Comparison of ceftibuten once daily and amoxicillin-clavulanate three times daily in the treatment of acute exacerbations of chronic bronchitis.

In medical practice, antibiotics are generally given empirically for the treatment of acute exacerbations of chronic bronchitis (AECB). To be effective, antibiotic therapy should be broad in spectrum, and it should also cover the common beta-lactamase-producing pathogens. In this multicenter, randomized, investigator-masked study, 469 patients with AECB were randomized (in a ratio of 2:1) to receive 400-mg oral ceftibuten capsules once daily or 500-mg amoxicillin-clavulanate tablets three times daily for 5 to 15 days. Patients receiving ceftibuten were further divided into those who took the capsule with a meal (fed) and those who took the capsule 1 hour before a meal (fasted). Clinical and microbiologic responses were evaluated after treatment at 0 to 6 days (end of treatment) and 7 to 21 days (follow-up). Overall clinical success was determined by cure/improvement of signs and symptoms of AECB at the end of treatment and at follow-up. Overall microbiologic assessment was graded as eradication, persistence, relapse, reinfection, colonization, superinfection, or unassessable. Tolerability was evaluated by grading observed adverse events. The mean duration of treatment was 10.4 days for patients who received ceftibuten and 10.1 days for patients who received amoxicillin-clavulanate. A total of 252 patients receiving ceftibuten and 117 patients receiving amoxicillin-clavulanate were evaluable for clinical efficacy, and 55 patients were evaluable for microbiologic response. Both treatments improved the signs and symptoms of bronchitis, and overall clinical success rates were equivalent for patients treated with ceftibuten (211 of 252 [84%]) and amoxicillin-clavulanate (93 of 117 [79%]) (95% confidence interval [CI], -4.5% to 13.6%). Overall microbiologic eradication rates were also similar for patients treated with ceftibuten (36 of 37 [97%]) and amoxicillin-clavulanate (12 of 14 [86%]) (95% CI, -5.2% to 21.2%). The most frequently reported treatment-related adverse events were gastrointestinal disturbances, which occurred in 15% (47 of 316) and 24% (36 of 152) of patients treated with ceftibuten and amoxicillin-clavulanate, respectively. No significant difference was observed in the ceftibutenfed and ceftibuten-fasted groups in overall clinical assessments of the clinical efficacy population and safety population. In conclusion, 400 mg oral ceftibuten once daily has a similar clinical success rate to 500 mg amoxicillin-clavulanate three times daily, with a trend toward fewer gastrointestinal side effects, in the treatment of patients with AECB.

Resistance to verapamil sensitization of multidrug-resistant cells grown as multicellular spheroids.

The ability of verapamil to overcome resistance to adriamycin in a multidrug-resistant derivative of the V79 cell line (LZ), grown as multicellular spheroids or as monolayers, was examined. Verapamil was much less effective in overcoming resistance to adriamycin in spheroids than in monolayers. Verapamil increased the adriamycin content of cells grown as monolayers, but had no significant effect on the drug content of spheroids. This occurred in spite of the same mdr-I mRNA and protein levels in monolayers and spheroids. When the surviving fraction of cells was normalized to the cellular adriamycin content, cells both in monolayers and spheroids treated with verapamil were still more sensitive to adriamycin than their counterparts not treated with verapamil. The observed resistance of spheroids to adriamycin and verapamil sensitization may be caused by a drug-resistance mechanism that is functional only in spheroids, in addition to the activity of P-glycoprotein. Multicellular tissue architecture and cell-cell contact may play significant roles in this type of multidrug-resistance mechanism.

Influence of prior freezing on arylamine acetylation by human liver preparations.

Human liver tissue is an important resource for use in xenobiotic metabolism studies. Because of the scarcity of human livers, studies are frequently conducted with subcellular preparations from previously frozen material. The influence of prior freezing on arylamine acetylation with human S-9 was studied and differences were found between S-9 preparations derived from fresh liver and from stored frozen liver. Specifically, the capacity for formation of diacetylated benzidine was lost as a result of tissue freezing, and the rate and extent of monoacetylation appeared to be reduced also, though not as dramatically. These results demonstrate that freezing of human tissue prior to conducting metabolism studies with arylamines may alter metabolite profiles and indicate that, for reliable data, before using frozen human tissue in metabolism studies with arylamines one should check first for this possibility.

Combined effects of solvents on the rat's auditory system: styrene and trichloroethylene.

Because exposures to toxic agents typically involve more than one substance, it is necessary to know if combined exposures pose different risks than those to single agents. Many solvents have been implicated in central nervous disorders and some of them are known to produce hearing loss, probably mediated by damage to cochlear hair cells. Hearing loss was studied by recording the brainstem auditory evoked response (BAER) in male Long Evans rats exposed 8 h/day for 5 days to mixtures of styrene (STY) and trichloroethylene (TCE). Dose groups included air or solvent pairs (STY/TCE) in the following concentrations (ppm): (0:3000), (250:2250), (500:1500), (750:750) and (1000:0). Decreased BAER amplitude, indicative of hearing loss, was correlated with blood levels of total solvent. The effects were as predicted by a linear dose-addition model, indicating neither synergistic nor antagonistic interactions at the concentrations studied.

Hypoxia-induced drug resistance: comparison to P-glycoprotein-associated drug resistance.

In this report, we investigate several examples of hypoxia-induced drug resistance and compare them with P-glycoprotein associated multidrug resistance (MDR). EMT6/Ro cells exposed to drugs in air immediately after hypoxic treatment developed resistance to adriamycin, 5-fluorouracil, and actinomycin D. However, these cells did not develop resistance to colchicine, vincristine or cisplatin. When the cells were returned to a normal oxygen environment, they lost resistance. There was no correlation between the content of adriamycin and the development of adriamycin resistance induced by hypoxia. There was no difference between the efflux of adriamycin from aerobic cells and that from hypoxia-treated cells. The mRNA for P-glycoprotein was not detected in the hypoxia-treated cells. These results suggest that hypoxia-induced drug resistance is different from P-glycoprotein associated multidrug resistance.

Polymorphic N-acetylation of sulfamethazine and benzidine by human liver: implication for cancer risk?

Extensive epidemiologic studies have indicated a relationship between bladder cancer in populations exposed to arylamines and the slow phenotype for acetylation of arylamines (e.g., sulfamethazine) and aryl hydrazides (e.g., isoniazid). In human liver preparations, we have examined the association between the capability for sulfamethazine acetylation and that for the human bladder arylamine carcinogen, benzidine. By the usual criteria for polymorphic acetylation, we classified the 10 donor subjects as four rapid and six slow acetylators of sulfamethazine. Concurrent tests of benzidine acetylation in the same liver preparations yielded capacities to acetylate benzidine that were directly and significantly correlated with those for sulfamethazine acetylation (r = 0.672; P less than 0.05). We suggest that acetylation of these two compounds is directly related and knowledge of the human acetylator phenotype may be a useful indicator of possible risk for bladder cancer due to exposure of certain arylamines and, perhaps for other cancers in man.

Combined chromatographic-isotopic dilution analysis of fecapentaenes in human feces.

Fecapentaene-12 (FP-12) and fecapentaene-14 (FP-14) are genotoxic unsaturated ether lipids produced by colonic bacteria in man. We have developed and applied to feces collections from normal volunteers direct isotopic dilution procedures using tritium-labeled (at C5) FP-12 and FP-14 for measuring these compounds. FPs were recovered from feces by solvent extraction, silica cartridge clean-up, and analytical liquid chromatography. Low levels of FP-12 and FP-14 (less than 0.1 to 2.4 micrograms/g of freeze-dried feces) were observed. Identity of chromatographic peaks was established by co-elution and by ultraviolet absorption spectra obtained via photodiode array scanning. Two unknown peaks were tentatively identified from absorption spectra as closely related compounds with increased (hexane?) or decreased (tetraene?) number of double bonds. Levels of FPs increased after incubation of feces at 37 degrees C for 96 h under anaerobic conditions and pre-FP-12 and pre-FP-14 peaks were observed, which showed identical spectra with authentic FPs. These were interpreted to be isomeric forms of the all-trans-[3H]FPs used for the isotopic dilution analysis. Total FPs (including pre-FP) yielded a range of 0.3-80 micrograms FP-12 and 2.8-44 micrograms FP-14 per g of freeze-dried feces from the study group.

Comparative redox activities of anthracyclines by microsomes from P388 cells and rat liver.

In comparative tests of the capabilities of various analogs of doxorubicin and daunorubicin to augment oxygen consumption in microsome preparations from P388 mouse leukemia cells and from rat liver cells, we found statistically significant positive correlations between the Km values for microsomes from the two sources for parent compounds and analogs containing morpholinyl, cyanomorpholinyl- or imino-substitutions. Km values ranged from 0.015 to 1.3 mM, or about 90-fold for all analogs tested. Thus, rat liver microsomes are predictive for this characteristic in P388 microsomes. Examinations of the relationships between Km values from either source and ED50 values for cytotoxicity of the compounds against the P-388 cells also showed statistically significant positive correlations. However, the ED50 values encompassed a range from 0.00020 to 0.22 microM, or about 1000-fold. Therefore, additional mechanism(s) of action besides toxic oxygen radical formation must explain the extremely high cytotoxicity of the cyanomorpholinyl anthracyclines compared with their parent drugs.

N-(2-hydroxyethyl)doxorubicin from hydrolysis of 3'-deamino-3'-(3-cyano-4-morpholinyl)doxorubicin.

The susceptibility of 3'-deamino-3'-(3-cyano-4-morpholinyl)doxorubicin to hydrolysis at pH 7, 4, and 2 has been compared with that of the typically stable morpholine analogue. At pH 7, 74% of the cyanomorpholine was unchanged after 24 h at room temperature, but at pH 2 only 10% remained. Products identified were aglycon (8%) and N-(2-hydroxyethyl)doxorubicin (7%). Most of the losses were to unidentified polar products not eluted from HPLC. Authentic hydroxyethyl was synthesized from doxorubicin by reductive alkylation with glycolaldehyde. Antitumor potency was comparable to that of doxorubicin rather than of cyanomorpholine.

Redox activities of antitumor anthracyclines determined by microsomal oxygen consumption and assays for superoxide anion and hydroxyl radical generation.

To explore the structural characteristics of various derivatives of the anticancer drugs, doxorubicin and daunorubicin, for exhibiting redox activities believed to be associated with toxic radical production, we tested over fifty derivatives in a rapid screening procedure for augmenting oxygen consumption by rat liver microsomes. Measurement of parent drug disappearance and of metabolite appearance for fourteen anthracyclines with a broad range of activities for augmenting oxygen consumption indicated that a single reaction, conversion to the 7-deoxyaglycone, occurred. Multiple tests of selected compounds showed that the liver microsome system exhibited saturation kinetics, and calculated values of Vmax/Km gave the same relative order of activities as did the screening test. The liver microsome system was not found to be stereoselective. Measurements of the abilities of a number of the anthracycline derivatives after chemical activation by reduction with sodium borohydride to convert oxygen to superoxide anion, or to the hydroxyl radical, were also made. The reactivities of the anthracyclines in these latter two assays were positively related to the activities obtained in the rat liver microsome screening test, suggesting that all three tests were measuring various steps in the sequence from anthracycline semiquinone radical formation through oxygen activation and radical formation. Superoxide anion generation from chemically reduced anthracyclines was inhibited by the addition of calf thymus DNA, and the extent of inhibition was positively correlated with the measured DNA association constants of the anthracyclines. However, the DNA association constants were unrelated to superoxide anion generation in the absence of DNA or to the augmentation of oxygen consumption in liver microsomes. Half-wave potentials were negatively correlated with both the results of the microsomal oxygen consumption test and the production of superoxide anion in the chemical test system. No relationships were discerned among the DNA association constants, half-wave potentials, or reoxidizabilities of the anthracyclines tested. Comparisons of the relatively low activities of certain of the anthracyclines in the biochemical and chemical tests for oxygen activation with their known high activities against murine tumors in vivo, but low cardiotoxicities in animal model systems, suggest that the separation of the cytotoxic antitumor and cardiotoxic actions of these derivatives may have been achieved.

Uptake and retention of morpholinyl anthracyclines by adriamycin-sensitive and -resistant P388 cells.

3'-Deamino-3'-(4-morpholinyl)adriamycin (MRA) and 3'-deamino-3'(3-cyano-4-morpholinyl)adriamycin (MRA-CN) were compared with adriamycin (ADR) in ADR-sensitive (P388/S) and -resistant (P388/ADR) murine leukemia cell lines with respect to cytotoxicity and cellular accumulation. MRA is only two- to threefold more cytotoxic to P388/S in culture than ADR, whereas MRA-CN is 500-fold more cytotoxic than ADR to this cell line. Yet both MRA and MRA-CN retain their potency against P388/ADR in spite of a 150-fold decrease in potency for ADR. The observed noncross-resistance of both MRA and MRA-CN in P388/ADR correlates with their increased cellular uptake and retention relative to ADR and the inability of P388/ADR to exclude these analogs as readily as it does ADR. The decreased uptake of MRA and MRA-CN in P388/ADR relative to P388/S (1.5 to 2.0-fold), the increased efflux, and the ability of verapamil to enhance cellular uptake of these analogs in P388/ADR, as it does with ADR, all indicate that the mechanism of ADR-resistance effects ADR and the morpholino analogs in a similar manner but to far different extents. The potent cytotoxicity of MRA-CN appears to be related to strong cellular interactions of the drug with macromolecules that are characterized by its nonextraction from cells by chloroform: methanol or 10 M urea and may therefore represent covalent binding.

Metabolic disposition of 5-iminodaunorubicin in the rat.

We determined the metabolic disposition of 5-iminodaunorubicin (IMD) in rats receiving acute doses of IMD (16 and 4 mg/kg) i.v., i.p., and p.o. Major compounds found in plasma, liver, heart, lung, and brain of the rats receiving the higher dose, i.v. or i.p., were IMD and 5-imino-13-dihydrodaunorubicin. The aglycones, 5-iminodaunorubicinone and 5-imino-13-dihydrodaunorubicinone, were minor metabolites. No deoxyaglycones of IMD were detected in any tissue. We could not detect daunorubicin or its metabolites indicating IMD was not a prodrug of daunorubicin. Highest levels of IMD and metabolites were found in lung, liver, and heart, and lowest levels were found in the plasma and brain. Plasma levels of IMD after the higher i.v. dose decayed in a biphasic manner, and we calculated alpha-phase and beta-phase halftimes of decay of 1.4 and 10 hr, respectively. Patterns of distribution of IMD and metabolites were very similar following i.v. and i.p. treatments, except for higher amounts of IMD and metabolites in the liver after the latter route. A p.o. dose of IMD (16 mg/kg) yielded plasma levels of IMD that were only about 20% of those found by the parenteral routes. Summations of all compounds in all tissues at all times after this treatment yielded less than 2% of the corresponding totals found by the other routes. Results obtained following IMD (4 mg/kg) by the three routes generally confirmed conclusions derived from the studies at the higher dose, and we found an approximate linear dose relationship between the results from the two studies. Our inability to detect the formation of deoxyaglycone metabolites of IMD in vivo is consistent with earlier in vitro findings that IMD does not participate in the oxygen-cycling phenomenon typical of daunorubicin and doxorubicin to form drug and oxygen radicals and deoxyaglycone metabolites.

Metabolic disposition of N,N-dibenzyldaunorubicin in the rat.

The more efficacious and less cardiotoxic analogue of daunorubicin, N,N-dibenzyldaunorubicin (B2D), was found to be metabolized in rats by stepwise debenzylation that was superimposed on the known anthracycline metabolism via 13-ketone reduction and deglycosidation. Using high-pressure liquid chromatography for resolution and fluorescence for detection, we observed a series of metabolites in plasma, liver, heart, muscle, and lungs of rats receiving 10 mg B2D per kg, i.v., i.p., and p.o. Rats receiving 40 mg B2D per kg, i.v., died immediately, but this dose given p.o. was not lethal during 24 hr. Patterns of B2D and metabolites varied quantitatively with tissue and route of administration. Rat liver perfusion studies indicated extensive metabolism of B2D compared with limited metabolism of doxorubicin. These observations were consistent with an observed major first-pass effect on B2D in intact rats given B2D p.o. The predominant metabolites of B2D were the glycosidic derivatives, N-benzyldaunorubicin, daunorubicin, and their 13-dihydro derivatives. These metabolites of B2D had exhibited activity against mouse leukemia P388 as did B2D and were active in in vitro tests in which B2D was essentially inactive. These results indicate that B2D acts as a prodrug for a series of active metabolites. Conversion of B2D to these metabolites was relatively more efficient after p.o. administration than following i.v. or i.p. treatments.

Disposition of prothionamide in rats and armadillos.

A new method for the sensitive and selective measurement of prothionamide (PTH) and its S-oxide metabolite (PTHSO) in biological fluids was described. The limit of sensitivity was approximately 0.01 microgram of drug/ml of plasma. Endogenous materials, 2-propylisonicotinamide, ethionamide, dapsone, or monoacetyl dapsone did not interfere or contribute. Rats receiving PTH, intravenously or orally, showed a sexual dimorphism in the ability to oxidize PTH to PTHSO, with males exhibiting greater capacities for this conversion. Both sexes cleared the administered PTH more rapidly from the plasma than the metabolite, PTHSO. Following oral or intravenous administration of equimolar doses of PTHSO, both sexes exhibited an ability to reduce the administered PTHSO to PTH, with the female showing greater capacities for this conversion. Clearances after oral PTHSO administration were again more rapid for PTH than for PTHSO in both sexes. However, the total of PTH and PTHSO in the plasma during 8 hr following PTHSO administration was consistently less than following PTH dosing. Therefore, although PTHSO is retained longer than PTH after either PTH or PTHSO administration, giving PTHSO yielded less total active drug in the circulation. Comparison of plasma patterns of PTH and PTHSO in unfasted rats receiving one oral or eight daily oral doses of PTH did not indicate that PTH induces its own metabolism. Limited studies in armadillos receiving PTH and PTHSO intravenously led to the same general conclusions as those we derived from the rat studies regarding the disposition of PTH and PTHSO.

Mutagenic activity of antileprosy drugs and their derivatives.

We tested the mutagenic activity of antileprosy drugs (clofazimine, ethionamide, prothionamide, prothionamide-S-oxide, rifampin, and dapsone and many of its derivatives) using the Ames Salmonella/microsome assay system. None of these, including N-acetylated and N-hydroxylated derivatives of dapsone, were found to be positive with or without metabolic activation of this test. However, the sulfoxide and sulfide analogs of dapsone were found to be mutagenic with metabolic activation. These two analogs could not be detected in pharmaceutical preparations of dapsone (less than 0.01%), nor could they be found (in either unconjugated or conjugated form) in urine from volunteers taking a single oral dose of 50 mg of dapsone or from patients receiving daily oral doses of 100 mg of dapsone. Also, urine concentrates from volunteers taking 50 mg of dapsone did not exhibit mutagenic activity in the Ames screen. These results indicate that patients receiving antileprosy therapy with clofazimine, dapsone, ethionamide, prothionamide, and/or rifampin are not being exposed to mutagenic (and thereby possible carcinogenic) drugs.