Takayoshi Hidaka

In vitro and in vivo activities of the benzoxazinorifamycin KRM-1648 against Mycobacterium tuberculosis.

The in vitro and in vivo activities of a new benzoxazinorifamycin, KRM-1648 (KRM), against Mycobacterium tuberculosis were studied. The MIC at which 50% of the isolates are inhibited (MIC50) and the MIC90 of KRM for 30 fresh isolates of M. tuberculosis measured by the BACTEC 460 TB System were 0.016 and 2 micrograms/ml, respectively. These values were much lower than those for rifampin (RMP), which were 4 and >128 micrograms/ml, respectively, and considerably lower than those for rifabutin (RBT), which were 0.125 and 8 micrograms/ml, respectively. A correlational analysis of the MICs of these drugs for the clinical isolates revealed the presence of cross-resistance of the organisms to KRM and either RMP or RBT although the MICs of KRM were distributed over a much lower range than were those of the other two drugs. KRM and RMP at concentrations of 1 to 10 micrograms/ml almost completely inhibited the bacterial growth of RMP-sensitive strains (H37Rv, Kurono, and Fujii) of M. tuberculosis phagocytosed in macrophage-derived J774.1 cells. KRM was more active than RMP in inhibiting the growth of the RMP-resistant (MIC = 8 micrograms/ml) Kurata strain but failed to show such an effect against the RMP-resistant (MIC >128 micrograms/ml) Watanabe stain. When KRM was given to M. tuberculosis-infected mice at dosages of 5 to 20 mg/kg of body weight by gavage, one daily six times per week from day 1 after infection, it was much more efficacious than RMP against infections induced in mice by the RMP-sensitive Kurono strain, as measured by a reduction of rates of mortality, a reduction of the frequency and extent of gross lung lesions, histopathological changes in lung tissues, and a decrease in the bacterial loads in the lungs and spleens of infected mice. KRM also displayed significant therapeutic efficacy against infection induced by the RMP-resistant Kurata strain, while neither KRM nor RMP was efficacious against infection by the RMP-resistant Watanabe strain. In the case of infection with the Kurono strain, the efficacy of the drugs in prolonging the time of survival was in the order KRM, RBT, RMP. KRM was much more efficacious than RMP, when given at 1- to 4-week intervals. These findings suggest that KRM may be useful for the clinical treatment of tuberculosis contracted through RMP-sensitive strains, even when it is administered at long intervals.

Chemotherapeutic efficacy of a newly synthesized benzoxazinorifamycin, KRM-1648, against Mycobacterium avium complex infection induced in mice.

Newly synthesized benzoxazinorifamycin, KRM-1648, was studied for its in vivo anti-Mycobacterium avium complex (MAC) activities. When the MICs were determined by the agar dilution method with Middlebrook 7H11 agar medium, KRM-1648 exhibited similarly potent in vitro antimicrobial activities against the MAC isolated from AIDS and non-AIDS patients, indicating possible usefulness of KRM-1648 against AIDS-associated MAC infections. KRM-1648 exhibited potent therapeutic activity against experimental murine infections induced by M. intracellulare N-260 (virulent strain) and N-478, which has much weaker virulence. Similarly, KRM-1648 exhibited an excellent therapeutic efficacy against M. intracellulare infection induced in NK-cell-deficient beige mice (as a plausible model for AIDS-associated MAC infection), in which a much more progressed state of gross lesions and bacterial loads at the sites of infection were observed. When the infected beige mice were killed at weeks 4 and 8, obvious therapeutic efficacy was seen on the basis of reduction in the incidence and degree of lung lesions and bacterial loads in the lungs and spleen with infections due to M. intracellulare N-241, N-256, and N-260. In this case, the efficacy was the highest in N-260 infection, followed by strain N-241. When mice were observed until infection-induced death, survival time of the infected beige mice was found to be prolonged by KRM treatment. However, KRM-1648 was not efficacious in suppressing the progression of pulmonary lesions and the increase in bacterial loads at the sites of infection, including lungs and spleen, at the late phase of infection. This may imply some difficulty with chemotherapy for AIDS-associated MAC infection, even with KRM-1648 treatment, which has excellent in vitro and in vivo anti-MAC activities, as shown in present study.

Effect of a new rifamycin derivative, rifalazil, on liver microsomal enzyme induction in rat and dog

1. The effect of a new rifamycin derivative, rifalazil (KRM-1648), on liver microsomal enzyme induction was studied in rat and dog with repeated oral administration of the compound. Relative liver weight, cytochrome b5 and P450 contents, enzyme activities of NADPH-cytochrome c reductase, aniline hydroxylase, p-nitroanisole O-demethylase, aminopyrine N-demethylase, and erythromycin N-demethylase were measured. 2. In rat, rifalazil treatment at 300 mg/kg/day for 10 days increased cytochrome b5 content but it did not affect liver weight, P450 content or enzyme activities. In contrast, rifampicin and rifabutin increased relative liver weights, cytochrome contents and enzyme activities under similar conditions. 3. In dog, rifalazil did not affect any parameters at 30 or 300 mg/kg/day for 13 weeks. 4. These findings indicate that rifalazil is not an enzyme inducer in rat and dog. This property differs from other rifamycin derivatives such as rifampicin and rifabutin.

Genotoxicity studies on licorice flavonoid oil (LFO)

Licorice flavonoid oil (LFO) is a new functional food ingredient. In this study, the genotoxicity of LFO was investigated using a test battery of three different methods. In a reverse mutation assay using four Salmonella typhimurium strains and Escherichia coli, LFO did not increase the number of revertant colonies in any tester strain with or without metabolic activation by rat liver S9 mix. In a chromosomal aberration test using Chinese hamster lung (CHL/IU) cells, LFO did not induce any chromosomal aberrations either in the short period test without rat liver S9 mix or in the continuous treatment (24 h or 48 h) test. However, in the short-period test with rat liver S9 mix, LFO induced structural chromosomal aberrations at concentrations higher than 0.6 mg/mL. A bone marrow micronucleus test using male F344 rats was initially conducted. The animals were dosed by oral gavage at doses up to 5000 mg/kg/day. No significant or dose-dependent increases in the frequency of micronucleated polychromatic erythrocytes (MNPCE) were observed and the high dose suppressed the ratio of polychromatic erythrocytes (PCE) to total erythrocytes. Subsequently, a liver and peripheral blood micronucleus test using male F344 rats was conducted. No micronuclei induction either in hepatocytes or PCE was observed even at the highest dose of 5000 mg/kg/day. From the findings obtained from the genotoxicity assays performed in this study and the published pharmacokinetic studies of LFO, it appears unlikely that dietary consumption of LFO will present any genotoxic hazard to humans.

90-Day repeated-dose toxicity study of licorice flavonoid oil (LFO) in rats

Licorice flavonoid oil (LFO) is a new functional food ingredient consisting of licorice hydrophobic polyphenols in medium-chain triglycerides (MCT). As part of a safety evaluation, a 90-day oral toxicity study in rats was conducted using an LFO concentrate solution (2.90% glabridin). Male and female animals were assigned to one of 12 groups (10 males or females per group) and received corn oil (negative control), MCT (vehicle control), or 400, 600, 800 or 1600 mg/kg of the LFO concentrate solution. In conclusion, LFO concentrate solution induced an anticoagulation effect in both sexes, although there was a clear sex difference. Based on these findings, it is concluded that the no-observed-adverse-effect level (NOAEL) for the LFO concentrate solution is estimated to be 800 mg/kg/day for female rats, and approximately 400 mg/kg/day for male rats.

Identification of enzymes responsible for rifalazil metabolism in human liver microsomes

1. The major metabolites of rifalazil in human are 25-deacetyl-rifalazil and 32- hydroxy-rifalazil. Biotransformation to these metabolites in pooled human liver microsomes, cytosol and supernatant 9000g (S9) fractions was studied, and the enzymes responsible for rifalazil metabolism were identified using inhibitors of esterases and cytochromes P450 (CYP). 2. The 25-deacetylation and 32-hydroxylation of rifalazil occurred in incubations with microsomes or S9 but not with cytosol, indicating that both the enzymes responsible for rifalazil metabolism were microsomal. Km and Vmax

Therapeutic effect of KRM-1648 with various antimicrobials against Mycobacterium avium complex infection in mice

A new benzoxazinorifamycin, KRM-1648 (KRM), was studied for its therapeutic efficacy in combination with other antimicrobials against Mycobacterium avium complex infections in mice. When M. intracellulare-infected (intravenously) mice were given KRM, clarithromycin (CAM), sparfloxacin (SPFX), or ethambutol (EB) each alone or in combination, by gavage, once daily 6 times per week (streptomycin [SM] was given subcutaneously twice per week) from day 1, KRM + CAM exhibited combined efficacy in terms of reducing the incidence of gross lung lesions and the bacterial loads in the lungs and spleens. The addition of either EB or EB + SPFX to KRM + CAM increased the efficacy. Moreover, the multi-drug regimen of KRM + CAM + EB + SPFX or ofloxacin [OFLX]) was more efficacious than rifampicin (RMP) + CAM + EB + SPFX (or OFLX). In M. avium infection, KRM + clofazimine was the most efficacious among two-drug combinations tested followed by KRM + SM. KRM + CAM was considerably less effective against M. avium than against M. intracellulare infection. KRM + EB and KRM+OFLX failed to show such a combined effect.

In vitro antimicrobial activity of benzoxazinorifamycin, KRM-1648, against Mycobacterium avium complex, determined by the radiometric method.

MICs of a newly developed benzoxazinorifamycin derivative, KRM-1648, for Mycobacterium avium complex (MAC) were determined by the BACTEC 460 TB system and compared with those of other known antimicrobial agents. The radiometric method gave a fast, accurate, and reproducible MIC for each antimicrobial agent. MICs of KRM-1648 for 30 strains of MAC (10 strains each of M. avium isolated from AIDS and non-AIDS patients and of Mycobacterium intracellulare isolated from non-AIDS patients) were measured. The MICs, ranging from 0.004 to 0.0625 microgram/ml, were the lowest of all tested drugs, including rifampin, rifabutin, streptomycin, kanamycin, isoniazid, ethambutol, ofloxacin, ciprofloxacin, sparfloxacin, and clarithromycin. The MICs were 2 to 512 and 1 to 32 times lower than those of rifampin and rifabutin, respectively. With rifampin and ethambutol, there were some differences between the MICs for M. avium isolated from AIDS patients (American) and those for M. avium from non-AIDS patients (Japanese). Moreover, appreciable differences between the MICs of some drugs against M. avium and M. intracellulare isolated from non-AIDS patients were found. Many strains of M. avium were more susceptible to ofloxacin than M. intracellulare, but, conversely, M. avium was more resistant to rifampin, streptomycin, ethambutol, and clarithromycin than M. intracellulare.

Isolation and identification of major metabolites of rifalazil in mouse and human

1. Three metabolites of rifalazil have been isolated from dog urine and identified as 25-deacetyl-rifalazil, 30-hydroxy-rifalazil and 25-deacetyl-30-hydroxy-rifalazil. In the current study major metabolites of rifalazil in mouse and human were isolated and identified, and their antimicrobial activities determined. 2. Urinary excretion of rifalazil and its metabolites in six mouse strains, CD-1 (ICR), BALB/c, C57BL/6, C3H/He, DBA/2 and CBA/J, was examined. Two major metabolites were detected in mouse urine obtained after several oral doses, and the proportion of rifalazil metabolites against total urinary excretion varied over a 2-fold range (4.8-8.7%) in the different mouse strains. 3. One of two major metabolites in mouse urine was 25-deacetyl-rifalazil and the other was unknown: it was isolated from mouse urine and identified by ms and 1H- and 13C-nmr as 32-hydroxy-rifalazil. 4. In human, two major metabolites of rifalazil were detected in urine obtained after administration of a single oral dose. These metabolites were also produced by incubation of rifalazil with pooled human liver microsomes, and identified by lc/ms and lc/ms/ms as 25-deacetyl-rifalazil and 32-hydroxy-rifalazil. 5. The antimicrobial activities of 32-hydroxy-rifalazil against gram-positive bacteria and mycobacteria were similar with those of the parent compound.

Therapeutic efficacy of the benzoxazinorifamycin KRM-1648 against experimental Mycobacterium avium infection induced in rabbits.

The therapeutic efficacy of the benzoxazinorifamycin KRM-1648 was studied in an experimental rabbit infection system with avian Mycobacterium avium. The infected rabbits died from Yersin type infections, a peculiar type of experimental bovine tuberculosis characterized by a very rapid course, enlargement of the spleen and liver, and septic infection, 14 to 20 days after bacterial challenge, as evidenced by bacteremia and severe bacterial loads in the visceral organs. Histopathologic studies of the visceral organs of the infected rabbits revealed the development of numerous typical granulomatous lesions. This experimental rabbit infection system, features of which resemble certain features of disseminated M. avium complex infections in AIDS patients, was used to evaluate the therapeutic efficacy of KRM-1648, a newly synthesized benzoxazinorifamycin. KRM-1648 given orally at 25 and 50 mg/kg of body weight reduced the incidence and degree of bacteremia in infected rabbits and protected against subsequent death. Moreover, the drug allowed almost complete recovery of infected rabbits by week 7. KRM-1648 cleared infections in the lungs, liver, spleen, and kidneys and restored histopathologic features of healthy tissue in the visceral organs. KRM-1648 exhibited a more potent therapeutic effect against M. avium infection than rifampin and clarithromycin. Images