Metabolism of novel anti-HIV agent 3-cyanomethyl-4-methyl-DCK by human liver microsomes and recombinant CYP enzymes.

AIM: To investigate the metabolism of 3-cyanomethyl-4-methyl-DCK (CMDCK), a novel anti-HIV agent, by human liver microsomes (HLMs) and recombinant cytochrome P450 enzymes (CYPs). METHODS: CMDCK was incubated with HLMs or a panel of recombinant cytochrome P450 enzymes including CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, and 3A5. LC-ion trap mass spectrometry was used to separate and identify CMDCK metabolites. In the experiments with recombinant cytochrome P450 enzymes, specific chemical inhibitors combined with CYP antibodies were used to identify the CYP isoforms involved in CMDCK metabolism. RESULTS: CMDCK was rapidly and extensively metabolized by HLMs. Its intrinsic hepatic clearance estimated from the in vitro data was 19.4 mL·min(-1)·kg(-1), which was comparable to the mean human hepatic blood flow rate (20.7 mL·min(-1)·kg(-1)). The major metabolic pathway of CMDCK was oxidation, and a total of 14 metabolites were detected. CYP3A4 and 3A5 were found to be the principal CYP enzymes responsible for CMDCK metabolism. CONCLUSION: CMDCK was metabolized rapidly and extensively in human hepatic microsomes to form a number of oxidative metabolites. CYP3A4 and 3A5 were the predominant enzymes responsible for the oxidation of CMDCK.

[Research progress of enhancing quantitative sensitivity by using LC-MS(n) with derivatization method in bio-matrices].

Liquid chromatography/mass spectrometry (LC-MS(n)) has been essential to a large number of quantitative analytical applications in drug research, and especially in the drug PK/PD research, due to its high sensitivity and high specificity. But following the appearance of drugs with high activity and low dosage and the especial structural compounds, a number of limitations of LC-MS(n) have been noted. Derivatization changes the structure of drugs and therefore changes their physical and chemical properties, resulting in high ionization efficiency, low matrix effect and low disturbance by inorganic salts and endogenous compounds in LC-MS(n). In this article, recent progress in the research of the chemical derivatization strategy with LC-MS(n) is reviewed on breakthrough of some LC-MS(n) limitations, in particular focusing on the applications involving some drugs in bio-matrices.

Plasma membrane as the target site of cholic acid analogs.

Although the mechanism is unknown, Calculus Bovis and its active components, cholic acid analogs (CAAs), have been used in China to treat a wide range of diseases. Based on the previous finding that the potency of CAA is strongly dependent on the intrinsic surface activity, this paper aimed to investigate the role of the plasma membrane in the pharmacological activity of CAAs. First, CAAs (0.1 mM) caused a surface activity-dependent depression on ATPase activity in the cell membrane extract, but it had no effects on other cellular extracts, suggesting an indispensable role of the membrane environment for pharmacological activity. Second, CAAs lowered the membrane fluidity of cultured Caco-2 cells with the same rank-order of potency sequence. Third, the hypothesis that any functional protein located on the membrane is influenced by changes in cellular membrane fluidity was supported by: ileal contraction that was induced by acetylcholine and mediated by the muscarinic receptor (M-receptor) or the relaxation induced by adrenaline and mediated by the ß-adrenergic receptor (ß-receptor) was inhibited by CAAs. They also had similar rank-order of potency and the effects on the plasma membrane. Collectively, the plasma membrane may be a target for the CAAs to exert the multiple pharmacological effects which are mediated by the alteration of the membrane mobility and the function of integral membrane proteins.

Injury of cell tight junctions and changes of actin level in acute lung injury caused by the perfluoroisobutylene exposure and the role of Myosin light chain kinase.

OBJECTIVES: To investigate the injury of cell tight junctions and change in actin level in the alveolus epithelial cells of the lung after perfluoroisobutylene (PFIB) exposure and the role of myosin light chain kinase (MLCK) in the injury. METHODS: Rats and mice were exposed to a sublethal dose of PFIB. The changes in tight junction zonula occludens-1 (ZO-1), actin and myosin light chain kinase (MLCK) were detected by immunofluorescence at 30 min, 1, 2, 4, 8, 16, 24, 48 and 72 h after PFIB exposure. The role of MLCK was analyzed by lung indices and the actin level. RESULTS: The normal ZO-1 immunofluorescence density and those after PFIB exposure were 71.63, 39.41, 37.59, 35.71, 33.22, 31.34, 31.61, 24.51, 40.03 and 44.71 respectively, The normal actin immunofluorescence density and those after PFIB exposure were 31.82, 36.46, 36.57, 41.60, 40.95, 35.41, 30.69, 19.96, 29.30 and 33.00 respectively, The normal MLCK immunofluorescence density and those after PFIB exposure were 61.21, 50.87, 48.37, 43.65, 41.96, 35.44, 31.77, 30.85, 33.10 and 38.20 respectively. When the MLCK inhibitor ML-7 was given in advance, pulmonary edema and actin degradation were suppressed. CONCLUSIONS: At an earlier stage, the increased permeability of the blood-air barrier after PFIB exposure is probably the result of injury of cell tight junctions that acts in concert with later changes in actin, resulting in an increase in permeability. MLCK could be a potential target for novel drug development for relief of acute lung injury.

Pharmacokinetic properties of albiflorin and paeoniflorin after oral administration of pure compound, Radix Paeoniae alba extract and danggui-shaoyao-san extract to rats.

This study compared the pharmacokinetics of albiflorin (ALB) and paeoniflorin (PAE), respectively, after oral administration of ALB, PAE, Radix Paeoniae alba (RPA) extract, and Danggui-Shaoyao-San (DSS) extract to rats on separate occasions. Analytes were detected simultaneously with liquid chromatography-tandem mass spectrometry. Noncompartmental pharmacokinetic parameters were calculated. After oral administration of RPA and DSS extract to rats, ALB reached maximum concentrations of 4637 ± 2774 ng/ml (0.40 ± 0.14 h) and 226 ± 122 ng/ml (0.35 ± 0.14 h) and PAE reached maximum concentrations of 2132 ± 560 ng/ml (0.40 ± 0.14 h) and 143 ± 65 ng/ml (0.45 ± 0.11 h), respectively. Compared to the AUC(0 - t) value (1122 ± 351 and 722 ± 158 ng h/ml for ALB and PAE, respectively) after administration of monomers, larger AUC(0 - t) value of ALB (4755 ± 2560 ng h/ml) and PAE (2259 ± 910 ng h/ml) after administration of RPA extract and smaller AUC(0 - t) value of ALB (411 ± 118 ng h/ml) and PAE (242 ± 126 ng h/ml) after administration of DSS extract were obtained. The C(max), AUC, and K(el) of ALB and PAE were remarkably increased (P < 0.05, 0.01 or 0.005) during oral administration of RPA extract in comparison to that of DSS extract.

Cell injuries of the blood-air barrier in acute lung injury caused by perfluoroisobutylene exposure.

OBJECTIVES: To investigate the complete process of cell injuries in the blood-air barrier after perfluoroisobutylene (PFIB) exposure. METHODS: Rats were exposed to PFIB (140 mg/m(3)) for 5 min. The pathological changes were evaluated by lung wet-to-dry weight ratio, total protein concentration of bronchoalveolar lavage fluid and HE stain. Ultrastructural changes were observed by transmission electron microscope. Apoptosis was detected by in situ apoptosis detection. Changes of actin in the lung tissue were evaluated by western blot assay. RESULTS: No significant pulmonary edema or increased permeability was observed within the first 4 h, post PFIB exposure. However, inflammatory cell infiltration and alveolar wall thickening were observed from 2 h. Destruction of the alveoli constitution integrity, edema and protein leakage were observed at 8 h. The injuries culminated at 24 h and then recovered gradually. The ultrastructural injuries of alveolar type I epithelial cells, alveolar type II epithelial cells and pulmonary microvascular endothelial cells were observed at 30 min post PFIB exposure. Some injuries were similar to apoptosis. Compared with control, more serious injuries were observed in PFIB-exposed rats after 30 min. At 8 h, some signs of cell necrosis were observed. The injuries culminated at 24 h and then ameliorated. The number of apoptotic cells abnormally increased at 30 min post PFIB exposure, the maximum appeared at 24 h, and then ameliorated gradually. Western blot analysis revealed that the level of actin in the lung showed no significant changes within the first 4 h post PFIB exposure. However, it decreased at 8 h, reached a nadir at 24 h, and then recovered gradually. CONCLUSIONS: The pathological processes were in progress persistently post PFIB exposure. The early injuries probably were the result of the direct attack of PFIB and the advanced injuries probably arose from the inflammatory reaction induced by PFIB.

Metabolic stability of human parathyroid hormone peptide hPTH (1-34) in rat tissue homogenates: kinetics and products of proteolytic degradation.

The present study aim to investigate the metabolic stability and degradation of cleavage sites of human parathyroid hormone peptide, hPTH (1-34), in rat tissue homogenate, and to identify the types of proteases involved in hPTH (1-34) processing degradation. The stability of hPTH (1-34) in rat kidney, lung and liver homogenates was evaluated by LC-ESI-MS, and the structures of the major degradation products were identified by MALDI-TOF MS and LC-ESI-MS/MS. The ability of protease inhibitors to inhibit hPTH (1-34) degradation was used to identify the class of proteases involved in the metabolism of hPTH (1-34). hPTH (1-34) peptide was readily degraded in rat kidney, liver, and lung homogenates, with half-lives of 5.7, 32.2, and 18.9 min, respectively. The degradation of hPTH (1-34) in each tissue can be inhibited by inhibitors of serine and metalloproteases. The major degradation products of hPTH (1-34) are similar in each tissue and suggest that hPTH (1-15) and hPTH (16-34) appear as the major degradation products. The degradation patterns of hPTH (1-34) incubated in rat kidney, liver and lung homogenates are largely overlapping, and a majority of the fragments are generated via cleavages at sites of Leu15-Asn16 peptide bond.

[Determination of decapeptide LXT-101 in plasma by HPLC-MS/MS and its pharmacokinetics in Beagle dogs].

This paper developed a sensitive and specific liquid chromatography-electrospray ionization mass spectrometry (HPLC-MS/MS) method for the determination of decapeptide LXT-101 in Beagle dog plasma. Plasma samples spiked with internal standard (IS) were treated with acetonitrile to precipitate the protein. Selected reaction monitoring (SRM) using the precursor --> product ion combinations of m/z 472.1-->587.9 and m/z 502.8-->633.8 were used to quantify LXT-101 and IS, respectively. The linear calibration curves were obtained in the concentration range of 0.5 - 500.0 ng x mL(-1). The limit of quantification (LOQ) was 0.5 ng x mL(-1). The inter-day and intra-day precision (RSD) across three validation run over the entire concentration range was below 10.9%, and the accuracy (RE) was within +/- 1.8%. The main pharmacokinetic parameters of LXT-101 after muscle injection of 20 microg x kg(-1) were as follows, AUC(0-t): (176.8 +/- 116.7) microg x h x L(-1), MRT(0-t): (2.52 +/- 0.53) h, T(1/2): (1.4 +/- 0.3) h; CL: (0.16 +/- 0.09) L x h(-1) x kg(-1), and Vd: (0.30 +/- 0.16) L x kg(-1), respectively. The method is proved to be specific, sensitive and suitable for the investigation of LXT-101 pharmacokinetics in Beagle dog.

Development and preclinical studies of broad-spectrum anti-HIV agent (3'R,4'R)-3-cyanomethyl-4-methyl-3',4'-di-O-(S)-camphanoyl-(+)-cis-khellactone (3-cyanomethyl-4-methyl-DCK).

In prior investigation, we discovered that (3'R,4'R)-3-cyanomethyl-4-methyl-3',4'-di-O-(S)-camphanoyl-(+)-cis-khellactone (4, 3-cyanomethyl-4-methyl-DCK) showed promising anti-HIV activity. In these current studies, we developed and optimized successfully a practical 10-step synthesis for scale-up preparation to increase the overall yield of 4 from 7.8% to 32%. Furthermore, compound 4 exhibited broad-spectrum anti-HIV activity against wild-type and drug-resistant viral infection of CD4+ T cell lines as well as peripheral blood mononuclear cells by both laboratory-adapted and primary HIV-1 isolates with distinct subtypes and tropisms. Compound 4 was further subjected to in vitro and in vivo pharmacokinetic studies. These studies indicated that 4 has moderate cell permeability, moderate oral bioavailability, and low systemic clearance. These results suggest that 4 should be developed as a promising anti-HIV agent for development as a clinical trial candidate.

Determination of 4-dimethylaminophenol concentrations in dog blood using LC-ESI/MS/MS combined with precolumn derivatization.

A sensitive and reproducible LC-ESI/MS/MS method, which was combined with the precolumn dansyl chloride derivatization to enhance the signal intensity of analytes, was developed to determine blood 4-dimethylaminophenol (DMAP) concentrations. The linearity of the method was observed within the concentration range of 2-2000 ng/mL. The precision, accuracy, stability, recovery and matrix effect of the method were also investigated and found to meet the requirements for pharmacokinetic studies of the drug. By using this method, pharmacokinetic studies were conducted in dogs after i.m. and i.v. administrations. The results showed that DMAP could not only be absorbed into blood quickly after i.m., but also can be eliminated rapidly. Both i.m. and i.v. routes are appropriate for DMAP to be used in field first-aid. It has been proved that this LC-MS/MS combined with precolumn derivatization method can be used as a routine analytical method to provide enhanced measurements for blood DMAP concentrations. It is also useful for DMAP pharmacokinetic evaluation.

Degradation of salmon calcitonin in rat kidney and liver homogenates.

Salmon calcitonin (sCT), a 32-amino-acid peptide, is the active component in many pharmaceuticals used for the management of bone diseases. In this study, the stability of sCT in rat kidney and liver homogenates were evaluated by LC-ESI-MS, and the structures of the major degradation products were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The results show that the half life of sCT was 13.18 min in rat kidney homogenate (2.5 mg/ml, protein concentration) and 43.07 min in rat liver homogenate (2.5 mg/ml, protein concentration). MALDI-TOF MS results indicated that sCT was initially cleaved at Leu9-Gly10 and Gly10-Lys11 bonds in rat kidney homogenate in vitro, at the same time, the major degradation fragment, Lys11-Pro32-NH2 Was metabolized at the C-terminal amide by deamidation, whereas in rat liver homogenate, the initial cleavage sites were at Val8-Leu9 and His17-Lys18. The results indicated that the metabolism of sCT proceeds by initial endoproteolytic cleavage and subsequent exoproteolytic digestion.

Simultaneous determination of harpagoside and cinnamic acid in rat plasma by liquid chromatography electrospray ionization mass spectrometry and its application to pharmacokinetic studies.

A simple and sensitive method was developed for the simultaneous quantification of harpagoside and cinnamic acid in rat plasma using high-performance liquid chromatography system coupled to a negative ion electrospray mass spectrometric analysis. The plasma sample preparation was a simple deproteinization by the addition of two volumes of acetonitrile. The analytes were separated on an Intersil C8-3 column (2.1 mm i.d.x250 mm, 5 microm) with acetonitrile-5 mm ammonium formate aqueous solution (60:40, v/v) as mobile phase at a flow-rate of 0.2 mL/min. Detection was performed on a quadrupole mass spectrometer equipped with electrospray ionization (ESI) source operated under selected ion monitoring (SIM) mode. [M+HCOO]- at m/z 539 for harpagoside, [M-H]- at m/z 147 for cinnamic acid and [M-H]- at m/z 137 for salylic acid (internal standard) were selected as detecting ions, respectively. The method was validated over the concentration range 7-250 ng/mL for harpagoside and 5-500 ng/mL for cinnamic acid. The lower limits of quantitation for harpagoside and cinnamic acid were 7 and 5 ng/mL, respectively. The intra- and inter-day precisions (RSD%) were within 9.5% and the assay accuracies (RE%) ranged from -5.3 to 3.0% for both analytes. Their average recoveries were greater than 86%. Both analytes were proved to be stable during all sample storage, preparation and analysis procedures. The method was successfully applied to the pharmacokinetic study of harpagoside and cinnamic acid following oral administration of Radix Scrophulariae extract to rats.

[Effect of Danxiongfang and its components on experimental liver injury models induced by CCl4 in mice].

OBJECTIVE: The study investigates the protective effect on liver of Danxionfang and its components. METHOD: Mice are injected with CCl4 to establish liver injured model. ALT, AST, serum albumin, globulin in serum and SOD, MDA in liver and liver histological changes were measured to confirm the ability of protecting liver of Danxiongfang. RESULT: The results show Danxiongfang can inhibit obviously the abnormal increase of ALT, AST in serum and MDA in liver, enhance SOD activity in liver, total protein, albumin, globulin in serum, and decrease liver pathological changes, which suggests Danxiongfang can protect injured liver induced by CCl4. CONCLUSION: Danxiongfang showed powerful protective effect against liver damage induced by CCl4.

Simultaneous quantification of chlorogenic acid and caffeic acid in rat plasma after an intravenous administration of mailuoning injection using liquid chromatography/mass spectrometry.

A simple, rapid and sensitive method was developed for the simultaneous quantification of chlorogenic acid (CGA) and caffeic acid (CA) in rat plasma using a high-performance liquid chromatography system coupled to a negative ion electrospray mass spectrometric analysis. The plasma sample preparation was a simple deproteinization by the addition of two volumes of acetonitrile followed by centrifugation. The analytes and internal standard ferulic acid were separated on an Intersil C8-3 column (5 mm; 250 x 2.1 mm) with acetonitrile/0.05% triethylamine solution (70:30, v/v) as mobile phase at a flow rate of 0.2 mL/min with an operating temperature of 30 degrees C. Detection was performed on a quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source operated in selected ion monitoring (SIM) mode. Negative ion ESI was used to form deprotonated molecules at m/z 353 for chlorogenic acid, m/z 179 for caffeic acid, and m/z 193 for the internal standard ferulic acid. Linear detection responses were obtained for CGA concentrations ranging from 0.005 to 2.0 microg/mL and for CA concentrations ranging from 0.010 to 2.0 microg/mL and the lower limits of quantitation (LLOQs) for CGA and CA were 0.005 and 0.01 microg/mL, respectively. The intra- and inter-day precisions (RSD%) were within 9.0% for both analytes. Deviation of the assay accuracies was within +/-10.0% for both analytes. Their average recoveries were greater than 88.0%. Both analytes were proved to be stable during all sample storage, preparation and analytic procedures. The method was successfully applied to the pharmacokinetic study of CGA and CA following an intravenous dose of 5 mL/kg mailuoning injection to rats.

A simplified model to predict P-glycoprotein interacting drugs from 3D molecular interaction field.

A new two components partial least squares discriminant analysis (PLS) model for the prediction of P-glycoprotein-associated ATPase activity of drugs by using VolSurf compute theoretical molecular descriptors derived from 3D molecular interaction field was reported in the present study. By using 27 diverse drugs from literature, two models were constructed (R(2)=0.9003, 0.8150; Q(2)=0.7165, 0.7630) in this paper, which were similar to models that utilized MolSurf parametrization (R(2)=0.7760, 0.7180; Q(2)=0.7420, 0.6950) by using 22 drugs reported in the same literature. The results investigated VolSurf software was superior to MolSurf in its simplicity. Properties associated with the volume, polarizability, and hydrogen bond could have important impact on the P-glycoprotein-associated ATPase activity.

Comparative study on pharmacological effects of DM-phencynonate hydrochloride and its optical isomers.

AIM: The 3-azabicyclo(3,3,1)nonanyl-9-alpha-yl-alpha-cyclopentyl-alpha-phenyl-alpha-glycolate (DM-phencynonate hydrochloride, DMCPG) is a demethylated metabolite of 3-methyl-3-azabicyclo(3,3,1)nonanyl-9-alpha-yl-alpha-cyclopentyl-alpha-phenyl-alpha-glycolate (phencynonate hydrochloride, CPG). (+/-)DMCPG had one chiral center and two enantiomers [R(-) and S(+)DMCPG]. Here we carried out a comparative study of the pharmacological profiles of these optical isomers. METHODS: Affinity and relative efficacy were tested using a radioligand-binding assay with muscarinic acetylcholine receptors from the rat cerebral cortex. Pharmacological activity was assessed in three individual experiments: (1) potentiating the effect of a sub-threshold hypnotic dose of sodium pentobarbital; (2) inhibiting oxotremorine-induced salivation; and (3) inhibiting the contractile response to carbachol. RESULTS: In the competitive binding assay, R(-)DMCPG (K(i)=763.75 nmol/L) was 4- and 2-fold more potent than (+/-)DMCPG (K(i)=3186 nmol/L) and S(+)DMCPG (K(i)=1699 nmol/L) in inhibiting the binding of [(3)H]QNB. The R(-) and S (+) configurations showed positive cooperation (n(H)>1) with the muscarinic receptor, whereas (+/-)DMCPG had a negative cooperation (n(H)<1) relationship with the muscarinic receptor in a radio-binding assay. Both the R(-) and S(+) configurations could potentiate the effect of sub-threshold hypnotic dose of sodium pentobarbital in a dose-dependent manner (the ED(50) values were 2.53 and 18.65 mg/kg, respectively), but (+/-)DMCPG did not display significant central depressant effects at doses from 10 to 29.15 mg/kg (P>0.05). (+/-)DMCPG and its optical isomers suppressed the guinea pig ileum contractile response to carbachol. The IC(50) values were 7.78 x 10(-9), 1.88 x 10(-7), and 1.038 x 10(-7) nmol/L, respectively. In the anti-salivation study, (+/-)DMCPG and its enantiomers depressed oxotremorine- induced salivation in a dose-dependent manner, and the order of potency was R(-)DMCPG (ED(50)=0.44 mg/kg) > (+/-)DMCPG (ED(50)=2.88 mg/kg) >S(+)DMCPG (ED(50)=5.05 mg/kg). CONCLUSION: (+/-)DMCPG and its optical isomers have differences in their pharmacological potencies as anticholinergic agents, and the R(-) configuration is more active than the S(+) configuration.

Anticonvulsant effects of phencynonate hydrochloride and other anticholinergic drugs in soman poisoning: neurochemical mechanisms.

Previous studies have paid little attention to the anticonvulsant effect of anticholinergic drugs that act on both muscarinic (M) and nicotinic (N) receptors during soman-induced seizures. Therefore, with the establishment of a soman-induced seizures model in rats, this study evaluated the efficacy in preventing soman-induced convulsions of two antagonists of both the M and N receptors, phencynonate hydrochloride (PCH) and penehyclidine hydrochloride (8018), which were synthesized by our institute, and of other anticholinergic drugs, and investigated the mechanisms of their antiseizures responses. Male rats, previously prepared with electrodes to record electroencephalographic (EEG) activity, were pretreated with the oxime HI-6 (125 mg kg-1, i.p.) 30 min before they were administered soman (180 microg kg-1, s.c.). All animals developed seizures subsequent to this treatment. Different drugs were given at different times (5, 20 and 40 min after seizures onset) and their anticonvulsant effects were monitored and compared using the two variables, i.e. the dose that could totally control the ongoing seizures, as well as the speed of seizures control. The anticonvulsant effects of atropine, scopolamine and 8018 decreased with the progression of the seizures, and they eventually lost their anticonvulsant activity when the seizures had progressed for 40 min. In contrast, PCH showed good anticonvulsant effectiveness at 5 and 20 min, and especially at 40 min after seizures onset. Of the anticholinergic drugs tested, atropine, scopolamine, and 8018 showed no obvious protection against pentylenetetrazol (PTZ)-induced convulsions or N-methyl-D-aspartate (NMDA)-induced lethality in mice. However, PCH antagonized the PTZ-induced convulsions in a dose-dependant manner with an ED50 of 10.8 mg kg-1, i.p. (range of 7.1-15.2 mg kg-1) and partly blocked the lethal effects of NMDA in mice. PCH also dose-dependently inhibited NMDA-induced injury in rat primary hippocampal neuronal cultures, suggesting a possible neuroprotective action in vivo. In conclusion, our study suggests that the mechanisms of PCH action against soman-induced seizures might differ from those of the M receptor antagonists atropine and scopolamine, and that of the antagonist of both the M and N receptors, 8018. The pharmacological profile of PCH might include anticholinergic and anti-NMDA properties. Compared with the currently recommended anticonvulsant drug diazepam, with known NMDA receptor antagonists such as MK-801 and with conventional anticholinergics such as scopolamine and atropine, the potent anticonvulsant effects of PCH during the entire initial 40 min period of soman poisoning, and its fewer adverse effects, all suggest that PCH might serve as a new type of anticonvulsant for the treatment of seizures induced by soman.

[Anticonvulsant effect of phencynonate hydrochloride on maximal electroshock seizure and the metrazol seizure threshold test in mice].

AIM: To test the antiepileptic effect of phencynonate hydrochloride and investigate its antiepileptic mechanism. METHODS: Through establishment of different epilepsy models, antiepileptic effects of phencynonate hydrochloride and other drugs were examined. Besides, the effect of phencynonate hydrochloride and other compounds against NMDA-induced lethality in mice, NMDA-induced injury in rat primary hippocampal neuronal cultures and NMDA-induced current were also observed. RESULTS: Phencynonate hydrochloride produced a significant anticonvulsant effect on different epilepsy models. Furthermore, phencynonate hydrochloride also exerted its obvious protection against the lethal effects of NMDA in mice, antagonized the NMDA-induced injury in rat primary hippocampal neuronal cultures and blocked NMDA-induced current in a dose-dependent manner. CONCLUSION: Phencynonate hydrochloride had a notable anticonvulsant effect on typical epilepsy models, its antiepileptic mechanism might relate to its antagonism against NMDA receptor.

Pharmacological profiles of an anticholinergic agent, phencynonate hydrochloride, and its optical isomers.

AIM: To comparatively study the pharmacological profiles of 3-methyl-3-azabicyclo(3,3,1)nonanyl-9-alpha-yl-alpha-cyclopentyl-alpha-phenyl-alpha-glycolate (phencynonate hydrochloride, CPG), an anticholinergic agent, and its enantiomers [R(-)-and S(+)-CPG]. METHODS: The affinity and relative efficacy were tested using radioligand-binding assay with muscarinic acetylcholine receptors from rat cerebral cortex. The pharmacological activities were assessed in three individual experiments: (1) potentiating the effect of subthreshold hypnotic dose of sodium pentobarbital; (2) inhibiting oxotremorine-induced salivation; and (3) inhibiting the contractile response to carbachol. RESULTS: The order of potency of phencynonate hydrochloride and its optical isomers to inhibit the binding of [3H]quinuclidinyl benzilate ([3H]QNB) was R(-)-CPG (K(i)=46.49+/-1.27 nmol/L)>CPG(K(i)=271.37+/-72.30nmol/L)>S(+)-CPG(K(i)=1263.12+/-131.64 nmol/L). The results showed that R(-)-CPG had the highest affinity to central muscarinic receptors among the three compounds, but did not show any central depressant effects at dose from 10.00 to 29.15 mg/kg. CPG increased the effects of subthreshold hypnotic dose of sodium pentobarbital induced-sleeping [the ED50+/-95% LC value was 21.06+/-3.04 mg/kg]. CPG and R(-)-CPG displayed nearly equipotent effect in depressing oxotremorine-induced salivation [the ED50 +/-95% LC for R(-) and CPG were 1.10+/-0.28 and 1.07+/-0.15 mg/kg, respectively], and the contractile response to carbachol (pA(2) values for R (-) and CPG were 6.84 and 6.80, respectively). S(+)-CPG presented the lowest anticholinergic profiles, but could potentate effects of its enantiomers in some manner. CONCLUSIONS: These data suggested that R(-)-CPG acted as an eutomer in racemate and a competitive antagonist to acetylcholine muscarinic receptors, but S(+)-CPG was less active in comparison to R(-)-CPG and its racemate. The central depressant effects of R(-)-CPG and S(+)-CPG were lower in comparison to its racemate.

Effects of pretreatment with 8018 on the toxicokinetics of soman in rabbits and distribution in mice.

The effects of 8018 [3-(2'-phenyl-2'-cyclopentyl-2'-hydroxyl-ethoxy)quinuclidine] on the elimination of soman in rabbits blood and distribution in mice brain and diaphragm were investigated using the chirasil capillary gas chromatographic analysis method. In all experiments, the concentration of P(+)soman was below the detection limit (<0.1 ng x mL(-1)). 8018 (1 mg x kg(-1), im, 10 min pre-treated) could significantly reduce the concentration of P(-)soman in rabbit blood from 53.6 +/- 13.3 to 26.2 +/- 9.70 ng x mL(-1) blood as compared to soman-treated control animal at 15 s following soman injection (43.2 microg x kg(-1), iv). Toxicokinetic parameters showed 8018 could increase clearance (CL((S))) from 20.8 +/- 1.54 to 38.2 +/- 15.3 mLx kg(-1) x s(-1) and reduce AUC of P(-)soman from 2.08 +/- 0.151 to 1.30 +/- 0.564 mg x s x L(-1). 8018 could reduce the concentration P(-)soman in diaphragm from 74.7, 70.5, 88.7 ng x g(-1) to 54.5 45.6, 50.0 ng x g(-1) at the time of 30, 90, 120 s after intoxication of soman subcutaneously vs. soman control respectively, but it had no influence on the concentration of free P(-)soman in brain. Isotope trace experiments showed that it could significantly increase the distribution amount of bound [3H]soman in mice plasma and small intestine during 0-120 min after mice received [3H]soman (0.544 GBq.119 microg x kg(-1), sc) compared to soman control group.