Toshio Awaya

N-methylation underlying Parkinson's disease.

The discovery of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) leads to the hypothesis that Parkinsons disease (PD) is maybe initiated or precipitated by environmental or endogenous toxins by the mechanism similar to that of MPTP in genetically-predisposed individuals. Endogenous analogs of MPTP, such as beta-carbolines (betaCs) and tetrahydroisoquinolines, have been proposed as possible causative candidates causing PD and are bioactivated into potential neurotoxins by N-methylation enzyme(s). These N-methylated betaCs and tetrahydroisoquinoline have been higher cerebrospinal levels in parkinsonian patients than age-matched controls. Thus, there is a hypotheses to influence the pathogenesis of PD, that is, the excess enzyme activity to activate neurotoxins, such as N-methyltransferase, might be higher in PDs. Indeed, simple betaCs, via N-methylation steps, induced bradykinesia with the decreased dopamine contents in the striatum and midbrain in C57/BL mice. In younger (65 years old) PD patients, the excretion amount of N(1)-methyl-nicotinamaide was significantly higher than that in younger controls. The protein amount of nicotinamide N-methyltransferase (NNMT) was also significantly higher in younger PD patients than that in younger controls. These findings described here would indicate that the excess N-methylation ability for azaheterocyclic amines, such as betaCs, before the onset had been implicated in PD pathogenesis. On the other hand, the contribution of aberrant cytochrome P450 or aldehyde oxidase activity acting on the pyridine ring, that could act as detoxification routes of endogenous neurotoxins, would be small in the etiology of PD.

l-Deprenyl prevents the cell hypoxia induced by dopaminergic neurotoxins, MPP+ and β-carbolinium: a microdialysis study in rats

N-Methyl-4-phenylpyridinium (MPP(+)) and 2,9-di-methyl-norharmanium (2,9-Me2NH(+)), which is a beta-carbolinium proposed as an endogenous MPP(+)-like toxin underlying Parkinsons disease, are strong mitochondrial toxins. We have measured the extracellular lactate levels as a marker for the in vivo cell hypoxia in the striatum of freely moving rats. The perfusions with MPP(+) and 2,9-Me2NH(+) increased extracellular lactate levels in a dose-dependent manner. These increases in lactate levels were significantly prevented by the co-perfusion with 10 microM L-deprenyl, a selective monoamine oxidase (MAO)-B inhibitor, but not by pargyline, a non-specific MAO inhibitor. The increase in extracellular lactate levels was considered to be the reflection of the cell damage resulted from the impairment of mitochondrial function. The present results suggested that L-deprenyl would rescue nerve cells from these toxins through the direct influence on the mitochondrial electron transport.

Tandospirone, a 5-HT1A agonist, ameliorates movement disorder via non-dopaminergic systems in rats with unilateral 6-hydroxydopamine-generated lesions.

Serotonin 1A (5-HT1A) receptors are distributed throughout the brain with their highest concentrations in the frontal cortex, subthalamic nucleus and entopeduncular nucleus as well as the dorsal and median raphe nucleus. There is growing evidence that 5-HT1A receptor agonists have an antidepressant effect in individuals with major depressive disorders. Recent clinical studies suggest that tandospirone, a highly potent and selective 5-HT1A receptor agonist used clinically as an antidepressant in Japan and China, may act as an antiparkinsonian drug. In the present study, we investigated the effect of tandospirone on contralateral rotational behavior in a unilateral hemiparkinsonian rat model produced with 6-hydroxydopamine (6-OHDA). Tandospirone, as well as 8-hydroxy-2-(di-n-propylamino) tetralin (8-OHDPAT), significantly increased contralateral turnings in a dose-dependent manner (0.5-10 mg/kg). Tandospirone also remarkably potentiated the contralateral turning induced by 0.025 mg/kg of apomorphine. Pretreatment with WAY-100635, a 5-HT1A receptor antagonist, almost completely blocked the contralateral turning behavior evoked by tandospirone and 8-OHDPAT, but not that by apomorphine. SCH-23390, a selective dopamine D1 receptor antagonist, did not affect on the tandospirone-induced rotational behavior. These results suggested that tandospirone could act on postsynaptic 5-HT1A receptors and modulate excitatory amino acid pathways in the basal ganglia. Thus, tandospirone could have therapeutic potential for the treatment of Parkinsons disease by modulating neuronal activities of non-dopaminergic pathways.

Sodium tauroursodeoxycholate prevents paraquat-induced cell death by suppressing endoplasmic reticulum stress responses in human lung epithelial A549 cells.

Paraquat is a commonly used herbicide; however, it is highly toxic to humans and animals. Exposure to paraquat causes severe lung damage, leading to pulmonary fibrosis. However, it has not been well clarified as how paraquat causes cellular damage, and there is no established standard therapy for paraquat poisoning. Meanwhile, endoplasmic reticulum stress (ERS) is reported to be one of the causative factors in many diseases, although mammalian cells have a defense mechanism against ERS-induced apoptosis (unfolded protein response). Here, we demonstrated that paraquat changed the expression levels of unfolded protein response-related molecules, resulting in ERS-related cell death in human lung epithelial A549 cells. Moreover, treatment with sodium tauroursodeoxycholate (TUDCA), a chemical chaperone, crucially rescued cells from death caused by exposure to paraquat. These results indicate that paraquat toxicity may be associated with ERS-related molecules/events. Through chemical chaperone activity, treatment with TUDCA reduced paraquat-induced ERS and mildly suppressed cell death. Our findings also suggest that TUDCA treatment represses the onset of pulmonary fibrosis caused by paraquat, and therefore chemical chaperones may have novel therapeutic potential for the treatment of paraquat poisoning.

Meloxicam ameliorates motor dysfunction and dopaminergic neurodegeneration by maintaining Akt-signaling in a mouse Parkinson's disease model.

A series of oxicam non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to be neuroprotective against 1-methyl-4-phenyl pyridinium in human neuroblastoma SH-SY5Y cells via the phosphatidylinositol 3-kinase (PI3K)/Akt pathway independent of cyclooxygenase (COX) inhibition. The present study endeavored to establish this novel effect of meloxicam (MLX), an oxicam NSAID, in a mouse Parkinsons disease (PD) model using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Male C57BL/6 mice, which received MPTP (30 mg/kg/day; s.c.) for 5 consecutive days (chronic model) with 10-day follow-up saline administrations, showed significant motor dysfunction in the pole test due to reduced tyrosine hydroxylase (TH) protein levels in the brain on day 16 after MPTP/saline treatment. Daily coadministrations of MLX (10mg/kg/day; i.p.) and MPTP for the first 5 days and follow-up 10 days with MLX administrations alone (MPTP/MLX treatment) significantly ameliorated MPTP-induced behavioral abnormalities in mice. Concomitant decreases of TH protein levels in the striatum and midbrain of MPTP/MLX-treated mice were not only significantly (p<0.01 and p<0.05, respectively) ameliorated but phosphorylated Akt (pAkt473) expression in the midbrain was also significantly (p<0.01) increased in the midbrain when compared with MPTP/saline-treated mice. These results suggest that MLX, an oxicam NSAID, attenuated dopaminergic neuronal death in the experimental MPTP-PD model by maintenance of the Akt-signaling. Oxicam NSAIDs may serve as potential drugs for PD treatment via a novel mechanism of action.

Cefoselis, a β-lactam antibiotic, easily penetrates the blood-brain barrier and causes seizure independently by glutamate release

Summary.Cefoselis is a widely used β-lactam antibiotic, but occasionally induces seizures and convulsion in elder and renal failure patients. However, β-lactams are known not to pass through the blood-brain barrier (BBB). In this study, we examined the BBB penetration of cefoselis in normal and renal failure rats by means of brain microdialysis. Cefoselis was dose-dependently appeared in brain extracellular fluid in proportion to its blood level. The elimination constant from brain extracellular fluid (apparent) was slightly lower than that from blood. These results indicated that cefoselis might penetrate the BBB or be discharged by a certain transport system. In contrast to the result of cefoselis, cefazolin, a leading drug of cephalosporins, could not be detected in the brain extracellular fluid after an intravenous injection. In renal dysfunction rats, the elimination half-lives of cefoselis from both blood and brain were extensively prolonged. This would be one of responsible factors inducing seizures seen in patients. However, the additional factor, such as decrease in brain function related to aging, would be involved in seizures in patient received cefoselis, because an extremely high dose was required to induce seizures even in renal failure rats. A local administration of cefoselis into the hippocampus through the microdialysis probe caused a striking elevation of extracellular glutamate, with a minimum increase in γ-aminobutyric acid (GABA). However, a systematic cefoselis administration via the tail vein did not elevate extracellular glutamate and GABA concentrations in the hippocampus of renal failure rats that exhibited marked seizures. These results suggested that not the stimulation of glutamate release, but the blockade of GABA receptors might be responsible for the seizure induced by cefoselis.

The tissue distribution of lidocaine in acute death due to overdosing

A 78-year-old woman committed double suicide with a bolus injection of 1 g of lidocaine hydrochloride. The tissue distribution of lidocaine in this fatal poisoning was investigated using an improved gas chromatographic method. The blood level was 42 microg/ml and the brain concentration was 70 miccro/g. Thus, the cause of death in this case was clearly diagnosed as lidocaine overdosing. The blood level of 42 microg/ml was higher than that calculated using the standard pharmacokinetic parameters. Lower blood/tissue ratios were also detected compared with the data described in the literature. These results indicated that the cardiac failure should have occurred immediately after the lidocaine injection. The present data show the distribution of lidocaine after acute death due to overdosing.

Recovery of susceptibility to penicillin G in clinical isolates of Streptococcus pneumoniae despite increased accumulation of pbp gene alterations

Two hundred consecutive clinical isolates of Streptococcus pneumoniae isolated in 2005 and 2006 were analysed for susceptibility to various antimicrobials, pbp gene alterations and macrolide resistance gene expression (2007 analysis) and the results were compared with previous data (2003 analysis). The average minimum inhibitory concentration (MIC) of penicillin G in isolates with 1a(m)/2x(m)/2b(m) decreased from 1.135+/-0.503 mg/L in the 2003 analysis to 0.872+/-0.540 mg/L in the 2007 analysis (P=0.0046). The prevalence of isolates with 1a(m)/2x(m)/2b(m) increased from 30.5% to 32.3%, but the difference was not statistically significant (P=0.6979). The prevalence of isolates with a clarithromycin MIC > or = 1.0mg/L increased from 65.9% to 80.0% (P=0.0005). Isolates expressing ermB increased from 46.6% to 62.6% (P=0.0004). We conclude that the decrease in penicillin resistance of S. pneumoniae does not correlate with a decrease in pbp mutations; on the contrary, the prevalence of isolates with pbp mutations increased. A decrease in penicillin resistance in S. pneumoniae with pbp mutations appears to explain the present results regarding the recovery of penicillin susceptibility. Our results suggest that the spread of mutated pbp genes among S. pneumoniae itself is not responsible for acquisition of the penicillin-resistant phenotype. Use of beta-lactams, especially oral cephalosporins, appears to be responsible for the acquisition of penicillin resistance.

Genotyping of cytochrome P450 isoform genes is useful for forensic identification of cadaver

Tailor-made medical treatment based on the polymorphism of genes encoding drug-metabolizing enzymes has been advocated and is being tried on an experimental basis at numerous centers. If DNA polymorphism analysis becomes routine in tailor-made medical treatment, it will be very useful in forensic identification. In this study, we determined the genotype frequencies of five p450 (CYP) isoform genes, CYP1A2, CYP2D6, CYP2E1, CYP2C9 and CYP2C19 in 196 Japanese individuals to evaluate their forensic usefulness. These genes encode the most important enzymes among the CYP superfamily that metabolize clinically used drug. The frequency of each allele agreed well with those reported previously and their genotype frequencies did not deviate from those expected from Hardy-Weinberg equilibrium. CYP2C subfamilies such as CYP2C9 and CYP2C19 on chromosome 10 showed high sequence homology, as high as over 95% in the regions flanking polymorphic sites. Although 3240 genotype combinations of these five CYP isoform genes are theoretically possible, 101 combinations were detected in this study. The genotype frequencies of these five isoform genes excluded their linkage. The following two genotype combinations showed the highest frequency of 0.036: CYP1A2*1A/*1A, CYP2D6*1/*10, CYP2E1*1/*1, CYP2C9*1/*1 and CYP2C19*1/*1 and CYP1A2*1A/*1C, CYP2D6*1/*10, CYP2E1*1/*1, CYP2C9*1/*1 and CYP2C19*1/*1. Thus, genotyping of CYP isoform genes should be useful in forensic identification.

Estimation of the duration after methamphetamine injection using a pharmacokinetic model in suspects who caused fatal traffic accidents

When the population parameters of drug pharmacokinetics in the human body system are known, the time-course of a certain drug in an individual can generally be estimated by pharmacokinetics. In the present two cases where methamphetamine abusers were suspected to have inflicted mortalities in traffic accidents, the time-elapse or duration immediately after methamphetamine injection to the time when the accidents occurred became points of contention. In each case, we estimated the time-course of blood methamphetamine after the self-administration in the suspects using a 2-compartment pharmacokinetic model with known pharmacokinetic parameters from the literatures. If the injected amount can be determined to a certain extent, it is easy to calculate the average time-elapse after injection by referring to reference values. However, there is considerable individual variability in the elimination rate based on genetic polymorphism and a considerably large error range in the estimated time-elapse results. To minimize estimation errors in such cases, we also analyzed genotype of CYP2D6, which influenced methamphetamine metabolism. Estimation based on two time-point blood samples would usefully benefit legal authorities in passing ruling sentences in cases involving similar personalities and circumstances as those involved in the present study.