Dexmedetomidine inhibits muscarinic type 3 receptors expressed in <Emphasis Type="Italic">Xenopus</Emphasis> oocytes and muscarine-induced intracellular Ca<Superscript>2+</Superscript> elevation in cultured rat dorsal root ganglia cells

Dexmedetomidine, an α₂-adrenoceptor agonist, has been approved for clinical use, although the mechanism of dexmedetomidine action has not been fully elucidated. Several studies have shown that G protein-coupled receptors (GPCRs) are recognized as targets for anesthetics and analgesics. Therefore, it is of interest to determine whether dexmedetomidine affects the function of GPCRs other than the α₂-adrenoceptor. We examined the effects of dexmedetomidine on M₁, M₃, 5-HT_2C, substance P, and orexin 1 receptors in Xenopus oocytes expressing individual receptors. In addition, we investigated the effects of dexmedetomidine on muscarinic receptor-mediated changes in [Ca²⁺]_i in the dorsal root ganglia (DRG) of 3-week-old Wister rats. Dexmedetomidine did not affect the 5-HT_2C-, or substance P-induced Cl⁻ currents and had little inhibition on the orexin A-induced current in oocytes expressing the respective receptors. The compound also had little effect on the acetylcholine (ACh, 1 μM)-induced Ca²⁺-activated Cl⁻ currents in Xenopus oocytes expressing M₁ receptors. In contrast, dexmedetomidine inhibited the ACh-induced currents in Xenopus oocytes expressing M₃ receptors; 1 nM, 10 nM, 100 nM, and 1 μM dexmedetomidine reduced the current to 66.5 ± 4.8, 51.3 ± 12, 34.6 ± 11, and 26.8 ± 6.4% of the control value, respectively (EC₅₀ = 3.5 ± 0.7 nM). Dexmedetomidine reduced the ACh-induced Cl⁻ currents after treatment with the selective protein kinase C inhibitor GF109203X. Moreover, the compound inhibited the muscarinic receptor-mediated increases in [Ca²⁺]_i in cultured DRG cells in a concentration-dependent manner. Dexmedetomidine inhibits the function of M₃ receptors, in addition to its agonistic effects on α₂-adrenoceptors, which provides further insight into the pharmacological properties of dexmedetomidine.     

The effects of the neurosteroids: pregnenolone,progesterone and dehydroepiandrosterone on muscarinic receptor-induced responses in <Emphasis Type="Italic">Xenopus</Emphasis> oocytes expressing M<Subscript>1</Subscript> and M<Subscript>3</Subscript> receptors

The neurosteroids pregnenolone, progesterone, and dehydroepiandrosterone (DHEA) occur naturally in the nervous system. They act on neural tissues, participate in neuronal signaling, and are reported to alter neuronal excitability via nongenomic mechanisms. Muscarinic receptors have important roles in neuronal functions in the brain and autonomic nervous system. In this study, we investigated the effects of pregnenolone, progesterone, and DHEA on M₁ and M₃ muscarinic receptors using the Xenopus oocyte expression system. Pregnenolone and progesterone inhibited the acetylcholine (ACh)-mediated responses of M₁ and M₃ receptors expressed in Xenopus oocytes, whereas DHEA did not. The half-maximal inhibitory concentrations (IC₅₀) for pregnenolone inhibition of M₁ receptor- and M₃ receptor-mediated currents were 11.4 and 6.0 M respectively; the IC₅₀ values for progesterone inhibition of M₁ receptor- and M₃ receptor-mediated currents were 2.5 and 3.0 M respectively. The selective protein kinase C (PKC) inhibitor GF109203X had little effect on the pregnenolone or progesterone inhibition of the ACh-induced currents in Xenopus oocytes expressing M₁ or M₃ receptors. The inhibitory effects of pregnenolone and progesterone were overcome at higher concentrations of ACh. Pregnenolone and progesterone inhibited the [³H]quinuclidinyl benzilate (QNB) binding to M₁ and M₃ receptor expressed in Xenopus oocytes, and Scatchard plot analysis of [³H]QNB binding revealed that pregnenolone and progesterone altered the K_d value and the B_max, indicating noncompetitive inhibition. In conclusion, pregnenolone and progesterone inhibited M₁ and M₃ receptor functions noncompetitively by the mechanism independent of PKC and by interfering with ACh binding to the receptors.     

Cocaine inhibits cromakalim-activated K<Superscript>+</Superscript> currents in follicle-enclosed<Emphasis Type="Italic"> Xenopus</Emphasis> oocytes

The effect of cocaine on K⁺ currents activated by the K_ATP channel opener cromakalim was investigated in follicular cells of Xenopus oocytes. The results indicate that cocaine in the concentration range of 3–500 M reversibly inhibits cromakalim-induced K⁺ currents. The IC₅₀ value for cocaine was 96 M. Inhibition of the cromakalim-activated K⁺ current by cocaine was noncompetitive and voltage independent. Pretreatment with the Ca²⁺ chelator BAPTA did not modify the cocaine-induced inhibition of cromakalim-induced K⁺ currents, suggesting that Ca²⁺-activated second messenger pathways are not involved in the actions of cocaine. Outward K⁺ currents activated by the application of 8-Br-cAMP or forskolin were also inhibited by cocaine. The EC₅₀ and slope values for the activation of K⁺ currents by cromakalim were 184±19 M and 1.14 in the absence of cocaine as compared to 191±23 M and 1.03 in the presence of cocaine (300 M). Cocaine also blocked K⁺ currents mediated through C-terminally deleted form of Kir6.2 (KirC26) in the absence of sulfonylurea receptor with an IC₅₀ value of 87 M, suggesting that cocaine interacts directly with the channel forming Kir6.2 subunit. Radioligand binding studies indicated that cocaine (100 M) did not affect the binding characteristics of the K_ATP ligand, [³H]glibenclamide. These results demonstrate that cromakalim-activated K⁺ currents in follicular cells of Xenopus oocytes are modulated by cocaine.     

<Emphasis Type="Italic">In Vitro</Emphasis> hepatoprotective compounds from <Emphasis Type="Italic">Suaeda glauca</Emphasis>

Bioassay-guided fractionation of the MeOH extract of Suaeda glauca yielded four phenolic compounds, methyl 3,5-di-O-caffeoyl quinate (1) and 3,5-di-O-caffeoyl quinic acid (2), isorhamnetin 3-O-beta-D-galactoside (3), and quercetin 3-O-beta-D-galactoside (4). Compounds 1 and 2 were hepatoprotective against tacrine-induced cytotoxicity in human liver-derived Hep G2 cells with the EC(50) values of 72.7+/-6.2 and 117.2+/-10.5 microM, respectively. Silybin as a positive control showed an EC(50) value of 82.4+/-4.1 microM.     

Two new antibacterial norabietane diterpenoids from cyanobacteria, <Emphasis Type="Italic">Microcoleous </Emphasis><Emphasis Type="Italic">lacustris</Emphasis>

Two abietane diterpenes were isolated from cyanobacteria Microcoleous lacustris, 20-nor-3α-acetoxyabieta-5,7,9,11,13-pentaene and 20-nor-3α-acetoxy-12-hydroxy-abieta-5,7,9,11,13-pentaene. These compounds were assayed against Staphylococcus aureus, Staphylococcus epidermidis, Salmonella typhi, Vibrio cholerae, Bacillus subtilis, Bacillus cereus, Escherichia coli, and Klebsiella pneumoniae. Both compounds showed activity against S. aureus, S. epidermidis, S. typhi, and V. cholerae, but not against the other bacteria.     

Characterisation of (<Emphasis Type="Italic">R</Emphasis>/<Emphasis Type="Italic">S</Emphasis>)-propafenone and its metabolites as substrates and inhibitors of P-glycoprotein

Digoxin is a drug with a narrow therapeutic index, which is a substrate of the ATP-dependent efflux pump P-glycoprotein. Increased or decreased digoxin plasma concentrations occur in humans due to the inhibition or induction of this drug transporter in organs with excretory function such as small intestine, liver and kidney. It is well known that serum concentrations of digoxin increase considerably in humans if propafenone is given simultaneously. However, it has not been investigated in detail whether propafenone and its metabolites are substrates and/or inhibitors of human P-glycoprotein. The aim of this study, therefore, was to investigate the P-glycoprotein-mediated transport and inhibition properties of propafenone and its major metabolites 5-hydroxypropafenone and N-desalkylpropafenone in Caco-2 cell monolayers. Inhibition of P-glycoprotein-mediated transport by propafenone and its metabolites was determined using digoxin as a P-glycoprotein substrate. No polarised transport was observed for propafenone and N-desalkylpropafenone in Caco-2 cell monolayers. However, 5-hydroxypropafenone translocation was significantly greater from basal-to-apical compared with apical-to-basal (P_app basal–apical vs. P_app apical–basal, 10.21±2.63×10^–6 vs. 4.34±1.84×10^–6 cm/s; P<0.01). Moreover, propafenone, 5-hydroxypropafenone and N-desalkylpropafenone inhibited P-glycoprotein-mediated digoxin transport with IC₅₀ values of 6.8, 19.9, and 21.3 M, respectively. In summary, whereas propafenone and N-desalkylpropafenone are not substrates of P-glycoprotein, 5-hydroxypropafenone is translocated by human P-glycoprotein across cell monolayers. In addition, propafenone and its two major metabolites 5-hydroxypropafenone and N-desalkylpropafenone are inhibitors of human P-glycoprotein and therefore contribute to the digoxin–propafenone interaction observed in humans.     

Interethnic differences of <Emphasis Type="Italic">PEPT2</Emphasis> (<Emphasis Type="Italic">SLC15A2</Emphasis>) polymorphism distribution and associations with cephalexin pharmacokinetics in healthy Asian subjects

Objectives  The aims of this study were to characterize the population frequency of PEPT2 (SLC15A2) polymorphic variants in three Asian ethnic populations, namely Chinese, Malay and Asian Indian, and to investigate the associations of ethnicity (Chinese vs. Asian Indian), PEPT2 haplotype and cephalexin pharmacokinetics in healthy Asian subjects. Methods   PEPT2 polymorphisms were screened from a cohort of 96 Chinese, 96 Malay and 96 Asian Indian subjects. Cephalexin (1000 mg, orally) pharmacokinetics was characterized in an additional 15 Chinese and 15 Asian Indian healthy subjects. These 30 subjects were subsequently genotyped for their PEPT2 polymorphisms. Results  In total, ten common single nucleotide polymorphisms (SNPs) were detected in the three populations, forming two PEPT2 haplotypes. There were significant ethnic differences in PEPT2 haplotype distribution: the frequencies of the *1 and *2 alleles were 0.307 and 0.693 in the Chinese population, 0.495 and 0.505 in the Malay population and 0.729 and 0.271 in Asian Indian population, respectively. The C _max of cephalexin was significantly lower in the Chinese (29.80 ± 4.09 μg ml⁻¹) population than in the Asian Indian one (33.29 ± 4.97 μg ml⁻¹; P = 0.045). This difference could be explained by the higher average body weight of the Chinese population. There was no other significant difference in cephalexin pharmacokinetics between either ethnic or PEPT2 genotype groups. Conclusion   PEPT2 polymorphism distributions differ significantly between Chinese, Malay and Asian Indian populations. However, cephalexin pharmacokinetics is not meaningfully different between Chinese and Asian Indians. The association between the PEPT2 haplotype and cephalexin pharmacokinetics could not be confirmed, and future studies under better controlled conditions are needed.     

Antimicrobial phenolic derivatives from<Emphasis Type="Italic">Dendranthema zawadskii</Emphasis> var.<Emphasis Type="Italic">latilobum</Emphasis> kitamura (Asteraceae)

A phytochemical study on the root ofDendranthema zawadskii var.latilobum Kitamura, using a series of silica gel column chromatography and reversed phase C-18 HPLC chromatography, led to the isolation of (1S, 2S)-1, 2, 3-trihydroxy-1-(3, 4-methylenedioxyphenyl)propane (1), 4-methoxycinnamic acid (2), acacetin (3) and caffeic acid methyl ester (4). The structures of these compounds were determined using spectroscopic analyses (UV, IR, HRTOFMS and NMR), with comparison of their spectral data with previously reported values. Compound1 was isolated for the first time, with compounds2 and4 from this plant reported for the first time. The antibacterial and antifungal activities of the isolated compounds were measured using the disc diffusion method. Also, their cytotoxicities against the cancer cell lines, A549, B16F1 and SK-Mel-2, and brine shrimp lethalities were evaluated.     

Prevalence of <Emphasis Type="Italic">UGT1A9</Emphasis> and <Emphasis Type="Italic">UGT2B7</Emphasis> nonsynonymous single nucleotide polymorphisms in West African,Papua New Guinean,and North American populations

Objective UDP-glucuronosyltransferases (UGTs) UGT1A9 and UGT2B7 are involved in the metabolism of antimalarial dihydroartemisinin and antiretroviral zidovudine. Our aim was to analyze the prevalence of UGT1A9 (chromosome 2) and UGT2B7 (chromosome 4) nonsynonymous single nucleotide polymorphisms (SNPs) in West African (WA), Papua New Guinean (PNG), and North American (NA) populations. Methods Using a post-PCR ligation detection reaction-fluorescent microsphere assay, frequencies of UGT1A9 (8G > A, 98T > C, 766G > A) and UGT2B7 (211G > T, 802C > T, 1192G > A) SNPs were determined in WA (n = 133, 5 countries), PNG (n = 153), and NA (n = 350, 4 ethnic groups) individuals. Results The UGT1A9 variant alleles were not common in the study populations. None of the SNPs were present in WA and PNG. Among NA, all 3 SNPs were present (1% each) in Asian-Americans, while 98T > C was present only in Caucasian-Americans (1%) and Hispanic-Americans (1%). Regarding UGT2B7 SNPs, the prevalence of 802C > T was 21% in WA, 28% in PNG, and 28–52% in NA. The SNP 211G > T was present only in Asian-Americans (9%) and Hispanic-Americans (2%), while 1192G > A was not present in any of the subjects. No significant linkage was observed at UGT1A9, UGT2B7, and between both the loci in any of the study populations. Conclusions Taken together, the UGT1A9-UGT2B7 polymorphism profile in WA and PNG populations is similar to African-Americans, but different from Asian-Americans. It is important to determine if these differences, along with previously reported differences in cytochrome P450 2B6 allele frequencies, are associated with altered metabolism/effectiveness of artemisinin drugs. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

<Emphasis Type="Italic">Meso</Emphasis>-dihydroguaiaretic acid isolated from <Emphasis Type="Italic">Saururus chinensis</Emphasis> inhibits cyclooxygenase-2 and 5-lipoxygenase in mouse bone marrow-derived mast cells

Meso-dihydroguaiaretic acid (MDGA) is a medicinal herbal product isolated from the aerial parts of Saururus chinensis that inhibits the cyclooxygenase-2 (COX-2)-dependent phase of prostaglandin D(2) (PGD(2)) generation in bone marrow-derived mast cells (BMMC) (IC(50) 9.8 microM). However, this compound did not inhibit COX-2 protein expression in BMMC at concentrations up to 30 microM, indicating that MDGA directly inhibits COX-2 activity. In addition, this compound consistently inhibited the production of leukotriene C(4) (IC(50) 1.3 microM). These results demonstrate that MDGA inhibits both COX-2 and 5-lipoxygenase. Furthermore, this compound strongly inhibited the degranulation reaction in BMMC (IC(50) 11.4 microM). Therefore, this compound might provide a basis for novel anti-inflammatory drug development.     

Flavonoids from<Emphasis Type="Italic">Spatholobus suberectus</Emphasis>

Two pterocarpans [(6aR,11aR)-maackiain, (6aR,11aR)-medicarpin], one flavanone [(2S)-7-hydroxy-6-methoxy-flavanone], one isoflavan (sativan) and two isoflavones (pseudobaptigenin, genistein) were isolated from the Spatholobus suberectus (Leguminosae). Their chemical structures were determined by comparison of their spectroscopic parameters of CD, EIMS, 1D-NMR and 2D-NMR with those reported in the literatures. All of these compounds are reported for the first time from this plant through the present study.     

Spray-Dried Amorphous Solid Dispersions of Simvastatin,a Low T<Subscript>g</Subscript> Drug: <Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">in Vivo</Emphasis> Evaluations

Purpose To obtain free flowing, stable, amorphous solid dispersions (SDs) of simvastatin (SIM), a drug with relatively lower glass transition temperature (T_g) by spray drying technique, and to perform comparative in vivo study in rats, which could justify the improvement in rate and extent of in vitro drug release.Methods Dichloromethane suspensions of SIM either alone or in combination with PVP (1:1 or 1:2 parts by weight) were spray dried with proposed quantity of Aerosil 200 (1:1, 1:1:1, 1:2:2 parts by weight of SIM, Aerosil 200 and PVP, respectively). SDs were characterized initially in comparison with pure drug and corresponding physical mixtures in same ratios by drug content, saturation solubility, SEM, DSC, XRPD, IR, and in vitro drug release. SD 1:2:2 was further subjected to accelerated stability testing and checked for in vitro drug release and presence of crystallinity using DSC and XRPD. In addition, improvement in rate and extent of in vitro drug release from SD 1:2:2 was justified by in vivo study in rats.Results Combination of SD and surface adsorption techniques has been attempted to overcome the limitations of spray drying technique for amorphization of low T_g drugs. Based on powder characteristics, drug content, saturation solubility, and feasibility of processing into tablets; SD 1:2:2 was selected as the optimized formulation. During initial characterization, SEM, DSC, and XRPD analyses confirmed the presence of amorphous form in SD 1:2:2. IR spectroscopy revealed possibility of hydrogen bonding interaction between SIM and PVP in SDs. Also, there was dramatical improvement in rate and extent of in vitro drug release of SD 1:2:2. Insignificant decrease in dissolution was observed with no evidence of crystallinity during accelerated stability studies of SD 1:2:2. Moreover in vivo study in rats also justified the improvement in therapeutic efficacy of SD 1:2:2 over pure SIM.Conclusions Thus, present study demonstrates high potential of spray drying technique for obtaining stable amorphous SDs of low T_g drugs.     

Cross-linked Small Polyethylenimines: While Still Nontoxic,Deliver DNA Efficiently to Mammalian Cells <Emphasis Type="Italic">in Vitro</Emphasis> and <Emphasis Type="Italic">in Vivo</Emphasis>

No HeadingPurpose. Polyethylenimine (PEI) is among the most efficient nonviral gene delivery vectors. Its efficiency and cytotoxicity depend on molecular weight, with the 25-kDa PEI being most efficient but cytotoxic. Smaller PEIs are noncytotoxic but less efficient. Enhancement in gene delivery efficiency with minimal cytotoxicity by cross-linking of small PEIs via potentially biodegradable linkages was explored herein. The hypothesis was that cross-linking would raise the polycations effective molecular weight and hence the transfection efficiency, while biodegradable linkages would undergo the intracellular breakdown after DNA delivery and hence not lead to cytotoxicity. Toward this goal, we carried out cross-linking of branched 2-kDa PEI and its 1:1 (w/w) mixture with a linear 423-Da PEI via ester- and/or amide-bearing linkages; the in vitro and in vivo gene delivery efficiency, as well as toxicity to mammalian cells, of the resultant cross-linked polycations were investigated.Methods. The efficiency of the cross-linked PEIs in delivering in vitro a plasmid containing -galactosidase gene and their cytotoxicity were investigated in monkey kidney cells (COS-7). Dynamic light scattering was used to compare the relative DNA condensation efficiency of the unmodified and cross-linked PEIs. In vivo gene delivery efficiency was evaluated by intratracheal delivery in mice of the complexes of a luciferase-encoding plasmid and the PEIs and estimating the luciferase expression in the lungs.Results. Cross-linking boosted the gene delivery efficiency of the small PEIs by 40- to 550-fold in vitro; the efficiency of the most potent conjugates even exceeded by an order of magnitude that of the branched 25-kDa PEI. Effective condensation of DNA was evident from the fact that the mean diameter of the complexes of the cross-linked PEIs was some 300 nm with a narrow size distribution, while the complexes of the unmodified small PEIs exhibited a mean size of >700 nm with a very broad size distribution. At concentrations where the 25-kDa PEI resulted in >95% cell death, the conjugates afforded nearly full cell viability. The cross-linked PEIs were 17 to 80 times m ore efficient than the unmodified ones in vivo; furthermore, their efficiencies were up to twice that of the 25-kDa PEI.Conclusions. Cross-linking of small PEIs with judiciously designed amide- and ester-bearing linkers boosts their gene delivery efficiency both in vitro and in vivo without increasing the cytotoxicity. The high efficiency is dependent on the nature of the linkages and the PEIs used.     

Plasma B-esterase and glutathione <Emphasis Type="Italic">S</Emphasis>-transferase activity in the toad <Emphasis Type="Italic">Chaunus schneideri</Emphasis> (Amphibia,Anura) inhabiting rice agroecosystems of Argentina

B-esterase (BChE: butyrylcholinesterase and CbE: carboxylesterase) and glutathione S-transferase (GST) activity were measured in the plasma of Chaunus schneideri collected in rice fields and surrounding environments and in a reference pristine forest. The chemical criterion based on in-vitro reactivation of BChE activity using pyridine-2-aldoxime methochloride (2-PAM) was also determined. Mean values of plasma BchE, CbE, and GST activity for samples from agricultural areas were different from those for samples from pristine forest. Plasma samples from the two agricultural areas showed positive reactivation of BChE activity after incubation with 2-PAM. Based on our experimental evidence we suggest B-esterases and gluthatione S-transferases can be used in field monitoring as biomarkers of exposure of wildlife to pesticides, because the analysis in non-destructive and is sensitive to anti-ChE agrochemicals. Chemical reactivation of BChE is also a complementary method for assessing the effects of pesticides on toads inhabiting rice fields. Further studies are urgently needed to investigate adverse effects of massive exposure to pesticides experienced by native populations of anurans.     

Chemopreventive potential of Epoxy clerodane diterpene from <Emphasis Type="Italic">Tinospora cordifolia</Emphasis> against diethylnitrosamine-induced hepatocellular carcinoma

Summary  Medicinal plants are a promising source for identification of lead molecules for cancer therapy. In our continuous search to discover bioactive compounds from natural products, we isolated (5R, 10R)-4R, 8R-dihydroxy-2S, 3R:15, 16-diepoxycleroda-13(16), 17, 12S:18,1S-dilactone (ECD), a diterpenoid from Tinospora cordifolia and studied its chemopreventive potential in diethylnitrosamine (DEN) induced hepatocellular carcinoma (HCC) rats. Fifty male Wistar rats were divided into five groups. Group I served as normal control. Group II–IV were given DEN (0.01% in drinking water) for twenty weeks. In addition, Group III (preventive treatment) received ECD (10 mg/kg body weight) throughout the study. Group IV (curative treatment) received ECD (10 mg/kg body weight) for the last 8 weeks. Group V received ECD alone (10 mg/kg body weight) throughout the experimental period. At the end of the experimental period all the animals were sacrificed and analyzed for biochemical end points to assess the effect of ECD treatment in DEN induced HCC. The animals treated with DEN showed a decrease in the activities of antioxidant (SOD, CAT) and detoxification enzymes (GSH, GPx) with increase in the activities of the hepatic markers (SGOT, SGPT, LDH). Treatment of ECD in both preventive and curative DEN induced animals increased the level of antioxidants and detoxification enzymes, and decreased serum transaminase level and hepatic marker enzymes to near normal. Histopathological and nodular incidence also confirmed that ECD remarkably reduced tumor incidence and reversed damaged hepatocytes to normal. Our findings confirm that ECD exhibits preventive effect against chemically induced HCC in rats. ECD can be a potent chemopreventive drug for HCC.     

<Emphasis Type="Italic">In vivo</Emphasis> anti-inflammatory effect of a new steroidal saponin,mannioside A,and its derivatives isolated from <Emphasis Type="Italic">Dracaena mannii</Emphasis>

A new steroidal saponin, mannioside A (1), was isolated from the stem bark of Dracaena mannii, together with the known pennogenin (2), pennogenin-3-O-beta-D-glucopyranoside (3) and pennogenin-3-O-alpha-L-rhamnopyranosyl-(1-->2)-[alpha-L-rhamnopyranosyl-(1-->3)]-beta-D-glucopyranoside (4). Their structures were determined using 1D-and 2D-NMR spectroscopy and mass spectrometry. Compounds 1 and 3 significantly inhibited carrageenan-induced paw edema in the rat; compound 4 was moderately active whereas 2 showed very weak activity.     

Relationship between <Emphasis Type="Italic">CYP2C8</Emphasis> genotypes and diclofenac 5-hydroxylation in healthy Spanish volunteers

Purpose  CYP2C8 seems to be involved in diclofenac 5-hydroxylation, while, in vitro, the 4′-hydroxylation and 3′-hydroxylation seem to be mediated mainly by CYP2C9. We have demonstrated the relevance of CYP2C9 genotypes for diclofenac 4′-hydroxylation in healthy volunteers, so that the present study was aimed at analyzing the role of both CYP2C8 and CYP2C9 genotypes on diclofenac metabolism, as well as determining the CYP2C8 allele frequencies and their relationship with CYP2C9 variants. Methods  A group of 142 healthy white Spanish volunteers was studied. Previously, 102 of these subjects had been phenotyped with diclofenac and genotyped for CYP2C9. The CYP2C8 genotypes were determined by allele-specific PCR-RFLP methods. The urinary concentrations of diclofenac and its main metabolites were analysed using an HPLC-UV method after the administration of a single oral dose of diclofenac as described previously for part of the population studied here. Results  The diclofenac/5-hydroxydiclofenac urinary concentration ratio was higher in individuals carrying a CYP2C8*3 or CYP2C8*4 allele than in those homozygous for wild-type allele CYP2C8*1 (P < 0.05). Moreover, approximately 93% of the subjects with a CYP2C8*3 allele also carried a CYP2C9*2, and 80% of the subjects that had CYP2C9*2 variant also carried a CYP2C8*3. In addition, the four CYP2C9*2/*2 individuals were CYP2C8*3/*3. Conclusions  This is the first study showing the influence of CYP2C8 genotypes on diclofenac metabolism in vivo. The linkage disequilibrium between CYP2C8*3 and CYP2C9*2 alleles was confirmed in this Spanish population.     

2,2-dimethyl-2<Emphasis Type="Italic">H</Emphasis>-pyran-derived alkaloids I. Practical synthesis of acronycine and benzo[<Emphasis Type="Italic">b</Emphasis>]acronycine and their biological properties

The 2,2-dimethyl-2H-pyran-derived alkaloids acronycine and its demethylated congeners were prepared in three steps from anthranilic acid and phloroglucinol. The phenylboronic acid-mediated interamolecular cyclization reaction of 1,3-dihydroxyacridone and 3-methylbut-2-enal was employed as a key step, which was also applied to the synthesis of related cytotoxic benzo[b]acronycine. Inhibitory activities of the compounds prepared on topoisomerase I and II as well as their cytotoxicities were evaluated. Cytotoxicity of 2 is closely related to the strong inhibitory activity against topo II at 20 microM level.     

Antinociceptive,antipyretic, and anti-inflammatory activities of <Emphasis Type="Italic">Putranjiva roxburghii</Emphasis> Wall. leaf extract in experimental animals

The effects of the ether extract from the leaves of Putranjiva roxburghii (P. roxburghii) Wall. were assessed on nociceptive responses in mice by using writhing, hot plate, and formalin tests and the antipyretic activity was determined in yeast-induced fever in rats. Anti-inflammatory activities were also investigated using carrageenin-induced paw edema in rats and croton oil-induced ear and anus edemas. The ether extract (100, 200, and 400 mg/kg, p.o.) of P. roxburghii dose-dependently produced analgesic activity in acetic acid-induced writhing in mice. The extract had no significant effect in the hot plate test in mice. At the dose of 400 mg/kg, the extract significantly suppressed the licking activity in the late phase of the formalin test in mice and decreased fever induced by yeast in rats. The extract exhibited moderate inhibitory activity of inflammation in carrageenin-induced paw edema in rats. The extract inhibited croton oil-induced ear edema in a dose-dependent manner (1.25, 2.5, and 5.0 mg/ear) in mice. The extract decreased anus edema induced by croton oil at the high dose of 800 mg/kg in rats. The results indicated that the ether extract of P. roxburghii leaves possesses analgesic, antipyretic, and anti-inflammatory activities.