USOO5872104A United States Patent (19) 11 Patent Number: 5,872,104 Vermeulen et al. (45) Date of Patent: Feb. 16, 1999 54) COMBINATIONS AND METHODS FOR OTHER PUBLICATIONS REDUCING ANTIMICROBAL RESISTANCE HaSobe et al., “The Synergism of Nucleoside Antibiotics 75 Inventors: Nicolaas M. J. Vermeulen Combined with Guanine 7-N-Oxide Against a Rhabdovi Woodinville: Dennis E. Schwartz rus, Infectious Hematopoietic Necrosis Virus (IHNV).” The Redmond both of Wash. s Journal of Antibiotics, vol.39, No. 9, 1291-1297, 1986. s Kovacs, et al,“Potent Effect of Trimetrexate, a Lipid 73 Assignee: Oridigm Corporation, Seattle, Wash. Soluble Antigolate, on Toxoplasma gondii.” The Journal of Infectious Diseases, vol. 155, No. 5, 1028–1032, May 1987. Touze, J.E." Actualites Et Renouveau En Therapeutique 21 Appl. No.: 364,246 Anti-Parasitaire,” La Presse Medicale, vol. 23, No. 26, 22 Filed: Dec. 27, 1994 1195–1198, Sep. 3-10, 1994. (51) Int. Cl. ............................................... A61K 31/70 Primary Examiner-Elli Peselev 52 U.S. Cl. ................................. 51429, 514/30, 514/35. Attorney, Agent, or Firm-Arnold, White & Durkee 51446 (57) ABSTRACT 58 Field of Search ................................ 514/29, 303, 30, 514/35, 46; 536/7.2 Disclosed are novel methods, combinations of agents and kits for use in killing, or inhibiting the growth of, microor 56) References Cited ganisms. Enhanced antimicrobial action is provided by using a methylation inhibitor, as exemplified by using an U.S. PATENT DOCUMENTS agent that inhibits methylation or maturation of bacterial 2,870,138 1/1959 Murray ...................................... 536/72 RNA in combination with, e.g., a macrollide lincosamide 4,497,794 2/1985 Klein et al. ............................... 514/29 streptogramin B (MLS) antibiotic. The methods and com 4,968,690 11/1990 Marquez et al. ... 514/303 positions described may be employed to reduce the resis 5,132,291 7/1992 Gruber ...................................... 574/43 tance of Susceptible microorganisms to antimicrobial agents FOREIGN PATENT DOCUMENTS and thus to treat animals or patients with infections. 0 052 705 6/1982 Germany. 132 Claims, 5 Drawing Sheets U.S. Patent Feb. 16, 1999 Sheet 1 of 5 5,872,104 P-AMNOBENZOIC ACD PTERIDINE (PABA) S-ADENOSYL DIHYDROPTEROATE METHONNE SYNTHETASE DECARBOXYLASE DIHYDROFOLATE REDUCTASE SAH HYDROLASE METHYL-B12 f HOMO Y-AMINOLEVULENIC CYSTEINE HADENOSINE ACD ADENOSNE DEAMNASE FG. A U.S. Patent Feb. 16, 1999 Sheet 2 of 5 5,872,104 ARGINNE DECARBOXYLASE DECARBOXYLATED AGMATINE SAM UREO HYDROLASE ORN THINE ORN THINE DECARBOXYLASE METHYL SPERMIDINE - ADENOSNE METHYL SPERMINE -- ADENOSNE FIG. B U.S. Patent Feb. 16, 1999 Sheet 3 of 5 5,872,104 N H N N H N N WUUNOZ WTO U.S. Patent Feb. 16, 1999 Sheet 4 of 5 5,872,104 F. s s CN O oo co r CN O y y- s U.S. Patent Feb. 16, 1999 Sheet 5 of 5 5,872,104 Wu 9NOZ WTO 5,872,104 1 2 COMBINATIONS AND METHODS FOR methods, combined compositions and kits, for use in reduc REDUCING ANTIMICROBAL RESISTANCE ing resistance to antimicrobials and antibiotics and for treating infections. The invention rests in the Surprising use BACKGROUND OF THE INVENTION of one or more inhibitors of methylation in conjunction with 1. Field of the Invention an antimicrobial agent. The combination is used to impair The present invention relates generally to the fields of the ability of a microorganism to methylate various Sub bacteria, antimicrobials and antibiotics. More particularly, it StrateS. provides novel methods, kits, and combinations of antimi AS the inventors contemplate that methylation, either at crobial agents and inhibitors, for use in reducing the resis the DNA, RNA, protein or small molecule level, plays a role tance of bacteria and other microorganisms to antimicrobial in all mechanisms of resistance of a microorganism to an agents. In particular, the invention provides for enhanced antimicrobial agent, inhibition of methylation will be widely bacterial killing using a macrollide, lincosamide and Strep applicable to reducing resistance in any given microorgan togramin B (MLS) antibiotic in combination with an agent ism or bacterium, and to treating a variety of diseases. For that inhibits methylation, as exemplified by inhibiting example, the organisms listed in Table 1 and Table 2 may be methylation or maturation of bacterial RNA. 15 targeted using the antibiotics also listed in Table 1 and Table 2. Description of the Related Art 2, amongst others, in combination with a methylation inhibi The first antibiotics were used clinically in the 1940s and tor. However, the invention is not limited to antibiotics and 195S, and their use has been increasing Significantly since to bacteria alone, and is widely applicable to all this period. Although an invaluable advance, antibiotic and microorganisms, including yeast and a variety of other antimicrobial therapy Suffers from Several problems, par pathogenic and even parasitic organisms. ticularly when Strains of various bacteria appear that are The invention therefore encompasses methods, composi resistant to antibiotics. Interestingly, bacteria resistant to tions and kits that include a methylation inhibitor capable of Streptomycin were isolated about a year after this antibiotic inhibiting any process listed in Table 1, as exemplified by the processes of antibiotic alteration, target Site alteration and was introduced (Waksman, 1945). 25 The development of antibiotic resistance is a Serious and antibiotic transport. Also, as methylation inhibitors act to life-threatening event of worldwide importance. For alter gene expression and protein Synthesis, the invention example, Strains of StaphylococcuS are known that are includes methylation inhibitors that prevent the by-pass of immune to all antibiotics except one (Travis, 1994). Such antibiotic Sensitive-steps, prevent an increase in the amount bacteria often cause fatal hospital infections. Among other of an inhibited enzyme, prevent the production of an alter drug resistant organisms are: pneumococci that cause pneu native endogenous or exogenous product, and prevent the monia and meningitis, Cryptosporidium and E. coli that production of a metabolite that antagonizes the action of cause diarrhea, and enterococci that cause blood-stream, inhibitor. Surgical wound and urinary tract infections (Berkelman et. One particular example of the applicability of the inven al., 1994). 35 tion is in providing methods and combinations for use in Davies (1986) described seven basic biochemical mecha reducing the resistance of bacteria to macrollide, nisms for naturally-occurring antibiotic resistance: (1) alter lincosamide, and streptogramin B (MLS) antibiotics, or in ation (inactivation) of the antibiotic; (2) alteration of the enhancing the Sensitivity of Susceptible Strains to Such target site; (3) blockage in the transport of the antibiotic; (4) antibiotics. In this case, the inhibitor will primarily inhibit by-pass of the antibiotic Sensitive-step (replacement); (5) 40 methylation of bacterial rRNA, thus rendering the bacterium increase in the level of the inhibited enzyme (titration of susceptible to antibiotic rRNA binding and allowing bacte drug), (6) Sparing the antibiotic-sensitive step by endog rial protein synthesis to be inhibited. enous or exogenous product; and (7) production of a The various embodiments of the invention include metabolite that antagonizes action of inhibitor. methods, combined compositions and kits for reducing the Certain bacteria become resistant to antibiotics by utiliz 45 resistance of a microorganism to an antimicrobial agent, as ing ribosomal mutations (Cunliffe, 1990), although some exemplified by reducing the antibiotic resistance of an reports have stated that this type of resistance is of doubtful MLS-susceptible bacterium to an MLS antibiotic. Also clinical significance (Spratt, 1994). Ribosomal mutations provided are methods, compositions and therapeutic kits for result in bacterial resistance to macrollide, lincosamide and killing microorganisms, Such as bacteria, and particularly, streptogramin B (MLS) antibiotics, as has been observed in 50 MLS-susceptible bacteria; and methods for treating infec the resistance of various Staphylococcus, Streptococcus, tions and diseases caused by microorganisms, including Enterococcus, Bacillus and Mycoplasma Strains to important treating MLS-Susceptible bacterial infections and diseases. antibiotics Such as erythromycin (LeClercq & Courvalin, By way of example only, certain infections that may be 1991). treated using the invention are listed in Table 3. The induction of erythromycin resistance generally leads 55 To reduce the resistance of a microorganism to an anti to bacterial strains that express rRNA which does not bind microbial agent, as exemplified by reducing the resistance of to this type of antibiotic. Mainly, erythromycin resistance is a bacterium to an antibiotic, or to kill a microorganism or due to the induction by macrollides of a methylase protein, bacterium, one would generally contact the microorganism which catalyzes the methylation of the binding site of or bacterium with an effective amount of the antibiotic or erythromycin on the rRNA, thus preventing antibiotic bind 60 antimicrobial agent in combination with an amount of a ing. Despite intensive Studies in this area, there remains few, Second agent effective to inhibit methylation in the micro if any, practical proposals as to how bacterial resistance to organism or bacterium. In terms of killing or reducing the MLS antibiotics