Synthesis and biological evaluation of 9-alkoxy-6,7-dihydro-5H-benzo[c] [1,2,4]triazolo[4,3-a]azepines as potential anticonvulsant agents.

A novel series of 9-alkoxy-6,7-dihydro-5H-benzo[c][1,2,4]triazolo[4,3-a]azepine derivatives was synthesized and screened for anticonvulsant activity by the maximal electroshock (MES) test and the subcutaneous pentylenetetrazol (scPTZ) test. Neurotoxic effects were also determined by the rotarod neurotoxicity test. The results revealed that all of the compounds exhibited anticonvulsant activity, Compound 5d was found to possess the most potential anticonvulsant activity in the anti-MES potency test; it had a median effective dose (ED50) of 12.3 mg/kg, a median toxicity dose (TD50) of 73.5 mg/kg, and a protective index (PI) of 6.0, which is slightly lower than the PI of the prototype drug carbamazepine (ED50=8.8, PI=8.1). In the scPTZ test, compound 5c was the most active, with an ED50 value of 19.8 mg/kg, a TD50 value of 80.8 mg/kg and a PI value of 4.1, which are much greater than the ED50 and the PI of the prototype drug carbamazepine (ED50>100, PI<0.72), Possible structure-activity relationships are also discussed.

Genotyping of human platelet antigen-15 by single closed-tube Tm-shift method.

INTRODUCTION: Genotyping of human platelet antigens (HPA) is useful for the diagnosis and prevention of platelet alloimmune syndromes. HPA-15 might play an important role in the development of platelet alloimmune syndromes. There are several disadvantages in the conventional methods for HPA-15 genotyping. The aim of this study was to develop a new method for HPA-15 genotyping by using single closed-tube melting temperature (T(m))-shift genotyping. METHODS: Two GC-rich tails of different lengths were attached to 5'-end of HPA-15 allele-specific PCR primers, such that HPA-15 alleles can be discriminated by the T(m)s of the PCR products. One hundred blood samples were genotyped for HPA-15 by the T(m)-shift and conventional polymerase chain reaction with sequence-specific primers (PCR-SSP). RESULTS: The comparison of the PCR-SSP and the T(m)-shift method showed four discordant results in one hundred samples tested. Confirmatory results demonstrated that the PCR-SSP produced several errors, whereas HPA-15 genotyping by T(m)-shift is correct. The retesting results of T(m)-shift method were consistent with those of the initial testing. CONCLUSION: The single closed-tube T(m)-shift method for HPA-15 genotyping is high-throughput, rapid, reliable, reproducible and cost-effective and it is superior to conventional PCR-SSP used in routine genotyping of HPA-15.

Delayed neuropathy and inhibition of soluble neuropathy target esterase following the administration of organophosphorus compounds to hens.

Delayed neuropathy and inhibition of soluble neuropathy target esterase (NTE) and acetylcholinesterase (AChE) activities in different regions of brain and spinal cord of adult hens were studied after the intravenous administration of leptophos (30 mg/kg), tri-o-cresyl phosphate (TOCP 40 mg/kg) or dipterex (200 mg/kg). The level of NTE activity varied according to the regions of the central nervous system (CNS) of the control (normal) hen, being higher in the cerebrum (74.1 micromol of phenyl valerate hydrolyzed/10 minutes/mg protein) and in the cerebellum (68.7), and lower in the spinal cord (44.5 in cervical, 55.6 in thoracic and 50.0 in lumbar cord). Hens given leptophos and TOCP demonstrated delayed neuropathy with obvious inhibition of NTE, but the times of onset and the degrees of peak inhibition of NTE activity were different: 6-24 hours after dosing and 73-82% of normal activity for leptophos, and 24-48 hours and 45-80% for TOCP, respectively. Furthermore, the average inhibition of NTE during 6-48 hours after dosing, (called here 'period average inhibition') was also significantly different between the leptophos group (63-73%) and TOCP group (40-64%). Hens given dipterex did not demonstrate delayed neuropathy, and had the least peak inhibition and period average inhibition of NTE activity among the 3 groups. Ratios of NTE inhibition/AChE inhibition were higher in the leptophos group (0.91-1.24) and TOCP group (1.13-2.45) than in the dipterex group (0.25-0.79). These results indicate that the distribution of NTE in the soluble fraction of membrane proteins is different in different regions of the CNS, and that the degree of peak inhibition of NTE activity and the time of onset of peak inhibition induced by organophosphorus compounds (OPs) also differ for different OPs. Thus, practical and useful NTE measurements should identify the peak inhibition and period inhibition in several nervous tissue regions.

Pharmacokinetics and neurotoxicity of dipterex in hens. A comparative study of administration methods.

We compared the tissue concentration of dipterex and the inhibition of the neuropathy target esterase (NTE) activity among groups of hens (n = 8 each) which were intravenously (i.v.), subcutaneously (s.c.) or orally (p.o.) administered the insecticide dipterex. The tissue concentrations of dipterex in the s.c. group were higher than those in the i.v. and p.o. groups. When dosed subcutaneously, the tissue concentration of dipterex was high in the brain, spinal cord and muscle at 3 hr after dosing and then concentrated in the spinal cord and muscle for the subsequent 3 hr. When dosed intravenously or orally, dipterex was evenly dispersed in various tissues. All hens treated with dipterex showed acute neurotoxic signs within 15 min after dosing. The hens dosed intravenously recovered from this acute poisoning within 3 hr, and the hens dosed orally recovered within 6 hr, while the hens dosed subcutaneously recovered within 24 hr after dosing. One hen in the s.c. group exhibited acute neurological sequelae following the acute poisoning. In addition, the loss of body weight was the largest in the s.c. group (157 +/- 49 g), moderate in the i.v. group (133 +/- 91 g), small in the p.o. group (96 +/- 54 g) and the smallest in the PMSF (phenylmethanesulfonyl fluoride, which was dosed to promote delayed neuropathy) group (80 +/- 49 g). In the untreated hens, the activity of NTE in both the cerebrum and cerebellum was higher than that in the midbrain (p < 0.01). There was no difference in NTE activity between the cerebrum and cerebellum. In both the cerebrum and midbrain, the inhibition of NTE activity in the p.o. group was less than that in the i.v. and s.c. groups, and no difference was found between the i.v. and s.c. groups. In the cerebellum, the inhibition of NTE activity in the s.c. group was larger than that in the i.v. and p.o. groups. These results indicate that the s.c. dosing of dipterex results in a stronger neurotoxicity compared to i.v. and p.o. dosing. However, it was difficult to induce the clinical signs of delayed neuropathy with any administration of dipterex in hens, even when the promotion of delayed neurotoxicity of dipterex was attempted with PMSF or double doses of dipterex itself.

Transfer of leptophos in hen eggs and tissues of embryonic rats.

To estimate the delayed neurotoxic effect of OPs on the next generation, we tried two examinations; one was on the distribution of leptophos in tissues and eggs of hens which are highly susceptible to the delayed neurotoxic effect of OPs but have no placenta, and the other was on the concentration of OPs in tissues of both pregnant and embryonic rats which are not susceptible to the delayed neurotoxic effect but have placenta, after leptophos was administered to the mother in both experiments. First, organophosphorus compound-induced delayed neurotoxicity (OPIDN) was checked in 4 hens and the concentration of leptophos was determined in the other 16 hens after 20 adult laying hens were given 30 mg/kg leptophos (iv), a neurotoxic organophosphate. Three out of 4 hens treated with leptophos showed OPIDN. The concentration of leptophos decreased sharply in the blood, liver, brain and spinal cord from 24 to 48 hr after leptophos administration, but clearance of leptophos was relatively slow in the ovary. Leptophos in laid egg yolk was detected every day for 10 days, and the highest concentration of leptophos in egg yolk was observed on the 6th day after administration to hens. Secondly, in order to investigate the transfer of leptophos to the embryo through the placenta, we divided the thirty-two pregnant rats into 2 groups. The first group received 10 mg/kg leptophos intraperitoneally on the 17th day of pregnancy and the second received 20 mg/kg leptophos on the same day. The time-course of leptophos concentration in the tissues of pregnant and embryonic rats was checked, and the correlation between findings in the pregnant rats and the embryos was determined. The time-course of leptophos concentration in the blood, liver, brain and placenta of the rats was similar to that in hens. Leptophos concentration in the liver and brain of the embryos was equal to approximately 60% of leptophos concentration in each tissue of the pregnant rats, and the concentration of leptophos in the liver and brain of embryonic rats correlated with that in the blood and placenta of pregnant rats (p < 0.01). In both groups treated with 10 and 20 mg/kg leptophos, the concentrations of leptophos in the liver and brain of embryos were lower than that of pregnant rats in the early period after dosing, but the concentrations in embryos were inversely higher than those in pregnant rats in the latter period (48 hr). Compared with the biological half-lives of leptophos in the liver and brain of pregnant rats, these parameters in embryonic rats were 1.58 and 1.87 times, respectively. These results indicate that some of the fat-soluble organophosphorus compounds readily pass through the blood-placenta barrier into the embryos and accumulate there. Therefore, the neurobehavioral development of F1 rats exposed to some organophosphorus compounds through the placenta of pregnant rats should be further examined.

Effect of phenylmethylsulfonyl fluoride administration prior to and following leptophos administration on electrolyte concentration and enzyme activity in hen serum.

We observed acute toxicity, delayed neurotoxicity, disappearance of leptophos from tisuues and biochemical changes in four groups of hens: a group given only 30 mg/kg leptophos (iv) as the 'leptophos group', two groups given a treatment of 30 mg/kg phenylmethylsulfonyl fluoride (PMSF) (sc) 24 hr prior to (as the 'pretreated group') and following (as the 'posttreated group') administration of the same dose of leptophos as the leptophos group, and a group given a vehicle only as the 'control group'. All groups other than the control group showed acute toxicity. The scores for organophosphate-induced delayed neurotoxicity (OPIDN) in the posttreated group reached the maximal level on the 16th day after leptophos administration and those in the leptophos group reached the maximal level on the 25th day. Serum acid phosphatase (AcP) activities in the leptophos group and the posttreated group were significantly lower than that in the control group (p<0.05) on the 6th day after leptophos administration and then recovered to the normal level on the 15th day. In these two groups, serum creatine phosphokinase (CPK) activity was significantly higher (p<0.01) and the concentration of serum Ca(2+) was significantly lower (p<0.05) than in the control group on the 15th day after leptophos administration. Serum leucine aminopeptidase (LAP) activity in the posttreated group was significantly lower than that in the control group (p<0.01). As for the significant changes by time interval between the 6th and the 15th days after leptophos administration, CPK activity was elevated and serum Ca(2+) reduced in both the leptophos group and the posttreated group, and LAP activity was also reduced in the posttreated group. The courses of leptophos disappearance in several tissues of these hens were similar in the 3 groups. These results suggest that the treatment by PMSF prior to or following the administration of leptophos can significantly modify not only clinical signs of OPIDN but also changes of several biochemical indices accompanied by OPIDN. Furthermore, it is possible to expect that these biochemical indices can provide some valuable clues for exploring the modification of OPIDN by PMSF treatment.

Effects of various post-treatment by phenylmethylsulfonyl fluoride on delayed neurotoxicity induced by leptophos.

Delayed neurotoxicity induced by leptophos, an organophosphorus insecticide, was intensified in hens when phenylmethylsulfonyl fluoride (PMSF) at dose of 30, 60, and 120 mg/kg body weight was administered at different time intervals (24 hr, 3 days, and 5 days) for each dose of PMSF after the hens were exposed to 30 mg/kg (i.v.) of leptophos. The scores for organophosphorus-induced delayed neuropathy (OPIDN) in all groups treated with 120 mg/kg PMSF were significantly higher than those in the group treated with leptophos only (P<0.05 or P<0.01) and the initial signs of OPIDN appeared 2 or 3 days earlier in the former groups than in the latter group. Further, the greater the PMSF post-treatment dose, the more severe were the signs of OPIDN. These findings indicate that post-treatment with PMSF promotes leptophos-induced OPIDN and reduces the period to OPIDN onset. We also examined the effects of various time intervals between PMSF administration and exposure to leptophos on the development of OPIDN. The OPIDN scores in the two groups of hen treated with PMSF on days 3 and 5 after leptophos exposure were high, especially the score of the 5 days treated group became significantly higher on the 18th and 19th day after leptophos administration than even that of the 24 hr treated group with PMSF (P<0.05). These findings suggest that variations in both the dose of PMSF and the time intervals of PMSF post-treatment may affect the delayed neurotoxicity induced by leptophos. Moreover, these results also indicate that PMSF should not be used for either the treatment or the prevention of OPIDN.