The inhibitory effects of roxatidine acetate hydrochloride (ROX), a new H₂-receptor antagonist, on the oxidative drug-metabolizing enzyme system in mouse hepatic microsomes and in man in vivo were compared with those of cimetidine (CIM). CIM markedly inhibited testosterone 6β-, 7α- and 16α-hydroxylase, aminopyrine N-demethylase and aniline hydroxylase activities in mouse hepatic microsomes with inhibition constants (K_i) of 0.2-3.49 mM. ROX exhibited much weaker inhibitory effects on each enzyme activity with 12 to 100-fold higher values of K_i than those of CIM. CIM gave type II difference spectra with dissociation constants (K_s) of 10.4 and 111 μM while ROX gave reverse type I difference spectra with K_s of 55.6 μM. The ratio of 6β-hydroxycortisol (6β-OHF) to 17-hydroxy corticosteroids (17-OHCS) in urine, used as an indicator of oxidative drugmetabolizing capacity in man, was decreased by 25-35% of the original level on 1-3 d after oral treatment with 800 mg/d of CIM. The ratio was not significantly changed during oral treatment with 150 mg/d of ROX. These results indicate that ROX exhibits a lower affinity for cytochrome P-450 and a lower inhibitory potency on the drug-metabolizing enzymes in hepatic microsomes than does CIM.

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Acetaminophen caused a reversible change in the fluorescence polarization of eosin maleimide and dansyl chloride labeled to rat liver microsomes, indicating that acetaminophen acts on microsomal membrane proteins. Acetaminophen in the presence of nicotinamide adenine dinucleotide phosphate (NADPH), brought about an irreversible change in the fluorescence polarization of the probes. Acetaminophen, with or without NADPH, was also found to change the fluorescence polarization of lipid-soluble fluorescent probes, 2- and 12-(9-anthroyloxy) stearic acid labeled to microsomes, indicating its action on microsomal membrane lipids.

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The activities of 2- and 4-hydroxylation enzymes of estradiol 17-sulfate in various rat tissues were measured by determining 2-hydroxyestradiol 17-sulfate and 4-hydroxyestradiol 17-sulfate by high performance liquid chromatography equipped with an electrochemical detector. Incubations were carried out in the presence of a nicotinamide adenine dinucleotide phosphate (NADPH)-generating system using microsomal fractions of each tissue. The livers of male and female rats, followed by the kidneys, had the highest 2-hydroxylase activities, while other tissues, including the brain, heart, lung, testis, ovary and uterus, possessed similar levels of activity. With regard to 4-hydroxylase, again the liver, followed by the ovary, had the highest activity. There was no significant difference in the activities of other tissues investigated. In addition, two catechol estrogens, 6β-, 7β- and 16α-hydroxylated metabolites of estradiol 17-sulfate, were formed when male heart and lung and female liver and kidney were used. Male kidney was found to have a weak 6β-hydroxylase activity, also. These results show that various rat tissues, including the liver, have 2- and 4-hydroxylase activities for estradiol 17-sulfate and that some of the tissues have additional metabolic abilities such as hydroxylation at 6β-, 7β- or 16α-positions of the substrate.

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Intestinal absorption, hepatic biotransformation and intestinal bacterial modification of the C₂₅ homolog of ursodeoxycholic acid, homoursodeoxycholic acid, and its glycine conjugate, glycohomoursodeoxycholic acid, were studied in rats. Homoursodeoxycholic acid, like ursodeoxycholic acid, was efficiently absorbed from the intestine and rapidly excreted into the bile. Most (>95%) of the absorbed homoursodeoxycholic acid was found to undergo β-oxidation to form two C₂₃ bile acids, norursodeoxycholic acid and nor-β-muricholic acid during passage through the liver. Bacterial modification of homoursodeoxycholic acid was very similar to that of ursodeoxycholic acid. In the rat intestinal tract, glycohomoursodexycholic acid was deconjugated to form unconjugated homoursodeoxycholic acid which was then 7β-dehydroxylated to form homolithocholic acid.

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The purpose of this study was to investigate age-related changes in in vivo dopamine (DA) synthesis in the brain of Wistar rats of ST and LWH substrains. There were no significant differences in the regional DA levels in aged versus mature rats of both substrains. In the LWH substrain, the in vivo DA synthesis as reflected in 3, 4-dihydroxyphenylalanine accumulation in the striatum and nucleus accumbens was significantly lower in the aged rats. In the ST substrain, however, no age-related changes in the regional in vivo DA synthesis were observed for the mature rats. These results suggested that the striatum and nucleus accumbens of the LWH rats may be more vulnerable to aging than those of ST substrain.

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The effect of a single intravenous or oral administration of mitomycin C (MMC), 5-fluorouracil (5-FU) or cyclophosphamide (CP) on drug absorption was studied in rats in relation to changes in membrane characteristics. At 48 h after pretreatment, a differential effect on the absorption of sulfanilamide and L-tryptophan was observed in in situ recirculation experiments. Intravenous MMC administration suppressed the absorption of both sulfanilamide and L-tryptophan to a similar extent as a higher oral dose of this agent. Dosing with 5-FU via both routes caused the largest but almost equal suppression of L-tryptophan absorption. However, CP had no effect on the absorption of the two drugs. Differences in these effects were considered to reflect their pharmacological and pharmacokinetic properties. Absorption of drugs from the small intestine had a positive correlation with small intestinal wet weight regardless of the antitumor drug used, pretreatment doses and routes of administration and the results indicated that the change in absorptive surface area played a major role in this phenomenon. Toxicity to intestinal mucosa was shown to derive from an effect on dividing cells in the crypts because MMC and 5-FU preferentially decreased thymidine kinase activity. However, at the membrane level, increased mucosal membrance permeability was also confirmed by measuring the release rates of D-glucose from liposomes consisting of mucosal total lipids obtained from the antitumor drug-treated rats. Pretreatment with lipophilic and polymeric prodrugs of MMC did not exhibit any effect on drug absorption and thus, the possibility of alleviation of toxicity and adverse reactions via the prodrug approach was suggested.

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The pharmacokinetics of inulin was studied following intravenous administration of ¹⁴C-inulin to 1-d, 1-, 3- and 8-week-old rats. The distribution volume of inulin varied 2-fold, from 689 ml/kg in 1-d-old rats to 340 ml/kg in 8-week-old rats in the growth process of rats. This result was similar to that of warfarin and there was a statistically significant correlation between the distributino volume of warfarin and inulin (r=0.984, p<0.02). In the growth process of rats, the K_P values of warfarin in muscle, which play an important role in the distribution kinetics of warfarin changed in parallel with those of inulin. These results and pharmacokinetic considerations indicated that in warfarin, which is highly bound to serum protein and shows a small distribution volume, the change in the distribution volume in the growth process of rats following administration of a pharmacologically realistic dose (1 mg/kg) is led by the change in the extracellular volume of tissues and that the change in serum protein binding of warfarin might play a minor role in the change in the distribution volume in the growth process.

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Radioactive N-ethyl [2, 3-¹⁴C] maleimide-S-glutathione (¹⁴C-NEM-S-G) was used as a model compound of glutathione conjugate. The metabolism of the conjugate in the extrarenal tissues was investigated in nephrectomized rats and perfused rat livers. When ¹⁴C-NEM-S-G was injected intravenously into nephrectomized rats, a significant amount of ¹⁴C-NEM-S-cysteine (¹⁴C-NEM-S-Cys) was detected in the blood, demonstrating that the conjugate could be metabolized to its cysteine form by extrarenal γ-glutamyl transferase and dehydropeptidase-I. In the isolated liver perfusion with Krebs-Henseleit buffer containing 25 mM Hepes, both ¹⁴C-NEM-S-Cys and ¹⁴C-NEM-S-cysteinylglycine (¹⁴C-NEM-S-Cys-Gly) were formed from ¹⁴C-NEM-S-G. Furthermore, ¹⁴C-NEM-S-Cys-Gly was converted to ¹⁴C-NEM-S-Cys in the plasma, although ¹⁴C-NEM-S-G was not metabolized by the plasma itself. These results demonstrate that the liver and plasma contribute to the metabolism of glutathione conjugates in vivo.

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In order to further evaluate the antitumor activity of 1-(2-chloroethyl)-3-isobutyl-3-(β-maltosyl)-1-nitrosourea (TA-077), the therapeutic ratios (a ratio of the optimal dose versus the dose showing 25% increase in life span) of TA-077 were examined using two L1210 leukemia systems and the results were compared with those of twenty-six antitumor agents for reference. In the i.p.-i.p. system, in which the tumor cells were inoculated intraperitoneally (i.p.) and the drugs were also administered i.p., TA-077 administered daily for five consecutive days showed a therapeutic ratio of 40.6, which was the third highest of all drugs tested. However, in the i.v.-i.v. system, in which the tumor cells were inoculated intravenously (i.v.) and the drugs were also administered i.v., TA-077 given on the same schedule showed a ratio of 13.7, which was the highest of all the drugs examined. The results of the present study also indicated that L1210 leukemia cells were highly sensitive to nitrosourea derivatives. Therefore, this should be taken into consideration for screening of candidates for new antitumor agents.

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The combined cytotoxic effect of forskolin with mitomycin C (MMC) was investigated using two cell lines, AH66 and AH66F. Forskolin significantly enhanced the cytotoxicity of MMC and increased the uptake of MMC and the intracellular adenosine 3', 5'-cyclic monophosphate (cyclic AMP) level in AH66 cells but similar results were not obtained with AH66F cells. Dibutyryl cyclic AMP enhanced the effect and uptake of MMC in both cell lines. These results confirm that elevated cyclic AMP in the cells increases the cellular uptake of MMC and enhances the cytotoxicity of MMC as reported in our previous papers. Therefore, forskolin may be suitable for antitumor combination therapy. On the other hand, the functional components associated with the cyclic AMP generating system were thought to be present in membranes of both AH66 cells and AH66F cells from results that showed that cholera toxin, islet-activating protein and prostaglandin E₁ increased the level of cyclic AMP in both cells. The elevation of cyclic AMP by these adenylate cyclase activators in AH66 cells was augmented by forskolin. However, the cyclic AMP level in AH66F cells was scarcely affected by forskolin and the elevation of cyclic AMP by the activators was inhibited by this diterpene. From these results, it appears that the AH66F cell line may be useful for the elucidation of the action mechanism of forskolin and of the transmission system from hormone receptors to adenylate cyclase.

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