Europeans outraged over expanded access.

AIDS: The growing chasm between therapeutic options in Europe and America is causing anger and desperation among HIV-infected Europeans and their physicians. Many European doctors and activists accuse Abbott Laboratories and Merck, with the help of Food and Drug Administration (FDA) decisionmaking, of being primarily committed to ensuring that the U.S. market is fully supplied with therapeutic drugs, no matter what the cost is to Europe. Both companies deny any such plans and reveal their manufacturing and supply plans for Norvir (Abbott) and Crixivan (Merck) for the near future in order to meet the needs of the entire AIDS community. The International Association of Physicians in AIDS Care is calling for the replacement of the expanded-access model with a global drug rationing agency which would approve and supervise the distribution of drugs for compassionate use.^ieng

Quinolone antibacterial agents: relationship between structure and in vitro inhibition of the human cytochrome P450 isoform CYP1A2.

The inhibitory effect of 44 quinolone antibacterials and derivatives (common structure, 4-oxoquinoline-3-carboxylic acid) on cytochrome P450 isoform CYP1A2 activity was tested using human liver microsomes and caffeine 3-demethylation as a specific test system for this enzyme. By direct comparison of molecules differing structurally in only one position, the following structure-activity relationships were found. 3'-Oxo derivatives had a reduced or similar activity and M1 metabolites (cleavage of piperazinyl substituent) had a greater inhibitory activity, compared with the parent molecule. Alkylation of the 7-piperazinyl substituent resulted in a reduced inhibitory potency. Naphthyridines with an unsubstituted piperazinyl group at position 7 displayed a greater inhibitory potency than did corresponding quinoline derivatives. Derivatives with a fluorine substitution at position 8 had only a minor effect. Molecular modeling studies with inhibitors and caffeine showed that it is possible to explain the potency of the quinolones to inhibit CYP1A2 on a molecular level. The keto group, the carboxylate group, and the core nitrogen at position 1 are likely to be the most important groups for binding to the active site of CYP1A2, because the molecular electrostatic potential of all inhibitors is very similar to that of caffeine in these regions. The presence of a piperazinyl substituent, however, seems to be no prerequisite for inhibitory potency. Finally, an equation to estimate the potency to inhibit CYP1A2 was developed by quantitative structure-activity relationship analysis.

Pharmacokinetics of temafloxacin in humans after multiple oral doses.

The multiple-dose pharmacokinetics and tolerance of temafloxacin, a new fluoroquinolone antibacterial agent, were evaluated in healthy volunteers. Temafloxacin was found to be well tolerated when administered orally every 12 h for 7 days at doses of 100, 200, 300, 400, 600, and 800 mg. Steady-state maximum and minimum concentrations in plasma were proportional to dose, averaging slightly over 1.0 and 0.5 microgram/ml/100 mg administered. Analyses of variance found no significant differences among the dosage groups in total apparent clearances (CLT/F), renal clearances (CLR), or nonrenal clearances, which averaged 197, 119, and 78 ml/min, respectively. The half-life increased slightly with dose, averaging 8.4 h overall. The extent of absorption of temafloxacin was quite reproducible, with day-to-day intrasubject variability in minima averaging under 10%. Renal glomerular filtration of unbound drug was the dominant elimination process; however, tubular secretion and reabsorption also appear to occur. Secretion was estimated to account for about 12% of CLT/F during a regimen of 600 mg every 12 h. CLR was relatively constant for urine flow rates above 1 ml/min, but reabsorption appeared to occur under low-flow conditions, resulting in day-versus-night differences in CLR. Intersubject variability in CLT/F over the eightfold range in dosage was only 20%, and 60% of this variance was accounted for by differences in body surface area (or lean body mass), concentration in plasma, and urine flow rate. Overall, it appears that the pharmacokinetics of temafloxacin are essentially linear, reproducible within a subject, and predictable among subjects.

Assessment of adverse events during drug development: experience with temafloxacin.

The safety of temafloxacin was evaluated in 753 subjects in phase I trials and in 2602 patients included in phase II and III studies. Comparative treatment was given to 153 subjects in phase I who received placebo and to 2031 patients in phase II and III trials who were given other quinolones (n = 1169) or non-quinolone comparators (n = 862). Adverse events were collated by spontaneous reporting by the patients or observations by investigators and, additionally in some studies, by the use of diary cards filled in by the patients. The results showed that temafloxacin was at least as safe as the comparators. Dose related gastrointestinal adverse reactions were found only in patients with reduced renal function who received a high temafloxacin dose. There was a low incidence of quinolone-related adverse reactions (photosensitivity, CNS-toxicity, or theophylline drug-drug interactions). The most common adverse reactions were gastrointestinal ones which occurred in 13.4% of the patients included in phase II and III trials. The corresponding frequencies in patients randomized to quinolone and non-quinolone comparators were 15.7% and 11.6% (P less than 0.05) [corrected], respectively. In conclusion, temafloxacin was shown to be at least as safe as the comparators used in the extensive phase I to III programme for evaluation of this new quinolone.

Temafloxacin: a safe antibiotic for the elderly, patients with comorbidities, and other special populations.

During the Phase I, II, and III clinical development program, temafloxacin has demonstrated an excellent safety profile, with reported overall rates of adverse events comparable to quinolone and nonquinolone reference agents. This favorable safety record was consistently observed when special populations, suspected to be at higher risk for drug-related adverse events, were evaluated. For instance, the rates of all adverse events as well as most adverse events by organ system were similar between elderly (greater than or equal to 66 years) and younger temafloxacin recipients. Temafloxacin does not significantly alter the pharmacokinetic/pharmacodynamic effects of numerous drugs often used in the elderly (for example, warfarin, theophylline, caffeine, and cimetidine). Based on an evaluation of adverse events among elderly patients with varying degrees of renal impairment, dosage adjustment of temafloxacin is necessary only for elderly patients with severe renal impairment. Temafloxacin appears to be comparably safe to other quinolones and nonquinolones for the treatment of infections among diabetics and patients with chronic lung disease. Because of its lack of interaction with theophylline clearance, temafloxacin may be preferred to selected other quinolones for the latter population.

Comparison of organ-specific toxicity of temafloxacin in animals and humans.

This article summarizes animal studies conducted to determine the toxic and mutagenic potential of temafloxacin. The four target tissues of potential concern with fluoroquinolone use are the kidney, the eye, the weight-bearing joints of young animals, and the central nervous system. Based on the results of these studies in rats and dogs, it appears unlikely that crystalluria or nephrotoxicity will occur in humans who receive temafloxacin. Pre-marketing clinical trials in humans (n = 5,308) correlate well with chronic toxicity animal studies, reporting no crystalluria or clinically significant nephrotoxicity. Reversible electroretinographic (ERG) changes in dog studies were demonstrated only with the administration of high temafloxacin dosages. A Phase I study evaluating the safety of temafloxacin at 600 mg b.i.d. for 14 days in human subjects reported no significant changes in ophthalmologic parameters. Evidence of cartilaginous joint damage was observed in puppies receiving oral temafloxacin, in young dogs receiving intravenous temafloxacin, and in a single dog receiving a lethal dosage in a dose range-finding study. However, these toxic findings were not evident in any dogs in the subacute or chronic oral toxicity studies or in a longer duration intravenous study. Although limited evidence would suggest that young children may not be at risk, thorough clinical investigations of quinolones in these patients have only recently been initiated. Signs of central nervous system toxicity caused by temafloxacin were absent in two rodent studies, during which clonic convulsions were induced by concomitant use of fenbufen plus enoxacin or ciprofloxacin, and in human subjects evaluated by positron emission tomography. Temafloxacin, contrary to most other quinolones, was considered nonmutagenic in all mutagenicity tests conducted. In reproductive studies, temafloxacin was not uniquely toxic to the developing conceptus in the laboratory rat, mouse, rabbit, or primate. Based on these animal studies, temafloxacin appears to be non-mutagenic and to have a low potential for producing renal or ocular toxicity; however, like other quinolones, it should not be routinely used in children or pregnant women because of evidence of cartilage damage reported in young dogs. Premarketing clinical trials to date confirm the safety of temafloxacin use in adults.

Pharmacokinetics of temafloxacin in humans after single oral doses.

Temafloxacin (A-63004) is a new quinolone antibacterial agent with a broad spectrum of activity against gram-positive and gram-negative aerobes and anaerobes. The pharmacokinetics and metabolism of temafloxacin were determined in healthy volunteers after administration of single oral doses of 100, 200, 400, 600, 800, and 1,000 mg. The corresponding peak concentrations in plasma (mean +/- standard deviation) were 0.98 +/- 0.26, 1.61 +/- 0.57, 2.43 +/- 0.56, 3.87 +/- 0.64, 4.54 +/- 1.03, and 6.67 +/- 0.74 micrograms/ml. The times that elapsed to attain peak levels ranged from 1.25 to 3.5 h. Statistical analyses of parameters related to the extent of absorption and the linearity of the dispositional pharmacokinetics detected no dose-related trends. Study-wide, total clearance (223 ml/min) and renal clearance (125 ml/min) showed low intersubject variability, with coefficients of variation near 20%. The terminal-phase rate constant of 0.090 +/- 0.008 h-1 corresponds to a half-life of 7.7 h. Temafloxacin was excreted mainly in the urine, with 57 +/- 11% of the dose appearing in the urine unchanged. Conjugated temafloxacin, oxidative metabolites, and conjugates thereof were minor components in urine, collectively accounting for 5 to 8% of the dose. Since intravenously dosed dogs eliminated 50% of the dose by nonrenal processes, urinary recoveries approaching two-thirds of the dose in humans were consistent with high, if not quantitative, absorption. Reported adverse events were generally mild, were randomly distributed between temafloxacin- and placebo-treated subjects, and were not dose related.

Synthesis, antibacterial activities, and pharmacological properties of enantiomers of temafloxacin hydrochloride.

Temafloxacin hydrochloride [(+/-)-7-(3-methylpiperazin-1-yl)-6-fluoro-1-(2,4-difluorophenyl)- 1,4-dihydro- 4-oxoquinoline-3-carboxylic acid hydrochloride] is a potent member of the 4-pyridone-3-carboxylic acid class of antibacterial agents and is currently under clinical development as a broad-spectrum antimicrobial agent. It is a racemate having a chiral center at the C3 of the 7-piperazin-1-yl group. The two enantiomers were synthesized and tested for their antibacterial activities. Although no difference in in vitro antibacterial activities was observed, a minor difference in in vivo antibacterial activities was observed. However, they both exhibited similar pharmacological profiles.

Synthesis and biological properties of A-71497: a prodrug of tosufloxacin.

Tosufloxacin (5, A-61827 tosylate or T-3262) is currently under product development by both Abbott Laboratories and Toyama Chemical Co. Its registration as antibacterial agent has been approved in Japan. It has been found to be extremely effective in treating several bacterial infections. However, due to its inherent low water solubility, the development of an intravenous formulation will be extremely difficult and may preclude its parenteral use. In search of a more water-soluble analog of tosufloxacin for parenteral use, the 3-formyl derivative of tosulfoxacin, A-71497 (13), was synthesized for evaluation. It was found to produce high plasma levels of tosufloxacin upon both oral and subcutaneous administration to mice. High plasma levels of tosufloxacin were also obtained when 13 was administered both orally and intravenously to dogs. It possesses increased water solubility and makes the development of intravenous formulation possible. The chemical synthesis as well as biological properties of A-71497 (13) are described.

Structure-activity relationship of quinolone antibacterial agents: the effects of C-2 substitution.

Very little is known about the structure-activity relationship of quinolone antibacterials at the 2-position. Because of the loss of biological activity with 2-methyl and 2-hydroxyl substitution, modifications at C-2 were generally considered to be unfavourable. Quinolone derivatives having a ring between positions 1 and 2 were recently shown to have biological activity. The sulfur-bridged analogs such as the benzothiazolo[3,2-a]quinolone, KB-5246 and NAD-394 have been reported to be highly active in vitro. The authors have synthesized 2-methylthiociprofloxacin, 2-methylofloxacin, the 5-oxopyrrolo[1,2-a]quinoline and isothiazolonaphthyridine to assess the importance of the sulfur atom on biological activity as well as the effect of C-2 substituent on the spatial arrangements of N-1 or the 3-carboxylic group. It was found that the planarity between the 4-keto and 3-carboxylic acid groups of quinoline molecules is the most important criterion for biological activity. The syntheses of the above four compounds are also described.

Mechanism of inhibition of DNA gyrase by quinolone antibacterials: specificity and cooperativity of drug binding to DNA.

Although the functional target of quinolone antibacterials such as nalidixic acid and norfloxacin has been identified as the enzyme DNA gyrase, the direct binding site of the drug is the DNA molecule [Shen, L. L., & Pernet, A. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 307-311]. As described in this paper, binding specificity and cooperativity of quinolones to DNA were further investigated with the use of a variety of DNA species of different structures and different base compositions. Results show that the drug binding specificity is controlled and determined largely by the DNA structure. The drug binds weakly and demonstrates no base preference when DNA strands are paired. The drug binds with much greater affinity when the strands are separated, and consequently, binding preference emerges: it binds better to poly(G) and poly(dG) over their counterparts including poly(dI). The results suggest that the drug binds to unpaired bases via hydrogen bonding and not via ring stacking with DNA bases. The weak binding to relaxed double-stranded DNA and the stronger binding to single-stranded DNA are both nonspecific as they do not demonstrate binding saturation and cooperativity. The specific type of binding, initially demonstrated in our previous publication with the supercoiled DNA and more recently with complex formed between linear DNA and DNA gyrase [Shen, L. L., Kohlbrenner, W. E., Weigl, D., & Baranowski, J. (1989) J. Biol. Chem. (in press)], occurs near the drug's supercoiling inhibition concentration. As shown in this paper, binding saturation curves of this type are highly cooperative (with Hill constant greater than 4).(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of inhibition of DNA gyrase by quinolone antibacterials: a cooperative drug--DNA binding model.

We have proposed a cooperative quinolone-DNA binding model for the inhibition of DNA gyrase. The essential feature of the model is that bound gyrase induces a specific quinolone binding site in the relaxed DNA substrate in the presence of ATP. The binding affinity and specificity are derived from two unique and equally important functional features: the specific conformation of the proposed single-stranded DNA pocket induced by the enzyme and the unique self-association phenomenon (from which the cooperativity is derived) of the drug molecules to fit the binding pocket with a high degree of flexibility. Supporting evidence for and implications of this model are provided.

Aromatic dienoyl tetramic acids. Novel antibacterial agents with activity against anaerobes and staphylococci.

Streptolydigin (1) and tirandamycin A (2) are typical members of the naturally occurring class of 3-dienoyl tetramic acids. These compounds, which possess potent antibacterial activity particularly against anaerobes, have been shown to inhibit bacterial RNA polymerase. In contrast, tenuazonic acid (5), which lacks a complex dioxabicyclononane moiety and diene chromophore present in 1 and 2, exhibits essentially no antimicrobial activity and has no effect on bacterial RNA polymerase, suggesting that one or both of these structural features may be critical for antibacterial activity. In this paper, we report on a novel series of synthetic dienoyl tetramic acids that lack a complex dioxabicyclononane unit. Several of these compounds, particularly 8T-W, exhibit potent antimicrobial activity against Gram-positive and Gram-negative anaerobes as well as staphylococci. We will discuss the structure-activity relationship for this series of compounds which, in contrast to their natural counterparts, do not inhibit significantly RNA polymerase. We will also discuss preliminary results on the biochemical and microbiological properties of this series of compounds, several of which moderately inhibit supercoiling by DNA gyrase isolated from E. coli H560, although this enzyme has not been established as their target in whole cells. Compound 8W, which is not cross-resistant with DNA gyrase subunit A or B inhibitors or tirandamycin, has also been demonstrated to be rapidly bactericidal.

Synthesis and activity of nonhydrolyzable pseudomonic acid analogues.

Several series of pseudomonic acid analogues have been prepared that incorporate modified functionalities in place of the C1-C3 alpha,beta-unsaturated ester group. The inhibition of isoleucyl-tRNA synthetase and the in vitro activity of these compounds against various Gram-positive and Gram-negative strains are described. Several derivatives showed enzyme inhibition equivalent to or better than that of methyl pseudomonate (3), while lacking the hydrolyzable ester group at C1. These analogues include the corresponding phenyl ketone and the ether 12. The long-chain ketone 24 exhibited similar in vitro activity as the parent ester.

Design, synthesis, and properties of (4S)-7-(4-amino-2-substituted-pyrrolidin-1-yl)quinolone-3-carboxylic acids.

The quinolonecarboxylic acids constitute a class of extremely potent and orally active broad-spectrum antibacterial agents. These compounds have been shown to inhibit DNA gyrase, a key enzyme in bacterial DNA replication. The 7-(3-aminopyrrolidinyl)quinolone A-60969 (1) is a particularly potent member of this class and is currently undergoing clinical evaluation. We have studied a series of enantiomerically homogeneous (4S)-7-(4-amino-2-substituted-pyrrolidinyl)quinolones in an effort to utilize the 2-position of the pyrrolidine moiety to improve upon the solubility and pharmacokinetic properties of this class of compounds while still maintaining potent antibacterial activity. We have found that the absolute stereochemistry at the 2-position of the pyrrolidine ring is critical to the maintenance of such activity. In this paper, we report the full details of the asymmetric synthesis and the in vitro and in vivo structure-activity relationships of this series of compounds as well as the physiochemical properties, such as water solubility and log P, associated with the structural modifications. We also discuss the pharmacokinetic properties of several of these compounds in mice and the pharmacokinetics of 59, which has the best overall properties of agents in this study, in dog.

Asymmetric synthesis and properties of the enantiomers of the antibacterial agent 7-(3-aminopyrrolidin-1-yl)-1-(2,4-difluorophenyl)-1,4-dihydro-6- fluoro-4-oxo-1,8-naphthyridine-3-carboxylic acid hydrochloride.

Compound 1 [7-(3-aminopyrrolidin-1-yl)-1-(2,4-difluorophenyl)-1,4-dihydro-6-f luoro-4-oxo-1,8-naphthyridine-3-carboxylic acid hydrochloride] is a potent member of the quinolonecarboxylic acid class of antibacterial agents and is currently undergoing clinical evaluation. We have developed efficient asymmetric syntheses of the enantiomers of this agent. The S-(+) enantiomer 1a is 1-2 log2 dilutions more active than the R-(-) enantiomer 1b against aerobic bacteria and 1-2 or more log2 dilutions more active against anaerobic bacteria in vitro. The enantiomer 1a shows significantly better in vivo activity in a Pseudomonas aeruginosa mouse protection model compared to racemic 1. Coupled with the improved solubility profile of 1a relative to racemic material, these features may be of practical significance from a clinical standpoint.

Structure-activity relationships in quinolone antibacterials: design, synthesis and biological activities of novel isothiazoloquinolones.

Over the past years it was found that modification of the 3-carboxylic acid group of quinolones generally produced compounds with a substantial decrease in antibacterial activity. The 3-carboxylic acid moiety together with the 4-carbonyl function are believed to be the most structurally critical sites for this class of compounds to DNA gyrase. The authors have designed and synthesized a series of quinolone analogues in which the 3-carboxylic acid group has been modified. These compounds, 2,3,4,9-tetrahydroisothiazolo[5,4-b]quinoline-3,4-diones, possess biological activities far superior to their parent counterparts. For example, the MICs (microgram/ml) for A-62824 (ciprofloxacin 3-carboxylic acid modified analogue) and ciprofloxacin against some organisms are as follows: S. aureus ATCC 6538P (0.02, 0.2); S. epidermidis 3519 (0.05, 0.2); E. coli Juhl (0.005, 0.01); P. aeruginosa A5007 (0.05, 0.1) and Acinetobacter sp. CMX 699 (0.05, 0.78). This investigation has produced the first successful modification of the 3-carboxylic acid group of quinolones resulting in a series of extremely potent antibacterials. The design and synthesis as well as the biological activities of these new derivatives are described.

Chiral DNA gyrase inhibitors. 2. Asymmetric synthesis and biological activity of the enantiomers of 9-fluoro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-2,3-dihydro-7H- pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid (ofloxacin).

A short and efficient synthesis, starting with (R)- and (S)-alaninol, of the two optical antipodes of the quinolone antimicrobial agent ofloxacin has been devised. Testing in vitro of the products against a range of bacteria and in an assay system incorporating purified DNA gyrase from different bacterial species demonstrates that the S-(-) enantiomer is substantially the more active.

Inhibition of exogenous 3-deoxy-D-manno-octulosonate incorporation into lipid A precursor of toluene-treated Salmonella typhimurium cells.

Analogs of 3-deoxy-D-manno-octulosonate (KDO) were designed to inhibit CTP:CMP-KDO cytidylyltransferase (CMP-KDO synthetase). Since these analogs lacked whole-cell antibacterial activity, a permeabilized-cell method was developed to measure intracellular compound activity directly. The method employed a mutant of Salmonella typhimurium defective in KDO-8-phosphate synthetase (kdsA), which accumulated lipid A precursor at 42 degrees C. Cells permeabilized with 1% toluene were used to evaluate inhibitor effect on [3H]KDO incorporation into preformed lipid A precursor. KDO incorporation proceeded through the enzymes CMP-KDO synthetase and CMP-KDO:lipid A KDO transferase. Optimum KDO incorporation occurred between pH 8 and 9 and required CTP, prior lipid A precursor accumulation, and a functional kdsB gene product, CMP-KDO synthetase. The apparent Km for KDO in this coupled system at pH 7.6 was 1.38 mM. The reaction products isolated and characterized contained 1 and 2 KDO residues per lipid A precursor molecule. Several KDO analogs produced concentration-related reductions of KDO incorporation in toluenized cells with 50% inhibitory concentrations comparable to those obtained in purified CMP-KDO synthetase systems. Two compounds, 8-amino-2-deoxy-KDO (A-60478) and 8-aminomethyl-2-deoxy-KDO (A-60821), competitively inhibited KDO incorporation, displaying Kis of 4.2 microM for A-60478 and 2.5 microM for A-60821. These data indicated that the inactivity of the KDO analogs on intact bacteria was the result of poor permeation into cells rather than intracellular inactivation.

Synthesis and structure-activity relationship of 1-aryl-6,8-difluoroquinolone antibacterial agents.

A series of new arylfluoroquinolones has been prepared. These derivatives are characterized by having fluorine atoms at the 6- and 8-positions, substituted amino groups at the 7-position, and substituted phenyl groups at the 1-position. The in vitro antibacterial potency is greatest when the 1-substituent is 2,4-difluorophenyl and the 7-substituent is a 3-amino-1-pyrrolidinyl group. 1-(4-Fluorophenyl)-6,8-difluoro-7-piperazin-1-yl-1,4-dihydro-4-oxo quinoline-3- carboxylic acid (22) was found to possess excellent in vitro potency and in vivo efficacy.