PNU-159548, a novel cytotoxic antitumor agent with a low cardiotoxic potential

PURPOSE: PNU-159548 (4-demethoxy-3'-deamino-3'aziridinyl-4'-methylsulphonyl-daunorubicin), a derivative of the anticancer idarubicin, has a broad spectrum of antitumoral activity in vitro and in vivo attributable to its DNA intercalating and alkylating properties. The present study was conducted to determine the cardiotoxic activity of PNU-159548 relative to doxorubicin in a chronic rat model sensitive to anthracycline-induced cardiomyopathy. METHODS: Young adult male rats were allocated to the following treatment groups: group 1, PNU-159548 vehicle control (colloidal dispersion); group 2, doxorubicin control (saline); groups 3, 4, 5, 6, and 7, PNU-159548 at 0.12, 0.25, 0.50, 0.75, and 1.0 mg/kg, respectively; and group 8, 1.0 mg/kg doxorubicin. Treatments were administered intravenously once weekly for 4 weeks (first sacrifice time) or for 7 weeks (rats killed at weeks 8, 12, 22, 27, or 35). Body weights, organ weights, serum chemistry, hematology, serum troponin-T, and cardiac histopathology were followed throughout the study. RESULTS: Doxorubicin caused irreversible cardiomyopathy evident at week 4 in some rats and progressing in severity in all rats by week 8. There were also marked myelotoxicity, increased liver and kidney weights, testicular atrophy, and about 20% mortality by week 27 in doxorubicin-treated rats. The deaths were attributed to cardiomyopathy and/or nephropathy. PNU-159548 caused a dose-dependent myelotoxicity, with the dose of 0.5 mg/kg per week being equimyelotoxic to 1.0 mg/kg per week doxorubicin. PNU-159548 also caused an increase in liver weight that was reversible and a non-reversible testicular atrophy but, unlike doxorubicin, had no effect on kidney weight. At equimyelotoxic doses, the cardiotoxicity caused by PNU-159548, expressed as the mean total score, was less than one-twentieth of that induced by doxorubicin, and much less than that predicted on the basis of its content of idarubicin, which is in turn markedly less cardiotoxic than doxorubicin. CONCLUSIONS: The novel cytotoxic antitumor derivative, PNU-159548, is significantly less cardiotoxic than doxorubicin at equimyelosuppressive doses. The combination of intercalating and alkylating activities within the same molecule without the cardiotoxic side effects of anthracyclines makes PNU-159548 an excellent candidate for clinical development in oncology.     

Inhibitory effects of anticancer drugs on dextromethorphan-O-demethylase activity in human liver microsomes

The dextromethorphan-O-demethylase activity determined in human liver microsomes was used to screen various anticancer drugs for their ability to inhibit this cytochrome CYP2D6-dependent activity. Competitive inhibition indicates that the drug binds the enzyme and is potentially subjected to a polymorphic metabolism. Among the 13 anticancer drugs tested, 4 compounds caused competitive inhibition of dextromethorphan-O-demethylation: lomustine (Ki=7.7 M), doxorubicin (Ki=75 M), vinorelbine (Ki=22 M), and vinblastine (Ki=42 M). The results of these studies indicate that the metabolism of the drugs concerned is possibly altered in poor metabolizers of debrisoquine and requires further investigation to study their specific routes of biotransformation. The metabolism of these drugs probably involves various biotransformation pathways, among which the CYP2D6-dependent route would be of minor importance. A second hypothesis is that these drugs could be inhibitors of the isozyme without being a substrate.     

Influence of supplemental ascorbate on the antitumor activity of 5-hydroxydopa, a purported cytotoxic metabolite

The present study was conducted to clarify the mechanism responsible for enhancement of the anti-melanoma activity of levodopa methylester by supplemental ascorbate in vivo. 5-Hydroxydopa, a known cytotoxic agent and the major metabolite formed from levodopa in the presence of ascorbate and mushroom tyrosinase in vitro, was assessed for its antitumor activity against i.p. and s.c. inoculated B16 melanoma, P388 leukemia, and L1210 leukemia in mice with and without supplemental ascorbate. Treatment with 5-hydroxydopa failed to significantly increase survival of mice bearing i.p. or s.c. pigmented and non-pigmented B16 melanomas even though it inhibited local tumor growth. Treatment increased survival of both P388 and L1210 leukemias, and this increase was more pronounced in mice bearing i.p. tumors than in mice bearing s.c. tumors. This treatment significantly decreased final tumor weight of both leukemias implanted s.c., and inhibited ascites formation in mice inoculated with i.p. tumors. Ascorbate supplementation decreased or abrogated the effect of 5-hydroxydopa on survival in mice bearing i.p. or s.c. leukemia tumors and decreased survival relative to control mice bearing i.p. or s.c. pigmented and s.c. non-pigmented tumors. It did not affect survival of treated mice bearing i.p. non-pigmented melanoma tumors. Ascorbate supplementation did not modify the effect of 5-hydroxydopa treatment on primary s.c. tumor growth in mice bearing melanoma or leukemia tumors nor did it affect ascites formation in treated mice bearing i.p. leukemia tumors. The lack of correlation between the observed inhibition of primary tumor growth and the absence of an effect on survival in 5-hydroxydopa treated mice bearing i.p. melanoma may relate to an inability of this drug to interfere with tumor metastasis. These data argue against a role for 5-hydroxydopa as a metabolically derived cytotoxic formed in situ during concurrent treatment with levodopa methylester and supplemental ascorbate.     

Dihydrofluorouracil, a fluorouracil catabolite with antitumor activity in murine and human cells

Dihydrofluorouracil (FUH2), the initial catabolite of 5-fluorouracil (FUra), was examined to determine whether this derivative had antitumor activity or host cell (bone marrow) toxicity. Studies were undertaken with Ehrlich ascites tumor and bone marrow cells isolated from CF-1 mice. Cells were exposed for 1 h either to no drug (control) or to varying concentrations, ranging from 1 to 250 microM, of either FUra, FUH2, or alpha-fluoro-beta-alanine. Cells were then cultured and colony formation was assessed after 10 to 14 days. Ehrlich ascites tumor cells were more sensitive to FUra [50% lethal dose (LD50) = 18 microM] than to FUH2 [LD50 = 50 microM], with no sensitivity to alpha-fluoro-beta-alanine even at 250 microM. Bone marrow cells had a toxicity profile similar to that of FUra (LD50 = 10 microM) but were relatively insensitive to FUH2 (LD50 greater than 250 microM), with no sensitivity to alpha-fluoro-beta-alanine. Subsequent studies examined colony formation of the human breast carcinoma cell line MCF-7 following 1 h exposure to varying concentrations of FUra and FUH2. These cells were less sensitive to both FUra (LD50 approximately 80 microM) and FUH2 (LD50 approximately 350 microM). Initial studies on the mechanism of toxicity of FUH2 demonstrated that this FUra catabolite could produce inhibition of thymidylate synthase activity in Ehrlich ascites tumor cells with a pattern similar to that resulting from exposure to FUra. This is the first study to demonstrate that FUH2 (a quantitatively important catabolite of FUra) is cytotoxic, and it suggests that FUH2 may contribute to the toxicity of FUra in vivo, possibly by being anabolized to FUra.     

Anticellular and antitumor activity of duocarmycins, novel antitumor antibiotics.

The anticellular and antitumor activities of novel antitumor antibiotics, duocarmycins (DUMs), were examined against human and murine tumor cells. DUMs consist of five compounds, A, B1, B2, C1 and C2, which possess a pharmacophore similar to that of CC-1065, a previously isolated antibiotic. Among them, DUMA exhibited ultrapotent growth-inhibitory activity with an IC50 value of 6 pM against human uterine cervix carcinoma HeLa S3 cells. DUMA and DUMB1 also inhibited the growth of adriamycin (ADM)-resistant lines of human nasopharynx carcinoma KB cells and breast carcinoma MCF-7 cells as well as their sensitive lines. DUMs inhibited the growth of s.c.-inoculated murine tumors such as B16 melanoma, sarcoma 180, M5076 sarcoma and colon 26. DUMs were also significantly effective in increasing the lifespan of i.p.-inoculated B16 melanoma-bearing mice, although their effect was marginal against other i.p.-inoculated tumors. As a whole, DUMB1 exhibited superior activity to the other four compounds. DUMB1 rapidly inhibited the incorporation of [3H]-TdR into macromolecules of HeLa S3 cells as compared with that of [3H]UR or [3H]leucine. DNA strand breaks were detected in DUMB1-treated HeLa S3 cells by agarose gel electrophoresis with a contour-clamped homogeneous electric field apparatus. These results indicate that DUMs possess interesting biological activities as DNA-targeting antitumor antibiotics.     

Metabolism of N-nitrosodialkylamines by human liver microsomes

The metabolism of N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine, N-nitrosobenzylmethylamine, and N-nitrosobutylmethylamine was investigated in incubations with human liver microsomes. All of the 16 microsomal samples studied were able to oxidize NDMA to both formaldehyde and nitrite at NDMA concentrations as low as 0.2 mM; the rates of product formation of the samples ranged from 0.18 to 2.99 nmol formaldehyde/min/mg microsomal protein (median, 0.53 nmol). At a concentration of 0.2 mM NDMA, the rates of denitrosation (nitrite formation) were 5 to 10% (median, 6.3%) those of demethylation (formaldehyde formation); the ratio of denitrosation to demethylation increased with increases in NDMA concentration, in a similar manner to rat liver microsomes. Immunoblot analysis with antibodies prepared against rat P-450ac (an acetone-inducible form of cytochrome P-450) indicated that the P-450ac [P-450j (isoniazid-inducible form)] orthologue in human liver microsomes had a slightly higher molecular weight than rat P-450ac and the amounts of P-450ac orthologue in human liver microsomes were highly correlated with NDMA demethylase activities (r = 0.971; P less than 0.001). Analysis of four selected microsomal samples showed that human liver microsomes exhibited at least three apparent Km and corresponding Vmax values for NDMA demethylase. This result, suggesting the metabolism of NDMA by different P-450 enzymes, is similar to that obtained with rat liver microsomes, even though most of the human samples had lower activities than did the rat liver microsomes. The high affinity Km values of the four human samples ranged from 27 to 48 microM (median, 35 microM), which were similar to or slightly lower than those observed in rat liver microsomes, indicating that human liver microsomes are as efficient as rat liver microsomes in the metabolism of NDMA. The human liver microsomes also catalyzed the dealkylation and denitrosation of other nitrosamines examined. The rates of product formation and the ratios of denitrosation to dealkylation varied with the structures and concentrations of the substrates as well as with the microsomal samples tested. The results indicate that human liver microsomes are capable of metabolizing N-nitrosodialkylamines via the pathways that have been established with rat liver microsomes.     

Pattern of antitumor activity of a novel camptothecin, ST1481, in a large panel of human tumor xenografts.

PURPOSE: ST1481 is the lead compound of a novel series of 7-modified camptothecins, the 7-oxyimino methyl derivatives, characterized by potent topoisomerase I inhibition and cytotoxic activity. Based on its therapeutic efficacy in a human non-small cell lung carcinoma model and its favorable pharmacological profile, the novel analogue was selected for further preclinical development. EXPERIMENTAL DESIGN: We investigated the growth-inhibitory effects of ST1481 and topotecan, used as a reference compound, in a panel of human tumor cell lines of various tumor types (ovarian carcinoma, glioblastoma, osteosarcoma, and melanoma), including sublines with acquired resistance to cisplatin. We explored the antitumor efficacy in a large panel of human tumor xenografts, with particular reference to intrinsically resistant tumor types, using oral administration and an intermittent treatment schedule. RESULTS: ST1481 showed a potent antiproliferative activity with comparable effects in all tested cell lines. Only U-87-MG glioma cells were less sensitive, presumably as a consequence of the efficiency of the S-phase checkpoint. ST1481 produced a remarkable antitumor effect (tumor volume inhibition > 85%) in 16 of 18 examined models, with an appreciable rate of complete tumor regressions in 11 of 18 models (despite the nonoptimal intermittent treatment schedule). The most impressive antitumor effects were observed against lung carcinoma, melanoma, and osteosarcoma models, as documented by the high rate of complete responses (up to 100%). The efficacy of ST1481 was significantly superior to that of topotecan in 9 of 17 tumors. The novel drug was also markedly effective against slowly growing tumors (A549 lung carcinoma and HT29 colon carcinoma) when a daily protracted treatment was used to fully exploit the therapeutic potential of camptothecins. CONCLUSIONS: The unusual potency of ST1481 in a variety of tumor cell lines suggests the ability of the drug to overcome several resistance factors. The profile of antitumor efficacy further supports the therapeutic interest in the novel analogue and provides a rational basis for clinical evaluation in selected tumor types.     

Drug interactions of paclitaxel metabolism in human liver microsomes

The human liver metabolism of paclitaxel (Taxol), an anticancer drug, leads to three metabolites: 6alpha-hydroxypaclitaxel, 3'-p-hydroxypaclitaxel and 6alpha,3'-p-dihydroxypaclitaxel. The inter-individual variability of paclitaxel metabolism was investigated first in vitro using 22 human liver microsomes. Three metabolites have been detected by HPLC. This preliminary work revealed marked inter-individual differences in paclitaxel metabolism. The amount of major metabolite 6alpha-hydroxypaclitaxel formed varied 16-fold (0.7 to 11.5 nmol/mg/h). We next studied the effect of 29 compounds (antineoplastics, antiemetics, histamine-2 receptor antagonist, antalgics, antifungals, antivirals, psychotropics, antibiotic, corticoid, antiarrhythmic, calcium channel blocker) on paclitaxel metabolism in human liver microsomes. Among the compounds studied, quercetin, antifungal drugs such as ketoconazole and miconazole, and the antineoplastic drug doxorubicin inhibited formation of 6alpha-hydroxypaclitaxel. Dixon plots indicated that quercetin and doxorubicin inhibited 6alpha-hydroxypaclitaxel formation through a competitive mechanism with a Ki of 10.1 microM and 64.8 microM, respectively. The inhibition of this metabolite by ketoconazole was through a noncompetitive mechanism with a Ki of 11.8 microM. Our data thus suggest that special attention should be paid when these drugs are combined in clinical practice.     

Gamma-hydroxybutyric acid, a metabolite of UFT, shows anti-angiogenic activities and antitumor effect

We investigated the antitumor vasculogenesis and antitumor activity of gamma-hydroxybutyric acid (GHB), a metabolite of UFT. In a mouse dorsal air sac (DAS) assay, UFT demonstrated a wide spectrum of anti-tumor vasculogenesis except for AZ-521 tumor. Although the expression of vascular endothelial growth factor (VEGF) was detected in almost all tumor cell lines used in the DAS assays, expression of basic fibroblast growth factor (bFGF) was only detected in the AZ-521 tumor. GHB inhibited the chemotactic migration and morphological changes of human umbilical vein endothelial cells (HUVECs) induced by VEGF at IC50 values of 2.8 and 0.31 microM respectively. In addition to these in vitro assays, GHB blocked tumor growth of MC-5, a human breast cancer, in a xenograft model at inhibition rate of 37%. Moreover, GHB showed an additive effect in combination with 5-FU in this model. These results indicate that the anti-tumor vasculogenesis activity of GHB is involved in part in the antitumor effect of UFT.     

Alpha-hydroxylation of N-nitrosopyrrolidine and N'-nitrosonornicotine by human liver microsomes

The environmental carcinogens N-nitrosopyrrolidine (NPYR) and N'-nitrosonornicotine (NNN) were incubated with human liver microsomes to test for the presence of enzymes catalyzing alpha-hydroxylation, which is likely to be an activation mechanism for these compounds. Both nitrosamines underwent alpha-hydroxylation; rates were higher for NPYR than for NNN, as observed in rats. The results indicate that both NPYR and NNN can be metabolically activated by human liver microsomes.     

In vitro metabolism of benzo(a)pyrene by human liver microsomes and lymphocytes.

The metabolism of benzo(a)pyrene by human liver microsomes and human lymphocytes has been analyzed by high-pressure liquid chromatography. Human liver forms seven known metabolites and at least five additional unidentified metabolites that migrate as distinct peaks. Lymphocytes incubated with benzo(a)pyrene for 30 min do not form dihydrodiols. Lymphocytes incubated for 24 hr with benzo(a)pyrene form all of the metabolites produced by liver including dihydrodiols as well as additional metabolites. The ratios of phenols formed by liver and lymphocytes are different, and preparations from humans form a different profile of metabolites than that formed by rat liver.     

Cytotoxicity and antitumor activity of carzelesin, a prodrug cyclopropylpyrroloindole analogue.

The cyclopropylpyrroloindole analogues are DNA minor-groove binders containing a cyclopropyl group, which mediates N3-adenine covalent adduct formation in a sequence-selective fashion. Carzelesin (U-80244) is a cyclopropylpyrroloindole prodrug containing a relatively nonreactive chloromethyl precursor to the cyclopropyl function. Activation of carzelesin requires two steps, (a) hydrolysis of a phenylurethane substituent to form U-76073, followed by (b) ring closure to form the cyclopropyl-containing DNA-reactive U-76074. The formation of the DNA-reactive U-76074, via U-76073, from carzelesin was shown to proceed very slowly in phosphate-buffered saline (t1/2 greater than 24 h) but to occur rapidly in plasma from mouse, rat, dog, and human (initial t1/2 values ranging from 18 min for mouse to 52 min for rat) and in cell culture medium (t1/2 approximately 40 min). Although carzelesin was less potent in terms of in vitro cytotoxicity and in vivo optimal dosage and showed low affinity for binding to DNA, it was therapeutically more efficacious against mouse L1210 leukemia than was U-76074 or adozelesin (U-73975), another cyclopropylpyrroloindole analogue which is currently in phase I clinical trials. Carzelesin also proved to be more efficacious than U-76074 or adozelesin against mouse pancreatic ductal 02 adenocarcinoma, a system reported to be resistant to every agent tested. Carzelesin was highly effective against this tumor and produced 97% tumor growth inhibition. In addition, i.v. administered carzelesin showed significant activity (National Cancer Institute criteria) against i.v. or s.c. implanted Lewis lung carcinoma, i.p. or s.c. implanted B16 melanoma, s.c. implanted colon 38 carcinoma, and five s.c. implanted human tumor xenografts, including clear cell Caki-1 carcinoma, colon CX-1 adenocarcinoma, lung LX-1 tumor, ovarian 2780 carcinoma, and prostatic DU-145 carcinoma. Carzelesin treatment produced 100% complete remissions (no palpable tumor mass at the termination of the experiment) in mice bearing early-stage human ovarian 2780. Pharmacologically, carzelesin proved to be relatively schedule and route independent and was highly active against i.p. implanted L1210 leukemia, regardless of whether the analogue was given i.v., i.p., s.c., or p.o. These results, collectively, suggest that carzelesin is absorbed and distributed well. Both carzelesin and adozelesin caused marked tumor shrinkage in mice bearing human lung LX-1 or advanced-stage human ovarian 2780 carcinoma; however, tumor regrowth occurred shortly after the treatment with adozelesin was stopped. Little or no apparent tumor regrowth occurred after treatment with carzelesin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Factors influencing the activity of dimethylnitrosamine demethylase in hamster, rat and chicken liver microsomes

In vitro metabolism of dimethylnitrosamine (DMN) by liver microsomal fractions of hamster, rat and chicken revealed that the three species under certain assay conditions, were capable of metabolizing DMN at different rates (hamster greater than rat greater than chicken). The magnitude of the demethylase activity was found to be dependent on the nature of the buffer, the concentration of cytochrome P-450 (P-450) and the concentration of the substrate DMN. Enzyme activity was higher in Hepes buffer than in the phosphate buffer. Concentrations of phosphate higher than 20 mM inhibited the activity of the rat and chicken enzymes. This effect of phosphate was not a consequence of increase in ionic strength since KCl over a wide range of concentration failed to inhibit the activity.     

Isoflavones modulate the glucuronidation of estradiol in human liver microsomes

Soy food has been associated with a reduced incidence of hormonal cancer in Asian countries, and the soy isoflavones daidzein and genistein are believed to protect against tumors induced by the endogenous hormone 17beta-estradiol (E2). In the present study, we have examined if daidzein and genistein as well as several structurally related isoflavones are able to modulate the in vitro glucuronidation of E2 in human hepatic microsomes. It is known that different isoforms of UDP-glucuronosyltransferase (UGT) are involved in E2 glucuronidation: UGT1A1 leads exclusively to the 3-glucuronide and is stimulated by E2 via homotropic kinetics, whereas UGT2B7 gives rise to the 17-glucuronide of E2 following Michaelis-Menten kinetics. In our study, daidzein markedly stimulated the 3-glucuronidation, thereby enhancing the metabolic clearance of E2. In contrast, genistein inhibited the 3-glucuronidation. The 17-glucuronidation of E2 was not affected by either compound. Formononetin and the daidzein metabolites equol, 3'-hydroxy-daidzein, 6-hydroxy-daidzein and glycitein behaved similar to daidzein, whereas biochanin A resembled genistein. The effect of daidzein on the 3-glucuronidation of E2 in human hepatic microsomes was also obtained with human recombinant UGT1A1. Since the only other compound known to stimulate E2 glucuronidation via allosteric kinetics is 17alpha-ethynylestradiol, our study is the first report of the heterotropic stimulation of a UGT by a non-steroidal and naturally occurring compound. An enhanced rate of glucuronidation of E2 by daidzein and its metabolites may contribute to the putative protection of soy against hormonal cancer.     

Benzene metabolism by human liver microsomes in relation to cytochrome P450 2E1 activity

Low levels of benzene from sources including cigarette smokeand automobile emissions are ubiquitous in the environment.Since the toxicity of benzene probably results from oxidativemetabolites, an understanding of the profile of biotransformationof low levels of benzene is critical in making a valid riskassessment. To that end, we have investigated metabolism ofa low concentration of [¹⁴C]benzene (3.4 µM) by microsomesfrom human, mouse and rat liver. The extent of phase I benzenemetabolism by microsomal preparations from 10 human liver samplesand single microsomal preparations from both mice and rats wasthen related to measured activities of cytochrome P450 (CYP)2E1. Measured CYP 2E1 activities, as determined by hydroxylationof p-nitrophenol, varied 13-fold (0.253–3.266 nmol/min/mg)for human samples. The fraction of benzene metabolized in 16min ranged from 10% to 59%. Also at 16 min, significant amountsof oxidative metabolites were formed. Phenol was the main metaboliteformed by all but two human microsomal preparations. In thosesamples, both of which had high CYP 2E1 activity, hydro-quinonewas the major metabolite formed. Both hydroquinone and catecholformation showed a direct correlation with CYP 2E1 activityover the range of activities present. A simulation model wasdeveloped based on a mechanism of competitive inhibition betweenbenzene and its oxidized metabolites, and was fit to time-coursedata for three human liver preparations. Model calculationsfor initial rates of benzene metabolism ranging from 0.344 to4.442 nmol/mg/min are directly proportional to measured CYP2E1 activities. The model predicted the dependence of benzenemetabolism on the measured CYP 2E1 activity in human liver samples,as well as in mouse and rat liver samples. These results suggestthat differences in measured hepatic CYP 2E1 activity may bea major factor contributing to both interindividual and interspeciesvariations in hepatic metabolism of benzene. Validation of thissystem in vivo should lead to more accurate assessment of therisk of benzene's toxicity following low-level exposure.     

A comparative study of tamoxifen metabolism in female rat, mouse and human liver microsomes

The metabolisms of tamoxifen in female rat, mouse and humanliver microsomal preparations were compared. Rat, mouse andhuman liver microsomes were incubated with tamoxifen in thepresence of NADPH and MgCl₂ and the metabolites formed wereanalysed by on-line HPLC electrospray ionization MS. The majormetabolites formed by rat liver microsomes were 4-hydroxytamoxifen,4'-hydroxytamoxifen, N-desmethyltamoxifen and tamoxifen N-oxide.In addition, two epoxide metabolites, 3,4-epoxytamoxifen and3',4'-epoxytamoxifen, and their hydrolysed derivatives, 3,4-dihydrodihydroxytamoxifenand 3',4'-dihydrodihydroxytamoxifen, have been identified. Thepattern of the main metabolites obtained with human liver microsomesresembles qualitatively that of rat liver microsomes. The majordifferences between rat and human liver microsomes were thatthe amount of hydroxylated metabolites were much lower in humanand only traces of 3,4-epoxytamoxifen and the correspondingdihydrodihydroxy derivative were detected. No 3',4'-epoxytamoxifendetected in human liver microsomes. The four major metaboliteswere also formed in much larger amounts and with faster ratesof formation by mouse liver microsomes, though tamoxifen N-oxideclearly predominated in this species. Polar metabolites, 3,4-dihydroxytamoxifenand 4-hydroxytamoxifen N-oxide, which were undetectable in ratand human, were formed in significant amounts in mouse microsomes.As in human microsomes, there was only one epoxide metabolite,3,4-epoxytamoxifen, produced by mouse liver microsomes at levelslower than that found in rat. The faster rate of metabolismand the production of polar metabolites may indicate the abilityof mouse to detoxify tamoxifen by rapid elimination comparedwith rat and human. The production of a larger amount of potentiallyreactive epoxide metabolites in rat may be responsible for theliver carcinogenesis in this species.     

Identification of benzene oxide as a product of benzene metabolism by mouse, rat, and human liver microsomes

Benzene is a ubiquitous environmental pollutant that is known to cause hematotoxicity and leukemia in humans. The initial oxidative metabolite of benzene has long been suspected to be benzene oxide (3,5- cyclohexadiene-1,2-oxide). During in vitro experiments designed to characterize the oxidative metabolism of [14C]benzene, a metabolite was detected by HPLC-radioactivity analysis that did not elute with other known oxidative metabolites. The purpose of our investigation was to prove the hypothesis that this metabolite was benzene oxide. Benzene (1 mM) was incubated with liver microsomes from human donors, male B6C3F1 mice, or male Fischer-344 rats, NADH (1 mM), and NADPH (1 mM) in 0.1 M sodium phosphate buffer (pH 7.4) and then extracted with methylene chloride. Gas chromatography-mass spectrometry analysis of incubation extracts for mice, rats, and humans detected a metabolite whose elution time and mass spectrum matched that of synthetic benzene oxide. The elution time of the benzene oxide peak was approximately 4.1 min, while phenol eluted at approximately 8 min. Benzene oxide also coeluted with the HPLC peak of the previously unidentified metabolite. Based on the 14C activity of this peak, the concentration of benzene oxide was determined to be approximately 18 microM, or 7% of total benzene metabolites, after 18 min of incubation of mouse microsomes with 1 mM benzene. The metabolite was not observed in incubations using heat- inactivated microsomes. This is the first demonstration that benzene oxide is a product of hepatic benzene metabolism in vitro. The level of benzene oxide detected suggests that benzene oxide is sufficiently stable to reach significant levels in the blood of mice, rats, and humans and may be translocated to the bone marrow. Therefore benzene oxide should not be excluded as a possible metabolite involved in benzene-induced leukemogenesis.      

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.     

重组人淋巴毒素抗肿瘤作用的研究

目的：探讨重组人淋巴毒素体内外抗肿瘤作用。方法：体外采用ＭＴＴ法测ｒｈＬＴ对人胃癌细胞ＭＫＮ－４５的细胞毒作用,体内采用小鼠移植性肿瘤研究其抗肿瘤活性。结果：ｒｈＬＴ对体外培养的ＭＫＮ－４５有较强的细胞毒作用,其ＩＣ５０为１．９８×１０＾－２μｇ／ｍｌ,ｒｈＬＴ对小鼠体内移植的Ｓ１８０实体瘤和小鼠Ｅｈｒｌｉｃｈ氏实体瘤均有一定的抑制作用,其抑制率分别为３４．１３％和３４．０８％;