Anticonvulsant activity, degradation into γ-aminobutyric acid (GABA), and concentration in brain of 1-dodecanoyl-2-pyrrolidinone (I), a lipophilic derivative of a lactam of GABA, were compared with those of N-dodecanoyl GABA (II) and 1-dodecyl-2-pyrrolidinone (III) to get information about their pharmacological mechanisms. Compounds I and II degraded into GABA in mouse liver homogenate, gradually into GABA in brain homogenate and more slowly in plasma. Compound III had no degradation in the biological media. The derivatives administered intraperitoneally had dose-dependent anticonvulsant activity on picrotoxin-induced seizure in mice. Their anticonvulsant activities were changed by the time intervals between pretreatment of derivatives and administration of picrotoxin. Compounds II and III showed anticonvulsant activity on pentylenetetrazole-induced seizure and a prolonged sleeping time induced by sodium pentobarbital in mice. However, these three derivatives never significantly increased the GABA level in mouse brain after intraperitoneal administration compared to the endogenous GABA level. They were detected as intact derivatives in the brain. In the previous report, we demonstrated the anticonvulsant activity of sodium dodecanoate. These results suggested that the dodecyl chain of derivatives may be important for their anticonvulsant activities and I does not act as GABA via prodrug.

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A (1-3)-β-D-glucan, grifolan (GRN), recovered from the peritoneal cavity after 1 d from i.p. injection contained a significant amount of anionic metabolite (s) having lower M_r than the parent GRN. In parallel with this observation, GRN induced peritoneal exudate cells exhibiting a higher level of oxidative burst than the non-stimulated, resident peritoneal cells. Chemical oxidation of GRN by active oxygen species such as (a) ascorbic acid-CuSO₄, (b) hydrogen peroxide, (c) hydrogen peroxide-CuSO₄, or (d) hypochlorous acid also produced anionic as well as lower M_r degradation products. Under these experimental conditions the structural changes were remarkable and in the order of a<b<c<d. The products formed under the conditions (a) and (b) retained significant antitumor activity but those of (c) and (d) lost the activity. However, oxidation product (s) of curdlan, an antitumor inactive (1-3)-β-D-glucan having no branch, by condition (d) induced significant antitumor activity. These results suggested that oxidative degradation of (1-3)-β-D-glucans produced some temporary active metabolites and then gradually changed to the inactive form. However, these active metabolites contribute less than the parent glucan on the whole activation mechanisms of the host by (1-3)-β-D-glucans.

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Effect of intravenous administration of protoporphyrin IX (PP) on lipid peroxidation was studied in rats. PP and/or PP-derived porphyrins were found to be mainly distributed in livers, spleen and lungs. Dose-dependent decreases in the Fe²⁺ and L-ascorbic acid-stimulated lipid peroxidation in homogenates of livers and dose-dependent increases in porphyrin concentration in livers were observed after the PP injection. In the experiments with a 20 mg/kg dose of PP, the peroxidation level in the liver homogenates reached its minimum level during the period of 3 to 24 h accompanying the high porphyrin concentration in livers after the administration. After 96 h, a relatively high porphyrin concentration was still retained, but decreases in the peroxidation levels had ceased. PP administration caused a dose-dependent decrease in the endogenous lipid peroxides in livers within 0.5 h and the low levels were maintained throughout the course of the 168-h study. These results clearly show that the administered PP is distributed in the liver and inhibits the lipid peroxidation in vivo.

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The localization of tissue plasminogen activator (t-PA) on microthrombi in various organs of disseminated intravascular coagulation rats (DIC rats) was investigated by using microautoradiographic technique. After the injection of [¹²⁵I] fibrinogen, experimental DIC rats induced by the infusion of thrombin for 1 h were submitted to microautoradiograms (MARGMs) of some major organs. The radioactivity of [¹²⁵I] fibrin thrombi, which were observed as silver grains, was localized in the glomeruli and parts of small vessels in the kidney. In the liver, microthrombi were seen in sinusoid vessels and on Kupffer cells. In addition, many microthrombi were noted in small vessels in the lung and marginal zones in the spleen. Two min after the intravenous administration of [¹²⁵I] t-PA to DIC rats, many silver grains were observed on each MARGM of the kidney, lung, liver and spleen showing the formation of microthrombi. From the identical results with the observations of MARGMs after the injection of [¹²⁵I] fibrinogen, we confirmed that t-PA was highly accumulated to microthrombi formed in small vessels of the organs. The scattered silver grains were widely observed on the hepatocytes. This result suggested that t-PA bound to the parenchymal cell surface might be transported into the hepatocytes by receptor-mediated endocytosis. On the other hand, when [¹²⁵I] urokinase plasminogen activator ([¹²⁵I] u-PA) was administered intravenously to DIC rats, many silver grains were observed on MARGM of the proximal tubules in the kidney but not seen on MARGMs of the glomeruli in the kidney, nor in the lung, liver, and spleen. This observation suggested that u-PA might not have a characteristic to accumulate to thrombi.

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A pharmacokinetic study of FK-506 (FK), a novel immunosuppressant being about hundred times more potent than cyclosporin A (CyA) in the in vitro experiment, has been performed in rats after intravenous and oral administrations at two doses, 1.0 and 5.0 mg/kg. As compared with CyA, plasma, bile, urine and lymph FK levels were determined by a fluorescence high-performance liquid chromatographic method with chemiluminescence detection. Non-compartment pharmacokinetic parameters were calculated by area/moment analysis. After the i.v. injection of FK at 1.0 and 5.0 mg/kg, the total plasma clearance, CL_tot, was 7.028±0.076 (mean±S.E.) and 7.651±0.755 ml/min, steady-state distribution volume, V_{d, ss}, was 4623±28 and 4201±529 ml/kg, elimination half-life at the terminal elimination phase, t<1/2β>, was 2.36±0.25 and 2.54±0.29 h, and the volume of the initial distribution space, V₁, was 1336±150 and 1065±115 ml/kg, respectively. By comparing the pharmacokinetic parameter with CyA, the CL_tot of FK is about three times greater than that of CyA. There is not a significant difference on t_1/2β between CyA and FK. V₁ and V_{d, ss} of FK are 4-5 times greater than that of CyA. Therefore, higher clearance of FK is ascribed not only to the faster elimination from the rat body but also to the greater distribution space in the body. The mean percentage of FK transferred into the thoracic lymphatics over 6 h were 0.09±0.02% (1.0 mg/kg) and 0.16±0.02% (5.0 mg/kg), respectively. As the mean percentages of FK excrcted into both the bile and urine for 6 h after i.v. injection were 0.0744±0.0177% and 0.0073±0.0018%, respectively, the main elimination pathway of this drug is thought to be the metabolism in the body. To the other groups of rats, FK was administered intraduodenally, 5.0 mg/kg, in two kinds of liquid preparations, and both the systemic and lymphatic availabilities were studied. The mean systemic availabilities of FK were 11.3% and 23.5% from the two preparations. The lymphatic availability of this drug over the experimental period, 6 h, was less than 0.2%. These results suggest that FK distributes more extensively in the rat body than CyA.

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As a part of the development of organic fluorescent reagents, bimane derivatives were coupled to guanosine 3'-monophosphate, and they were shown to be useful fluorogenic substrates for the assay of ribonuclease T₁

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The healing effect of human epidermal growth factor (hEGF) on second degree burn was studied in rats. No improvement in wound healing was found on topical application of EGF alone to burn sites, but an ointment containing EGF and nafamostat mesilate (NM), a protease inhibitor, accelerated the healing rate of burns. The dry weight of the granulation tissue on the wound site in the group treated with EGF plus NM ointment did not change, although that in other groups decreased. After treatment with EGF ointment containing NM, the content of uronic acid, as an index of acid mucopolysaccharide, at 3 d after burn rapidly increased and had recovered to nearly normal levels at 7 d after burn. However, the uronic acid content in the other groups (control, EGF alone, and NM alone) showed a higher value at 7 d than at 3 d. When compared with the control values significant increases in hydroxyproline, as an index of collagen, in the wound site were observed at 7 d after treatment with EGF ointment containing NM. The degradation of [¹²⁵I] EGF in burned tissue homogenate decreased significantly in a concentration-dependent manner in the presence of NM. Body weights did not change after treatment with EGF plus NM ointment, although the body weights of other treatment groups decreased after burn, suggesting that EGF ointment containing the protease inhibitor, NM, alleviated the effects of burn shock. These findings indicate that the stabilization of EGF at the wound site is an important factor for the expression of its healing effects.

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Prostaglandin E₁ (PGE₁) specifically bound to the membrane prepared from murine renal outer medulla. The extent of binding of [³H] PGE₁ to the membrane was increased about 4-fold by guanosine triphosphate (GTP) and its analogs, but the dissociation of bound [³H] PGE₁ from the membrane was in turn enhanced by GTPγS. Scatchard plot analyses revealed that GTPγS increased the binding affinity more than 2-fold without a major change in the number of binding sites. When [³H] PGE₁-bound proteins were cross-linked in the membrane by dithiobis (succinimidyl propionate), bound [³H] PGE₁ was no longer dissociated by GTPγS treatment, suggesting that cross-linking produced a stable complex of PGE receptor with a GTP-binding protein. The cross-linked [³H] PGE₁-specifically bound proteins solubilized from the membranes labeled with [³H] PGE₁ in the presence or absence of GTPγS were eluted as an apparently single radioactive peak at the same position of M_r=150000 by gel filtration, indicating that the PGE receptor forms a complex with a GTP-binding protein regardless of the treatment with GTPγS by which [³H] PGE₁ binding is promoted. The ability of GTPγS to stimulate [³H] PGE₁ binding was eliminated by pretreatment of the membrane with pertussis toxin, but not cholera toxin, indicating that the PGE receptor is coupled to a pertussis toxin-sensitive GTP-binding protein.

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