The current developments in liquid chromatography-mass spectrometry (LC-MS) and its applications to the analysis of pharmaceuticals are reviewed. Various mass spectrometric techniques, including electrospray and nanospray ionization, atmospheric pressure chemical ionization and photoionization and their interface with liquid chromatographic techniques are described. These include high performance liquid chromatography, capillary electrophoresis and capillary electrochromatography and the advantages and disadvantages of each technique are discussed. The applications of LC-MS to the studies of in vitro and in vivo drug metabolism, identification and characterization of impurities in pharmaceuticals, analysis of chiral impurities in drug substances and high-throughput LC-MS-MS systems for applications in the “accelerated drug discovery” process are described.

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In the course of studies on the metabolism of active components of Huangqin-Tang by human intestinal flora (HIF), Huangqin-Tang and all individual herbs in the decoctions were incubated with a human fecal suspension separately. By using a high-performance liquid chromatographic (HPLC) method which was previously established in our laboratory, the metabolites in both the compound prescription and all the single herb decoctions were identified and determined both qualitatively and quantitatively. We found that the constituents of Huangqin-Tang, incluing baicalin (baicalein 7-glucuronide; BG), wogonoside (wogoninoglucuronide; WG), oroxylin-A-glucuronide (OG) from Scutellariae Radix, paeoniflorin (PF) from Paeoniae Radix, liquiritin (liquiritigenin 4′-O-glucoside; LG), isoliquirtin (isoliquiritigenin 4-glucoside; ILG) and glycyrrhizic acid (GL) from Glycyhhizea Radix, were converted to their metabolites baicalein (B), wogonin (W), oroxylin-A (O), paeonimetabolin-I (PM-I), liquiritigenin (L), isoliquiritigenin (IL) and glycyrrhetinic acid (GA) by HIF. The contents of the metabolites in Huangqin-Tang and in each single herb decoction increased significantly after incubation with intestinal flora. Comparing with single herb decoctions, the transformation of BG, WG, OG, LG and ILG in the compound prescription was promoted, however, that of PF and GL was inhibited. All the results suggested that the glycosides of many medicinal herbs could be converted to aglycones by HIF, and the metabolism of most glycosides was improved in the compound prescription.

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Four novel peptides that inhibit cathepsin B, designated as Cabin-1, -2, -3, and -4, were isolated from a thermolysin digest of human plasma. After gel filtration and cation-exchange chromatography, the peptide mixture was purified by reverse-phase HPLC to isolate Cabin-1, -2, -3, and 4, with the amino acid sequences LGPVTQE, VLQSSGLYS, VVSVLT, and LVYDAY, respectively. These peptides correspond to f(64—70) of human apolipoprotein A-I for Cabin-1, f(56—64) and f(185—190) of the human immunoglobulin G γ chain for Cabin-2 and -3, and f(66—71) of human transferrin for Cabin-4. Synthetic Cabin-1, -2, -3, and -4 showed dose-dependent inhibition of cathepsin B. Their IC₅₀ values were 450, 500, 20, and 5.0 μmol/l, respectively. Lineweaver–Burk plots suggested that Cabin-3 is a noncompetitive inhibitor, while Cabin-4 is a competitive inhibitor. Among the N- and C-terminal deletion peptides of Cabin-2 and -4, Cabin-2(1—8), VLQSSGLY, was found to have the most potent inhibitory activity, with an IC₅₀ of 3.8 μmol/l.

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We developed a rapid and convenient extraction procedure of human hair proteins to examine their biochemical properties in detail. This procedure is based upon the fact that the combination of thiourea and urea in the presence of a reductant can effectively remove proteins from the cortex part of human hair. The extracted fraction mainly consisted of hard α-keratins with molecular masses of 40—60 kDa, matrix proteins with 12—18 kDa, and minor components with 110—115 kDa and 125—135 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein phosphorylation in human hair was investigated by immunoblotting with antibodies against phosphoserine, phosphothreonine and phosphotyrosine. We found serine phosphorylation in α-keratins and matrix proteins and threonine phosphorylation in α-keratins. The extraction was also found to be effective when wool, chicken feathers, rat hair and human nails were used as starting materials.

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Recombinant acidic calponin, a member of the calponin family, interacted with F-actin, but not with microtubules, desmin filaments, tropomyosin, calmodulin, S100 and phosphatidylserine (PS) vesicles with significant affinity. The bindings of acidic calponin to F-actin occurred in a concentration-dependent manner and were saturated at a molar ratio of about 1 acidic calponin to 1—2 actin molecules. The apparent Kd value of acidic calponin to F-actin was calculated to be 1.6×10⁵ M⁻¹. Chemical cross-linking experiments indicated that a 1 : 1 molar covalent complex of acidic calponin and actin monomer was produced as in the case of basic calponin–actin binding. No significant morphologic change of F-actin was observed by the addition of acidic calponin. Acidic calponin had little effect on actomyosin Mg²⁺-ATPase activity unlike basic calponin. Basic calponin partially competed with acidic calponin for binding to F-actin. Domain mapping with V8 protease revealed that acidic calponin binding site resided within the C-terminal 16 kDa fragment of actin, where the binding of basic calponin also occurs. However, both calponins showed reversal effects on fluorescence intensity of pyrene-labeled F-actin. Fragments of acidic calponin with 30 and 22 kDa, lacking the C-terminal acidic tail, were bound to F-actin. Interestingly, both the fragments became bound to PS vesicles, but not to other components. Circular dichroism studies showed that limited digestion of acidic calponin resulted in about 30% decrease of α-helix and β contents. The present results suggest that acidic calponin is functionally distinct from basic calponin and expresses a novel characteristic after removal of the acidic tail region.

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Recently, there has been many investigations on the relationship between leptin and obesity, which is the main health problem in developed countries. In some reports, it has been claimed that the adrenalectomy has lead to weight loss and thus prevented obesity induced in rodents in various ways. It has also been accepted that diet-induced obesity in animals is very similar to obesity in humans beings. In this study, obesity has been developed with high-calorie diet given for 8 weeks in Sprague–Dawley rats. Then, it has been investigated how leptin and some metabolic parameters change in blood samples obtained from rats 15 d after adrenalectomy. Leptin levels was determined with Radio Immun Assay (RIA, Linco Research Co^®) method. Our study showed that, there were statistically significant increases in leptin (p<0.001), glucose (p<0.05), triglyceride (p<0.01) levels in diet-induced obese rats (n=19) when compared with the findings of control rats, lean ones (n=16), (Tables 3, 4). Adrenalectomy led to decreased serum leptin (p<0.001) and triglyceride (p<0.01) levels both in the obese and lean rats (Table 5). As a conclusion, it could be claimed that the decrease in leptin levels may be attributed to reduced adipose tissue due to adrenalectomy. On the other hand, the decreases in glucose and triglyceride levels might be the consequence of reduced lipogenesis and impaired gluconeogenesis with the effect of adrenalectomy. It was concluded that adrenalectomy might prevent obesity by affecting leptin and intermediate metabolism.

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Two basic proteins enhancing vascular permeability have been purified from the exudate of the chronic phase of carrageenan-induced inflammation in rats. One major and one minor peak on reversed-phase HPLC showed molecular masses of 9.3 kDa and 7.6 kDa, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. NH₂-terminal amino acid sequencing analysis of the purified proteins revealed that the major peak is identical to C3a, while the main sequence of the minor peak is identical to NH₂-terminal 11 amino acids truncated C3a. In addition, plasmin was able to cleave C3a into the N-truncated C3a. Intradermal injection of both purified C3a and N-truncated C3a into rat skin enhanced vascular permeability, and the increased permeability was suppressed by the pretreatment with cyproheptadine. Our results suggest that the purified C3a and N-truncated C3a have the characteristics of anaphylatoxins and may contribute to exudation in the chronic phase of carrageenan-induced inflammation in rats.

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We examined effects of adenine-nucleotides on the sequence-specificity of origin recognition complex (ORC)-binding to DNA, using a gel electrophoretic mobility shift assay. The sequence-specific DNA binding of ORC was observed in the presence of ATP or ATP-γ-S but not in the presence of adenosine 5′-diphosphate (ADP) or in the absence of any adenine-nucleotides. In contrast, the sequence-independent DNA binding of ORC was observed under any one of these conditions. These results suggest that ATP increases the sequence-specificity of ORC-binding to DNA. In relation to the requirement for incubation at high temperature and inhibition by cardiolipin, there was no significant difference between the sequence-specific and the sequence-independent DNA binding activities of ORC.

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Interactions of serotonin (5-HT) with different specific 5-HT receptors that can coexist in the same blood vessel sometimes generate opposite effects. The aim of this study was to characterize the functional role of previously described mRNAs for 5-HT receptors in the rat aorta (5-HT_2A, 5-HT_2B, 5-HT_1B, 5-HT₇) as well as to study the known 5-HT_2A receptor-mediated constrictor response and investigate the influences of endothelium and preconstriction on the tissue in that response. A slight endothelium- and concentration-dependent relaxant effect was observed for 5-HT in aorta precontracted with either 5 μM phenylephrine (PE) or 1 μM prostaglandin F_2α (PGF_2α) in the presence of 0.3 μM ketanserin. EC₅₀ values for 5-HT and α-methyl-5-HT relaxant responses after PE were 43.10±4.00 and 57.11±8.01 nM, respectively. pK_B values for antagonists cyproheptadine and rauwolscine were 8.92±0.22 and 7.15±0.12, respectively. In nonprecontracted tissues, the contractile potency of 5-HT was higher in the absence of endothelium (EC₅₀, μM): 2.60±0.28 and 4.12±0.21 in the absence and in the presence of endothelium, respectively. The differences were statistically significant (p<0.05). In precontracted tissues, the differences in EC₅₀ values (2.22±0.40 and 4.65±0.60 μM without and with endothelium, respectively) were also statistically significant (p<0.05). pK_B values for the 5-HT_2A antagonist ketanserin were similar under all conditions tested. In conclusion, under our experimental conditions there are two functional 5-HT receptors in rat aorta: 5-HT_2A contractile receptor in smooth muscle and a high-affinity relaxant receptor that mediates a very slight response and the pharmacology of which could be compatible with an endothelial 5-HT_2B receptor.

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The biochemical effects of the 2-nitroimidazole hypoxic cell radiosensitizers KIN-804, KIN-806, and their analogues KIN-844 and TX-1877 on brain acetylcholinesterase (AChE) and hepatic free radical scavenging systems, such as reduced glutathione (GSH) and glucose-6-phosphate dehydrogenase (G-6-PDH) levels, and hepatic antioxidants, such as superoxide dismutase (SOD) and catalase, were evaluated in Ehrlich ascites carcinoma (EAC)-bearing Swiss albino mice. The assay of brain AChE revealed nonsignificant changes with all drugs examined. To evaluate the hepatic metabolic capacity, groups of mice were divided into control, EAC-inoculated, 10-Gy local gamma-irradiated, and KIN-804, KIN-844, KIN-806, or TX-1877 (50 mg/kg body weight, i.p.) groups, and gamma-irradiation was combined with each drug. EAC inoculation markedly suppressed GSH, G-6-PDH, SOD, and catalase levels. On the other hand, treatment with gamma-irradiation significantly enhanced them. The treatment of EAC-bearing mice with each drug alone in the absence of gamma-irradiation revealed that KIN-806 and its derivative TX-1877 showed antitumor activity through their significant recovery of GSH and SOD levels, respectively, in the EAC-bearing mice group. Similarly, the combined treatment of EAC-bearing mice with gamma-irradiation with each of the drugs tested showed that KIN-806 and TX-1877 significantly increased GSH and SOD, and to a lesser extent G-6-PDH and catalase levels. On the other hand, KIN-804 and KIN-844 had only a nonsignificant effect on all parameters examined. In conclusion, these data reveal that the administration of KIN-806 and TX-1877 with or without subsequent gamma-irradiation, resulted in significant recovery of GSH and SOD activities that were inhibited by EAC inoculation.

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Angiogenesis, in particular anti-angiogenesis, is an area of particular therapeutic interest in cancer treatment. Several anti-angiogenic agents are in the final stages of clinical trials. One of these agents, thalidomide, best known for its teratogenic potential, is showing promise against several tumor types. Thalidomide has been shown previously to require bio-activation to exert its anti-angiogenic effect in isolated blood vessels and endothelial cells. In this work, we confirmed these findings using the in utero chicken embryo chorioallantoic membrane (CAM) system. In particular, the anti-angiogenic effect of thalidomide is significantly enhanced by activation by human but not by rat liver microsomes. We also showed in the CAM assay that hydroxylation of thalidomide at either the 1′- or 5-position retained anti-angiogenic activity whereas its hydroxylation at the 4-position led to an inactive compound. We further demonstrated that thalidomide shows weak anti-proliferative activity against MDA-MB-231 human breast cancer cells in culture. Thalidomide showed slightly more anti-proliferative activity, however, against the SH-SY5Y human neuroblastoma and human umbilical vein endothelial cell (HUVEC) types. Furthermore, incubation of thalidomide with human liver microsomes added no additional anti-proliferative effect in these cell types versus thalidomide given alone. Finally, we report that none of the thalidomide metabolites tested had any anti-proliferative effect against the breast or neuroblastoma cells, but do possess appreciable anti-proliferative activity against the endothelial cells. In summary, this work suggests that hydroxylated thalidomide analogs based on putative metabolites of the drug possess significant anti-angiogenic activity and that exploring further derivatives of these as potential anti-angiogenic agents warrants further merit.

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Melatonin, has been reported to participate in the regulation of a number of important physiological and pathological process. It has also the ability to protect the genetic material of hematopoietic cells of mice from damaging effects of acute total body irradiation. The objective of this study was to the potential radioprotective effects of pharmacological doses of melatonin in total body irradiated rat's peripheral blood cells. Forty adult rats were divided into 4 equal groups. Group 1 received no melatonin or irradiation (control group), while group 2 received only melatonin (5 mg/kg, i.p.). Group 3 received only total body irradiation (RT) by 5 Gy of γ irradiation only and group 4 received RT plus melatonin (5 mg/kg, i.p., 30 min before RT). An hour and a half following RT, blood samples were taken. Leukocytes and thrombocytes number and hemoglobin levels were measured in all groups. Five mg/kg dose of melatonin significantly protected leukocytes and as well as thrombocytes number against γ irradiation. There were no significant differences between Hb levels. Our results suggest that melatonin administration prior to irradiation prevented radiation damage on peripheral blood cells. Melatonin radioprotection is achieved by its ability as a scavenger for free radicals generated by ionizing radiation and acts probably as a growth factor, especially for granulocytes in bone marrow.

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In order to clarify that squalene monohydroperoxide (SQOOH) correlates with changes in morphology through cytotoxicity and establish in vitro evaluation of the cytotoxicity of lipid hydroperoxide, the effect of SQOOH on cytotoxicity and morphology in normal human epidermal keratinocytes (NHEK(B)) and the Gunze three-dimensional cultured human skin model (Vitrolife-skin) was investigated. Additionally, the effect of radical scavengers (mannitol, vitamin C and (+)-catechin) on the cytotoxicity in Vitrolife-skin was studied. The level of lipid hydroperoxide (phoshatidylcholine hydroperoxide: PCOOH) in the cellular membrane was increased with the concentration of SQOOH, and the rise in cytotoxicity in NHEK(B) was associated with changes to the cellular membrane. A concentration-dependent and protective effect on the increase in cytotoxicity and PCOOH content was observed. To clarify the effect of SQOOH on the release of cytokine from cells, IL-2 level from NHEK(B) and Vitrolife-skin were investigated. IL-2 release from the cells was enhanced by SQOOH and increased at a non-cytotoxic dose.
These results suggest that the increase in lipid hydroperoxides resulting from the auto-oxidation of lipids within cellular membranes in the presence of SQOOH correlates with changes in morphology due to cytotoxicity. SQOOH enhanced the release from cells at a non-cytotoxic dose. A method for assessing the protective effect on the cytotoxicity of lipid hydroperoxides using cells would be useful for in vitro evaluation of the cytotoxicity.

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The mechanism of aggregation and insolubilization of lens proteins was examined based on the kinetics of crystallin–crystallin interaction determined by the surface plasmon resonance method on a BIAcore system. Lens proteins are composed mainly of three types crystallins, α-, β-, and γ-crystallin. The present study indicated that α-crystallin shows marked self-interaction. Furthermore, this interaction was shown to be due to αA-crystallin, which is a subunit of α-crystallin. It was also clarified that this mutual interaction of αA-crystallin decreases abruptly after the age of 20 years. On the other hand, it was assumed that αB-crystallin, the other subunit of α-crystallin, may play an important role in interactions with β- and γ-crystallin, while α-crystallin shows chaperone-like activity. Based on the present results, αA- and βB-crystallin may play different roles when α-crystallin displays chaperone-like activity, and also that the decreased chaperone-like activity of α-crystallin may finally result in cataract formation following aggregation and insolubilization of lens proteins.

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In order to develop new imaging markers for brain hypoxia, 4-bromo-1-(3-fluoropropyl)-2-nitroimidazole (4-BrFPN) was designed based on molecular orbital calculations, synthesized and labeled with fluorine-18 as a lipophilic nitroimidazole analog with a lower energy LUMO orbital than those for fluoromisonidazole (FMISO) and 1-(3-fluoropropyl)-2-nitroimidazole (FPN). In an in vitro radiosensitization study, the sensitizer enhancement ratio for 4-BrFPN was found to be 1.65 at a 1 mM concentration, in comparison to 1.81 for FMISO. The preparation of ¹⁸F-labeled 4-BrFPN (4-Br¹⁸FPN) was achieved by [¹⁸F]fluoride ion displacement reaction of the tosylate precursor, in a reasonable radiochemical yield (33%, not corrected for decay). Metabolites in tumor and muscle extracts from methylcholanthrene-induced fibrosarcoma mice, as well as the tissue distribution of 4-Br¹⁸FPN in normal rats, were studied. The initial uptake into rat brain of 4-Br¹⁸FPN was significantly higher relative to ¹⁸F-labeled FMISO (¹⁸FMISO), followed by a rapid washout from the brain. The tumor uptake of 4-Br¹⁸FPN was somewhat enhanced compared to those obtained with ¹⁸FMISO and ¹⁸F-labeled FPN (¹⁸FPN), but with lower tumor localization than ¹⁸FMISO. Analyses of tumor and muscle extracts showed metabolites remaining base line on the radio-TLC plates, and they were produced to a greater extent in tumor than muscle. The use of two drugs which increase hypoxic cell fraction in tumor, hydralazine or nitro-L-arginine, produced a significant increase in tumor levels of 4-Br¹⁸FPN, suggestive of a hypoxic mechanism of accumulation. The results imply that lowering of the LUMO energy of a molecule alone is not sufficient to improve its biodistribution properties for better imaging of regions of hypoxia.

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The aqueous ethanol extract of Lygodii Spora (spore of Lygodium japonicum (THUNB.) SW.) showed in vitro testosterone 5α-reductase inhibitory activity and in vivo anti-androgenic activity using growth of flank organ in castrated Syrian hamsters and hair regrowth after shaving in testosterone-treated C57Black/6CrSlc mice. From the lipophilic constituents of Lygodii Spora, oleic, linoleic and palmitic acids were identified as the main active principles inhibiting testosterone 5α-reductase.

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The 80% aqueous acetone extract of Zedoariae Rhizoma was found to show a protective effect against D-galactosamine (D-GalN)/lipopolysaccharide-induced acute liver injury in mice. To clarify the active compounds, the principal constituents were examined and 11 sesquiterpenes (furanodiene, curdione, neocurdrione, dehydrocurdione, germacrone, 13-hydroxygermacrone, curcumenol, isocurcumenol, aerugidiol, zedoarondiol, and curcumenone) and a diarylheptanoid (curcumin) were found to inhibit the increase in serum aspartate aminotransaminase and alanine aminotransaminase at a dose of 50 mg/kg p.o. in agreement with the previous in vitro studies, except for dehydrocurdione, aerugidiol, and zedoarondiol. In particular, curdione, neocurdione, curcumenol, and isocurcumenol potently inhibited the increase at a dose of 12.5 mg/kg p.o. Furthermore, the eight sesquiterpenes, furanodiene, curdione, neocurdione, dehydrocurdione, germacrone, 13-hydroxygermacrone, curcumenol, and curcumenone, also showed a protective effect against D-GalN/tumor necrosis factor-α-induced liver injury in mice at a dose of 50 mg/kg p.o.

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Significant selective cyclooxygenase-2 (COX-2) inhibitory activities were observed for two new 1,4-naphthoquinone sodium salts, sodium 3-hydroxide-2{[sodium 3-hydroxide-1,4-dioxo(2-naphthyl)]ethyl}naphthalene-1,4-dione (impatienolate) (1) and sodium 2-hydroxide-3-(2-hydroxyethyl)naphthalene-1,4-dione (balsaminolate) (2), which were isolated from the corolla of Impatiens balsamina L. (Balsaminaceae). Their structures were elucidated by spectral techniques. Our results offer evidence supporting the use of I. balsamina L. to treat articular rheumatism, pain, and swelling.

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We cloned the gene of d-limonene synthase (ArLMS) from Agastache rugosa (Labiatae). The function of ArLMS was elucidated by the preparation of recombinant protein and subsequent enzyme assay. ArLMS consisted of 2077 nucleotides including 1839 bp of coding sequence that encodes a protein of 613 amino acids. This protein has a 60 kDa molecular weight, which is identical to that of d-limonene synthase from Schizonepeta tenuifolia (Labiatae). The deduced amino acid sequence of ArLMS shows high homology with the known d- and l-limonene synthases from Labiatae plants. Here, we discussed the amino acid residues responsible for the stereochemical regulation in limonene biosynthesis.

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We examined the antimetastatic effect of acteoside, a phenylethanoid glycoside widely distributed in the plant kingdom, on lung metastasis using a mouse model injected with B16 melanoma cells intravenously. Male C57BL/6 mice were injected intravenously with 2×10⁵ of B16 melanoma cells, while acteoside at a dose of 50 mg/kg was administered intraperitoneally every other day from 13 d before B16 melanoma cell injection until all mice had succumbed to the metastatic tumor burden in the lung. Administration of acteoside prolonged survival time significantly and the average survival time was 63.3±3.4 d compared with 52.1±2.5 d in control mice. This result suggests that acteoside showed suppressive effect on lung metastasis of B16 melanoma cells.

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In vitro antifungal activities of naphtoquinone-derivatives, which are constituents of Shikon, roots of Lithospermum erythrorhizon, were investigated against several fungal pathogens. When the biological activity of these compounds was tested against fungi, a wide range of sensitivity was recorded. Shikonin was found to have a stronger than fluconazole against yeast-like fungi: four-fold against Candida krusei (minimal inhibitory concentration (MIC); 4 μg/ml) and two-fold (MIC; 4 μg/ml) against Saccharomyces cerevisiae, though it showed the same potency as fluconazole against C. glabrata. Deoxyshikonin also exhibited four-fold stronger activity against C. krusei (MIC; 4 μg/ml) and three-fold (MIC; 2 μg/ml) stronger against S. cerevisiae. Acetylshikonin and β-hydroxyisovaleryl shikonin showed lower activities against all fungal pathogens except for C. krusei compared with the standard. Against the filamentous fungus, Trichosporon cutaneum, all naphthoquinones were found to have a range of activity with lower potency than standard. This result provides a rational basis for the clinical use of shikon and shows the possibility of its use in medicinal treatment as an anti-inflammatory agent with antifungal activity.

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Paclitaxel was bound via its hydroxyl group to carboxymethyldextran (CMDex, 150 kDa) by means of an amino acid linker; the linker was introduced into the 2′- or 7-hydroxyl group of the paclitaxel through an ester bond. These conjugates—CMDex-2′-paclitaxel and CMDex-7-paclitaxel—were designed to be water-soluble with a paclitaxel content between 6—8% (w/w) with a degree of subsititution (DS) of the CM groups at 0.6 per sugar residue. The release of the paclitaxel from the conjugates was influenced by the hydroxyl group (2′- or 7-) of paclitaxel to which the amino acid linker was introduced, and by what amino acid was used as the linker. In mouse plasma incubated at 37 °C for 72 h, the most paclitaxel was released using CMDex-paclitaxel conjugate with 2′-gly followed by, in descending order, 2′-ala, 2′-leu, 2′-ile, and 7-gly as the amino linkers. Colon 26, a Taxol^® resistant cancer, was introduced into mice and the conjugates were intravenously administered by bolus injection for a tumor distribution study, and intermittently intravenously administered for a tumor growth regression study. In both studies the highest amount of paclitaxel release was found in the CMDex-2′-gly-paclitaxel followed by CMDex-2′-ala-paclitaxel, CMDex-2′-leu-paclitaxel and paclitaxel. There was a direct correlation between the amount of paclitaxel released and the observed efficacy. CMDex-2′-ile-paclitaxel and CMDex-7-gly-paclitaxel did not show any anti-tumor activity. These results clearly demonstrate that a CMDex-paclitaxel with an appropriate amino acid linker has significant anti-tumor activity against colon 26, and that these anti-tumor effects appear to correlate with the amounts of paclitaxel released in the tumor.

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We evaluated the in vitro susceptibility of four serotypes of enterohaemorrhagic Escherichia coli (E. coli O26, E. coli O111, E. coli O157, and E. coli O165) with diverse DNA patterns to antimicrobial agents. The minimum inhibitory concentrations (MIC) determined in a total of 83 strains using Mueller–Hinton agar under aerobic and anaerobic conditions were 0.015—0.12 μg/ml for ciprofloxacin, 0.06—1 μg/ml for norfloxacin, 2—64 μg/ml for fosfomycin without glucose-6-phosphate (G-6-P), 0.25—32 μg/ml for fosfomycin with G-6-P, 2—256 μg/ml for kanamycin, 0.125—2 μg/ml for cefoperazone, and 0.06—1 μg/ml for ceftazidime. The MIC of ciprofloxacin, norfloxacin, cefoperazone, and ceftazidime were low in all strains examined.

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The stereoselective pharmacokinetics of BOF-4272 enantiomers in rats and dogs was investigated by simultaneously measuring concentrations in arterial, portal, and venous plasma, the liver, and the kidney at 2 h after the oral administration of the racemic drug. The concentrations of BOF-4272 enantiomers were measured using high-performance liquid chromatography. The concentrations of the S(−) enantiomer in arterial, portal, and venous plasma were higher than those of the R(+) enantiomer in rats, but the opposite was found in dogs. In rats, absorption from the intestinal tract into the portal system was almost the same for the two enantiomers, whereas the hepatic uptake of the R(+) enantiomer was greater than that of the S(−) enantiomer. In dogs, absorption from the intestinal tract into the portal system was greater for the R(+) enantiomer than for the S(−) enantiomer, whereas hepatic uptake was comparable for the two enantiomers. The stereoselectivity of the renal uptake of BOF-4272 enantiomers had little effect on the stereoselectivity of enantiomers in the systemic circulation in both rats and dogs. The stereoselectivity in the systemic circulation of BOF-4272 enantiomers is therefore related to hepatic uptake in rats, and to absorption from the intestinal tract into the portal system in dogs.

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To determine the effectiveness of human chorionic gonadotropin (hCG) administered rectally, we studied the pharmacokinetics and pharmacodynamics of hCG using a hollow-type suppository. HCG was not detected in plasma when only hCG was administered rectally, even at a higher dose (4000 IU/kg body weight) than intravenous injection, because of its low bioavailability due to high molecular weight or degradation by proteolytic activity. To enhance the rectal absorption of hCG, the effectiveness of its coadministration with α-cyclodextrin (α-CyD), an absorption-enhancing agent, was investigated in male rabbits. HCG was detected in plasma following coadministration of hCG and α-CyD (10 mg/kg body weight) into the rectum. The plasma hCG concentration increased with increasing dose of α-CyD. The AUC_0→48 observed after coadministration of hCG and α-CyD at 30 mg/kg body weight was approximately four times higher than that of hCG and α-CyD at 10 mg/kg body weight. HCG at a high concentration induced a rapid increase in the plasma testosterone concentration (74.2±3.4 ng/ml) 2 h after intravenous administration. However, the testosterone concentration 24 h after intravenous administration decreased to the physiological level (approximately 20 ng/ml) which had been observed before such administration. On the other hand, the maximum level of testosterone concentration (40.0±12.6 ng/ml) was observed 24 h after rectal administration of hCG (400 IU/kg body weight) in combination with α-CyD (30 mg/kg body weight). Moreover, the plasma testosterone concentration (31.0±11.4 ng/ml) obtained 72 h after rectal administration tended to be maintained at a higher level than that (14.4±0.9 ng/ml) observed before the administration. These results suggest that the hollow-type suppository as a rectal delivery system of hCG is promising as a new mode of hCG therapy.

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The effects of buprenorphine, a powerful mixed agonist/antagonist analgesic, on several cytochrome P450 (CYP) isoform specific reactions in human liver microsomes were investigated to predict drug interaction of buprenorphine in vivo from in vitro data. The following eight CYP-catalytic reactions were used in this study: CYP1A1/2-mediated 7-ethoxyresorufin O-deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated 7-benzyloxyresorufin O-debenzylation, CYP2C8/9-mediated tolbutamide methylhydroxylation, CYP2C19-mediated S-mephenytoin 4-hydroxylation, CYP2D6-mediated bufuralol 1′-hydroxylation, CYP2E1-mediated chlorzoxazone 6-hydroxylation, and CYP3A4-mediated testosterone 6β-hydroxylation. Buprenorphine strongly inhibited the CYP3A4- and CYP2D6-catalyzed reactions with Ki values of 14.7 μM and 21.4 μM, respectively. The analgesic also weakly inhibited specific reactions catalyzed by CYP1A1/2 (Ki=132 μM), CYP2B6 (Ki=133 μM), CYP2C19 (Ki=146 μM), CYP2C8/9 (IC₅₀>300 μM), and CYP2E1 (IC₅₀>300 μM), but not CYP2A6 mediated pathway. In consideration of the Ki values obtained in this study and the therapeutic concentration of buprenorphine in human plasma, buprenorphine would not be predicted to cause clinically significant interactions with other CYP-metabolized drugs.

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The effects of p-phenylbenzoic acid (PPBA) on sulfation and glucuronidation was studied in rats. Following intravenous injection of ¹⁴C-p-tert-butylphenol (¹⁴C-TB, 50 μmol/kg) with PPBA (50 μmol/kg), the sulfation of ¹⁴C-TB was decreased by 38.8% and its glucuronidation increased by 62.3%. In a system of isolated hepatocytes, the sulfation of ¹⁴C-TB increased and its glucuronidation decreased following the addition of 100 μM of PPBA, suggesting competitive inhibition by the glucuronidation of PPBA. On the other hand, the sulfation of ¹⁴C-TB decreased and its glucuronidation increased following the addition of 400 μM of PPBA, which represents the plasma concentration of PPBA in vivo. At this concentration, significant leakage of GOT, GPT and LDH into the medium was observed, whereas, no significant change in the ATP synthesis was noted. Microsomes preincubated with 60 μM of PPBA, which represents the intracellular concentration of PPBA in the liver in vivo, showed no change in the level of glucuronidation of ¹⁴C-TB. These results suggest that PPBA in the plasma injures cell membranes and causes a leakage of cytosolic sulfotransferase, whereas UDPGT, embedded in the microsomal membrane, does not leak from the injured cell membrane, resulting in the decrease in sulfation and increase in glucuronidation. Similar in vivo effects on the sulfation/glucuronidation ratio were observed for benzoic acids substituted with benzoyl and phenoxy groups.

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We investigated the effects of the particle size of indomethacin-loaded poly-L-lactic acid microspheres (IDM-loaded PLA MS), the helium pressure used to accelerate the particles, and the bombardment dose of PLA MS on the plasma concentration of IDM after bombarding with IDM-loaded PLA MS of different particle size ranges, 20—38, 44—53 and 75—100 μm, the abdomen of hairless rats using the Helios^TM gene gun system (Helios^TM gun system). Using larger particles and a higher helium pressure, produced an increase in the plasma IDM concentration and the area under the plasma concentration–time curve (AUC) and resultant F (relative bioavailability with respect to intracutaneous injection) of IDM increased by an amount depending on the particle size and helium pressure. Although a reduction in the bombardment dose led to a decrease in C_max and AUC, F increased on decreasing the bombardment dose. In addition, a more efficient F was obtained after bombarding with IDM-loaded PLA MS of 75—100 μm in diameter at each low dose in different sites of the abdomen compared with that after bolus bombardment with a high dose (dose equivalent). These results suggest that the bombardment injection of drug-loaded microspheres by the Helios^TM gun system is a very useful tool for delivering a variety of drugs in powder form into the skin and systemic circulation.

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The aim of this study is to determine the effects of 1,25(OH)₂D₃ and its analogues on tumor growth and body weight, changes in plasma ionized calcium, parathyroid hormone-related protein (PTHrP) production, bone resorption, and the distribution of the 1,25(OH)₂D₃ receptor (VDR) on tumors in nude mice-bearing the canine adenocarcinoma (CAC-8). Thirty-seven nude mice were implanted subcutaneously with CAC-8. Two weeks after implantation, the mice were divided into 5 groups and injected intraperitoneally 3 times/week for 4 weeks with 5 different substrates. Group I (nontumor-bearing mice) were injected with vehicle. Groups II through V were CAC-8-bearing mice injected with the following: Grp. II, vehicle; Grp. III, analog V; Grp. IV, 1,25(OH)₂D₃; and Grp. V, EB1089. Our results showed that mice body weight (% change) of CAC-8-bearing mice was significantly lower than those of nontumor-bearing mice (p<0.05). CAC-8-bearing mice treated with analog V maintained their body weight better than CAC-8-bearing mice treated with either vehicle, 1,25(OH)₂D₃, or EB1089. A reduction of tumor growth was observed in CAC-8-bearing mice treated with 1,25(OH)₂D₃ and its analogues; however, the reduction was not statistically significant compared to the vehicle-treated CAC-8-bearing mice. All CAC-8-bearing mice increased osteoclastic bone resorption and hypercalcemia. Immunohistochemical staining of CAC-8 with VDR antibody demonstrated a positive reaction in nuclei of tumor cells. In conclusion, CAC-8-bearing mice treated with analog V were more active and maintained their body weight better than other CAC-8-bearing groups. Analog V-treated mice also showed no toxic side effects of hypercalcemia despite an increase in plasma-ionized calcium comparable to nontumor-bearing mice. Tumor volumes of CAC-8-bearing mice treated with 1,25(OH)₂D₃ and its analogues were smaller than vehicle-treated CAC-8-bearing mice. This finding suggested an inhibitory effect on tumor cell growth.

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