1-(2-Chloroethyl)-3-isobutyl-3-(β-maltosyl)-1-nitrosourea (TA-077) is a new masked antitumor agent, which is hydrolyzed by maltase to give rise to a more cell-permeable and, hence, more cytotoxic metabolite, TA-G. TA-077 was intravenously administered to mice, rats, guinea pigs, rabbits and dogs and the time-courses of blood concentrations of TA-077 and TA-G were followed. The time-course patterns of blood TA-077 in these animals were markedly different from one another depending on the level of plasma maltase, while those of blood TA-G were all similar except in the early stage after the injection ; i.e., the blood concentration of TA-G reached a peak shortly after the injection and decreased rapidly with a half-life of 6 to 15 min, and the higher the level of plasma maltase, the earlier the time of peak appearance. Tissue concentrations of TA-077 and TA-G in major organs were also measured at various times after intravenous injection of TA-077 to guinea pigs and VX-2 tumor-bearing rabbits. In both species, tissue concentrations of TA-077 did not exceed the blood concentration and rapidly decreased to insignificant levels by 30 to 45 min. The tissue level of TA-G in the kidney, a maltase-rich organ, was always the highest among the organs examined in both species. The concentration of TA-G in the VX-2 tumor was relatively high. Possible significance of tissue maltase to the cellular uptake and antitumor effect of TA-077 is also discussed.

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1-(2-Chloroethyl)-3-isobutyl-3-(β-maltosyl)-1-nitrosourea (TA-077) was hydrolyzed to its glucosyl metabolite TA-G by homogenates of guinea pig organs, rabbit VX-2 carcinoma and rat Yoshida sarcoma. The rate of TA-G formation by the kidney was the highest among the tissues examined and that by the diluted blood was undetectably low, reflecting their maltase activities. TA-077 was also hydrolyzed by suspensions of Yoshida sarcoma, AH130 hepatoma, L1210 leukemia, P388 leukemia and DBLA-10/C leukemia cells. The rate of TA-G formation was increased 10 fold by homogenizing the tumor cells. TA-G was taken up by the tumor cells much more efficiently than TA-077, explaining the higher sensitivities of the cultured tumor cells to TA-G than to TA-077. However, neither the maltase activity nor the membrane permeability to the drugs was a factor influential enough to explain the differences in drug sensitivity among the tumor cell lines.

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A new nitrosourea antitumor agent TA-077, 1-(2-chloroethyl)-3-isobutyl-3-(β-maltosyl)-1-nitrosourea, was intravenously administered to 15 cancer patients at doses ranging from 7 to 100 N (1 N=30 mg/m²) in a phase I clinical trial. Time courses of blood concentrations of TA-077 and its active metabolite TA-G, 3-β-D-glucopyranosyl analog of TA-077, were followed. The TA-G concentration reached a maximum at 7.0±2.3 min, and decreased thereafter with a half-life of 12.9±2.8 min. The time-course patterns and various pharmacokinetic parameters of TA-077 and TA-G were similar to those in the guinea pig, which, like humans, lacks plasma maltase activity. The 2 h-urinary excretion rate of TA-G in the above patients ranged from 0.15 to 7.7% of the dose. The areas under the concentration-time curve and maximal concentration values were both linearly correlated to the dose with correlation coefficients of 0.78 and 0.82, respectively. Repeated administration of TA-077 (29 to 40 N) for 5 or 6 consecutive days did not affect the pharmacokinetic parameters of TA-077 and TA-G in 7 cancer patients except for slight increases in the half-life and area under the curve of blood TA-G.

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The changes in glycosaminoglycan (GAG) synthesis during the differentiation of HL-60 cells to macrophage-like cells by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) were studied by using ³⁵S-sulfate or ³H-glucosamine as a tracer. The incorporation of ³⁵S-sulfate into GAGs of HL-60 cells treated with TPA (TPA-treated HL-60 cells) decreased by 30% compared with untreated HL-60 cells (HL-60 cells). The profiles of the Sepharose CL-6B chromatography showed that the molecular weight size of proteoglycans (PGs) in the medium fraction of TPA-treated HL-60 cells was larger than that of HL-60 cells although the PGs in the cell fractions of both HL-60 and TPA-treated HL-60 cells were of the same molecular weight size. On the other hand, the molecular weight sizes of GAGs in both the medium and the cell fractions of TPA-treated HL-60 cells were larger than those of HL-60 cells. In order to examine the synthesis of GAG chains, HL-60 and TPA-treated HL-60 cells were incubated with ³⁵S-sulfate in the presence of 4-methylumbelliferyl-β-D-xyloside, an exogenous initiator. The incorporation of ³⁵S-sulfate into total GAGs of the TPA-treated HL-60 cells exposed to the β-D-xyloside increased 3-fold over that of HL-60 cells. HL-60 cells synthesized core protein-initiated GAGs and β-D-xyloside-initiated GAGs but TPA-treated HL-60 cells synthesized β-D-xyloside-initiated GAGs only. β-D-Xyloside-initiated GAGs synthesized by both cells were obtained in the same fraction by Sepharose CL-6B chromatography. In addition, alterations in the chain length and synthetic activity of GAGs gradually occurred with the morphological changes and came to constant values 36 h after treatment with TPA. These results suggest a very close relationship with the monocytic differentiation of HL-60 cells. However, it is not possible at present to decide whether such changes in PG synthesis result from chain length only or induction of different PGs.

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In order to develop a percutaneous (p.c.) dosage form of valproic acid (VPA), ointments containing VPA and both VPA and its calcium salt (VPA-Ca) were prepared. The p.c. absorption was studied by determining the plasma concentrations of VPA in rabbits following application of either ointment. In addition, the in vitro penetration study with excised full-thickness rabbit skin was done to evaluate the pharmaceutical availability. VPA was readily and rapidly absorbed through the skin from the gel ointment. The plasma concentrations of VPA higher than 30 μg/ml were maintained for 6 h after application of 5% VPA·5% VPA-Ca gel ointment, and for 4 h after application of 5% VPA ointment. The bioavailability of VPA following dosing of both ointments was above 97%. The in vitro penetration rate through the skin and skin/solvent partition coefficient were significantly lower with 5% VPA·5% VPA-Ca solution than with 5% VPA, suggesting the slower penetration of VPA from the 5% VPA·5% VPA-Ca. A small difference was observed in the release rate of drugs from these ointments. The present results demonstrated the pharmaceutical effectiveness of the VPA VPA-Ca ointment for seizure disorders.

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A comparative pharmacokinetic study of theophylline between the first and repeated oral administration and the assessment of clinical utility of theophylline test-dose concept were performed in 6 (study I) and 4 (study II) healthy male volunteers with different dosing schedules. In study I, although the average of theophylline systemic clearance (Cl_sys) was significantly (p<0.05) lower in the repeated dosing than in the first dosing, large intersubject variations were observed. Plasma free fatty acids which inhibit drug metabolizing enzyme activity were not influenced by theophylline chronic administration. In study II, the volunteers received oral multiple doses of a theophylline powder preparation to maintain 5 to 15 μg/ml plasma concentration on the basis of the pharmacokinetic parameters calculated from the single oral test-dose. A good prediction of the plasma concentration was observed only in one case and the maximum levels in the rest exceeded 20 μg/ml, a toxic concentration. Throughout the Studies, a stable Cl_sys was obtained in smokers, but the Cl_sys in non-smokers decreased by one third to a half during multiple dosing. These findings suggest that theophylline showed time-dependent pharmacokinetics and that test-dose concept for theophylline may not be applicable in all cases because of a large intersubject variation in the Cl_sys change between single and multiple dosing.

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The application and usefulness of resealed erythrocytes as cell carriers of human urokinase (UK) were studied in rabbits. UK purified by affinity chromatography were used to load erythrocytes by a dialysis method. The amount of UK entrapped in the erythrocytes was 2064±416 IU per ml of packed cells, with efficiency of encapsulation being 20.0±4.2%. When the UK loaded erythrocytes were incubated in Hank's solution or rabbit plasma for 3 h at 37°C, the UK activities in the resealed cells declined similarly to first-order kinetics. The UK activities released into Hank's solution increased cumulatively during the incubation but those in plasma decreased after a peak concentration at 15 min. After i.v. administration of free UK, the activity decreased according to a biexponential function, suggesting its uptake in the liver and kidney and the formation of UK-proteinase inhibitor complexes and the in vivo decline of UK in loaded cells also indicated a biexponential kinetics. The AUC ratio of UK after an intravenous injection of the loaded erythrocytes was calculated to be about 8.0%. These results indicated that the UK loaded erythrocytes may be useful as a dosage form for treatment of patients with thrombosis.

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The effects of oral activated carbon beads on the total serum cholesterol and triglyceride levels and on the fecal bile acid excretion in rats fed a basal or high cholesterol diet containing 5% activated carbon beads for 6 weeks were investigated. The beads appeared to exert little effect on the growth rate of rats and no tendency towards constipation was also observed. Although treatment with activated carbon beads gave little influence on the total serum cholesterol and serum triglyceride levels in rats fed either a basal or high cholesterol diet, the beads did produce a significant rise in the total fecal bile acid excretion. The extra fecal bile acids resulted chiefly from an increase in lithocholic acid. Furthermore, the beads showed a tendency to inhibit absorption of dietary cholesterol from the gastrointestinal tract in rats fed a high cholesterol diet. These results suggested the possibility of activated carbon beads as a hypocholesterolemic agent through binding with bile acids in the intestine.

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Intracellular recordings of the CA3 pyramidal neurons in the guinea pig hippocampus were made in vitro. U-50 488H (100 μM), a selective opioid κ agonist, decreased the synaptic response produced by stimulation of the mossy fibers but did not affect the membrane potential, the input resistance and the generation of the Na⁺ spikes or the Ca²⁺ spikes. Iontophoretically applied U-50 488H depressed the depolarization produced by L-glutamate. U-50 488H (100 μM) also depressed the consistent depolarization produced by veratrine (3×10^-5 g/ml) and this effect was partially antagonized by naloxone. Moreover, application of U-50 488H led to a disappearance of the anomalous rectification. These results suggest that U-50 488H depresses the synaptic activities of the CA3 pyramidal neurons by inhibiting a subtype of the Na⁺ channel, "Na⁺ channel type II"which slowly closes, of the soma and/or the dendrites of the neurons.

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The intestinal absorption of choline, an endogenous quaternary ammonium, from the rat jejunum has been investigated with an in situ ligated loop method and an in vitro everted sac method. Choline was absorbed rapidly from the ligated jejunum and structural analogs inhibited choline absorption competitively. In in vitro experiments, choline was transported from the mucosal fluid to the intracellular fluid against a concentration gradient and the rate of tissue uptake was highly affected by incubation temperature, aerobic condition and the presence of a metabolic inhibitor, 2, 4-dinitrophenol. The tissue accumulation of choline was saturable at concentrations below 100 μM and, above this concentration the uptake ratio of choline (medium to tissue) was almost constant. One mM hemicholinium-3, which is well known to inhibit choline uptake by neurons through the choline specific carrier, also significantly inhibited choline uptake, especially at concentrations of choline below 100 μM. The fact that the choline uptake is linear in the presence of hemicholinium-3 shows that choline is partially absorbed by passive diffusion. The difference between the total tissue accumulation and choline uptake by the passive diffusional pathway followed Michaelis-Menten kinetics and the apparent K_r of 47 μM and V_max of 4.1 nmol/ml intracellular fluid/min were determined by an in vitro everted sac method. These findings suggested that, at lower concentrations, choline was absorbed from the rat intestine mainly by an active transport system.

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In vivo effects of tetrahydrocannabinols (THCs) and their eight monooxygenated metabolites on the hepatic microsomal drug-metabolizing enzymes in mice were studied. Δ⁸-THC and its metabolites (7α-hydroxy-, 7β-hydroxy- and 7-oxo-Δ⁸-THC, and 8α, 9α- and 8β, 9β-epoxyhexahydrocannabinol) tended to increase the enzyme contents or activities except for 7β-hydroxy-Δ⁸-THC which affected the microsomal enzymes in a different manner between the single and subchronic treatments. Single administration (5 mg/kg, i.v.) of 7-oxo-Δ⁸-THC, 8α, 9α- and 8β, 9β-epoxyhexahydrocannabinol led to a significant increase in hepatic microsomal p-nitroanisole O-demethylase and aniline hydroxylase activities accompanying a significant increase in cytochrome P-450 content in hepatic microsomes. The same results were obtained with subchronic treatment of mice with these metabolites (5 mg/kg/d, i.v. for 7 d), although the effect of 8β, 9β-epoxyhexahydrocannabinol on cytochrome P-450 was not statistically significant. 7β-Hydroxy-Δ⁸-THC significantly increased nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome creductase and aniline hydroxylase activities by single administration, while the metabolite significantly decreased the contents of cytochrome b₅ and P-450 and p-nitrophenol uridine diphosphate-glucuronyltransferase activity by the subchronic treatment. In contrast, Δ⁹-THC and its metabolites (8α-hydroxy-, 8β-hydroxy- and 8-oxo-Δ⁹-THC) did not significantly affect the microsomal enzymes by both treatments except that the single administration of 8α-hydroxy-Δ⁹-THC and the subchronic treatment of Δ⁹-THC significantly decreased NADPH-cytochrome c reductase activity. These results indicated that monooxygenated metabolites of Δ⁸- and Δ⁹-THC affect the hepatic microsomal drug-metabolizing enzyme systems of mice differently.

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In vitro techniques were used to test certain concepts regarding the enhancement of the transdermal delivery of nifedipine from topical vehicles. Tested vehicles include the volatile solvent acetone, nonvolatile solvents such as propylene glycol and isopropyl myristate, volatile/nonvolatile mixtures, and those mixtures, with a polymer additive. An ethylene-vinyl acetate (EVA) copolymer membrane was used as the lipoidal barrier for the diffusing drug. Not merely the rate of transport per unit area, but the activity coefficient and the diffusion coefficient of the penetrating agent in the barrier were obtained. Despite the 10000-fold difference in the vehicle concentration, water and several hydrophilic vehicles containing finely ground suspensions of the drug produced nearly the same rate of penetration. Some lipophilic solvents affected the barrier function of the EVA membrane to promote penetration of the drug. It has been found that the activity coefficient in the EVA membrane is very susceptible to wide variations by imbibition of such solvents. From volatile/nonvolatile mixtures, a transient enhancement in the transport of nifedipine across the membrane was observed. The increase in the flux was accounted for by the increase in the thermodynamic activity of the drug in the nonvolatile vehicle caused by the evaporation of the volatile component. The eventual decrease in penetration was the result of the drug precipitation from the supersaturated solution. The precipitation was inhibited and/or retarded during the entire time course of the experiments when a polymer additive was present. The steady-state fluxes from mixtures with a polymer additive were higher by about 3 to 5 times than that of the control experiment.

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Production of superoxide anion (O^-₂) induced by tetradecanoyl phorbol acetate (TPA) in intact guinea pig polymorphonuclear leukocytes (PMNL) was markedly inhibited by a seleno-organic compound, 2-phenyl-1, 2-benzisoselenazol-3 (2H)-one (Ebselen), with glutathione peroxidase-like activity. The compound almost completely inhibited O^-₂ production by a particulate fraction prepared from TPA-treated PMNL at a concentration as low as 250 nM.

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