Yaichiro Kotake

Regional distribution of parkinsonism-preventing endogenous tetrahydroisoquinoline derivatives and an endogenous parkinsonism-preventing substance-synthesizing enzyme in monkey brain.

1,2,3,4-Tetrahydroisoquinoline (TIQ) and 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1BnTIQ), which exist in the brain of several mammalian species, are parkinsonism-inducing substances, and 1-methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ), which is enzymatically synthesized in rat brain, is a parkinsonism-preventing substance. In this study, we examined the regional distribution of contents of TIQ, 1MeTIQ, and 1BnTIQ, and activity of 1MeTIQ-synthesizing enzyme in monkey brain. The TIQ and 1BnTIQ contents in cerebrum and substantia nigra, and the 1MeTIQ contents in striatum and substantia nigra were higher than those in other brain regions, and 1MeTIQ-synthetic activity was high in cerebrum and thalamus. We speculate that 1MeTIQ-synthesizing enzyme may play an important role in idiopathic Parkinsons disease.

Prediction of in vivo hepatic clearance and half-life of drug candidates in human using chimeric mice with humanized liver

Accurate prediction of pharmacokinetics (PK) parameters in humans from animal data is difficult for various reasons, including species differences. However, chimeric mice with humanized liver (PXB mice; urokinase-type plasminogen activator/severe combined immunodeficiency mice repopulated with approximately 80% human hepatocytes) have been developed. The expression levels and metabolic activities of cytochrome P450 (P450) and non-P450 enzymes in the livers of PXB mice are similar to those in humans. In this study, we examined the predictability for human PK parameters from data obtained in PXB mice. Elimination of selected drugs involves multiple metabolic pathways mediated not only by P450 but also by non-P450 enzymes, such as UDP-glucuronosyltransferase, sulfotransferase, and aldehyde oxidase in liver. Direct comparison between in vitro intrinsic clearance (CLint,in vitro) in PXB mice hepatocytes and in vivo intrinsic clearance (CLint,in vivo) in humans, calculated based on a well stirred model, showed a moderate correlation (r2 = 0.475, p = 0.009). However, when CLint,in vivo values in humans and PXB mice were compared similarly, there was a good correlation (r2 = 0.754, p = 1.174 × 10−4). Elimination half-life (t1/2) after intravenous administration also showed a good correlation (r2 = 0.886, p = 1.506 × 10−4) between humans and PXB mice. The rank order of CL and t1/2 in human could be predicted at least, although it may not be possible to predict absolute values due to rather large prediction errors. Our results indicate that in vitro and in vivo experiments with PXB mice should be useful at least for semiquantitative prediction of the PK characteristics of candidate drugs in humans.

Modulation of connexin 43 in rotenone-induced model of Parkinson's disease.

Gap junctional communication plays an important role in various models of brain pathology, but the changes of gap junctions in Parkinsonism are still not understood. In this study, we show that a major gap junctional protein, connexin43 (Cx43), in astrocytes is enhanced both in a rat Parkinsons disease (PD) model induced with rotenone, a widely used pesticide that inhibits mitochondrial complex I, and in vitro in cultured astrocytes stimulated with rotenone. Enhancement of Cx43 protein levels in rotenone-treated cultured astrocytes occurred in parallel with an increase in gap junctional intercellular communication, but was not accompanied with an increase in Cx43 mRNA levels. Furthermore, the rotenone-induced increase of Cx43 protein levels both in vitro and in vivo was associated with increased levels of phosphorylated Cx43, which is required for gap junctional intercellular communication. In our rat PD model, phosphorylated Cx43 was selectively enhanced in the basal ganglia regions, which contain DA neurons or their terminal areas. The increase of Cx43 levels was lower in the substantia nigra pars compacta and the striatum than in the substantia nigra pars reticulata and the globus pallidus. Our findings indicate that modulation of Cx43 protein, and consequently gap junctional cellular communication, in astrocytes may play an important role in PD pathology.

Neuroprotective effect of 1-methyl-1,2,3,4-tetrahydroisoquinoline on cultured rat mesencephalic neurons in the presence or absence of various neurotoxins

1-Methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) is an endogenous brain amine and its content in parkinsonian brain is decreased compared with that in control brain. There is some evidence that 1MeTIQ protects dopaminergic neurons against dysfunction such as that seen in Parkinsons disease. In this study, we examined the neuroprotective effect of 1MeTIQ against four dopaminergic neurotoxins, 1-methyl-4-phenylpyridinuim ion, 6-hydroxydopamine, rotenone, and l-benzyl-1,2,3,4-tetrahydroisoquinoline, in cultured rat mesencephalic neurons. 1MeTIQ exerted neuroprotective action against all these toxins. Furthermore, (R)-1MeTIQ was neuroprotective, while (S)-1MeTIQ had little effect, indicating that the effect is stereoselective. The protective action of 1MeTIQ was most effective in mesencephalic neurons, especially in tyrosine hydroxylase-positive neurons. 1MeTIQ showed no affinity for dopamine receptors and did not influence the inhibition of mitochondrial respiratory complex I by rotenone, 1-methyl-4-phenylpyridinuim ion, or 1-benzyl-1,2,3,4-tetrahydroisoquinoline. These results raise the possibility that 1MeTIQ indirectly acts as an anti-oxidant such as the induction of anti-oxidative enzymes, because all these four neurotoxins can burden oxidative stress in common. This is the first report to confirm a protective effect of 1MeTIQ at the cultured neuron level, and it may have potential as a lead compound for the development of new agents to treat Parkinsons disease.

Tributyltin-induced endoplasmic reticulum stress and its Ca2 +-mediated mechanism

Organotin compounds, especially tributyltin chloride (TBT), have been widely used in antifouling paints for marine vessels, but exhibit various toxicities in mammals. The endoplasmic reticulum (ER) is a multifunctional organelle that controls post-translational modification and intracellular Ca(2+) signaling. When the capacity of the quality control system of ER is exceeded under stress including ER Ca(2+) homeostasis disruption, ER functions are impaired and unfolded proteins are accumulated in ER lumen, which is called ER stress. Here, we examined whether TBT causes ER stress in human neuroblastoma SH-SY5Y cells. We found that 700nM TBT induced ER stress markers such as CHOP, GRP78, spliced XBP1 mRNA and phosphorylated eIF2α. TBT also decreased the cell viability both concentration- and time-dependently. Dibutyltin and monobutyltin did not induce ER stress markers. We hypothesized that TBT induces ER stress via Ca(2+) depletion, and to test this idea, we examined the effect of TBT on intracellular Ca(2+) concentration using fura-2 AM, a Ca(2+) fluorescent probe. TBT increased intracellular Ca(2+) concentration in a TBT-concentration-dependent manner, and Ca(2+) increase in 700nM TBT was mainly blocked by 50μM dantrolene, a ryanodine receptor antagonist (about 70% inhibition). Dantrolene also partially but significantly inhibited TBT-induced GRP78 expression and cell death. These results suggest that TBT increases intracellular Ca(2+) concentration by releasing Ca(2+) from ER, thereby causing ER stress.

Neuroprotective or neurotoxic activity of 1-methyl-1,2,3,4-tetrahydroisoquinoline and related compounds.

1-Methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) 1 and various 5- or 6,7-substituted analogues were synthesized and assayed for neurotoxicity towards SH-SY5Y cells. Among mono-substituted derivatives of 1, hydroxyl substitution decreased the toxicity, while methoxyl substitution increased it. Disubstituted derivatives of 1, 5a and 5b, showed the opposite tendency. Hydroxy-1MeTIQ derivatives were tested for neuroprotective activity, and 3b and 4b exhibited greater efficacy than 1. We suggest that hydroxy-1MeTIQ derivatives, especially 4b, may have potential for the treatment of Parkinsons disease.

1‐Benzyl‐1,2,3,4‐tetrahydroisoquinoline binds with tubulin β, a substrate of parkin, and reduces its polyubiquitination

J. Neurochem. (2010) 114, 1291–1301.

Predictability of plasma concentration–time curves in humans using single-species allometric scaling of chimeric mice with humanized liver

Abstract 1. We used chimeric mice (PXB mice®), which were repopulated with human hepatocytes, to evaluate their predictabilities of human pharmacokinetics. 2. The relationships of total clearance (CLt) and the volume of distribution at steady state (Vdss) between that predicted from single-species allometric scaling (SSS) of PXB mice and the observed human values indicated good correlations for various drugs metabolized by cytochrome P450s (CYPs) and non-CYPs. 3. We examined the Dedrick plot with which the plasma concentration–time curves can exhibit superimposability using SSS of PXB mice for CLt and Vdss. The predicted plasma concentration–time curves using the complex Dedrick plot from PXB mice were generally superimposed with the observed human data. 4. However, the predicted curve of diazepam was not superimposable with the observed profile. Residual mouse hepatocytes in the livers of PXB mice may affect predictability of CLt of diazepam because significant discrepancy of in vitro intrinsic clearance in PXB mouse liver microsomes consisted of low and high replacement of human hepatocytes were observed. 5. The complex Dedrick plot with SSS from PXB mice is useful for predicting the plasma concentration–time curve in drug discovery, although there are some limitations.

Methoxychlor and fenvalerate induce neuronal death by reducing GluR2 expression

GluR2, an α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit, plays important roles in neuronal survival. We previously showed that exposure of cultured rat cortical neurons to several chemicals decreases GluR2 protein expression, leading to neuronal toxicity. Methoxychlor, the bis-p-methoxy derivative of dichlorodiphenyltrichloroethane, and fenvalerate, a synthetic pyrethroid chemical, have been used commercially as agricultural pesticides in several countries. In this study, we investigated the effects of long-term methoxychlor and fenvalerate exposure on neuronal glutamate receptors. Treatment of cultured rat cortical neurons with 1 or 10 µM methoxychlor and fenvalerate for 9 days selectively decreased GluR2 protein expression; the expression of other AMPA receptor subunits GluR1, GluR3, and GluR4 did not change under the same conditions. Importantly, the decreases in GluR2 protein expression were also observed on the cell surface membrane where AMPA receptors typically function. In addition, both chemicals decreased neuronal viability, which was blocked by pretreatment with 1-naphtylacetylspermine, an antagonist of GluR2-lacking AMPA receptors, and MK-801, an N-methyl-d-aspartate (NMDA) receptor antagonist. These results suggest that long-term exposure to methoxychlor and fenvalerate decreases GluR2 protein expression, leading to neuronal death via overactivation of GluR2-lacking AMPA and NMDA receptors.

Inhibition of dopamine receptors by endogenous amines : Binding to striatal receptors and pharmacological effects on locomotor activity

Endogenous amine 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1BnTIQ) derivatives are synthesized. and their activity for dopaminergic systems are evaluated in vitro and in vivo by receptor binding assay and pharmacological tests. It is proposed that 1BnTIQ derivatives can act as endogenous dopaminergic antagonists.