4-Hydroxy-1-[2-(4-hydroxyphenoxy)ethyl]-4-(4-methylbenzyl)piperidine: a novel, potent, and selective NR1/2B NMDA receptor antagonist.

A structure-based search and screen of our compound library identified N-(2-phenoxyethyl)-4-benzylpiperidine (8) as a novel N-methyl-D-aspartate (NMDA) receptor antagonist that has high selectivity for the NR1/2B subunit combination (IC(50) = 0.63 microM). We report on the optimization of this lead compound in terms of potency, side effect liability, and in vivo activity. Potency was assayed by electrical recordings in Xenopus oocytes expressing cloned rat NMDA receptors. Side effect liability was assessed by measuring affinity for alpha(1)-adrenergic receptors and inhibition of neuronal K(+) channels. Central bioavailability was gauged indirectly by determining anticonvulsant activity in a mouse maximal electroshock (MES) assay. Making progressive modifications to 8, a hydroxyl substituent on the phenyl ring para to the oxyethyl tether (10a) resulted in a approximately 25-fold increase in NR1A/2B potency (IC(50) = 0.025 microM). p-Methyl substitution on the benzyl ring (10b) produced a approximately 3-fold increase in MES activity (ED(50) = 0.7 mg/kg iv). Introduction of a second hydroxyl group into the C-4 position on the piperidine ring (10e) resulted in a substantial decrease in affinity for alpha(1) receptors and reduction in inhibition of K(+) channels with only a modest decrease in NR1A/2B and MES potencies. Among the compounds described, 10e (4-hydroxy-N-[2-(4-hydroxyphenoxy)ethyl]-4-(4-methylbenzyl)piperid ine, Co 101244/PD 174494) had the optimum pharmacological profile and was selected for further biological evaluation.     

Effects of PNU-109,291, a selective 5-HT1D receptor agonist, on electrically induced dural plasma extravasation and capsaicin-evoked c-fos immunoreactivity within trigeminal nucleus caudalis.

We studied the effects of PNU-109291 [(S)-(-)-1-[2-[4-(4-methoxyphenyl)-1-piperazinyl]ethyl]-N-methyl-isoc hroman-6-carboxamide], a receptor agonist showing 5000-fold selectivity for primate 5-HT1D versus 5-HT1B receptors (Ennis et al., J. Med. Chem. 41, 2180-2183), on dural neurogenic inflammation and on c-fos like immunoreactivity within trigeminal nucleus caudalis evoked by electrical and chemical activation of trigeminal afferents, respectively. Subcutaneous injection of PNU-109291 in male guinea pigs dose-dependently reduced dural extravasation of [125I]-labeled bovine serum albumin evoked by trigeminal ganglion stimulation with an IC50 of 4.2 nmol kg(-1). A dose of 73.3 nmol kg(-1) blocked the response completely. The selective 5-HT1B/1D receptor antagonist GR-127935 (> or = 2 micromol kg(-1) i.v.) prevented this effect. In addition, the number of c-fos immunoreactive cells within guinea pig trigeminal nucleus caudalis induced by chemical meningeal stimulation (intracisternally administered capsaicin) was reduced by more than 50% with PNU-109291 (> or = 122.2 nmol kg(-1) administered s.c. 45 min before and 15 min after capsaicin). These data indicate that the 5-HT1D receptor subtype plays a significant role in suppressing meningeal neurogenic inflammation and attenuating trigeminal nociception in these guinea pig models. Since 5-HT1D receptor mRNA and protein are expressed in trigeminal ganglia but not vascular smooth muscle, the 5-HT1D receptor subtype may become a useful therapeutic target for migraine and related headaches.     

Xanthine oxidase inhibitors from Brandisia hancei

Xanthine oxidase is a key enzyme associated with the incidence of hyperuricemia-related disorders. Repeated chromatography of the enzyme inhibitory part of the water extract of the twigs and leaves of Brandisia hancei (Scrophulariaceae) gave a flavone luteolin, an iridoid glycoside mussaenoside, two beta-sitosterol glycosides daucosterol and beta-sitosterol gentiobioside, and five phenylethanoids arenarioside, brandioside, acteoside, 2'-O-acetylacteoside and isoacteoside. Luteolin and isoacteoside inhibited the xanthine oxidase (XO, EC 1.2.3.2) with the IC50 values at 7.83 and 45.48 microM, respectively. Isoacteoside was found to be the first phenylethanoid that decreased substantially the formation of uric acid by inhibiting competitively xanthine oxidase (Ki value: 10.08 microM). Furthermore, the study suggested that the caffeoylation of the 6'-hydroxyl group of the phenylethanoids was essential for the enzyme inhibitory action.     

Analysis of differential effects of Pb2+ on protein kinase C isozymes

Protein kinase C has been implicated as a cellular target for Pb2+ toxicity. We have previously proposed that Pb2+ modulates PKC activity by interacting with multiple sites within the enzyme. In order to further characterize the Pb-PKC interactions we compared the effects of Pb2+ on the CA-dependent and -independent protein kinase C isozymes using recombinant human PKC-alpha, PKC-epsilon, and PKC-zeta as well as the catalytic fragment of bovine brain protein kinase C, the PKC-M. The results demonstrate that, whereas at pM concentrations Pb2+ activates PKC-alpha half maximally (KAct approximately 2 pM), it has no effect on PKC-epsilon, PKC-zeta, or PKC-M activities. The activation of PKC-alpha by Pb2+ is additive with Ca2+ in a manner indicating interaction with half of the calcium activation sites. In the micromolar range of concentrations, Pb2+ inhibits all PKCs with estimated K0.5 of 1.0, 2.3, 28, and 93 microM for PKC-M, PKC-alpha, PKC-epsilon, and PKC-zeta, respectively. Examination of Pb2+ effects on PKC-M kinetics indicates a mixed type inhibition with respect to ATP and noncompetitive inhibition with respect to histone. Taken together with the results of our previous study (Tomsig and Suszkiw, J. Neurochem. 64, 2667-2673, 1995) and the evidence for the existence of two Ca2+ coordination sites Ca1 and Ca2 within the C2 domain (Shao et al., Science [Washington, D.C.] 273, 248-251, 1996), the results of the current study provide further support for a multisite Pb-PKC interaction scheme wherein lead (1) partially activates the enzyme through pM-affinity interactions with the Ca1 site and inhibits the divalent cation-dependent activity through nM-affinity interactions with Ca2 site in the C2 domain and (2) inhibits the constitutive kinase activity through microM-affinity interactions with the catalytic domain. The concentration dependence of the differential effects of Pb2+ on the calcium-dependent and -independent PKCs underscores the importance of the C2 motif as a high affinity molecular target for Pb2+.     

Gene cloning and sequencing of BmK AS and BmK AS-1, two novel neurotoxins from the scorpion Buthus martensi Karsch.

Based on the known amino acid sequences of BmK AS and BmK AS-1, the gene specific primers were designed and synthesized for 3' and 5' RACE (Rapid Amplification of cDNA Ends). Their partial cDNA fragments obtained by 3' and 5' RACE were cloned and sequenced, and the full length cDNA sequences of BmK AS and BmK AS-1 were then completed by overlapping their two partial cDNA sequences, respectively. The predicted amino acid sequences both consist of 85 amino acid residues including a putative signal peptide of 19 residues and a mature toxin of 66 residues. They are different in 17 amino acid residues, among them 11 residues in the mature toxin. The predicted amino acid sequences of BmK AS and BmK AS-1 were almost consistent with those determined and revised (personal communication), only different in one and two residues at their COO-terminal parts, respectively. Based on the determined cDNA sequences, and using the total DNAs isolated from the scorpion venom glands as a template, the genomic DNAs of BmK AS and BmK AS-1 were also amplified by PCR and sequenced. It showed that no intron was inserted in their open reading frames, while in the exon of signal peptide sequences of other Na+, K+ and Cl- channel toxins from the same scorpion, an intron is usually found. However, the Northern blot hybridization results indicated that the sizes of their mRNA should be around 800 bp. Their extra sequences around 400 bp which might function as an intron should be located at their 5' untranslated regions.     

Molecular characterization of a new excitatory insect neurotoxin with an analgesic effect on mice from the scorpion Buthus martensi Karsch.

Besides the neurotoxins active on mammals, a new excitatory insect selective toxin with a mice analgesic activity was found and purified from the venom of the scorpion Buthus martensi Karsch (BmK) (Ji, Y.H., Mansuelle, P., Terakawa, S., Kopeyan, C., Yanaihara, N., Hsu, K., Rochat, H., 1996. Toxicon 34, 987; Luo, M.J., Xiong, Y.M., Wang, M., Wang, D.C., Chi, C.W., 1997. Toxicon 35, 723.). This peptide (designated as BmK IT-AP) is composed of 72 amino acid residues. Its primary structure was determined by automated Edman degradation of the N-terminal part of the reduced and S-carboxamidemethylated protein and its lysylendopeptidase degraded fragments. Based on the determined sequence, the gene specific primers were designed and synthesized for 3' and 5' RACE (rapid amplification of cDNA ends). Their partial cDNA fragments obtained by 3' and 5' RACEwere cloned and sequenced and the full length cDNA sequence of BmK IT-AP was then completed by overlapping their two partial cDNA sequences. It encodes a precursor of 90 amino acid residues: a signal peptide of 18 residues and a mature peptide of 72 residues which are consistent with the determined protein sequence of BmK IT-AP. The genomic DNA of the peptide was also amplified by PCR from the scorpion genomic DNA and sequenced, which is a first report on the genomic structure of a scorpion toxin specific for insects. Its sequence revealed an intron of 590 bp inserted in the end part of the signal peptide. The peptide caused a fast excitatory contraction paralysis on house fly larvae. Furthermore, the peptide also showed an obvious analgesic effect on mice, as assayed by using a twisting test model. This effect of BmK IT-AP well characterized at molecular level is first reported among the known scorpion insect neurotoxins.     

Possible involvement of P-glycoprotein in biliary excretion of CPT-11 in rats.

In our previous work, we found that the biliary excretion of the carboxylate form of irinotecan, CPT-11, on rat bile canalicular membrane consists of two components, the low-affinity one being canalicular multispecific organic anion transporter (cMOAT). In the present study, we have investigated the high-affinity component by studying the uptake in canalicular membrane vesicles. The ATP-dependent uptake of the carboxylate form of CPT-11 was inhibited significantly by several substrates and/or modulators of P-glycoprotein, including PSC-833, verapamil, and cyclosporin A, at a substrate concentration of 5 microM, at which the high-affinity component is involved predominantly in CPT-11 transport. When the concentration of the carboxylate form of CPT-11 was 250 microM, at which the low-affinity component (cMOAT) is involved predominantly in its transport, the inhibitory effect of the above compounds was reduced greatly. Similarly, there was also much lower inhibition of the ATP-dependent uptake of S-(2,4-dinitrophenyl)-glutathione, a substrate of cMOAT, by the above compounds. Taurocholic acid, a substrate of canalicular bile acid transporter, failed to inhibit the uptake of CPT-11 at the substrate concentration of both 5 and 250 microM. These results suggest that P-glycoprotein may act as the high-affinity component in the biliary excretion of the carboxylate form of CPT-11 in rats.     

Correlation of biliary excretion in sandwich-cultured rat hepatocytes and in vivo in rats.

The relationship between biliary excretion in sandwich-cultured rat hepatocytes and in vivo in rats was examined. The biliary excretion of seven model substrates in 96-h sandwich-cultured rat hepatocytes was determined by differential cumulative uptake of substrate in the monolayers preincubated in standard buffer (intact bile canaliculi) and Ca2+-free buffer (disrupted bile canaliculi). Biliary excretion in vivo was quantitated in bile duct-cannulated rats. The biliary excretion index of model substrates, equivalent to the percentage of retained substrate in the canalicular networks, was consistent with the percentage of the dose excreted in bile from in vivo experiments. The in vitro biliary clearance of inulin, salicylate, methotrexate, [D-pen2,5]enkephalin, and taurocholate, calculated as the ratio of the amount excreted into the bile canalicular networks and the area under the incubation medium concentration-time profile ( approximately 0, approximately 0, 4.1 +/- 1.0, 12.6 +/- 2.2, and 56. 2 +/- 6.0 ml/min/kg, respectively), correlated with their intrinsic in vivo biliary clearance (0.04, 0, 17.3, 34.4, and 116.9 ml/min/kg, respectively; r2 = 0.99). The model compound 264W94 was not excreted in bile either in vivo or in vitro. The glucuronide conjugate of 2169W94, the O-demethylated metabolite of 264W94, was excreted into bile in vitro when 2169W94, but not 264W94, was incubated with the monolayers; 2169W94 glucuronide undergoes extensive biliary excretion after administration of 264W94 or 2169W94 in vivo. Biliary excretion in long-term sandwich-cultured rat hepatocytes correlates with in vivo biliary excretion. The study of biliary excretion of metabolites in the hepatocyte monolayers requires consideration of the status of metabolic activities.     

Female gender is a risk factor for torsades de pointes in an in vitro animal model.

Recent clinical observations indicate that female gender is associated with a higher risk of developing torsades de pointes (TdP) cardiac arrhythmia. In this study, we used the Langendorff technique in isolated perfused rabbit hearts and the whole-cell patch-clamp technique in ventricular myocytes to examine the gender difference in TdP incidence and gain insight into the underlying mechanisms. Isolated rabbit hearts were perfused by using the Langendorff technique. TdP was induced by abrupt changes of cycle length (deltaCL) in the presence of Tyrode's solution containing 1 mM 4-aminopyridine (4AP) and 50% reduced Mg2+ and K+ (low K/Mg). The effects of 1 mM 4AP on cardiac potassium currents were characterized by using the patch-clamp technique. Results demonstrated that (a) no significant gender difference was observed in the absolute QT interval before or after 4AP perfusion in the presence of low K/Mg; (b) 4AP caused marked QT prolongation in the ECG; (c) a significantly higher TdP incidence (nine of 12) was found in female hearts compared with male hearts (three of 12; p < 0.05); (d) 1 mM 4AP primarily inhibited Ito, although a slight inhibition of IKr also occurred in low-K/Mg Tyrode's solution. (e) No inhibition of IK1 was observed. (f) No gender difference was found in the potassium current block produced by 4AP. Female gender is associated with a higher incidence of TdP in an experimental isolated heart model and mechanisms subsequent to QT prolongation may contribute to such a gender difference.