INTRODUCTION

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The antimicrobial era has reached the point where the emergence of resistant microbes is accelerating while the pace of discovery of new drugs is decelerating (11). Until recently, a new drug or combination arrived just in time to overcome the problem of resistance. Few novel chemical entities have been brought to the market during the past decade. Most new drugs are derivatives of older compounds. Some have increased activity, a broader spectrum of activity, or improved pharmacological properties but can only temporarily overcome the problem of resistance. There is a need for new classes of antimicrobial compounds. This need is particularly critical for infections caused by methicillin- and vancomycin-resistant strains of Staphylococcus aureus, coagulase-negative staphylococci, vancomycin-resistant enterococci, and penicillin-resistant pneumococci.

Quinine and several other antimalarial drugs, including mefloquine, have been reported to exhibit activity against Streptococcus pneumoniae, S. aureus, and Escherichia coli (2, 3, 7, 8). Mefloquine is also active against Mycobacterium avium complex (1). In preliminary experiments, one of the authors (C.M.K.) found that mefloquine exhibited in vitro activity against gram-positive bacteria, including methicillin-resistant staphylococci, pneumococci, and streptococci, but was much less active against gram-negative bacteria. MIC of mefloquine for staphylococci and enterococci was 16 µg/ml, and the minimal bactericidal concentration (MBC) ranged from 16 to 32 µg/ml. These concentrations cannot be achieved in human serum. Mefloquine is highly lipid soluble and has a large volume of distribution (5). In an attempt to find more active derivatives, a collaborative arrangement was developed between The Ohio State University (OSU) and The Walter Reed Army Institute of Research (WRAIR). W.Y.E. selected about 400 mefloquine-related compounds from the WRAIR chemical inventory. Among these were compounds containing a piperidine attached to methanol at its 2 position, which in turn was attached to pyridine, quinoline, or benzylquinoline ring systems. These were found to be far more active than mefloquine against gram-positive bacteria. Some were also active against Candida albicans and Cryptococcus neoformans.

In this report, we describe the antimicrobial spectra of mefloquine and the related compounds, their synergy with gentamicin against staphylococci and pneumococci, and preliminary studies of their mode of action (4a).

	MATERIALS AND METHODS

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Microorganisms. Microorganisms consisted of American Type Culture Collection strains and clinical isolates of methicillin-susceptible and -resistant S. aureus and Staphylococcus epidermidis, Streptococcus pyogenes, gentamicin- and vancomycin-resistant Enterococcus faecalis and Enterococcus faecium, penicillin-susceptible and -resistant strains of S. pneumoniae, E. coli, Enterobacter cloacae, Proteus mirabilis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Haemophilus influenzae, Neisseria gonorrheae, Neisseria meningitidis, C. albicans, Candida spp., and C. neoformans. In addition, 21 clinical isolates of penicillin-susceptible and -resistant strains of S. pneumoniae and 24 clinical isolates of methicillin-susceptible and -resistant strains of S. aureus were provided by Christian Parker of Procter & Gamble Pharmaceuticals, Mason, Ohio.

Chemicals. Mefloquine (Ro 21-5998-000) was provided by Roche Laboratories, Nutley, N.J. All the antimicrobial drugs were obtained from Sigma Chemical Co., St. Louis, Mo.

Susceptibility tests. Bacteria were grown overnight at 35°C in Mueller-Hinton broth (MHB) (Difco Laboratories, Detroit, Mich.) and streaked on blood agar plates containing 5% sheep cells. A single colony was isolated and grown in MHB as recommended by the National Committee for Clinical Laboratory Standards (NCCLS) (9) for rapidly growing bacteria. Candida spp. and C. neoformans were incubated overnight at 35°C and assayed according to the NCCLS method (10) in RPMI 1640 medium (YeastOne; Trek Diagnostic Systems, Westlake, Ohio). Neisseriae were tested in Fildes medium (Difco).

Screening. WRAIR shipped the compounds to OSU under code. Some identical compounds were shipped with different code numbers to check reproducibility. The structures were not revealed until screening was completed. The compounds were dissolved in 1 ml of methanol or dimethyl sulfoxide, diluted in distilled water, and used the same day. Antimicrobial activity was determined with two strains of S. aureus. S. aureus ATCC 29213 is highly susceptible to penicillin and other antibacterial drugs. S. aureus T67738 is highly resistant to penicillin, oxacillin, gentamicin, trimethoprim-sulfamethoxazole, and ciprofloxacin but susceptible to imipenem, clindamycin, erythromycin, and vancomycin. The MIC was determined by the twofold dilution microtiter plate method. After 24 h of incubation, 0.01 ml was taken from the last two tubes or wells without visible growth and streaked on Trypticase soy agar plates (Difco). The highest dilution at which 99.9% of the bacterial inoculum was killed was considered the MBC.

Synergy and antagonism. Serial dilutions of the compounds alone or in combination with tetracycline, trimethoprim, cefazolin, ofloxacin, clarithromycin, rifampin, gentamicin, and vancomycin were prepared in microtiter wells by the checkerboard pattern method. Combinations were considered synergistic if the sum of the fractional MICs was 0.5 or less. They were considered additive if the MIC was half the MIC for both drugs (4). Intermediate results were considered additive.

Effect of compounds on optical density and release of nucleic acids. S. aureus T67738 was grown for 14 h at 35°C in MHB. The cells were collected by centrifugation and resuspended in 5 ml of a 1:10 dilution of MHB to yield an optical density at 550 nm of 0.400 in a Spectronic 601 spectrophotometer (Milton Roy, Rochester, N.Y.). Compounds at five times the MIC or the same volume of water was added to 2 ml of the suspension. The optical density was determined at intervals of up to 24 h. Samples were removed at 0, 1, and 3 h and centrifuged at 10,000 × g for 5 min. DNA was extracted from the supernatant, applied to an 0.8% agarose gel, and stained with ethidium bromide (12).

DNA gyrase inhibition. DNA gyrase inhibition was determined as described by Inoue et al. (6) with Micrococcus luteus DNA gyrase, topoisomerase I, and the supercoiled plasmid pBR322 (Gibco BRL, Rockville, Md.). Ciprofloxacin served as the positive control.

	RESULTS

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Antimicrobial activity of mefloquine. The MIC of mefloquine was 16 µg/ml for all strains of staphylococci, regardless of susceptibility or resistance to penicillin, oxacillin, gentamicin, imipenem, ciprofloxacin, or other antimicrobial drugs within the ranges published by the NCCLS (Table 1). The MBC for all strains of staphylococci was either 16 or 32 µg/ml. At concentrations of 32 µg/ml, mefloquine reduced the count of two strains each of S. aureus and S. epidermidis from 2 × 10⁸ to less than 1 × 10¹ CFU/ml after 24 h of incubation. Mefloquine was highly active against S. pneumoniae (0.2 to 1.5 µg/ml), was less active against Streptococcus faecalis, and showed poor or no activity against gram-negative bacteria and C. albicans.

Screening of mefloquine-related compounds. Thirty-six compounds were found in the screening program to be active against both penicillin-susceptible S. aureus ATCC 29213 and methicillin-resistant S. aureus T67738 at concentrations of 0.8 to 1.56 µg/ml and bactericidal at 1.56 to 2.5 µg/ml. These compounds resembled mefloquine. The chemical structures of some of the most active compounds are shown in Fig. 1. They are designated by WRAIR (WR) numbers. The OSU code numbers are used to simplify presentation in the tables.

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Structures of mefloquine and related compounds (WR, WRAIR catalog number; OSU, OSU code number).

Antibacterial spectrum of mefloquine-related compounds. The activities of the compounds against gram-positive bacteria are summarized in Table 2. The MICs and MBCs were usually the same. In some instances, particularly with the enterococci, the MBC was one twofold dilution higher. The MIC ranges for penicillin-susceptible and -resistant S. pneumoniae and methicillin-susceptible and -resistant S. aureus were virtually identical and were pooled in Table 2.

Antifungal spectrum of mefloquine-related compounds. The compounds were about as active as amphotericin B against yeasts (Table 3). The MBCs were equal to and in some cases twofold higher than the MICs. In killing curve experiments, the cell counts fell 4 to 5 log₁₀ units within 4 h.

Interactions of mefloquine-related compounds with gentamicin and vancomycin. The antimicrobial activities of compounds 95 and 99 in combination with gentamicin and vancomycin were examined by checkerboard pattern experiments with methicillin-susceptible and -resistant strains of S. aureus. The MICs and MBCs of the compounds were decreased fourfold when the compounds were combined with limiting dilutions of gentamicin. The MICs and MBCs of gentamicin were decreased four- to eightfold when gentamicin was combined with limiting dilutions of the compounds. In contrast, there was only a twofold decrease in the MICs and MBCs of the compounds combined with vancomycin.

View larger version (22K): [in this window] [in a new window]   FIG. 2.   Antibacterial activities of compound 95, gentamicin, and vancomycin alone or in combination against penicillin-susceptible S. aureus ATCC 29213. Symbols: , control; , vancomycin at 1 µg/ml; , gentamicin at 1 µg/ml; , compound 95 at 1 µg/ml; , compound 95 at 4 µg/ml; , compound 95 at 6 µg/ml; , compound 95 at 1 µg/ml plus vancomycin at 1 µg/ml; and , compound 95 at 1 µg/ml plus gentamicin at 1 µg/ml.

Preliminary studies of mode of action. The compounds appeared to lyse staphylococci, as evidenced by a rapid fall in optical spectroscopy and release of DNA into the medium (Fig. 3 and 4). None of six compounds tested inhibited DNA gyrase activity.

View larger version (11K): [in this window] [in a new window]   FIG. 3.   Change in the optical density at 550 nm (OD550) of methicillin-resistant S. aureus T67738 incubated at 37°C in MHB. Symbols: , control; , compound 199 at 15 µg/ml (five times the MIC).

View larger version (105K): [in this window] [in a new window]   FIG. 4.   Agarose gel stained with ethidium bromide to visualize nucleic acids that leaked from methicillin-resistant S. aureus T67738 incubated at 37°C in MHB (control) and with 15 µg of compound 199 per ml (three times the MIC). The numbers refer to hours of incubation. C, nucleic acid standards.

	DISCUSSION

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We found that mefloquine was bactericidal against gram-positive bacteria, including staphylococci, pneumococci, and enterococci. It had poor activity against gram-negative enteric bacteria and yeasts. There was no cross-resistance or antagonism with beta-lactam antibiotics, quinolones, and other antimicrobial drugs. No intrinsically resistant strains were isolated from large inocula exposed to the drug. Mefloquine is highly lipid soluble. It has a large volume of distribution and a long serum half-life, is bound to serum proteins, and accumulates intracellularly (1, 5). Peak levels in blood following standard therapeutic and prophylactic doses weekly are in the range of 979 to 1,500 ng/ml (13, 14). These values are far lower than the MIC of 16 µg/ml for staphylococci but are close to the MIC for S. pneumoniae.

The WRAIR screening program yielded a series of mefloquine-related compounds that possess a piperidine attached to methanol at its 2 position, which in turn was attached to a variety of ring structures. Compounds without the piperidine methanol group were inactive. Their antimicrobial spectrum resembled that of mefloquine. Their activity against S. pneumoniae was similar to that of mefloquine, but they were more active against staphylococci, enterococci, and yeasts.

The compounds appear to act by disrupting the microbial cell membrane. Increased membrane permeability may account for their ability to augment the activity of gentamicin. The precise mechanism of action is unclear, but quinine has been shown to specifically inhibit the membrane-associated F₀F₁ H⁺-ATPase of S. pneumoniae (8). The compounds did not inhibit DNA gyrase activity by the method used in this study and were active against quinolone-resistant staphylococci.

High lipid solubility and serum protein binding may limit the potential therapeutic efficacy of mefloquine and its related compounds when used alone against extracellular infections. Subinhibitory concentrations of the mefloquine-related compounds in combination with gentamicin might be effective against pneumococcal, staphylococcal, and enterococcal infections.

The mefloquine-related compounds were highly active against C. neoformans at MICs similar to those of amphotericin B. Their lipid solubility favors penetration into the central nervous system and cells. Mefloquine has already been shown to be active against intracellular infections caused by M. avium (1). The activities of the mefloquine-related compounds against Histoplasma capsulatum and other intracellular microorganisms warrant further study.

The pharmacology, toxicology, and potential efficacy of the mefloquine-related compounds need to be determined. Water-soluble derivatives, metabolites, or prodrugs might be advantageous. Combinations of the compounds with aminoglycoside antibiotics offer the greatest potential for therapeutic use. The WRAIR inventory should be further explored for interesting antibacterial and antifungal compounds.