Kazuyuki Tomisawa

Improvement in cholesterol metabolism in mice given chronic treatment of taurine and fed a high-fat diet.

The effects of chronic treatment of taurine on hypercholesterolemia and atherosclerosis were examined in C57BL/6J mice fed a high-fat diet containing 15% fat and 1.25% cholesterol. Taurine was dissolved in drinking water at 1% (w/v) and was given to mice ad libitum during 6 months-feeding of a high-fat diet. Hypercholesterolemia occurred and lipid accumulation on the aortic valve was evident. Taurine treatment lowered serum LDL + VLDL cholesterol by 44% in mice fed a high-fat diet, while it elevated serum HDL cholesterol by 25%. As a result, the atherogenic index, the ratio of HDL to LDL + VLDL was markedly improved. Cholesterol content in the liver also decreased by 19% with taurine. Similar tendencies were seen in mice fed regular chow, but the changes were not significant. The area of aortic lipid accumulation, which served as an index of atherosclerosis, was reduced by 20% with taurine. In the liver, taurine doubled the activity of cholesterol 7alpha-hydroxylase. These observations, together with prior findings, suggest that the cholesterol-lowering action of taurine may relate to the increased conversion of cholesterol to bile acids via stimulation of cholesterol 7a-hydroxylase activity. Thus, chronic treatment of high-fat mice with taurine improves the abnormal profile of the serum lipoproteins, and thereby retards the progression of atherosclerosis.

Neuropharmacological profile of peripheral benzodiazepine receptor agonists, DAA1097 and DAA1106.

Receptor binding and behavioral profiles of N-(4-chloro-2-phenoxyphenyl)-N-(2-isopropoxybenzyl)acetamide (DAA1097) and N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)acetamide (DAA1106), novel, selective agonists for the peripheral benzodiazepine receptor (PBR) were examined. DAA1097 and DAA1106 inhibited [3H]PK 11195 binding to crude mitochondrial preparations of rat whole brain, with IC50 values of 0.92 and 0.28 nM. Likewise, DAA1097 and DAA1106 inhibited [3H]Ro 5-4864 binding to the same mitochondrial preparation, with IC50 values of 0.64 and 0.21 nM. In contrast, DAA1097 and DAA1106 did not inhibit [3H]-flunitrazepam, the central benzodiazepine receptor (CBR) ligand, binding to membranes of rat whole brain (IC50>10,000nM). Oral administration of DAA1097 and DAA1106 had anxiolytic effects in the mouse light/dark exploration test and in the rat elevated plus- maze test. Oral administration of DAA1106, diazepam and buspirone but not DAA1097 significantly increased sleeping time in hexobarbital-induced anesthesia in mice. The order of potency of potentiation of hexobarbital anesthesia was diazepam> buspirone> DAA1106> DAA1097. Oral administration of DAA1097 and DAA1106 but not diazepam and buspirone did not affect spontaneous locomotor activity in mice. These findings indicate that DAA1097 and DAA1106 are PBR selective ligands with potent anxiolytic-like properties, in laboratory animals.

Binding characteristics of [3H]DAA1106, a novel and selective ligand for peripheral benzodiazepine receptors

Here, we investigated the binding characteristics of [3H]N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)acetamide ([3H]DAA1106), a potent and selective ligand for peripheral benzodiazepine receptors, in mitochondrial fractions of the rat brain. [3H]DAA1106 bound to the mitochondrial fraction of the rat brain in a saturable manner. The dissociation constant (Kd) and maximal number of binding sites (Bmax) obtained from Scatchard plot analysis of the saturation curve of [3H]DAA1106 binding were 0.12 +/- 0.03 nM and 161.03 +/- 5.80 fmol/mg protein, respectively. [3H]DAA1106 binding to mitochondrial preparations of the rat cerebral cortex was inhibited by several peripheral benzodiapine receptor ligands, and DAA1106 was the most potent inhibitor in inhibiting [3H]DAA1106 binding among the peripheral benzodiazepine receptor ligands we tested. The binding of [3H]DAA1106 was not affected by several neurotransmitter-related compounds, including adrenoceptor, gamma-aminobutyric acid (GABA), dopamine, 5-hydroxytryptamine (5-HT), acetylcholine, histamine, glutamate and central benzodiazepine receptor ligands even at a concentration of 10 microM. In the cerebral cortex of rhesus monkeys, DAA1106 and 1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3-isoquinoline carboxamide (PK11195) potently inhibited [3H]DAA1106 binding, while 7-chloro-5-(4-chlorophenyl)-1-methyl-1,3-dihydrobenzo[e][1,4]diazepin -2-one (Ro5-4864) did not. The highest [3H]DAA1106 binding was observed in the olfactory bulb, followed by the cerebellum. In autoradiographic studies, practically the same results were obtained, in that the highest binding of [3H]DAA1106 was in the olfactory bulb. Potent labeling was also noted in ventricular structures such as the choroid plexus. Thus, [3H]DAA1106 is a potent and selective ligand for peripheral benzodiazepine receptors and should prove useful for elucidating the physiological relevance of events mediated through peripheral benzodiazepine receptors.

Inhibition of Gastric H+,K+-ATPase by Flavonoids: A Structure-Activity Study

Gastric H+, K(+)-ATPase plays a pivotal role in the final step of gastric acid secretion. Over 80 flavonoids, including flavones, flavanones, isoflavones and anthocyanidins were examined for their in vitro effect on gastric H+, K(+)-ATPase and some were found to be inhibitors of this enzyme. Kinetic studies showed that the inhibition of H+, K(+)-ATPase by flavonoids was competitive with respect to ATP, and non-competitive with respect to K+. Structure-activity analysis revealed the following: (1) The inhibitory potency of flavonoids depends on the number of hydroxyl groups up to four per molecule and that above this, no marked enhancement is seen; (2) The hydroxylation pattern is an important determinant of inhibitory potency. Two adjacent hydroxyl groups (catechol-type), three adjacent hydroxyl groups (pyrogallol-type) or hydroxyl groups at C-3, C-5 and C-7 are a minimum requirement for high potency inhibition; (3) Protection of the hydroxyl group(s) by glycosylation or methylation decreases potency; (4) Saturation of the C-2-C-3 double bond results in a decrease in potency; and (5) A ketone at C-4 is not essential for inhibition.

The atypical antipsychotic profile of NRA0045, a novel dopamine D4 and 5-hydroxytryptamine2A receptor antagonist, in rats

The atypical antipsychotic profile of (R)‐(+)‐2‐amino‐4‐(4‐fluorophenyl)‐5‐[1‐[4‐(4‐fluorophenyl)‐4‐oxobutyl] pyrrolidin‐3‐yl] thiazole (NRA0045), a potent dopamine D4 and 5‐hydroxytryptamine (5‐HT)2A receptor antagonist, was examined in rats. Spontaneous locomotor activity was decreased dose‐dependently with i.p. administration of clozapine (ED50 3.7 mg kg−1), haloperidol (ED50 0.1 mg kg−1) and chlorpromazine (ED50 0.9 mg kg−1), whereas inhibition of this type of behaviour induced by i.p. administration of NRA0045, at doses up to 10 mg kg−1, did not exceed 50%. Locomotor hyperactivity induced by methamphetamine (MAP, 2 mg kg−1, i.p.) in rats (a model of antipsychotic activity) was dose‐dependently antagonized by NRA0045 (ED50 0.4 mg kg−1, i.p., and 0.3 mg kg−1, p.o., respectively), clozapine (ED50 0.3 mg kg−1, i.p. and 0.8 mg kg−1, p.o., respectively), haloperidol (ED50 0.02 mg kg−1, i.p. and 0.1 mg kg−1, p.o., respectively), chlorpromazine (ED50 0.3 mg kg−1, i.p. and 3.3 mg kg−1, p.o., respectively). In contrast, the MAP (3 mg kg−1, i.v.)‐induced stereotyped behaviour in rats (a model of extrapyramidal symptoms) was not affected by NRA0045 or clozapine, at the highest dose given (30 mg kg−1, i.p.). Haloperidol (ED50 0.3 mg kg−1, i.p.) and chlorpromazine (ED50 4.8 mg kg−1, i.p.) strongly blocked the MAP‐induced stereotyped behaviour. NRA0045 and clozapine selectively blocked behaviour associated with activation of the mesolimbic/mesocortical dopamine neurones rather than nigrostriatal dopamine neurones. Extracellular single‐unit recording studies demonstrated that MAP (1 mg kg−1, i.v.) decreased the firing rate in the substantia nigra (A9) and ventral tegmental area (A10) dopamine neurones in anaesthetized rats. NRA0045 completely reversed the inhibitory effects of MAP on A10 dopamine neurones (ED50 0.1 mg kg−1, i.v.), whereas the inhibitory effects of MAP on A9 dopamine neurones were not affected by NRA0045, in doses up to 1 mg kg−1 (i.v.). Clozapine completely reversed the inhibitory effects of MAP on A10 dopamine neurones (ED50 1.9 mg kg−1, i.v.) and on A9 dopamine neurones (ED50 2.5 mg kg−1, i.v.). Haloperidol completely reversed the inhibitory effects of MAP on A10 (ED50 0.03 mg kg−1, i.v.) and on A9 dopamine neurones (0.02 mg kg−1, i.v.). NRA0045, like clozapine, was more potent in reversing the effects of MAP on A10 than A9 dopamine neurones. Prepulse inhibition (PPI) is impaired markedly in humans with schizophrenia. The disruption of PPI in rats by apomorphine (0.5 mg kg−1, s.c.) was reversed significantly by NRA0045 (3 mg kg−1, i.p.), clozapine (3 mg kg−1, i.p.) and haloperidol (0.3 mg kg−1, i.p.). Phencyclidine (PCP) elicits predominantly psychotic symptoms in normal humans and in schizophrenics. NRA0045 (0.03–0.3 mg kg−1, i.p.) and clozapine (0.1–1 mg kg−1, i.p.) significantly and dose‐dependently shortened the PCP(1.25 mg kg−1, i.p.)‐induced prolonged swimming latency in rats in a water maze task, whereas haloperidol (0.01–0.1 mg kg−1, i.p.) did not significantly alter swimming latency. These findings suggest that NRA0045 may have unique antipsychotic activities without the liability of motor side effects typical of classical antipsychotics.

In vitro pharmacological profile of nonpeptide CRF1 receptor antagonists, CRA1000 and CRA1001

We investigated pharmacological properties of CRA1000 (2-(N-(2-methylthio-4-isopropylphenyl)-N-ethylamino-4-(4-(3-fluoro phenyl)-1,2,3,6-tetrahydropyridin-1-yl)-6-methylpyrimidine) and CRA1001 (2-( N-(2-bromo-4-isopropylphenyl)-N-ethylamino-4-(4-(3-fluorophenyl)-1 ,2,3,6-tetrahydropyridin-1-yl)-6-methylpyrimidine), novel and selective antagonists for the corticotropin-releasing factor1 (CRF1) receptor. Both CRA1000 and CRA1001 inhibited [125I]ovine CRF binding to membranes of COS-7 cells expressing the rat CRF1 receptor with IC50 values of 30 and 38 nM, respectively, without affecting [125I]sauvagine binding to membranes of COS-7 cells expressing the rat CRF2alpha receptor. CRF elicited intracellular cyclic AMP (cAMP) accumulation in AtT-20 cells which express the CRF1 receptor but not the CRF2 receptor, and COS-7 cells expressing CRF1 or CRF2alpha receptors. The CRF-induced cAMP accumulation was inhibited by both CRA1000 and CRA1001, concentration-dependently, in AtT-20 cells and COS-7 cells expressing the CRF1 receptor, while these compounds did not attenuate the CRF response in COS-7 cells expressing the CRF2alpha receptor. CRF increased adrenocorticotropin (ACTH) secretion from AtT-20 cells, and CRA1000 and CRA1001 inhibited CRF-induced ACTH secretion, concentration-dependently, as did other CRF1 receptor antagonists. These results show that both CRA1000 and CRA1001 are potent and selective CRF1 receptor antagonists.

A selective dopamine D4 receptor antagonist, NRA0160: a preclinical neuropharmacological profile.

NRA0160, 5 - [2- ( 4- ( 3 - fluorobenzylidene) piperidin-1-yl) ethyl] - 4 -(4-fluorophenyl) thiazole-2-carboxamide, has a high affinity for human cloned dopamine D4.2, D4.4 and D4.7 receptors, with Ki values of 0.5, 0.9 and 2.7 nM, respectively. NRA0160 is over 20,000fold more potent at the dopamine D4.2 receptor compared with the human cloned dopamine D2L receptor. NRA0160 has negligible affinity for the human cloned dopamine D3 receptor (Ki=39 nM), rat serotonin (5-HT)2A receptors (Ki=180 nM) and rat alpha1 adrenoceptor (Ki=237 nM). NRA0160 and clozapine antagonized locomotor hyperactivity induced by methamphetamine (MAP) in mice. NRA0160 and clozapine antagonized MAP-induced stereotyped behavior in mice, although their effects did not exceed 50% inhibition, even at the highest dose given. NRA0160 and clozapine significantly induced catalepsy in rats, although their effects did not exceed 50% induction even at the highest dose given. NRA0160 and clozapine significantly reversed the disruption of prepulse inhibition (PPI) in rats produced by apomorphine. NRA0160 and clozapine significantly shortened the phencyclidine (PCP)-induced prolonged swimming latency in rats in a water maze task. These findings suggest that NRA0160 may have unique antipsychotic activities without the liability of motor side effects typical of classical antipsychotics.

Design, synthesis and structure-affinity relationships of 4-methylidenepiperidine and 4-aryl-1,2,3,6-tetrahydropyridine derivatives as corticotropin-releasing factor1 receptor antagonists.

Recently, various non-peptide corticotropin-releasing factor1 (CRF1) receptor antagonists have been reported. Structure-affinity relationships (SARs) of non-peptide CRF antagonists suggest that such antagonists can be constructed of three units: a hydrophobic unit (Up-Area), a proton accepting unit (Central-Area), and an aromatic unit (Down-Area). Our interest focused on the Up-Area in deriving the novel methylidenepiperidine derivatives 8-10 and 4-aryl-1,2,3,6-tetrahydropyridine derivatives 11-13 as non-peptide CRF1 receptor antagonists. Compounds 8a and 11a had moderate affinity for CRF1 receptor, but compounds 9, 10, 12 and 13 did not exhibit CRF1 receptor affinity. Modification of derivatives 11 afforded compounds 11i (CRA1001) and 11x (CRA1000), which had high affinity and selectivity for CRF1 receptors with potent anxiolytic-like and antidepressant-like properties in some experimental animal models. These findings suggest that the hydrophonic unit (Up-Area) may be useful for design of CRF1 antagonists. We report here the design, synthesis and SARs of the derivatives 8 and 11 and isosteres 9, 10, 12 and 13.

ACAT inhibitor HL-004 accelerates the regression of hypercholesterolemia in stroke-prone spontaneously hypertensive rats (SHRSP): stimulation of bile acid production by HL-004

The effect of the acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor HL-004 on bile acid production was studied during the regression phase of pre-established hypercholesterolemia in stroke-prone spontaneously hypertensive rats (SHRSP). These rats were fed a hypercholesterolemic diet containing 5% cholesterol, 2% cholic acid, and 20% suet for 30 days to induce hypercholesterolemia. The regression phase was started by switching the diet to normal chow, followed by another 30 days of the diet. The decrease in serum cholesterol level was accelerated by treatment with 0.09% HL-004. At the end of regression, hepatic ACAT activity was significantly lower in the HL-004 treated animals, an event concomitant with the significant decrease in cholesteryl ester content in the liver. In contrast hepatic cholesterol 7 alpha-hydroxylase activity was maintained at a higher level in the HL-004 treated animals. HL-004 increased the secretion of bile acid and biliary lipids in bile duct-cannulated SHRSP. In HepG2:cells, HL-004 at 1-30 microM dose-dependently stimulated bile acid synthesis from [3H]cholesterol. When cholesterol 7 alpha-hydroxylase activity of the liver was compared ex vivo in the presence and in the absence of exogenous cholesterol, it was suggested that the higher 7 alpha-hydroxylase activity of the HL-004 group could be attributed not only to expansion of the endogenous cholesterol pool, which may be the result of hepatic ACAT inhibition by HL-004 but to the direct effect of HL-004 on bile acid production. Thus, HL-004 accelerates the regression of hypercholesterolemia, an event which may be related to the stimulation of bile acid production in the liver.

The hypolipidemic action of the ACAT inhibitor HL-004 in hamsters fed normal chow.

1. A novel ACAT (acyl-CoA: cholesterol acyltransferase) inhibitor, HL-004, exhibited a strong inhibitory effect on the hepatic and intestinal ACAT, but was less effective on the adrenal ACAT in vitro. 2. HL-004 selectively decreased serum VLDL cholesterol, and inhibited hepatic ACAT activity in hamsters fed normal chow. 3. These results suggest that the cholesterol-lowering effect of HL-004 can be attributed to a decrease in hepatic VLDL secretion via inhibition of ACAT.