Different effects of FK317 on multidrug-resistant tumor in vivo and in vitro.

FK317, a novel substituted dihydrobenzoxazine, was examined for antitumor effects on multidrug-resistant (MDR) tumor cells in vitro and in vivo. In nude mice, FK317 markedly inhibited the growth of s.c. implanted KB-V1 vinblastine (VLB)-resistant human epidermal carcinoma KB cells, as well as the parent cells (KB-3-1). However, KB-V1 showed much greater resistance to FK317 than to VLB and adriamycin (ADM) in the in vitro study. This resistance was reversed by the addition of verapamil, whereby intracellular accumulation of FK317 in the KB-V1 cells was also decreased. After incubation of FK317 in human and mouse blood, it was shown to be rapidly metabolized to a monodeacetylated form, and slowly metabolized further to a dideacetylated form. With the removal of the acetyl groups from FK317, resistance indexes in KB-V1 and SBC-3/ADM, ADM-resistant human lung carcinoma, decreased. In addition, photolabeling of P-glycoprotein with [3H]azidopine in KB-V1 plasma membrane was completely inhibited by FK317, but not by the deacetylated metabolites. These results indicate that FK317 is metabolized to deacetylated forms, which do not bind to P-glycoprotein and are incorporated into MDR cells, causing cytotoxic effects.

The development of a sensitive and specific enzyme immunoassay for FK480, a novel cholecystokinin type-A receptor antagonist, in human plasma.

A sensitive and specific enzyme immunoassay for FK480, a novel cholecystokinin type-A (CCK-A) receptor antagonist, was developed to study the pharmacokinetics of the drug at low-dose administration using a specific monoclonal antibody. The high performance liquid chromatography (HPLC) method had been used for studying toxicokinetics, but its determination limit (2.5 ng ml-1) was too high for use in clinical studies. Subsequently we developed an enzyme immunoassay (EIA) using rabbit anti-FK480 serum (polyclonal antibody). It had higher sensitivity (0.1 ng ml-1) when 0.5 ml of plasma was used but its specificity was low because of the cross-reactivity of the metabolites of FK480. Therefore we produced several monoclonal antibodies for FK480 by cell fusion, and selected the antibody which was least cross-reactive for the isolated metabolites of FK480. Finally we developed a sensitive and specific EIA using this monoclonal antibody. The lower limit of quantification of this method was 0.2 ng ml-1 when 0.2 ml of human plasma was used. The coefficient of variation over the calibration range (0.2-10 ng ml-1) was less than 15%. We used this method for clinical studies, and it showed a good correlation to the HPLC method when plasma concentration was 2.5 ng ml-1 or more.

FK317, a novel substituted dihydrobenzoxazine, exhibits potent antitumor activity against human tumor xenografts in nude mice.

The antitumor effects of FK317, a novel substituted dihydrobenzoxazine, were evaluated using human tumor xenografts (small cell lung cancer, non-small cell lung cancer, stomach cancer, colon cancer, pancreatic cancer, breast cancer, cervical cancer and ovarian cancer). Tumor growth-inhibitory effects and the effective dose-range of FK317 were much stronger and broader, respectively, than those of reference drugs such as mitomycin C, adriamycin, cisplatin, taxol and irinotecan. Furthermore, the body weight decrease and myelosuppression in FK317-treated mice were less than in the animals given any of the reference drugs. To explain this tumor selectivity, the distribution of FK317 was investigated after dosing tumor-bearing mice with the 14C-labelled compound. The concentration of FK317 in tumor tissues was relatively low, and long tumor retention was not observed. However, thin-layer chromatographic separation revealed that the radioactivity in the tumor resided mainly in strongly cytotoxic metabolites, while that in other tissues resided mainly in non-cytotoxic metabolites. These results suggest that FK317 shows strong antitumor activity without side effects, and one reason for this is its specific metabolite pattern. FK317 is now undergoing phase I clinical trials.

Determination of the R,R- and S,S-enantiomers of vamicamide in human serum and urine by high-performance liquid chromatography on a Chiral-AGP column.

An enantioselective liquid chromatographic assay for the determinations of the R,R- and S,S-enantiomers of vamicamide, a potent anticholinergic drug, in human serum and urine is described. Racemic vamicamide and internal standard were purified from biological fluids using a two-step extraction procedure involving diethyl ether and 0.1% phosphoric acid. The overall recoveries of racemic vamicamide and internal standard were greater than 80%. The purified samples were measured by high-performance liquid chromatography on a Chiral-AGP column with ultraviolet absorbance detection at 260 nm. The standard curves for the analytes were linear from 10 to 200 ng/ml in serum and from 0.25 to 50 micrograms/ml in urine. The quantification limit of both enantiomers was 10 ng/ml for serum and 250 ng/ml for urine. Both intra-day and inter-day accuracy and precision data showed good reproducibility of the method. The assay has been applied for the analysis of vamicamide enantiomers in serum and urine samples from a healthy volunteer.

A new antitumor antibiotic, FK973: its metabolism in the blood and the antitumor effects of its metabolites on experimental models.

Our previous studies showed that a new, substituted dihydrobenzoxazine, FK973 (11-acetyl-8-carbamoyloxymethyl-4-formyl-14-oxa-1,11-diazatetracyclo+ ++- [7.4.1.0(2,7).0(10,12)tetradeca-2,4,6-trien-6,9-diyl diacetate), which is a triacetylated derivative of the fermentation product FR900482 of Streptomyces sandaensis No. 6897, had potent antitumor effects on experimental tumors in vivo and in vitro. In the present study, we investigated the metabolism of FK973 in the blood of human and animals and the antitumor effects of its metabolites. After the incubation of FK973 in the blood (hemolysate) or serum of humans, dogs, rats and mice, it was rapidly metabolized to two diacetates and a monoacetate, and slowly to FR900482. FK973 and all its deacetylated metabolites showed strong cytotoxicity on in vitro cultured murine L1210 leukemia cells, and the cytotoxicity of FK973 was the most potent. In the vivo experiments, FK973 and its metabolites prolonged the life of mice bearing ascitic P388 leukemia, and it potently inhibited the growth of murine B16 melanoma and Colon 38 adenocarcinoma implanted subcutaneously in mice. FK973 was the most effective compound. Thus, these results suggest that the antitumor effects of FK973 are stronger than those of its deacetylated metabolites produced in the blood of humans and animals.