MXPA00011359A - Enhancement of oxazolidinone antibacterial agents activity by using arginine derivatives - Google Patents

Abstract

The present invention provides methods and compositions for enhancing the effectiveness of oxazolidinone antibacerial agents against gram-negative organisms infection by using an arginine derivative.

Description

IMPROVEMENT OF THE ACTIVITY OF ANTIBACTERIAL AGENTS OF OXAZOLIDINONE THROUGH THE USE OF ARGININE DERIVATIVES FIELD OF THE INVENTION The present invention relates to methods and compositions for improving the effectiveness of the antibacterial agents of oxazolidinone against gram-negative organisms, by the use of an arginine derivative.

BACKGROUND OF THE INVENTION Oxazolidinone antibacterial agents are a new synthetic class of anti-microbial agents with potent activity against a number of human and veterinary pathogens, including gram-positive aerobic bacteria such as staphylococci and multi-resistant streptococci, anaerobic organisms such as bacteroids and species. of clostridia, and acid-fast organisms such as Mycobacterium tuberculosis and MycoJbacterium avi m. In particular, the oxazolidinone compounds of structures I-V have been found to be especially effective.

However, certain oxazolidinones generally have a poor activity at a useful level against aerobic gram-negative organisms such as E. Coli, Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, or Klebsiella pneumoniae. Thus, the use of these oxazolidinone antibacterial agents is limited only to infectious states due to gram-positive bacteria. Accordingly, it is among the objects of the present invention to provide a process to improve the inhibitory spectrum of oxazolidmones. Now, it has been discovered that when the oxazolidinone antibacterial agents are administered with an argimine derivative, a pronounced smergistic effect against gram-negative organisms is produced. The effective amount of an antibacterial agent of oxazolidmone that will be completely effective against gram-negative aerobic organisms is much less than would be needed if an oxazolidinone were administered without these arginine derivatives.

DESCRIPTION OF INFORMATION International publication number WO 96/33 85 describes methods for detecting inhibitors of microbial exit pumps, including those that export antibiotics. Detection methods are based on increasing the intracellular concentration of a compound, such as an antibiotic or a dye, when the bacterial cells are contacted with an exit pump inhibitor. In addition, this invention provides pharmaceutical compositions containing exit pump inhibitors, including certain arginine derivatives, and methods for treating microbial infections and improving the antimicrobial activity of certain antimicrobial agents.

The extract of the 36th ICAAC, presented by Pharmacia and Upjohn, Inc., describes the mutation of the antibiotic exit pump AcrAB in E. Coll confers susceptibility to oxazolídmone antibacterial agents.

SUMMARY OF THE INVENTION The present invention features a method and composition for treating infections by gram-negative organisms in mammals, which comprises the administration of an effective amount of an antibacterial agent of oxazolidmone and an argimine derivative of formula A wherein i is a) aryl, optionally substituted with alkyl of 1 to carbon atoms, alkoxy of 1 to carbon atoms, alkylthio of 1 to carbon atoms, halo, or -NH2, b) - (CH2), -ar? Lo, in which aplo is substituted with alkyl of 1 to carbon atoms, alkoxy from 1 to carbon atoms, alkylthio having 1 to 4 carbon atoms, halo, or -NH 2, c) thienyl, fuplo, pipdoyl, benzofuranyl, or benzothienyl; Z is R2, or -CHWR2; R2 is a) aryl, optionally substituted with one or two alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, alkylthio of 1 to 4 carbon atoms, halo, -NH2, alkylammon of 1 to 4 atoms carbon, dialkylammon of 1 to 4 carbon atoms, or -NHOH, b) alkyl of 1 to 4 carbon atoms, optionally substituted by fluoro, c) alkoxy of 1 to 4 carbon atoms, alkylthio of 1 to 4 carbon atoms carbon, d) halo, e) thienyl, furanyl, or pyridyl; W is H, -NH2, alkylammon of 1 to 4 carbon atoms, dialkylammon of 1 to 4 carbon atoms, halo, hydroxy, alkoxy of 1 to 4 carbon atoms, alkylthio or azaheterocycle; aryl is phenyl or naphthyl, azaheterocycle is n-morpholyl, n-piperazyl, n-pyrrolidyl, n-imidazolyl, n-pyrrolyl, n-pyrazolyl, n-tpazolyl, or n-tetrazolyl; i is O, 1 or 2, and pharmaceutically acceptable salts DETAILED DESCRIPTION OF THE INVENTION The present invention teaches that when oxazolidinone antibacterial agents are administered with an arginine derivative, the oxazolidinones are effective against aerobic gram-negative organisms such as E. Coll, Hae ophilus influenzae, Moraxella catarrhalis, Pseudomonas aerugmosa, or Klebsiella pneumomae, as well as gram-positive aerobic bacteria such as multiresistant staphylococci and streptococci, anaerobic organisms such as bacteroids and clostpid species, and acid-fast organisms such as Mycobacterium tuberculosis and Mycobacterium avium. The effective amount of an oxazolidinone antibacterial agent that will be completely effective against aerobic gram-negative organisms is much less than would be required if an oxazolidmone were administered without these argmam derivatives. For the purpose of the present invention, the term "C 1-4 alkyl" refers to an alkyl group having one to four carbon atoms such as, methyl, ethyl, propyl, butyl, and their isomeric forms thereof. The term "1-alkoxy" 4 carbon atoms "refers to an alkyl group having from one to four atoms of carbon bonded to an oxygen atom of the hydroxyl group, such as, methoxy, ethoxy, propyloxy, butyloxy, and their isomeric forms thereof The term "alkylthio of 1 to 4 carbon atoms" refers to an alkyl group having from one to four carbon atoms and isomeric forms thereof bound to a sulfur atom. The term "alkyl of 1 to 4 carbon atoms" refers to an alkyl group having one to four carbon atoms attached to an amino moiety; for example, methylamine, ethylamino, n-propylammon, n-butylamino and isomeric forms thereof. The term "dialkylamino of 1 to 4 carbon atoms" refers to two alkyl groups having one to four carbon atoms attached to an ammo moiety, for example, dimethylamine, methylethylamm; diethylamm, dipropylamino, methylpropylammo, ethylpropylamino, dibutylamino and isomeric forms thereof. The term "halo" refers to fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine. The term "aryl" refers to phenyl or naphthyl; The term "azaheterocycle" refers to n-morpholyl, n-piperazimyl, n-pyrrolidyl, n-ylidazolyl, n-pyrrolyl, n-pyrazolyl, n-tpazolyl, or n-tetrazolyl, The term "pharmaceutically acceptable salts" refers to salts useful for administering the compounds of this invention and include hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, acetate, propionate, lactate, mesylate, maleate, malate, succrate, tartrate, citrate, and the like. -hydroxyethyl sulfonate, fumarate and the like. These salts may be in the hydrated form. The term "mammal" refers to a man or animals of veterinary interest. Oxazolidinone antibacterial agents refer to compounds of formula B wherein R_ is methyl, ethyl, cyclopropyl or dichloromethyl; R2 is hydrogen or fluorine; Het is a saturated 6-membered heterocyclic moiety having one to two atoms selected from the group consisting of sulfur, nitrogen and oxygen. Optionally, the nitrogen atom of the heterocyclic may be substituted by a suitable group such as hydroxyacetyl, and the sulfur atom may be oxidized. In addition, the compounds of the formula Y encompass all possible stereoisomers and geometric shapes. Preferably, the oxazolidone antibacterial agents are the compounds of the formulas I-V as defined above. There are numerous references in the art describing a variety of oxazolidone derivatives and the methods for making them. The oxazolydamone antibacterial agents described above can be made according to the procedures described in U.S. Patent Nos. 5,652,238 and 5,688., 792, International Publications Nos. WO 93/23384, WO 97/09328 and WO 98/54161, incorporated herein by reference. The arginine derivatives of formula A are known and readily available or can be prepared by synthetic chemistry methods known to those skilled in the art. Preferably, an argimine derivative of formula A is L-phenylalanyl-L-arginyl-β-naphthylamide. The pharmaceutical compositions of this invention comprise an antibacterial agent of oxazolidmone and an arginine derivative of formula A, together with one or more pharmaceutically acceptable carriers, solid or liquid and optionally pharmaceutically acceptable adjuvants and excipients. Compositions in solid form include powders, tablets, dispersible granules, capsules and suppositories. A solid carrier can be at least one substance that can also function as a diluent, sabotage agent, solubilizer, lubricant, dispersing agent, binder, tablet disintegrating agent, and encapsulating agent. Inert solid carriers include magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextpna, starch, gelatin, cellulosic materials, ba wax or melting point, cocoa butter and the like. Liquid form compositions include solutions, suspensions and emulsions. For example, solutions of the compounds of this invention dissolved in water, water-propylene glycol and water-polyethylene glycol systems, optionally containing conventional coloring agents, sabotagers, stabilizers and thickening agents, can be provided. The pharmaceutical compositions are provided by employing conventional techniques. Preferably, the compositions are in the unit dose form containing an effective amount of an antibacterial agent of oxazolidmone, of the compounds of formula B. The amount of an antibacterial agent of oxazolidmone in the pharmaceutical composition and the dosage form unit of the same can be varied or adjusted Widely depending on the particular application method, the potency of the particular compound, the condition to be treated and the desired concentration. In general, the amount of an antibacterial agent of oxazolidmone will vary between 0 5% to 90% by weight of the total composition. In the therapeutic use for treating bacterial infections in humans and other animals that have been diagnosed with infections by aerobic gram-negative organisms, the antibacterial agent of oxazolidmone and the arginine derivatives and pharmaceutical compositions of the present invention thereof will be administered from oral, parenteral, transdermal and / or topical form at a dose to obtain and maintain a concentration, that is, an amount, or blood level of the active component in the animal that is subjected to the treatment that will be effective in an antibacterial manner. The preferred form of administration is oral In general, the antibacterially effective dose amount of the oxazolidone active antibacterial agent will be in the range of about 0 1 to about 100 mg / kg of normal body weight, more preferably about 3.0 to approximately 50 mg / kg of body weight / day. It should be understood that the doses may vary depending on the requirements of the patient, the severity of the bacterial infection that is treat, and the particular compounds that are used. Also, it should be understood that the individual dose administered may be increased beyond the previous higher level in order to quickly achieve the desired blood level, or the initial dose may be smaller than optimal, and the daily dose may be progressively increased by the course of treatment depending on the particular situation. If desired, the daily dose can also be divided into multiple doses for administration, for example, two to four times per day. The amount of an argimine derivative of the formula A to be used varies with the improvement of the activity of the particular argimine derivative and its absorption by the organism in question. Sufficient amounts of the arginine derivatives must be shown to render the aerobic organisms gram-negative susceptible to a pharmaceutically acceptable level of an antibacterial agent of oxazolidmone in the mammals to be treated. Sufficient amount of a particular argmam derivative can be determined simply by testing the minimum inhibitory concentration (MIC) of an antibacterial agent of oxazolidmone, and comparing the MIC of that antibactepane agent alone, with the MIC of that antibacterial agent used in combination with the derivative of arginma. In general, the molar ratio of a derivative of Arginine to the oxazolidinone antibacterial agent that is administered may be from about 0.01 to 10, preferably from about 0.1 to 1.0. Therefore, the daily dose of * an arginine derivative to improve the activity of oxazolidinone antibacterial agents against aerobic gram-negative organisms in mammals can vary from about 0.01 to 100 mg / kg of normal body weight, preferably in a amount of about 0.3 to 50 mg / kg of body weight. The arginine derivatives can be administered one to four hours before the oxazolidinone antibacterial agents are administered, or administered simultaneously with the oxazolidinone antibacterial agents.

BIOLOGICAL TEST The potentiation of the activity of oxazolidinone antibacterial agents against aerobic gram-negative organisms when combined with an arginine derivative of formula A uses two titration techniques: a) conventional board technique, and b) centrifugation method with silicone oil to quantitate an radiolabeled antibacterial agent of oxazolidinone in E. coli.

I. Determination of fractional inhibitory concentration index (FIC) by using the board technique. The "dashboard" method is the most commonly used technique to assess antimicrobial combinations (Lorian, V., editor, Antibiotics in Laboratory Medicine, Third Edition, p 432, Williams &Wilkms, Baltimore, Maryland 21201, USA). In the microdilution method, a board pattern is formed in the microtiter plate cavities containing multiple dilutions at twice the two agents tested. The test dilutions encompass a range of concentrations that is above and below the minimum inhibitory concentration (MIC) of each test agent for the test organism. The response to each test relationship (growth or non-growth) is used to calculate the Fractional Inhibitory Concentration (FIC) index. The drug-drug interaction is defined as an additive when the result for the two drugs together is equal to the sum of the results for each of the two drugs used separately (FIC index = 1.0). The interaction is described as Antagonism when the result for the two drugs is significantly lower than the additive response (FIC index> 1.0). The interaction is described as synergistic when the result for the two drugs combined is significantly greater than the Additive response (FIC index £ 0.50).

II. Quantification of a Radiolabeled Antibacterial Agent of Oxazolidmone of Formula I Accumulation in E. Coll. After Pre-Treatment of Bacterial Cells with an Arginine Derivative of Formula A. The measurement of the accumulation of the radiolabelled antibacterial agent of oxazolidone of formula 1 in E. Coli is carried out by the procedures described by Thanassi, DG , GSB Suh, H. Nikaido, J. Bactepology, 1995, p. 177, (4): 998-1007. Briefly, cells were cultured at log (OD530 0.5-0.7) medium in 0.2% LB-glucose at 37 ° C, harvested by centrifugation, washed twice and redispersed at an OD530 of 8.0 in potassium phosphate 50 mM pH 7.0, 1 mM MgSO4 and 0.2% glucose. 1.0 ml aliquots of the cell suspension were preincubated at 37 ° C for 10 minutes before the addition of an arginine derivative. Carbonyl-cyanide-m-chlorophenylhydrazone (CCCP) was used as the positive control. After the addition of an argimine derivative, L-phenylalanyl-L-argmil-β-naphthylamide, the cells were incubated for 30 minutes before addition of the agent radiolabeled antibactepane of oxazolidinone of formula I at a final concentration of 25 TM. The cells were then incubated for an additional 15 minutes. An aliquot of 50 TI was removed and stratified in a 300 TI silicone oil pad (70% fluid number 70% and 510 fluid number 30% silicone oil), Dow Corning Corp. Midland, MI). The tubes were centrifuged at 12,000 rpm for 3 minutes, 22 ° C, then frozen by immersion in liquid N2. The tips of each tube containing the cell pellets were cut and placed in scintillation flasks. After thawing, the cell pellets were dispersed in 200 TI distilled water and 4 ml of scintillation fluid was added. The samples were mixed well and counted in a liquid scintillation counter. To correct non-specific adhesion of the labeled drug to the cell surface, a control experiment was performed on cells completely incubated with only the addition of vehicle and radiolabeled antibacterial agent of oxazolidinone of the formula I.

III. Results The oxazolidone antibacterial agent of formula I alone showed very poor antibacterial activity, requiring a concentration of 256 mg / ml to inhibit E.

Coli 31700. L-phenylalanyl-L-arginyl-β-naphthylamide alone also showed very little antibacterial activity as evidenced by the non-inhibition of growth to a concentration of 256 mg / ml. However, when the two agents were combined in the "board" pattern, there was dramatic evidence of potentiation of the activity of the oxazolidinone antibacterial agent of formula I by L-phenylalanyl-L-arginyl-β-naphthylamide. For example, in the presence of 16 mg / ml of L-phenylalanyl-L-arginyl-β-naphthylamide, an oxazolidinone antibacterial agent of formula I at a concentration of 16 mg / ml was inhibitory to bacterial growth. The calculation of the FIC index produced a value of 0.23, clearly indicative of a synergistic interaction.

Claims (19)

1. CLAIMS: 1. A method for treating infections by gram-negative organisms in mammals, the method comprising administering an effective amount of an oxazolidinone antibacterial agent and an arginine derivative of the formula A Where Ri is: a) aryl, optionally substituted with alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, alkylthio of 1 to 4 carbon atoms, halo or -NH2, b) - (CH2) i-aryl, in which the aryl is substituted with alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, alkylthio of 1 to 4 carbon atoms, halo or -NH2, or c) thienyl, furyl , pyridyl, benzofuranyl or benzothienyl; Z is R2 or -CHWR2; R2 is a) aryl, optionally substituted with one or two alkyls of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, alkylthio of 1 to 4 carbon atoms, halo, -NH 2, alkylamino of 1 to 4 carbon atoms, dialkylamino of 1 to 4 carbon atoms or -NHOH, b) alkyl of 1 to 4 carbon atoms, optionally substituted with fluorine, c) alkoxy of 1 to 4 carbon atoms, d) alkylthio of 1 to 4 carbon atoms, e) halo or f) thienyl, furanyl or pyridyl; W is H, -NH2, alkylamino of 1 to 4 carbon atoms, dialkylamino of 1 to 4 carbon atoms, halo, hydroxy, alkoxy of 1 to 4 carbon atoms, alkylthio or azaheterocycle; aryl is phenyl or naphthyl; Azaheterocycle is n-morpholinyl, n-piperazinyl, n-pyrrolidinyl, n-imidazolyl, n-pyrrolyl, n-pyrazolyl, n-triazolyl or n-tetrazolyl; i is 0, 1 or 2, and pharmaceutically acceptable salts.
2. 2. The method according to claim 1, wherein the oxazolidinone antibacterial agent includes the compounds of structure B. Het XU N A0 ° i. B wherein R_ is methyl, ethyl, cyclopropyl or dichloromethyl; R2 is hydrogen or fluorine; and Het is a saturated 6-membered heterocyclic moiety having 1 to 2 atoms selected from the group consisting of sulfur, nitrogen and oxygen.
3. 3. The method according to claim 2 wherein the oxazolidinone antibacterial agent is a compound of structures I, II, III, IV or V.
4. 4. The method according to claim 2 wherein the oxazolidinone antibacterial agent includes a compound of structure II.
5. 5. The method according to claim 1 wherein the arginine derivative of formula A is L-phenylalanyl-L-arginyl-β-naphthylamide.
6. The method according to claim 1 wherein the oxazolidinone antibacterial agent and the arginine derivative of the formula A are administered in a ratio of 10 (oxazolidinone antibacterial agent): 0.01 (arginine derivative of the formula A).
7. The method according to claim 1 wherein the oxazolidinone antibacterial agent and the arginine derivative of the formula A are administered in a ratio of 1: 1.
8. The method according to claim 1 wherein the effective amount of the oxazolidinone antibacterial agent is from about 0.1 to about 100 mg / kg of body weight / day.
9. 9. The method according to claim 1 wherein the effective amount of the oxazolidinone antibacterial agent is from about 3 to about 50 mg / kg of body weight / day.
10. The method according to claim 1 wherein the amount of the arginine derivative of the formula A is from about 0.01 to about 100 mg / kg of body weight / day.
11. The method according to claim 1 wherein the amount of the arginine derivative of the formula A is from about 0.3 to about 50 mg / kg of body weight / day.
12. The method according to claim 1, wherein the oxazolidinone antibacterial agent and the arginine derivative of the formula A are administered simultaneously.
13. The method according to claim 1 wherein the amount of the arginine derivative of formula A is administered approximately one to four hours before the oxazolidinone antibacterial agent is administered.
14. The method according to claim 1 wherein the effective amount of an oxazolidinone antibacterial agent and an arginine derivative of formula A are administered orally, parenterally, transdermally or topically.
15. 15. The method according to claim 1, wherein the gram-negative organisms are aerobic gram-negative organisms.
16. 16. The method according to claim 15, wherein the gram-negative aerobic organism is E. coli, Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, or Klebsiella pneumoniae.
17. 17. A composition for treating infections by gram-negative organisms in a mammal, the composition comprising an oxazolidinone antibacterial agent of the formula B, an arginine derivative of the formula A and a pharmaceutically acceptable carrier.
18. The composition according to claim 17, wherein the oxazolidinone antibacterial agent includes the compounds of structures I, II, III, IV or V.
19. 19. The composition according to claim 17, wherein the arginine derivative of the formula A is L-phenylalanyl-L-arginyl-β-naphthylamide.