Evaluation of animal models for intestinal first-pass metabolism of drug candidates to be metabolized by CYP3A enzymes via in vivo and in vitro oxidation of midazolam and triazolam.

1. To search an appropriate evaluation methodology for the intestinal first-pass metabolism of new drug candidates, grapefruit juice (GFJ)- and vehicle (tap water)-pretreated mice or rats were orally administered midazolam (MDZ) or triazolam (TRZ), and blood levels of the parent compounds and their metabolites were measured by liquid chromatography/MS/MS. A significant effect of GFJ to elevate the blood levels was observed only for TRZ in mice. 2. In vitro experiments using mouse, rat and human intestinal and hepatic microsomal fractions demonstrated that GFJ suppressed the intestinal microsomal oxidation of MDZ and especially TRZ. Substrate inhibition by MDZ caused reduction in 1'-hydroxylation but not 4-hydroxylation in both intestinal and hepatic microsomal fractions. The kinetic profiles of MDZ oxidation and the substrate inhibition in mouse intestinal and hepatic microsomal fractions were very similar to those in human microsomes but were different from those in rat microsomes. Furthermore, MDZ caused mechanism-based inactivation of cytochrome P450 3A-dependent TRZ 1'-hydroxylation in mouse, rat and human intestinal microsomes with similar potencies. 3. These results are useful information in the analysis of data obtained in mouse and rat for the evaluation of first-pass effects of drug candidates to be metabolized by CYP3A enzymes.

Reduction of Alzheimer's disease amyloid-ß in plasma by hemodialysis and its relation to cognitive functions.

BACKGROUND/AIMS: Rapid removal of plasma amyloid-ß (Aß) by blood purification may serve as a peripheral Aß sink from the brain for Alzheimer's disease therapy. We investigated the reduction of plasma Aß during hemodialysis and cognitive states. METHODS: Aß concentrations and Mini-Mental State Examinations (MMSE) were investigated in 37 hemodialysis patients (68.9 ± 4.1 years). RESULTS: The dialyzers effectively removed Aß(1-40) and Aß(1-42), 63.9 ± 14.4 and 51.6 ± 17.0% at 4 h dialysis, resulting in the reduction of Aßs in whole-body circulation by 51.1 ± 8.9 and 32.7 ± 12.0%, respectively. Although the plasma Aßs before dialysis (750.8 ± 171.3 pg/ml for Aß(1-40)) were higher than those reported for Alzheimer's disease patients, the cognitive states of hemodialysis patients were relatively normal, especially of longer dialysis vintages. CONCLUSIONS: Dialyzers effectively reduced Aßs in whole-body circulation. Repeated rapid decrease of plasma Aßs might maintain cognitive state.

Separate evaluation of intestinal and hepatic metabolism of three benzodiazepines in rats with cannulated portal and jugular veins: comparison with the profile in non-cannulated mice.

Pharmacokinetic analyses of three kinds of benzodiazepines--midazolam (MDZ), triazolam (TRZ) and alprezolam (APZ)--were performed in rats with cannulated portal and jugular veins. Each drug was administered to the double-cannulated rats, and pharmacokinetic data for the parent drugs and their 1'- and 4-hydroxylated metabolites were compared with those obtained in non-cannulated mice. In bioavailability, the drugs ranked APZ >> TRZ = MDZ in rats, and APZ > TRZ >> MDZ in mice, with the values for MDZ remarkably different between rats and mice (19% in rats versus 2.3% in mice). In contrast, hepatic availability (Fh) was similar (APZ > TRZ > MDZ) in both species. Highly significant relationships were found between the ratio of the area under the plasma concentration-time curve (AUC) for the parent drugs in portal blood (AUC(por)) to that in systemic blood (AUC(sys)) and Fh in rats and mice. The double-cannulated rat is useful for estimating the hepatic availability of drug candidates by determining the AUC values for the parent drugs in portal and systemic blood samples.

Effects of premedication with fentanyl and midazolam on mask induction of anaesthesia of dogs with sevoflurane.

Fourteen beagles were used to determine the effects of fentanyl and midazolam as a premedicant for mask induction of anaesthesia with sevoflurane. The drugs were administered to each dog in a randomised cross-over design with a seven-day washout period between experiments. After a 15-minute equilibration period, a treatment consisting of fentanyl (10 mug/kg bodyweight) and midazolam (0.2 mg/kg) was given either intravenously or intramuscularly. Anaesthesia was then induced by the use of a facemask with sevoflurane in 100 per cent oxygen at a flow rate of 4 l/minute. Vaporiser settings were increased by 0.8 per cent at 15-second intervals until the value corresponding to 4.8 per cent sevoflurane was achieved. The time to the onset and cessation of involuntary movements, loss of the palpebral reflex, negative response to tail-clamp stimulation, and endotracheal intubation and cardiopulmonary variables were measured. Both the treatments with tentanyl and midazolam resulted in a shorter and smoother induction of anaesthesia than treatment with saline, and the cardiopulmonary changes were smaller and milder.

Detection of new anti-neutral glycosphingolipids antibodies and their effects on Trk neurotrophin receptors.

We screened sera from patients with various neurological disorders for the presence of anti-neutral glycosphingolipids antibodies and only found them in sera from relapsing polychondritis with limbic encephalitis patients. Neutral glycosphingolipids are resident in membrane lipid rafts where high affinity nerve growth factor (NGF) receptor, Trk is co-localized. Therefore, we examined whether these antibodies influence the action of NGF in NGF-responsive cells. The results strongly suggest that these antibodies enhance NGF-induced Trk autophosphorylation and neurite outgrowth as well as neurofilament M expression. These data strongly indicate that these anti-neutral glycosphingolipids antibodies have a functional impact on NGF-Trk-mediated intracellular signal transduction pathway.

Injection barrel with a tapered structure for a low speed and small size cryogenic hydrogen pellet in medium-sized plasma fusion devices.

An injection barrel was designed and fabricated for a small size 0.8 mm cryogenic pellet with a low speed of 200-300 m/s in medium-sized plasma fusion devices. Pellet injection with pneumatic acceleration was examined using a conventional in situ technique. A tapered structure was applied in the downstream side of the injection barrel to satisfy the requirement of pellet speed reduction by expansion of the propellant gas. Shadowgraph and light gate measurements show that the intact pellets have speeds of 260 ± 30 m/s and a typical size of 1.1-1.2 mm. The pellet ablation code based on a neutral gas shielding model shows that the penetration depth of the measured pellet parameters does not cross the plasma center, even in medium-sized plasma devices such as the Heliotron J helical device. The injection barrel with a tapered structure developed in this study is feasible for low speed pellet injection.

Purification and characterization of human liver beta-galactosidase from a patient with the adult form of GM1 gangliosidosis and a normal control.

beta-Galactosidases were purified to homogeneity from livers of a normal control and a patient with the adult form of GM1 gangliosidosis. The purification was achieved by chromatography on DEAE-Sepharose fast flow, Con A-Sepharose, p-aminophenyl-1-thio-beta-D-galactopyranoside-Sepharose, and QAE-Mono Q. The normal and mutant enzymes were purified about 5000-fold with a yield of 10% and 1800-fold with a yield of 34%, respectively, and could hydrolyze 4-methylumbelliferyl-beta-D-galactoside, GM1 ganglioside, and asialofetuin. The purified normal enzyme was eluted from a TSK gel G-4000SW column as three symmetrical peaks of protein which were coincident with the three peaks of enzyme activity. The enzyme in these three peaks had apparent molecular weights of 800,000 (polymer), 140,000 (dimer), and 65,000 (monomer), whereas the mutant enzyme was eluted as two symmetrical peaks of protein and enzyme activity. The apparent molecular weight of a major monomeric form of the enzyme (beta-galactosidase A) was 60,000, and no dimeric form of the enzyme existed. Normal and mutant purified enzyme preparations migrated as a single major protein band with apparent molecular weights of 65,000 or 60,000, respectively, by SDS-polyacrylamide gel electrophoresis after treatment with mercaptoethanol. On isoelectric focussing, the mutant enzyme migrated more anodally than the normal enzyme. The mutant enzyme also had altered enzyme properties, such as pH optimum, Km values, substrate specificity and heat-stability. These data on the characteristics of the purified enzyme preparations provide the first direct evidence that patients with the adult form of GM1 gangliosidosis have a structurally altered beta-galactosidase.

Design of polarizers for a mega-watt long-pulse millimeter-wave transmission line on the large helical device.

The polarizer is one of the critical components in a high-power millimeter-wave transmission line. It requires full and highly efficient coverage of any polarization states, high-power tolerance, and low-loss feature. Polarizers with rounded shape at the edge of the periodic groove surface are designed and fabricated by the machining process for a mega-watt long-pulse millimeter-wave transmission line of the electron cyclotron resonance heating system in the large helical device. The groove shape of λ/8- and λ/4-type polarizers for an 82.7 GHz transmission line is optimally designed in an integral method developed in the vector theories of diffraction gratings so that the efficiency to realize any polarization state can be maximized. The dependence of the polarization states on the combination of the two polarizer rotation angles (Φλ/8, Φλ/4) is examined experimentally in a low-power test with the newly developed polarization monitor. The results show that the measured polarization characteristics are in good agreement with the calculated ones.

Tyrosine phosphorylation of the catalytic subunit p110 of phosphatidylinositol-3 kinase induced by HMG-CoA reductase inhibitor inhibits its kinase activity in L6 myoblasts.

Previous studies from this laboratory have shown that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (HCRI) causes apoptotic cell death of a muscle cell-derived cell line, L6 myoblasts, by involving the phosphatidylinositol-3 (PI-3) kinase pathway and tyrosine phosphorylation of several cellular proteins, although the relationship between PI-3 kinase pathway and tyrosine phosphorylation responses remained to be elucidated. Here, we show that HCRI induces tyrosine phosphorylation of catalytic subunit p110 of PI-3 kinase as early as 5 min after addition of HCRI into culture medium. We could not detect the tyrosine phosphorylation of the regulatory subunit p85 of PI-3 kinase under the present experimental conditions. Concomitantly, the kinase activity toward PI in p110 immunoprecipitates was decreased with a similar time course. Furthermore, both herbimycin A and genistein, potent inhibitors of tyrosine kinase activity, inhibited HCRI-induced inhibition of PI-3 kinase activity as well as HCRI-induced apoptotic cell death. Once the catalytic subunit p110 becomes tyrosine-phosphorylated, the regulatory subunit p85 appears to be dissociated from the catalytic subunit, because we observed a decreasing amount of p85 regulatory subunits in p110 immunoprecipitates in response to HCRI treatment. These results strongly suggest the novel function of tyrosine phosphorylation of catalytic subunit p110 of PI-3 kinase in the regulation of its kinase activity. The tyrosine phosphorylation of these catalytic subunits may play an important role in the intracellular signal transduction of apoptotic cell death. To our knowledge, this is the first report that tyrosine phosphorylation of p110 catalytic subunit acts as a negative regulator of its kinase activity.

HMG-CoA reductase inhibitor-induced L6 myoblast cell death: involvement of the phosphatidylinositol 3-kinase pathway.

Our previous studies have shown that the HMG-CoA reductase inhibitor (HCRI) causes rhabdomyolysis and electrical myotonia in rabbits and also kills L6 myoblasts in culture. In the present study, we analyzed the intracellular signal transduction pathway of HCRI-induced cell death using L6 myoblasts as a model system. Here, we report that simvastatin, a lipophilic HCRI, efficiently inhibited isoprenylation of Ras proteins and therefore induced translocation of a significant part of Ras proteins from the membrane fraction into the cytosolic fraction within 10 min. With this translocation, PI 3-kinase activity of the Ras-bound form both in total and in the membrane fraction was also decreased profoundly. Furthermore, various PI 3-kinase inhibitors also caused cell death with morphological changes similar to those caused by simvastatin. These results might represent the molecular events of HCRI-induced cell death, and suggest the significance of PI 3-kinase activity of the Ras-bound form in the maintenance of cell viability.

Involvement of tyrosine phosphorylation in HMG-CoA reductase inhibitor-induced cell death in L6 myoblasts.

Our previous studies have shown that the HMG-CoA reductase (HCR) inhibitor (HCRI), simvastatin, causes myopathy in rabbits and kills L6 myoblasts. The present study was designed to elucidate the molecular mechanism of HCRI-induced cell death. We have demonstrated that simvastatin induces the tyrosine phosphorylation of several cellular proteins within 10 min. These phosphorylations were followed by apoptosis, as evidenced by the occurrence of internucleosomal DNA fragmentation and by morphological changes detected with Nomarski optics. Simvastatin-induced cell death was prevented by tyrosine kinase inhibitors. The MTT assay revealed that the addition of mevalonic acid into the culture medium partially inhibited simvastatin-induced cell death. Thus, these results suggested that protein tyrosine phosphorylation might play an important role in the intracellular signal transduction pathway mediating the HCRI-induced death of myoblasts.

Role of tyrosine phosphorylation of phospholipase C gamma1 in the signaling pathway of HMG-CoA reductase inhibitor-induced cell death of L6 myoblasts.

Our previous studies have shown that the HMG-CoA reductase (HCR) inhibitor (HCRI), simvastatin, kills L6 myoblasts by involving Ca2+ mobilization from the Ca2+ pool in the cells but not by influx from extracellular space. More recently, we found that HCRI induced tyrosine phosphorylation of several cellular proteins, followed by apoptotic cell death of L6 myoblasts. The present study was aimed to elucidate the molecular target(s) of these tyrosine phosphorylations induced by HCRI and demonstrated that simvastatin induces tyrosine phosphorylation of phospholipase C (PLC) gamma1. This tyrosine phosphorylation of PLC-gamma1 caused the increment of the intracellular inositol triphosphate (IP3) levels in L6 myoblasts. Pretreatment of the cells with herbimycin A, a specific inhibitor of protein tyrosine kinase, inhibited a simvastatin-induced increase in IP3 level in the cells as well as tyrosine phosphorylation of PLC-gamma1. Interestingly, pretreatment of the cells with U-73122, a specific inhibitor of PLC, prevented simvastatin-induced cell death. Thus, these results strongly suggest that simvastatin-induced tyrosine phosphorylation of PLC-gamma1 plays, at least in part, an important role for the development of simvastatin-induced cell death.

Influence of endogenous GM1 ganglioside on TrkB activity, in cultured neurons.

We verified the hypothesis that changes in the endogenous GM1 ganglioside density in the environment of TrkB, receptor of brain-derived neurotrophic factor, can affect receptor activity, and focused on rat cerebellar granule cells expressing both GM1 and TrkB. Changes of the amount of GM1 associated to immunoprecipitated TrkB and of receptor tyrosine phosphorylation were evaluated after treatment with phorbol-12-myristate-13-acetate (1 microM, 7 min), reported to affect the plasma membrane distribution of endogenous gangliosides in the same cells. After treatment, the amount of GM1 associated to receptor and TrkB phosphorylation decreased by about 40%. The amount of associated GM1 decreased by about 33% also after concomitant treatment with phorbol ester and brain-derived neurotrophic factor, but in this case the neurotrophin was unable to enhance receptor tyrosine phosphorylation. These results for the first time suggest that changes in the amount of endogenous GM1 in the environment of TrkB can modulate receptor activity, and offer new clues for a better understanding of physiological and pathological events of the nervous system.

Severe orthostatic hypotension in a female carrier of Fabry's disease.

A 21-year-old woman in a family with a history of Fabry's disease showed orthostatic hypotension and whorl-like corneal opacity typical for Fabry's disease. Biochemical studies revealed that she was a heterozygote of the Fabry gene. A variety of autonomic function tests demonstrated both sympathetic and parasympathetic dysfunction. To our knowledge, the present case is the first report of a heterozygous female carrier of Fabry's disease presenting dysfunction of the autonomic nervous system.

Phosphoglycerate kinase deficiency: an adult myopathic form with a novel mutation.

The authors report a 36-year-old man with exertional myoglobinuria and muscle cramp without hemolytic anemia or CNS symptoms. They found a deficiency of phosphoglycerate kinase (PGK) activity in muscle and erythrocytes and a 4-base pair deletion in exon 6 of the PGK gene. This mutation may cause a frameshift, yielding an abnormal stop codon in exon 6 by which a truncated PGK protein was produced. This phenotype is caused by a novel mutation of the PGK gene.

A family with beta-galactosidase deficiency: three adults with atypical clinical patterns.

Three adult patients in a single family showed severe myoclonus, ataxia, and pyramidal signs. Enzymatic analysis of lymphocytes, plasma, and cultured skin fibroblasts showed marked deficiency of beta-galactosidase activity, more profound with GM1 ganglioside than with another natural substrate, asialofetuin. Other lysosomal hydrolases were normal. Although the physical signs were similar to those of types 1 and 2 GM1 gangliosidosis, none had bony abnormalities.

Atypical adult GM1 gangliosidosis: biochemical comparison with other forms of primary beta-galactosidase deficiency.

We studied beta-galactosidase in skin fibroblasts from patients with different forms of beta-galactosidase deficiency: adult GM1 gangliosidosis, type 1 GM1 gangliosidosis, and Morquio B syndrome. Enzyme properties in the adult cases differed from the other disorders and also from normal controls. Genetic hybridization studies indicated that all three forms belong to the same complementation group. Therefore, the adult disorder must be due to a mutation of the structural gene for beta-galactosidase, which is allelic to the mutations in type 1 GM1 gangliosidosis and Morquio B syndrome.

Putative gap junctional communication between axon and regenerating Schwann cells during mammalian peripheral nerve regeneration.

Gap junctions are intercellular channels which mediate the traffic of ions and a variety of molecular messengers between contiguous cells. Here, we report on the possibility that atypical gap junctions develop between heterologous tissues, such as regenerating nerve axons and Schwann cells, during peripheral nerve regeneration in adult rats. After a complete transection and subsequent regeneration in the rat sciatic nerve distal segment, a small scale gap junction-like structure was observed between the regenerating axons and adjoining Schwann cells. Immunoelectron microscopy showed that one of the gap junctional proteins, connexin32, was located at a small region of contact between the axon and Schwann cells. Biocytin, a small molecular weight dye, was transported from regenerating axons into adjoining Schwann cells. The present findings suggest that regenerating axons communicate directly with adjacent Schwann cells through small gap junctions, which may play a role in the mechanism of regeneration following nerve transection.

Lung C-fiber CNS reflex: role in the respiratory consequences of extended environmental tobacco smoke exposure in young guinea pigs.

Environmental tobacco smoke (ETS) exposure harms the respiratory health of children and is associated with an increased risk of asthma and sudden infant death syndrome (SIDS). The mechanisms by which ETS causes these effects are not understood. We hypothesized that one mechanism is an upregulation of the lung C-fiber central nervous system (CNS) reflex responses, which would result in exaggerated reflex responses of apnea, bronchoconstriction, and mucous hypersecretion. The purpose of this work is to highlight evidence obtained in an animal model of postnatal ETS exposure supporting the hypothesis and present data suggesting that actions of the neuropeptide substance P in the nucleus tractus solitarius (NTS) may contribute. Exposing young guinea pigs to sidestream smoke, the surrogate for ETS, for 5 weeks during the equivalent of human childhood, increased the excitability of afferent lung C fibers and NTS neurons in the CNS reflex pathway and prolonged the expiratory apnea. The findings suggest that an increased excitability of NTS neurons that can augment reflex output may contribute to respiratory symptoms in children exposed to ETS. Besides ETS exposure, substance P can also excite NTS neurons and augment lung C-fiber CNS reflex responses. Others have shown that substance P synthesis in lung C fibers is upregulated by another environmental stimulant, allergen. Thus, an upregulation of the substance P system at NTS synapses could contribute to the increased NTS excitability and enhanced reflex responses to lung C-fiber stimulation, providing a potential mechanism to help explain the association of ETS exposure with respiratory symptoms and SIDS.

Ion heating and high-energy-particle production by ion-cyclotron heating in the large helical device

Ion-cyclotron heating was applied to the Large Helical Device. When the proton-cyclotron resonance was near the saddle point of the magnetic field-strength plane, strong ion-cyclotron damping occurred. Under these conditions efficient plasma heating was achieved for more than one minute. A high-energy ion tail was observed, and the effective tail temperature was determined by a balance between the wave acceleration and the electron-drag relaxation. There was no apparent sign of particle orbit loss effect in the investigated density range of 0.8-1.3x10(19) m(-3).