R₂or-CHWR₂；

R₂Is that

a) Aryl, optionally substituted by one or two C_1-4Alkyl radical, C_1-4Alkoxy radical, C_1-4Alkylthio, halogen, -NH₂、C_1-4Alkylamino radical, C_1-4Dialkylamino or-NHOH, or a substituted,

b)C_1-4alkyl, optionally substituted with fluorine atoms,

c)C_1-4alkoxy radical, C_1-4An alkylthio group is a group of one or more,

d) the halogen(s) are selected from the group consisting of,

e) thienyl, furyl or pyridyl;

w is H, -NH₂、C_1-4Alkylamino radical, C_1-4Dialkylamino, halogen, hydroxy, C_1-4Alkoxy, alkylthio or nitrogen heterocycle;

aryl is phenyl or naphthyl;

the azacyclo is n-morpholinyl, n-piperazinyl, n-pyrrolidinyl, n-imidazolyl, n-pyrrolyl, n-pyrazolyl, n-triazolyl or n-tetrazolyl;

i is 0, 1 or 2.

Detailed description of the invention

The present invention teaches that oxazolidinones are effective against aerobic gram negative organisms such as escherichia coli, haemophilus influenzae, moraxella catarrhalis, pseudomonas aeruginosa or klebsiella pneumoniae, as well as gram positive aerobic organisms such as multi-tolerant staphylococci and streptococci, anaerobic organisms such as bacteroides and clostridia species, and acid resistant organisms such as mycobacterium tuberculosis and mycobacterium avium when the oxazolidinone antibacterial agent is administered with an arginine derivative. The effective amount of oxazolidinone antibacterial agent that is fully effective against aerobic gram-negative organisms is much less than the amount required when the oxazolidinone is not administered with these arginine derivatives.

For the purposes of the present invention, the term "C_1-4Alkyl "refers to alkyl groups having one to four carbon atoms, such as methyl, ethyl, propyl, butyl, and their isomeric forms.

The term "C_1-4Alkoxy "refers to an alkyl group having one to four carbon atoms attached to one hydroxyl oxygen atom, such as methoxy, ethoxy, propoxy, butoxy, and the isomeric forms thereof.

The term "C_1-4Alkylthio "refers to alkyl groups having one to four carbon atoms attached to one sulfur atom and their isomeric forms.

The term "C_1-4Alkylamino "refers to an alkyl group having one to four carbon atoms attached to an amino moiety, such as methylamino, ethylamino, n-propylamino, n-butylamino, and their isomeric forms.

The term "C_1-4Dialkylamino "refers to two alkyl groups having one to four carbon atoms attached to an amino moiety, such as dimethylamino, methylethylamino, diethylamino, dipropylamino, methylpropylamino, ethylpropylamino, dibutylamino and the isomeric forms thereof.

The term "halogen" means fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine.

The term "aryl" refers to phenyl or naphthyl.

The term "azacyclo" refers to n-morpholinyl, n-piperazinyl, n-pyrrolidinyl, n-imidazolyl, n-pyrrolyl, n-pyrazolyl, n-triazolyl or n-tetrazolyl.

The term "pharmaceutically acceptable salt" refers to salts that may be used to administer the compounds of the present invention, including hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, acetate, propionate, lactate, methanesulfonate, maleate, malate, succinate, tartrate, citrate, 2-hydroxyethanesulfonate, fumarate, and the like. These salts may be in hydrated form.

The term "mammal" refers to an animal in the human or veterinary sense.

Oxazolidinone antibacterial agents refer to compounds of formula B

Wherein R is₁Is methyl, ethyl, cyclopropyl or dichloromethyl; r₂Is hydrogen or fluorine; het is a 6-membered saturated heterocyclic moiety having one to two atoms selected from the group consisting of sulfur, nitrogen and oxygen atoms. Alternatively, the nitrogen atom of the heterocyclic ring may be substituted with a suitable group, such as a hydroxyacetyl group, and the sulfur atom may be oxidized. In addition, the compounds of formula Y include all possible stereoisomers and geometric forms. Preferably, the oxazolidinone antibacterial agent is a compound of formula I-V as described above.

There are numerous references in the art that disclose various oxazolidinone derivatives and methods for their preparation. The oxazolidinone antibacterial agents described above may be prepared according to the procedures described in U.S. Pat. Nos. 5652238 and 5688792, International patent publications WO93/23384, WO97/09328 and WO98/54161, which are incorporated herein by reference.

Arginine derivatives of formula a are known, readily available or can be prepared by synthetic chemical methods known to those skilled in the art. Preferably, the arginine derivative of formula a is L-phenylalanyl-L-arginyl- β -naphthylamide.

The pharmaceutical compositions of the present invention comprise an oxazolidinone antibacterial agent and an arginine derivative of formula a, together with one or more solid or liquid pharmaceutically acceptable carriers and optionally pharmaceutically acceptable adjuvants and excipients. Solid form compositions include powders, tablets, dispersible granules, capsules and suppositories. A solid carrier can be at least one substance that also acts as a diluent, flavoring agent, solubilizer, lubricant, suspending agent, binder, tablet disintegrant, and encapsulating agent. Inert solid carriers include magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, cellulosic materials, low melting waxes, cocoa butter, and the like. Liquid form compositions include solutions, suspensions, and emulsions. For example, solutions of the compounds of the present invention dissolved in water, water-propylene glycol, and water-polyethylene glycol systems may be provided, optionally with conventional coloring agents, flavoring agents, stabilizing agents, and thickening agents.

The pharmaceutical composition is provided using conventional processes. Preferably, the compositions are in unit dosage form containing an effective amount of the oxazolidinone antibacterial agent, i.e., a compound of formula B.

The amount of oxazolidinone antibacterial agent in the pharmaceutical compositions and unit dosage forms thereof can vary or can be widely adjusted depending upon the particular method of administration, the potency of the particular compound, the condition being treated and the desired concentration. Generally, the amount of oxazolidinone antibacterial agent will range from 0.5% to 90% by weight of the total composition.

In therapeutic use for the treatment of bacterial infections in humans and other animals that have been diagnosed with aerobic gram-negative biological infections, the oxazolidinone antibacterial agent and arginine derivative or the pharmaceutical composition of the invention will be administered orally, parenterally, transdermally and/or topically at doses that achieve and maintain the concentration, i.e., amount, or blood level of the active ingredient in the mammal being treated will be antibacterially effective. The preferred mode of administration is oral. Generally, the dosage of such an antibacterially effective amount of an active oxazolidinone antibacterial agent will be in the range of from about 0.1 to about 100mg/kg normal body weight, more preferably from about 3.0 to about 50mg/kg body weight per day. It is understood that the dosage may vary depending on the needs of the patient, the severity of the bacterial infection being treated and the particular compound used. It is also understood that the initial dose may exceed the above upper limits in order to reach the desired blood level quickly, or the initial dose may be less than optimal, and that the daily dose may be gradually increased as the case may be during the course of treatment. If desired, the daily dose may also be divided into multiple doses, for example two to four times daily.

The amount of the arginine derivative of formula a used varies with the activity of the particular arginine derivative being increased and the absorption by the organism being treated. Sufficient arginine derivatives should be used to sensitize aerobic gram-negative organisms to pharmaceutically acceptable levels of oxazolidinone antibacterial agents in the mammal being treated. Sufficient quantities of a particular arginine derivative can be determined simply as follows: the Minimum Inhibitory Concentration (MIC) of the oxazolidinone antibacterial agent was tested and the MIC of the antibacterial agent alone was compared to the MIC of the combination of the antibacterial agent and the arginine derivative. In general, the molar ratio of arginine derivative to oxazolidinone antibacterial agent administered may be from about 0.01 to 10, preferably from about 0.1 to 1.0. Thus, the daily dosage of arginine derivatives for enhancing the efficacy of oxazolidinone antibacterial agents against aerobic gram-negative organisms in mammals may be from about 0.01 to 100mg/kg of normal body weight, preferably from about 0.3 to 50mg/kg of body weight. The arginine derivative may be administered one to four hours prior to, or simultaneously with, the oxazolidinone antibacterial agent.

Biological assay

The potentiation of the activity of oxazolidinone antibacterial agents against aerobic gram-negative organisms when combined with an arginine derivative of formula a two assay procedures were used:

a) a conventional checkerboard method, and

b) silicone oil centrifugation for quantification of radiolabeled oxazolidinone antibacterial agent in escherichia coli.

I. The Fractional Inhibitory Concentration (FIC) index was determined using the checkerboard method.

The "checkerboard" method is the most commonly used in vitro technique for assessing antimicrobial combinations (Lorian, V., editor. antibiotics in Laboratory Medicine, Third Edition, p.432, Williams & Wilkins. Baltimore, Maryland 21202, USA). In the microdilution method, a checkerboard pattern is formed in the wells of a microtiter plate containing multiple 2-fold dilutions of the test agent. The test dilutions span a range of concentrations above and below the Minimum Inhibitory Concentration (MIC) for each test agent used in the test organism. The response (growth or no growth) at each experimental ratio was used to calculate the Fractional Inhibitory Concentration (FIC) index. Drug-drug interactions are defined as additive effects when the results obtained with two drugs together are equal to the sum of the results obtained with the two drugs separately (FIC index 1.0). When the results for both drugs were significantly less than additive responses (FIC index > 1.0), the interaction was described as antagonistic. When the results of the two drug combinations were significantly greater than the additive response (FIC index < 0.50), the interaction was described as synergistic.

Quantification of radiolabeled oxazolidinone antibacterial agent of formula I

Accumulation in Escherichia coli following pretreatment of bacterial cells with arginine derivatives of formula A

Accumulation of radiolabeled oxazolidinone antibacterial agent of formula I in escherichia coli was measured according to Thanassi, d.g., g.s.b.suh, h.nikaido journal of bacteriology 1995, p.177, (4): 998 + 1007. Briefly, cells were grown to mid-log phase (OD) in LB/0.2% glucose at 37 ℃₅₃₀0.5-0.7), harvested by centrifugation, washed twice, again at 50mM potassium phosphate pH7.0, 1mM MgSO₄And 0.2% glucose to OD₅₃₀It was 8.0. A1.0 ml aliquot of the cell suspension was pre-incubated at 37 ℃ for 10 minutes before addition of the arginine derivative. Carbonyl Cyanometachlorophenylhydrazone (CCCP) was used as a positive control. After addition of the arginine derivative, L-phenylalanyl-L-arginyl-beta-naphthylamide, the cells were cultured for 30 minutes, followed by addition of the radiolabeled oxazolidinone antibacterial agent of formula I to a final concentration of 25 TM. The cells were then incubated for a further 15 minutes. A 50Tl aliquot was removed and layered on a 300Tl silicone oil pad (70% fluid No. 550 and 30% fluid No. 510 silicone oil, dow corning corp.midland, MI). Placing the test tube inCentrifuging at 12000rpm at 22 deg.C for 3 min, and soaking in solution N₂Freezing is carried out. The top of each tube containing the cell particles was removed and placed in a scintillation vial. After thawing, the cell particles were suspended in 200Tl distilled water and 4ml scintillation fluid was added. The samples were mixed well and counted in a liquid scintillation counter. To correct for non-specific adhesion of the labeled drug to the cell surface, control experiments were performed with cells cultured with only the addition of the carrier and radiolabeled oxazolidinone antibacterial agent of formula I throughout.

Results III

The oxazolidinone antibacterial agent alone demonstrated very poor antibacterial activity, requiring a concentration of 256mg/ml to inhibit escherichia coli 31700. The antibacterial activity was also demonstrated to be very poor with L-phenylalanyl-L-arginyl-beta-naphthylamide alone, with no growth inhibition up to concentrations of 256 mg/ml. However, when these two agents are used in combination in a "checkerboard" pattern, the activity of the oxazolidinone antibacterial agent of formula I is dramatically demonstrated to be potentiated by L-phenylalanyl-L-arginyl- β -naphthylamide. For example, the inhibitory concentration of the oxazolidinone antibacterial agent of formula I against bacterial growth in the presence of 16mg/ml L-phenylalanyl-L-arginyl- β -naphthylamide is 16 mg/ml. The calculated FIC index is 0.23, apparently indicating synergistic interaction.

Claims (19)

1. Use of an oxazolidinone antibacterial agent and a compound of formula a, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of an infection by a gram-negative organism in a mammal,

wherein

R₁Is that

a) Aryl, optionally substituted by C_1-4Alkyl radical, C_1-4Alkoxy radical, C_1-4Alkylthio, halogenElement or-NH₂Is substituted, or

b)-(CH₂)_iAryl, wherein aryl is substituted by C_1-4Alkyl radical, C_1-4Alkoxy radical, C_1-4Alkylthio, halogen or-NH₂Is substituted, or

c) Thienyl, furyl, pyridyl, benzofuryl or benzothienyl;

z is R₂or-CHWR₂；

R₂Is that

a) Aryl, optionally substituted by one or two C_1-4Alkyl radical, C_1-4Alkoxy radical, C_1-4Alkylthio, halogen, -NH₂、C_1-4Alkylamino radical, C_1-4Dialkylamino or-NHOH, or

b)C_1-4Alkyl, optionally substituted by fluorine atoms, or

c)C_1-4Alkoxy radical, C_1-4Alkylthio, or

d) Halogen, or

e) Thienyl, furyl or pyridyl;

w is H, -NH₂、C_1-4Alkylamino radical, C_1-4Dialkylamino, halogen, hydroxy, C_1-4Alkoxy radical, C_1-4An alkylthio or an azacyclic group;

aryl is phenyl or naphthyl;

the nitrogen heterocycle is n-morpholinyl, n-piperazinyl, n-pyrrolidinyl, n-imidazolyl, n-pyrrolyl, n-pyrazolyl, n-triazolyl or n-tetrazolyl;

i is 0, 1 or 2.

2. The use of claim 1 wherein the oxazolidinone antibacterial agent is a compound of structure B

Wherein R is₁Is methyl, ethyl, cyclopropyl or dichloromethyl;

R₂is hydrogen or fluorine;

het is a 6-membered saturated heterocyclic moiety having one to two atoms selected from the group consisting of sulfur, nitrogen and oxygen atoms.

3. The use of claim 2 wherein the oxazolidinone antibacterial agent is a compound of structure I, II, III, IV or V

4. The use of claim 2 wherein the oxazolidinone antibacterial agent is a compound of structure II

5. The use of claim 1, wherein the compound of formula a is L-phenylalanyl-L-arginyl- β -naphthylamide.

6. Use according to claim 1 wherein the ratio of oxazolidinone antibacterial agent to compound of formula a is 10: 0.01.

7. Use according to claim 1 wherein the ratio of oxazolidinone antibacterial agent to compound of formula a is 1: 1.

8. The use of claim 1 wherein the effective amount of the oxazolidinone antibacterial agent is 0.1 to 100mg/kg body weight/day.

9. The use of claim 1 wherein the effective amount of the oxazolidinone antibacterial agent is from 3 to 50mg/kg body weight/day.

10. The use of claim 1, wherein the amount of the compound of formula a is 0.01 to 100mg/kg body weight/day.

11. The use of claim 1, wherein the amount of the compound of formula a is 0.3 to 50mg/kg body weight/day.

12. The use of claim 1 wherein the oxazolidinone antibacterial agent and the compound of formula a are administered simultaneously.

13. The use of claim 1 wherein the compound of formula a is administered one to four hours prior to the administration of the oxazolidinone antibacterial agent.

14. The use of claim 1 wherein the effective amount of the oxazolidinone antibacterial agent and the compound of formula a are administered orally, parenterally, transdermally or topically.

15. The use of claim 1, wherein the gram-negative organism is an aerobic gram-negative organism.

16. The use of claim 15, wherein the aerobic gram-negative organism is escherichia coli, haemophilus influenzae, moraxella catarrhalis, pseudomonas aeruginosa or klebsiella pneumoniae.

17. A composition for the treatment of a gram-negative biological infection in a mammal comprising an oxazolidinone antibacterial agent of formula B as defined in claim 2, a compound of formula a as defined in claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

18. The composition of claim 17 wherein the oxazolidinone antibacterial agent is a compound of structure I, II, III, IV or V

19. The composition of claim 17, wherein the compound of formula a is L-phenylalanyl-L-arginyl- β -naphthalamide.