Comparison of the stability of some major cytochrome P450 and conjugation reactions in rat, dog and human hepatocyte monolayers.

The stability of four major cytochrome P450 isoenzymes (CYPIA, CYP2B, CYP2E1 and CYP3A) and of two phase II conjugation enzymes (glucuronyl- and sulfotransferases) was investigated in primary cultures of rat, dog and human hepatocytes in the same conditions. 7-ethoxyresorufin deethylation (EROD), 7-methoxycoumarin demethylation (MCOD), chlorzoxazone (CLOX) 6-hydroxylation, 1'- and 4-hydroxylation of midazolam (MDZ), and p-nitrophenol glucuronidation and sulfation, were used respectively. The EROD activity was stable over 72 hours in rat and dog and only 48 hours in human hepatocytes. The MCOD activity was also stable in rat but decreased in dog by 30% within 72 hours The CLOX hydroxylase activity was most stable in human whereas in rat and dog it fell down to 30% within 72 and 24 hours, respectively. The MDZ hydroxylase activity showed the same unstability profile in the three species investigated. Both conjugation reactions were either stable or showed an increase by up to 60-70% in all three species over 72 hours. The enzymes tested showed different stabilities in rat, dog and human hepatocytes over 72 hours, thus demonstrating the limitations of hepatocyte monolayers as models for metabolic investigations and emphasising the need for validation/characterization studies before routine use.

Estimation of flavin-containing monooxygenase activity in intact hepatocyte monolayers of rat, hamster, rabbit, dog and human by using N-oxidation of benzydamine.

The flavin-containing monooxygenase (FMO)-dependent N-oxidation of benzydamine has been assessed as a method for monitoring the activity of FMOs in monolayer cultures of hepatocytes from rat, dog, rabbit, hamster and human. The advantage of this substrate is that benzydamine N-oxide formation can be measured directly in extracts of cellular incubations without an intensive work-up procedure. Benzydamine and its N-oxide are readily separated by HPLC with fluorometric detection. This assay proved sensitive enough to monitor FMOs activity in intact monolayer of cultured hepatocytes. The formation of benzydamine N-oxide was inhibited when hepatocytes were coincubated with methimazole (another FMO substrate) in a dose-dependent manner, whereas N-octylamine (an inhibitor of cytochrome P450) had no inhibitory effect. In contrast to cytochrome P450, FMO activity assessed by benzydamine N-oxidation was relatively stable for all species studied during 72-h cultures.

Animal pharmacokinetics of the tumor necrosis factor receptor-immunoglobulin fusion protein lenercept and their extrapolation to humans.

The pharmacokinetics of the tumor necrosis factor receptorimmunoglobulin fusion protein, lenercept, were assessed in rats, rabbits, dogs and cynomolgus monkeys. Pharmacokinetic parameters were extrapolated to humans by allometric scaling. Lenercept was dosed i.v. at doses ranging from 0.1 to 5 mg/kg. Consistent with its all-human sequence, lenercept elicits an immune response in laboratory animals usually 6 to 10 days after dosing. The resulting period of more rapid clearance caused by the immune response was excluded from the pharmacokinetic evaluation. Lenercept showed a very low and similar clearance in all species tested (0. 0071-0.0097 ml.min/kg). The volume of distribution was estimated at values between 61 and 90 ml/kg, whereas the terminal half-life ranged from 3.4 days in rabbits to 6.5 days in rats. Thus, lenercept was characterized by similar pharmacokinetic properties across species, irrespective of their particular body weight. Accordingly, both clearance (ml/min) and volume of distribution (ml) scaled with an allometric exponent close to 1, whereas half-lives (including literature data in mice) yielded an allometric exponent close to 0. The predicted parameters in humans agree well with the observed values. Overall, the results demonstrate an allometric scaling for lenercept different from that for other therapeutic proteins, in that lenercept displays a similar pharmacokinetic behavior across species. Despite an early and pronounced immune response against this all-human protein in laboratory animals, the pharmacokinetic data were found to be predictive for humans, given that the more rapid immune-modulated clearance component in animals could be identified and excluded from the pharmacokinetic evaluation.

Protection against lethal Escherichia coli bacteremia in baboons (Papio anubis) by pretreatment with a 55-kDa TNF receptor (CD120a)-Ig fusion protein, Ro 45-2081.

Fusion proteins of the human 55-kDa TNF receptor extracellular domain with hinge and C2/C3 constant domains of human IgG1 or IgG3 heavy chains were tested in a primate sepsis model. Twenty-four baboons received 4.6, or 0.2 mg/kg of TNFR5-G1,3, or placebo, before the administration of a lethal dose of live Escherichia coli. Treatment with TNFR5-G1,3 decreased 5-day mortality from 88% in the placebo group to 12% in the TNFR5-G1,3-treated animals (p < 0.01 by Fisher's exact test). Treatments with TNR5-G1 and TNFR5-G3 in doses from 0.2 to 4.6 mg/kg were efficacious. Free plasma TNF was neutralized by all treatments, but inactive TNF/TNFR5-G1,3 complexes remained in circulation for prolonged periods. TNFR5-1,3 treatments attenuated the hemodynamic disturbances, reduced fluid requirements, and decreased the systemic IL-1 beta, IL-6, and IL-8 responses. In addition, TNFR5-G1,3 treatment shortened the granulocytopenia and reduced the loss of cellular TNF receptors from granulocytes. The decrease in fibrinogen concentrations and increase in prothrombin and partial thromboplastin times were significantly attenuated by TNFR5-G1,3 treatment. TNFR5-G1,3 treatment markedly attenuated the rise in plasma lactate concentration. Histologic studies of TNFR5-G1,3 revealed dose-dependent protection against tissue injury by Escherichia coli administration.

Metabolism of mofarotene in hepatocytes and liver microsomes from different species. Comparison with in vivo data and evaluation of the cytochrome P450 isoenzymes involved in human biotransformation.

The arotinoid mofarotene is a novel potent anticancer compound. The metabolic profiles obtained from rat, dog, and human plasma showed a good correlation with the corresponding in vitro profiles observed with liver microsomes and hepatocytes. Interspecies differences in its metabolism were investigated using microsomes prepared from the livers of the mouse, rat, dog, cynomolgus monkey, and humans. These in vitro experiments showed that, both qualitatively and quantitatively, the metabolic profiles obtained with cynomolgus monkey liver samples were similar to those observed with human liver material. However, rat and dog were also confirmed to be suitable species for assessing the safety of mofarotene, and were used in toxicology. The involvement of cytochrome P450 (CYP) in the metabolism of mofarotene was examined with human liver microsomes. CYP3A4 plays a major role in the metabolism, and CYP1A2 might be responsible for a minor pathway. Finally, the potential induction by mofarotene of four major CYP isoenzymes was investigated in rats. These experiments showed that CYP1A1 was clearly induced, whereas a slight induction of CYP3A and CYP2B was observed. Repeated administration of mofarotene had no effect on CYP2E1. These studies with liver microsomes and hepatocytes aided the selection of appropriate species for toxicology, and have provided information that will help to predict potential drug-drug interactions in clinical trials.

A highly sensitive tool for the assay of cytochrome P450 enzyme activity in rat, dog and man. Direct fluorescence monitoring of the deethylation of 7-ethoxy-4-trifluoromethylcoumarin.

The O-deethylation of 7-ethoxy-4-trifluoromethylcoumarin (EFC) by liver microsomes has been assessed as a method for monitoring the activity of cytochrome P450. The principle advantage of this substrate is the formation of a fluorescent product 7-hydroxy-4-trifluoromethylcoumarin (HFC) which can be assayed directly in the reaction medium. For rat microsomes the deethylated product was confirmed as the main metabolite, the reaction rate was linear with respect to both time and microsomal protein concentration and was independent of small changes in the added co-factors. A linear formation rate for the deethylated metabolite was also confirmed with dog and human microsomes. The intra-assay precision for rat, dog and human microsomes was 3, 5 and 4%, respectively. Hanes transformations of the dog and human data showed two phases, in contrast to a linear decline seen for the rat. Hybrid parameters for Vmax and Km, calculated from the apparently linear portions of these curves, gave interday SD for the Vmax of rat, dog and man of 2, 14 and 4%, respectively, and approximately 15% for the Km in all species. The Vmax in rat, dog and human microsomes was 1.4 +/- 0.2, 4.3 +/- 1.5 and 0.9 +/- 0.5 nmol HFC/min/nmol P450, respectively. The Km was 11.0 +/- 3.1, 67 +/- 19 and 6.8 +/- 2.5 microM, respectively. Direct evidence that at least two isoenzymes (cytochrome P450 1A2 and 2E1) metabolize EFC was obtained by experiments with competitive, suicide and immuno-inhibitors. Compared with ethoxycoumarin, the involvement of P450 2E1 in O-deethylation seemed similar in the rat. In conclusion, EFC provides a straightforward and reproducible assay for microsomal enzyme activity, requiring at most 25 pmol/mL of cytochrome P450.

Acetoxime is metabolized by human and rodent hepatic cytochrome P450 enzymes to the genotoxicant and carcinogen propane 2-nitronate.

The hepatocarcinogenicity of acetoxime has been tentatively linked with its metabolic oxidation to the potent genotoxicant and carcinogen propane 2-nitronate (P2-N). In order to test the hypothesis that acetoxime is metabolized to P2-N, the oxime (20 mM) was incubated with liver microsomes from mice, rats and two humans. Ion-pair HPLC analysis of the incubates afforded a peak that co-eluted with P2-N. P2-N exists in tautomeric equilibrium with 2-nitropropane (2-NP). Samples of the microsomal incubates, which had been adjusted to pH 5.5 and kept for 24 h in order to allow maximal tautomeric equilibration of P2-N to 2-NP to occur, were extracted with hexane. GLC analysis of the extracts yielded a peak that co-eluted with 2-NP, and gave a mass spectrum identical to that of authentic 2-NP. The metabolite peak obtained on HPLC was isolated and its hexane extract contained also 2-NP when investigated by GLC. P2-N was found by HPLC in the urine of rats that had received acetoxime (3.36 mmol/kg i.p.). Hexane extracts of urine samples, which had been adjusted to pH 5.5 and left for 24 h, contained 2-NP as demonstrated by GLC analysis. The results are consistent with the suggestion that the toxicity of acetoxime is associated with its biotransformation to P2-N.

Assessment of viability of hepatocytes in suspension using the MTT assay.

The viability and state of proliferation of cells in culture is conveniently assessed using MTT, which is metabolically reduced to stain functionally intact cells. The hypothesis was tested that this assay can also be used in the quantitation of effects of toxicants on hepatocytes in suspension. Hepatocytes isolated from BALB/c mice were incubated without or with menadione, rotenone, N-methylformamide or paracetamol. Cellular damage was measured by either MTT assay or release into the medium of lactate dehydrogenase (LDH). Results obtained with the two tests were compatible in the case of toxicity inflicted by menadione or N-methylformamide. Rotenone decreased cell viability as indicated by the MTT assay immediately after addition of the agent, whereas measurement of LDH release did not detect this rapid toxic effect. The MTT assay detected paracetamol-induced damage within the first hour of exposure, but this was not detected by the LDH assay until 3 hr had elapsed.

6,7-Dihydro-4H-indolones: synthesis and biological properties.

Syntheses of 6,7-dihydro-4H-indolones 6 and 10 by a selective Birch reduction of the benzene ring of the indole system are described. The antiestrogen zindoxifene and the 2-phenylindole 2 as well as 6 and 10 were tested for their relative binding affinities at the androgen receptor as well as for antiandrogenic and estrogenic properties. Both compounds 6 and 10 showed potent indirect antiandrogenic activity which were similar to those of the 2-phenyl-indoles zindoxifene and 2.

Growth-stimulating effect of adrenal androgens on the R3327 Dunning prostatic carcinoma.

Adrenal androgens are discussed as a reason for tumor progression after androgen ablation therapy. Because of the difference in the secretion of androgens by the adrenals of humans and rats, there is no reliable tumor model to study the role of adrenal androgens in tumor progression. Therefore, the main adrenal androgens were administered to rats in order to mimic human endocrine conditions. Application of dehydroepiandrosteron-sulfate (DHEA-S) alone or a mixture of androstendione (A), 11 beta-hydroxyandrostendione (OHA), dehydroepiandrosterone (DHEA), and its sulfate (DHEA-S) to castrated rats caused only a slight increase of prostate and seminal vesicle weight. Contrary to these findings, growth of the R3327 prostatic carcinoma in castrated rats was greatly stimulated by these adrenal androgens up to the level of the intact control. Thus, in spite of androgen ablation, tumor progression could be induced by exogenous adrenal androgens.

Mechanism of toxicity of the antimelanoma drug 4-hydroxyanisole in mouse hepatocytes.

To elucidate the mechanism of the hepatotoxicity of 4-hydroxyanisole (4-HA), its effect on the viability of mouse hepatocytes in suspension was investigated. Cell viability was assessed by measurement of release of lactate dehydrogenase into the medium. 4-HA was cytotoxic in a concentration-dependent and time-dependent fashion with an IC50 of 0.26 mmol/l after 4 h incubation. Almost all cells were killed after exposure to 4-HA for 4 h at 0.5 mmol/l or for 2 h at 1.0 mmol/l. At 5 and 10 mmol/l, 4-HA caused less cytotoxicity and 1 mmol/l or below. On coincubation with the P450 inhibitor octylamine, 4-HA cytotoxcity was reduced, which suggests the involvement of cytochrome P450 in the hepatocytotoxicity of this drug. Induction of P450 isoenzymes IA, IIB and IIE1 by pretreatment of mice with phenobarbitone, 3-methylcholanthrene or acetone had no significant effect on the toxicity of 4-HA towards hepatocytes. Depletion of hepatic glutathione by pretreatment of mice with buthionine sulphoximine (1.6 g/kg, intraperitoneally) 4 h before cell isolation led to an increase in 4-HA cytotoxicity. Incubation with N-acetylcysteine (10 mmol/l) abolished the cytotoxicity of 4-HA (1 mmol/l). Both these results are consistent with the intermediacy of a reactive metabolite of 4-HA. Production of hydroquinone by oxidative demethylation of 4-HA as toxication mechanism can be excluded as formation of formaldehyde was not observed on incubation of 4-HA with mouse liver microsomes. 3,4-diacetoxyanisole, a prodrug of the known 4-HA metabolite 3,4-dihydroxyanisole, was not more cytotoxic towards hepatocytes than 4-HA.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of zindoxifene on experimental prostatic tumours of the rat.

The anti-oestrogen zindoxifene was originally developed as a drug for the treatment of hormone-dependent mammary carcinomas. Experiments with rats bearing androgen-dependent prostatic tumours revealed anti-neoplastic activity of zindoxifene on these tumours also. Therefore, the inhibitory effect of this drug was studied in various prostatic tumour models in comparison to the anti-oestrogen tamoxifen and to castration. The growth of the hormone-dependent Dunning R3327 H tumour was strongly inhibited by zindoxifene (4 mg/kg), which was more effective than tamoxifen (43% T/C vs 87% T/C, the ratios of tumour weights in control and drug-treated rats). Zindoxifene was able to delay the relapse of these tumours by 7 weeks in comparison to castration. The experiments with Noble Nb-R prostatic tumours showed that administration of zindoxifene (5 mg) is superior to castration (5% T/C vs 52% T/C). The growth of tumours in castrated rats was completely inhibited by administration of zindoxifene. Therefore a peripheral mode of action has to be assumed.

3-(Cyclohexenonyl)-4-(4-hydroxyphenyl)-hexanes: antiandrogenes derived from the estrogen hexestrol.

Selective Birch-reduction of one of the phenolic rings of meso- or d,l-HES led to compounds 6a and 6b which exert a higher binding affinity to the androgen receptor as the respective parent HES. In vivo testing of 6a and 6b shows that 6a has the same potency in reducing accessory sex organ weights and testosterone levels in the intact mouse as has meso-HES, but strongly decreased estrogenic activity. Syntheses and testing of 7 having no ethyl side chains revealed the necessity of these groups for biological activity.

[Hydroxy substituted phenyltetralines: compounds with affinity for estrogen and androgen receptors]. / Hydroxy-substituierte Phenyltetraline: Verbindungen mit Affinität zu Ostrogen- und Androgenrezeptoren.

Among nonsteroidal antiandrogens, mostly compounds of the Flutamide-type are described. For the development of new compounds with affinity to androgen receptors and antiandrogenic activity five new hydroxy-substituted phenyltetralines were prepared and tested. Synthesis was achieved by reaction of the respective 1-tetralones with phenyl magnesium bromides, dehydration of the carbinols, hydrogenation and ether cleavage. In addition to the expected affinity to estrogen receptors, especially the 6-OH, 4'-OH-substituted compound 11 showed an affinity to androgen receptors, too. Whereas none of the tested compounds had a marked estrogenicity, 11 and 15 (6-OH, 3'-OH) exerted antiestrogenic effects. Androgenic properties were not given. In the adult, intact mouse, 11 and 15 exhibited a pronounced inhibition of seminal vesicle weights concomitant with a reduction of the testosterone level. Since in castrated, androgen-substituted animals only relatively weak direct antiandrogenic effects were demonstrable, the antiandrogenic activity of these new phenyltetralines is more due to an inhibition of testosterone biosynthesis than to a direct, receptor-mediated effect.

Carrier mediated action of platinum complexes on estrogen receptor positive tumors.

Three 1,2-diaminoethane-dichloro-platinum(II) complexes linked to 5-hydroxy-2-(4-hydroxyphenyl)-3-methylindole by spacer groups of varying length were evaluated for cytostatic activity in estrogen receptor (ER) positive and negative tumor cells. In vitro, only the growth of ER positive MCF-7 mammary tumor cells was inhibited whereas hormone independent MDA-MB 231 cells did not respond. In vivo, a strong inhibitory effect was only observed in ER positive MXT mammary tumors of the mouse. The complex with a hexyl group as spacer reduced the tumor weight by 89% after 6 weeks of treatment. The R 3327 Dunning prostatic tumor of the rat, which also contains ER was inhibited, too. Generally, the effect in ER negative tumors was weak. These findings can be rationalized by the high binding affinities of the complexes for ER. By the mouse uterine weight test it was shown that the endocrine activity of the complexes is very low. Therefore, a mode of action different from that exerted by estrogens or antiestrogens has to be assumed.

[1,2-Bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine] dichloro-platinum(II): an endocrine-active platinum complex with a specific prostatic tumor-inhibiting activity.

[1,2-Bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine]dichloro-platinum (II), (C), a platinum complex with endocrine activity and a specific effect on hormone-dependent mammary tumors, was tested for its tumor-inhibitory activity in the hormone-sensitive R 3327 and Nb prostate carcinoma models of the rat and for its endocrine activities in comparison to the ligand L and diethylstilbestrol (DES). Established tumors of the R 3327 prostate tumor were strongly inhibited by C. Its effect equaled that of DES and was significantly better than that of L. Accessory sex organ weights and testosterone levels were strongly reduced by C as well as L. This antigonadotrophic effect, which is almost comparable to DES, was confirmed in 10 day experiments with intact, mature mice and rats, whereas a direct antiandrogenic activity was not given. A part of the antitumor action of C is therefore due to this antigonadotrophic activity. Affinities to estrogen, progesterone, and androgen receptors, however, were very low. The hormone-sensitive Noble Nb-R prostatic carcinoma was almost completely inhibited by C, whereas L had only a weak effect. As C has no significant effect on the hormone-independent R 3327 HI prostate tumor and as its effect on hormone-dependent tumors is significantly better than that of the ligand L in spite of their similar endocrine properties, an apparently specific antiproliferative effect of C only on hormone-dependent prostate tumors is obvious. This was further shown in a long-term experiment with the R 3327 prostate carcinoma. Whereas tumors in the castration group relapsed from androgen ablation and exerted a progressive tumor growth, therapy with C almost completely prevented this relapse phenomenon. After 25 weeks of treatment, C inhibited tumor growth by 90% compared to castration. Owing to these results, this new endocrine active platinum complex with an apparently specific effect on hormone-dependent prostate tumors can be of value for the therapy of the prostatic carcinoma.

Catechol estrogens of the 1,1,2-triphenylbut-1-ene type: relationship between structure, estradiol receptor affinity, estrogenic and antiestrogenic properties, and mammary tumor inhibiting activities.

1,1,2-Triphenylbut-1-enes (26-35), which are substituted with one or two 3,4-diacetoxy groups or with one 3,4-diacetoxy and one 3- or 4-acetoxy group in two aromatic rings, were synthesized. The occurring E and Z isomers were isolated, and their identity was established by 1H NMR spectroscopy. A study on structure-activity relationship was carried out with regard to estradiol receptor affinity in vitro, estrogenic and antiestrogenic properties in the immature mouse, and inhibition of the hormone-dependent MXT mammary tumor of the mouse in vivo. Among the tested compounds, most of the 1,1-disubstituted 1,1,2-triphenylbut-1-enes (29, Z-30, Z,E-31) and (E)-1-(3-acetoxyphenyl)-1-phenyl-2-(3,4-diacetoxyphenyl)but- 1-ene (E-35) as well as its respective Z isomer (Z-35) exerted antiestrogenic properties. Compounds Z-30, Z,E-31, Z-35, and E-35 inhibited the growth of the hormone-dependent MXT tumor. The best antitumor effect without estrogenic side effects during therapy was shown by E-35.