The possibility of clinical application of test-dose concept for theophylline was assessed in 11 patients with serious underlying disease who required theophylline. Based on the pharmacokinetic parameters calculated from the single intravenous aminophylline administration, they received a continuous infusion of aminophylline in order to maintain about 10 μg/ml plasma which is considered to be the lowest therapeutic level. Plasma levels during a constant rate infusion were assayed at 6 : 00, noon, 18 : 00 and midnight on the 3rd or 4th day after the infusion had started. There were no significant differences among plasma levels at each sampling time, but plasma levels varied ranging from 5.1 to 24.8 μg/ml (12.1±5.5 μg/ml : mean±S.D.), which values were in disagreement with the predicted value in some cases. The correlations of the theophylline clearance ratio to dose, pH, arterial partial pressures of oxygen (Pao₂) and carbon dioxide (Paco₂) ratios, which were calculated by dividing the value during continuous infusion by the value at the test dose, were investigated to evaluate which factor largely contributed to the failure of this dosing method. Although the clearance ratio did not correlate to pH and Pao₂ ratios, significant negative relationships were observed between the clearance ratio and the dose (p < 0.05) or Paco₂ ratios (p < 0.02). In other words, the alternation of dose or Paco₂ resulted in the change of theophylline clearance. These findings suggest that the test-dose concept should not be used in the seriously ill patients whose theophylline clearance can change easily in relation to dose and/or Paco₂ change.

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The initiation process of effector mechanism of the antitumor activity of (1→3)-β-D-glucans has been examined in detail by applying treatment of mice with (1→3)-β-D-glucanases, kitalase (KIT) and zymolyase (ZYM), via i.p. route after i.p. administration of (1→3)-β-D-glucans, grifolan (GRN) and CM-curdlan (CM-CUD). A significant decrease of antitumor activity caused by GRN was observed by treatment with KIT within 48 h, and that caused by GRN administered via i.v. route was also reduced by the i.v. injection of KIT, although it was less effective than i.p. treatment. Pretreatment of mice with thioglycollate reduced the period of time to abrogate antitumor activity by the sequential treatment of KIT. Similar results were also observed on ZYM treatment after i.p. treatment of CM-CUD. These facts suggest that the period of manifestation of the antitumor activity by (1→3)-β-D-glucans must be longer than a few days.

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The sensitivity of Chinese hamster ovary cells to ethidium bromide, a deoxyribonucleic acid (DNA)-intercalating cationic dye, was reduced in the presence of caffeine, dimethylsulfoxide (DMSO) or 3-aminobenzamide (3AB). As drug resistance is frequently attained by decreased permeability of the drug, the effects of caffeine, DMSO and 3AB on the intracellular accumulation of ethidium were examined. These chemicals were found to decrease the intracellular dose of ethidium via their suppressing effects on the dye uptake with no effect on the efflux. The dye uptake was also suppressed by ouabain and K⁺, suggesting that ethidium was taken up by the K⁺ transport system. Although the exact mechanism implicated in the inhibitory effects of caffeine, DMSO and 3AB on the dye uptake is not yet clear, these chemicals may interfere with this transport system. The rate of decrease in intracellular dose caused by caffeine, DMSO and 3AB correlated well with that of the reduction in respective cellular sensitivity. This strongly suggests that the decrease in intracellular dye dose in the presence of these chemicals is mainly responsible for the reduced sensitivity. Caffeine, DMSO and 3AB caused a decrease in the binding of ethidium to DNA in vitro. The release of ethidium from DNA molecules may lessen DNA damage, thereby contributing to the reduction in cellular sensitivity to ethidium. However, correlation between inhibitory effects of these chemicals on the dye binding and those on cellular sensitivity is not as good as that between the uptake and the sensitivity. This indicates that effects on dye binding to DNA may not be a major reason for the reduction of cellular sensitivity to ethidium.

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Effects of the addition of serum to the perfusate on hepatic uptake of multilamellar vesicles (MLV) were examined in recirculating perfused rat liver. MLV was labelled with both membrane lipid marker ([¹⁴C] cholesteryl oleate) and aqueous phase marker ([³H] inulin or 5 (6)-carboxyfluorescein (CF)). The uptake rates of [¹⁴C] cholesteryl oleate and CF at 37°C coincided well, indicating both markers to be taken up as MLV. Inulin was released from MLV at 37°C but its uptake rate tended to approach that of [¹⁴C] cholesteryl oleate at 4°C or at high MLV dose. Some factor in serum promoted MLV uptake at 37°C, but its effect was inhibited at 4°C. The promoting effect was indicated to be possibly due to activation of adsorption of MLV to the hepatic Kupffer cell surface and/or that of phagocytosis by the cells. The saturation of MLV uptake was observed with increase in MLV dose, suggesting the saturation of MLV adsorption to the hepatic cell or the consumption of serum factor which promotes MLV uptake.

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The effect of fasting on the hydrolysis of salicyluric acid in rabbit intestinal microorganisms was investigated. The blood concentration of salicyluric acid and salicylic acid following oral, intracecal and rectal administration of salicyluric acid was determined. In fasted rabbits (24 and 48 h), the blood concentration of salicylic acid after oral administration was changed compared to the control. However, a significant effect of fasting was not observed in the blood concentration of salicylic acid after rectal administration. Following intracecal administration, the blood concentration of salicylic acid was increased in fasted rabbits compared to the control. From these results, it seems that the slow rate of stomach emptying due to coprophagy during fasting is the principal reason for the change of blood concentration of salicylic acid following oral administration of salicyluric acid.

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The difference in disposition of quinidine (Qd) and its diastereomer quinine (Qn) after intravenous administration was examined in rats at doses ranging from 5 to 20 mg/kg. Dose-dependent kinetics in total clearance and in distribution volume of tissue based on a two-compartment model was observed for Qd ; there was no evidence of nonlinearity for Qn. However, there was no significant difference between Qd and Qn for blood clearance at doses of 5 and 10 mg/kg, at which the blood clearances were almost equal to hepatic blood flow for both Qd and Qn since the excretion of Qd and Qn into the urine and bile was minimal. This indicates the elimination of these diastereomers to be nonrestrictive in the liver. A concentration dependence in unbound volume of tissue distribution and in plasma protein binding was observed for Qd ; there was no concentration dependence for Qn. Although affinity of the drug for components on or within the blood cells was not concentrationdependent for either Qd or Qn, a significantly higher binding capacity for Qn than for Qd was observed, attributable to blood cell binding. Based on these results, it is suggested that a larger number of binding sites exist for Qn than for Qd in the body. However, the dissociation binding constant for Qd is much lower than for Qn, resulting in a higher binding of Qd than Qn at low concentrations, with a reversal at high concentrations.

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Sizofiran (SPG), an antitumor glucan isolated from Shizophyllum commune FRIES was examined for its effect on the lymphoid cell functions related to production of cytokines or responses to these cytokines. Lymphoid cells were isolated from mice injected intramuscularly with SPG and then examined for responses to the cytokines, interleukin (IL)-1, IL-2 or IL-3 or the production of these cytokines in response to the stimuli of mitogens, and the following results were obtained. (1) The thymocytes isolated from the SPG-treated mice proliferated in response to stimulation by IL-1 in combination with concanavalin A (ConA) to a much greater degree than was the case with control mice. (2) When the spleen cells isolated from the mice were cultured with IL-2 or IL-3, augmented proliferative response was observed in either case. (3) Peritoneal macrophages, when stimulated with lipopolysaccharide (LPS), produced IL-1 to the levels much higher than those of control mice. (4) When the spleen cells were cultured with ConA, augmented production of IL-2 and IL-3 were also observed. (5) In addition, we found that activity stimulating the bone marrow cells in in vitro culture was reproducibly detected in the serum of mice 20 h after injection with SPG. Overall, these results indicate that SPG injected into mice has the ability to produce the bone marrow stimulating factor (s) in the serum early after injection and stimulate lymphoid cells to become much more responsive to various stimuli such as lectins or cytokines.

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Human plasma fibronectin (FN) was reduced and carboxamidemethylated, and its binding ability to several matrices was analyzed in vitro. The binding of S-carboxamidemethyl (Cam)-FN to heparin-Sepharose was not influenced by either 4M urea, 0.5 M NaCl or 0.5% heparin, but was disrupted by the coexistence of urea and NaCl or heparin. S-Cam-FN, compared with intact FN, obviously had a more potent ability to bind heparin, while it had little or no binding ability to gelatin, fibrin and thrombin-stimulated platelets. A conformational change of S-Cam-FN by heparin-binding has been proposed as a possible mechanism from the result of circular dichroic spectrum measurement.

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The mucosa-to-serosa permeability of non-electrolytes through the stripped colonic rat mucosa was examined in a Ussing-type chamber. The permeation clearances for inulin (12-15 Å radius) to erythritol (3.2 Å radius) increased linearly with the increase in their free diffusion coefficients. On the other hand, the clearances of glycerol, thiourea and urea of less than 3 Å radius increased in excess of what would be expected considering the above linearity. This suggests that there are large pores that do not restrict diffusive flow and small pores of 3 Å radius or greater that restrict diffusion in the paracellular channel. The transcellular permeation also occurred in a tracer efflux experiment with urea preloaded in colonic stripped mucosa. It was not ruled out that the higher permeability of the nonelectolytes less than 3 Å in radius was due to a transcellular route in parallel with the paracellular one.

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Four metabolites of menaquinone-4 [MQ-4] were isolated from rat urine, bile and liver. From rat urine following intravenous or oral administration of [¹⁴C] MQ-4, two major metabolites were isolated and their aglycones were identified as 2-methyl-3-(5'-carboxy-3'-methyl-2'-pentyl)-1, 4-naphthoquinone [K acid 1] and 3-(3'-carboxybutyl)-2-methyl-1, 4-naphthoquinone [K acid 2]. The aglycone of a third minor metabolite isolated from bile was tentatively identified as 2-methyl-3-(15'-carboxy-3', 7', 11'-trimethyl-2', 6', 10', 14'-hexadecatetranyl)-1, 4-naphthoquinone [MQ-4-COOH]. The structures of the three aglycones, which were excreted into the urine or bile mainly as glucuronide conjugates, indicated that oxidative degradation of the alkyl side chain of MQ-4 had occurred by ω-and β-oxidation. In addition, 2, 3-epoxy-MQ-4 was identified in the liver of rats which were pretreated with warfarin and then dosed with [¹⁴C] MQ-4.

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Following intravenous administration to rats of all-trans [¹⁴C] menaquinone-4 solubilized with purified soybean lecithin [L] or with HCO-60 [H], we examined the effect of the solubilizers on the distribution and excretion of menaquinone-4 [MQ-4]. The level of radioactivity in the liver after dosing with L was about 2 times higher than in dosing with H, and a similar result was obtained in the hepatic microsomal fraction, a target of MQ-4. The rate and amount of biliary excretion of radioactivity after dosing with L were greater than in dosing with H. In addition, the uptake of [¹⁴C] MQ-4 by the isolated perfused rat liver was greater with L than H, consistent with the in vivo observation. Further, upon incubation of L or H with hepatic microsomes, the MQ-4 metabolizing enzyme was more highly active toward L than H. These results show that L is more easily transported to the target region, and more rapidly metabolized and excreted into the bile than H, suggesting that the lecithin-solubilized preparation of MQ-4 may be more effective clinically.

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