Biomedical subjects

K Higaki

Publications and source records attributed to K Higaki.

At least 19 recordsLinked to original sources

PubMed2006

Prevalence of hepatitis C virus infection in cases of B-cell lymphoma in Japan.

AIMS: To determine the prevalence of hepatitis C virus (HCV) infection in B-cell lymphoma in Japan. HCV infection and type II (monoclonal IgM) cryoglobulinaemia (CG) may be involved in the pathogenesis of low-grade B-cell lymphoma (ML) in southern Europe. METHODS AND RESULTS: Forty-five (11.3%) of 400 B-cell ML cases were HCV antibody (Ab) positive, which was significantly (P < 0.01) higher than the blood donors (2.5%). Among them, 28 diffuse large B-cell lymphoma (DLBCL) cases were included. In the primary sites, 10 (47.6%) of 21 splenic DLBCL and seven (23.3%) of 30 gastric DLBCL were HCV Ab positive, which were significantly (P < 0.05) higher than the myeloma cases (4.9%). HCV infection was rarely (4.2%) detected in 24 lymphoplasmacytic and salivary gland low-grade B-cell ML cases. Type II CG was detected in one myeloma case (3.5%) of 29 HCV+ B-cell ML. By real-time polymerase chain reaction, HCV RNA was detected in fresh tumour tissues of all 11 B-cell ML cases examined. Lymphoma cells were positive for the envelope HCV non-structural (NS)3 and envelope (E2) proteins in six of eight examined B-cell ML cases. CONCLUSIONS: The rare incidence of type II CG is characteristic of Japanese HCV+ ML patients and may influence the low incidence of low-grade B-cell ML. HCV infection may play a role in lymphomagenesis of splenic and gastric DLBCL.

Adolescent↗

PubMed2004

Enhancement of topical delivery of drugs via direct penetration by reducing blood flow rate in skin.

The purpose of this work was to investigate the effect of blood flow in the skin on the direct penetration of topically applied drugs into the muscular layer, and to show that the skin blood flow could also be one of the important factors determining the direct penetration of drugs to the muscular layer. In vivo percutaneous absorption study was performed for antipyrine, salicylic acid or diclofenac by using rats with tape-stripped skin. Phenylephrine, which is well known to reduce the local blood flow by vasoconstrictor action, was topically applied to decrease the local blood flow in the skin. The concentrations of drugs in viable skin and muscle, and the local blood flow in the skin under the applied and the contralateral sites were determined to evaluate the effect of the local blood flow on the delivery of topically applied drugs into the muscular layer. Dose dependency for the effect of phenylephrine was, first of all, investigated for antipyrine in the range from 0.4 to 10 micromol. The distribution of antipyrine into the viable skin and muscular layer 2 h after topical application significantly increased, but the effect of phenylephrine was saturated around 2 micromol and the dose-dependent profiles for both tissues were almost superimposed. On the other hand, the fraction dose absorbed, plasma concentration and concentrations in viable skin and muscular layer under the contralateral site showed the decreasing tendency and the saturation of the effect around 2 micromol. To confirm the effect of phenylephrine on the local blood flow in the skin, the skin blood flow was measured 2 h after topical application of 2 micromol phenylephrine, and the significant decrease in the blood flow was recognized. In vivo percutaneous absorption studies were performed for salicylic acid and diclofenac, too. Extensive enhancement of penetration into the viable skin and muscular layer was observed for both drugs, although total absorption from the donor cell showed the decreasing tendency. In conclusion, direct penetration of drugs applied topically is enhanced by reducing the local blood flow in the skin, which would be a possible approach to improve the local delivery of drugs applied topically.

Administration, Topical↗

PubMed2004

Pharmacokinetic analysis of factors determining elimination pathways for sulfate and glucuronide metabolites of xenobiotics. III: mechanisms for sinusoidal efflux of 4-methylumbelliferone sulfate.

1. To elucidate the mechanisms involved in the sinusoidal efflux of sulfate and glucuronide metabolites of 4-methylumbelliferone (4MU), isolated rat liver perfusion studies were performed under several conditions. 2. The effect of sodium azide on the hepatic handling of both conjugates was examined. The net sinusoidal efflux clearance (CL(eff)) based on the unbound concentration in the liver did not change for 4MU glucuronide (4MUG) or significantly increase for 4MU sulfate (4MUS), suggesting that the sinusoidal efflux of both conjugates is not mediated by the transport systems dependent on adenosine triphosphate. 3. Under Cl(-)-depleted conditions, the CL(eff) of 4MUG significantly decreased, but the saturation of its sinusoidal efflux rather than the transport system dependent on Cl(-) might be involved because the hepatic concentration of 4MUG was extensively higher than that of the control study due to the extremely attenuated biliary excretion. The CL(eff) of 4MUS also significantly decreased, but its hepatic concentration was not different from that in the control study, suggesting that the transport system using Cl(-) is involved in the sinusoidal efflux of 4MUS. 4. The effect of glutathione was examined. CL(eff) of 4MUG was not affected by the additional glutathione, but CL(eff) of 4MUS decreased significantly, suggesting that some transport system sensitive to glutathione is involved in the sinusoidal efflux of 4MUS, but not of 4MUG. 5. Transporters such as Oatp1, Oatp2 and/or Npt1 might be involved in the sinusoidal efflux of 4MUS, but 4MUG is secreted from the sinusoidal membrane via the systems that are totally different from those for 4MUS.

Animals↗

PubMed2003

Differential metabolism of midazolam in mouse liver and intestine microsomes: a comparison of cytochrome P450 activity and expression.

1. Although multiple cytochrome P450s (CYP) contribute to hepatic phase I metabolism, CYP3A is the principal subfamily present in human and mouse small intestine. 2. Differences in phase I metabolism were investigated using midazolam (MDZ) hydroxylation in mouse liver and intestinal microsomes. The net MDZ metabolite formation rate in intestinal microsomes was approximately 30% that of liver microsomes (at 250 micro M MDZ). 3. Quantitative Western blotting with anti-CYP3A1 antibody detected two bands of immunoreactive protein in both liver and intestinal samples, 2.24 +/- 0.27 (mean +/- SD, n = 3) and 0.64 +/- 0.08 pmol mg(-1) protein, respectively. Qualitative Western blotting with anti-CYP2C11 antibody detected a single band of immunoreactive protein in liver microsomes and no signal in intestinal samples (1 micro g sample). 4. Ketoconazole potently inhibited formation of both alpha- and 4-OH-MDZ metabolites in intestinal microsomes (IC(50)' of 0.126 +/- 0.010 and 0.0955 +/- 0.014 micro M, respectively) and of 4-OH-MDZ formation in mouse liver microsomes (IC(50) of 0.041 +/- 0.003 micro M). However, ketoconazole (5 micro M) did not produce 50% inhibition of alpha-OH-MDZ formation in mouse liver microsomes. Inhibition by ritonavir (5 micro M) produced similar results. 5. MDZ hydroxylation is predominately CYP3A dependent in mouse intestine (compared with mouse liver) since CYP2C is not expressed in the intestine. The importance of CYP3A in the mouse intestine appears to mirror that in humans.

Algorithms↗

PubMed2003

Pharmacokinetic analysis of factors determining elimination pathways for sulfate and glucuronide metabolites of xenobiotics II: Studies with isolated perfused rat liver.

1. To elucidate the determining factors for elimination pathways of sulfate and glucuronide metabolites of xenobiotics, a single-pass perfusion of 4-methylumbelliferone (4MU) or p-nitrophenol (pNP) was performed with an isolated rat liver preparation. 2. Without bovine serum albumin in the perfusion system, clearance calculated based on the unbound concentration in the liver clearly showed that the net efflux clearances (CLeff) of sulfates from the sinusoidal membrane were much higher than those of glucuronides and that the biliary excretion clearances (CLb) of glucuronides were approximately two times larger than those of sulfates. 3. The ratios of CLeff to CLb were much higher for sulfates than those for glucuronides. The bile-oriented elimination of glucuronides or sinusoidal efflux-oriented elimination of sulfates was observed even using the perfusate including 3% bovine serum albumin, but the sinusoidal efflux of sulfates was extensively enhanced by bovine serum albumin in the perfusate. The mechanisms behind this stimulatory effect remain to be elucidated. 4. For both compounds, CLb of glucuronide was comparable with CLb of sulfate, meaning that CLb is not responsible for the biliary excretion of glucuronides at extensively higher rate than sulfates. 5. Higher concentration of glucuronides in the liver, partly caused by much lower CLeff of glucuronides than that of sulfates, is likely responsible for the bile-oriented excretion of glucuronides. The extensive sinusoidal efflux of sulfates, leading to the urine-oriented excretion, is attributed to the substantially higher CLeff than CLb. 6. In conclusion, the sinusoidal efflux is an important factor for determining elimination pathways of both sulfates and glucuronides, although further studies are needed to clarify the mechanisms of the sinusoidal efflux.

Acetaminophen↗

PubMed2002

Pharmacokinetic analysis of factors determining elimination pathways for sulfate and glucuronide metabolites of drugs. I: studies by in vivo constant infusion.

1. The hepatic and renal handling of glucuronides and sulphates of three phenolic compounds, 4-methylumbelliferone (4-MU), p-nitrophenol (pNP) and acetaminophen (APAP), were evaluated pharmacokinetically by in vivo constant infusion experiments in rat. It was shown that the urinary excretion rate at steady-state was larger than the biliary excretion rate for both glucuronides and sulfates, and sulfates, in particular, were extensively excreted into the urine. 2. For each glucuronide, however, biliary excretion clearances (CL(b)) calculated based on the total concentration and unbound concentration in the liver were much larger than the corresponding renal excretion clearances (CL(r)). Even in the case of sulfates, there was not any large difference between CL(r) and CL(b) based on the total and unbound concentration in tissues, which could not explain their extensive urinary excretion. From these results, these excretion clearances were recognized not to reflect necessarily the actual excretion rate obtained. 3. On the other hand, the tissue-to-plasma concentration ratio (K(p)) of both glucuronides and sulfates for every phenolic compound was much higher in the kidney than that in the liver. The results suggested that one of the most important determinants for the preferential excretion of these conjugates into the bile or urine is the extent of disposition of each compound to the liver or kidney. 4. In addition, K(p) of both glucuronides and sulfates in the liver, where these conjugates are mainly formed, was small. The K(p) of sulfates was quite low, suggesting that sulfates generated in the liver were subject to extensive sinusoidal efflux.

Acetaminophen↗

PubMed2002

Pharmacokinetic evaluation of high pulmonary disposition of clarithromycin after systemic administration.

1. The distribution characteristics of clarithromycin to the lung were investigated in vivo and in isolated lung perfusion experiments. The in-vivo integration plot analysis showed that the pulmonary uptake and extracellular distribution in the lung were significantly higher for clarithromycin than for erythromycin. 2. In the rat lung single-pass perfusion study, the pulmonary extraction ratio (E(ss)) of clarithromycin at steady-state was significantly higher than that of erythromycin, and the E(ss) of clarithromycin tended to decrease as the inflow concentration increased, suggesting the involvement of carrier-mediated transport in the pulmonary disposition of clarithromycin. 3. The outflow patterns of clarithromycin or erythromycin at various inflow concentrations were simultaneously analysed based on a pharmacokinetic model, which consists of the non-specific binding site, the specific binding site and the subsequent uptake process. The parameters obtained suggested that clarithromycin would have the higher affinity and higher capacity for the specific binding site, and the higher equilibrium constant for the non-specific binding site than erythromycin. 4. The simulation study using those parameters demonstrated that clarithromycin could be bound to the specific binding site and subsequently taken up more extensively than erythromycin. 5. A multiple-indicator dilution study also indicated that clarithromycin was more readily associated and extracted with the lung than with erythromycin. In the inhibition study, it was suggested that the pulmonary uptake of clarithromycin could be ascribed not only to the non-specific binding depending on its lipophilic nature, but also in part to some specialized mechanisms such as organic cation transporters.

Animals↗

PubMed2001

Surface hydrophobicity of particles is not necessarily the most important determinant in their in vivo disposition after intravenous administration in rats.

The in vivo disposition of polystyrene microsphere (MS) with the particle size of 50 nm (MS-50) and lecithin-coated MS-50 (LMS-50) after intravenous administration to rats was characterized. While a rapid elimination from the systemic circulation was observed for MS-50, much more prolonged circulating property was observed for LMS-50. In addition, this in vivo disposition property of LMS-50 was suggested to be ascribed to its lower affinity to the liver, which is the determining organ of the in vivo disposition of MS-50. The evaluation of surface hydrophobicity of MS-50 and LMS-50 in buffer solution revealed that the surface of MS-50 is more hydrophobic than that of LMS-50. However, LMS-50 was oppositely found to be more hydrophobic than that of MS-50 in rat serum. The profiles of serum proteins associated with MS-50 and LMS-50 were also examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that the amounts of some adsorbed proteins are greatly different between MS-50 and LMS-50. From these findings, it was suggested that the substantial difference in the in vivo disposition between MS-50 and LMS-50 would not be attributed to the difference in their surface hydrophobicity in the blood, but the difference in the type of serum proteins associated with them.

Animals↗

PubMed2001

Accumulation of cholera toxin and GM1 ganglioside in the early endosome of Niemann-Pick C1-deficient cells.

We investigated intracellular trafficking of GM1 ganglioside in Niemann-Pick C1 (NPC1)-deficient Chinese hamster ovary cells [NPC1(-) cells] by using cholera toxin (CT) as a probe. Both the holotoxin and the B subunit (CTB) accumulated in GM1-enriched intracellular vesicles of NPC1(-) cells. CTB-labeled vesicles contained the early endosome marker Rab5 but not lysosome-associated membrane protein 2 and were not labeled with either Texas red-transferrin or Lysotracker, indicating that they represent early endosomes. Similarly, CT accumulated in intracellular vesicles of human NPC fibroblasts that contained both Rab5 and early endosomal antigen 1. CTB accumulation in NPC1(-) cells was abolished by expression of wild-type NPC1 but not by mutant proteins with a mutation either in the NPC domain or the sterol-sensing domain. A part of these mutant NPC1 proteins expressed in NPC1(-) cells was localized on CTB-labeled vesicles. U18666A treatment of "knock in" cells [NPC1(-) cells that stably expressed wild-type NPC1] caused CTB accumulation similar to that in NPC1(-) cells, and a part of wild-type NPC1was localized on CTB-labeled vesicles in drug-treated cells. Finally, CT tracer experiments in NPC1(-) cells revealed retarded excretion of internalized toxin into the culture medium and an increase in the intracellular release of A subunits. In accordance with the latter result, CT was more effective in stimulating cAMP formation in NPC1(-) than in wild-type cells. These results suggest that transport of CT/GM1 complexes from the early endosome to the plasma membrane depends on the function of NPC1, whereas transport to the Golgi apparatus/endoplasmic reticulum does not.

Animals↗

PubMed2001

Niemann-Pick disease type C: spectrum of HE1 mutations and genotype/phenotype correlations in the NPC2 group.

In Niemann-Pick disease type C (NPC), a genetic heterogeneity with two complementation groups--NPC1, comprising > or =95% of the families, and NPC2--has been demonstrated. Mutations in the NPC1 gene have now been well characterized. HE1 was recently identified as the gene underlying the very rare NPC2. Here we report the first comprehensive study of eight unrelated families with NPC2, originating from France, Algeria, Italy, Germany, the Czech Republic, and Turkey. These cases represent essentially all patients with NPC2 who have been reported in the literature, as well as those known to us. All 16 mutant alleles were identified, but only five different mutations, all with a severe impact on the protein, were found; these five mutations were as follows: two nonsense mutations (E20X and E118X), a 1-bp deletion (27delG), a splice mutation (IVS2+5G-->A), and a missense mutation (S67P) resulting in reduced amounts of abnormal HE1 protein. E20X, with an overall allele frequency of 56%, was established as the common mutant allele. Prenatal diagnosis was achieved by mutation analysis of an uncultured chorionic-villus sample. All mutations except 27delG were observed in a homozygous state, allowing genotype/phenotype correlations. In seven families (with E20X, E118X, S67P, and E20X/27delG mutations), patients suffered a severe and rapid disease course, with age at death being 6 mo-4 years. A remarkable feature was the pronounced lung involvement, leading, in six patients, to early death caused by respiratory failure. Two patients also developed a severe neurological disease with onset during infancy. Conversely, the splice mutation corresponded to a very different clinical presentation, with juvenile onset of neurological symptoms and prolonged survival. This mutation generated multiple transcripts, including a minute proportion of normally spliced RNA, which may explain the milder phenotype.

Adult↗

PubMed2001

Biliary excretion of polystyrene microspheres depends on the type of receptor-mediated uptake in rat liver.

Hepatic uptake and biliary excretion of fluorescein isothiocyanate-labeled polystyrene microspheres with a particle size of 50 nm (MS-50) were studied in rats. Liver perfusion studies revealed that not only apo-E-mediated but also asialoglycoprotein receptor-mediated uptake is involved in the mechanism of the serum protein-dependent uptake of MS-50 in the liver. The uptake of MS-50 mediated by apo-E contributes more to the total uptake of MS-50 by the hepatocytes than that via asialoglycoprotein receptor in the presence of serum in the perfusate. Furthermore, it was found that MS-50 is substantially excreted into the bile by transcytosis. The extent of exocytosis of MS-50 taken up by the hepatocytes was much higher after MS-50 was endocytosed via asialoglycoprotein receptor than after taken up via the process mediated by apo-E. On the basis of these results, a possible regulation of the intracellular sorting of ligands, depending on the receptor-mediated uptake mechanism, was inferred.

Animals↗

PubMed2001

Expression of angiogenic factors, basic fibroblast growth factor and vascular endothelial growth factor, in human biliary tract carcinoma cell lines.

In order to clarify angiogenic mechanism in biliary tract carcinoma, expressions and functions of basic fibroblast growth factor (bFGF) and its receptors (FGFR-1-4), and vascular endothelial growth factor (VEGF) and its receptors were investigated by using human biliary tract carcinoma cell lines (KMC-1, KMC-2, KMBC and KMG-C). Expression of bFGF was confirmed in KMC-1 and KMC-2, and that of FGFR-1-4 in all the cell lines except no FGFR-2 in KMC-2. Expression of VEGF was detected in all the cell lines, whereas the cell lines did not express VEGF receptors. Addition of anti-bFGF neutralizing antibody to the medium did not suppress cell proliferation, whereas exogenous bFGF with or without heparin accelerated cell proliferation in all cell lines. Addition of anti-bFGF neutralizing antibody or anti-VEGF neutralizing antibody to the co-culture of human umbilical vascular endothelial cells (HUVEC) and KMC-2 suppressed the proliferation of HUVEC. Surgically obtained cholangiocarcinoma tissues (n=7) were immunohistochemically negative to bFGF, while six of the seven were positive to VEGF. These findings suggested that human biliary tract carcinoma cells express both bFGF and VEGF not as autocrine growth factors but as angiogenic factors. On the other hand, expression of VEGF was found at a higher frequency than bFGF both in the cell lines and tissues.

Journal Article↗

PubMed2001

Cloning and functional expression of rat kidney dipeptidyl peptidase II.

Dipeptidyl peptidase II (DPP II; EC 3.4.14.2) from rat kidney was purified to a specific activity of 65.4 micromol/min per mg of protein for Lys-Ala-beta-naphthylamide. The N-terminal and partial amino acid sequences of the enzyme were determined. The peptide sequences were used to identify expressed sequence tag (EST) clones. By using the cDNA fragment of one of the EST clones as a probe, we isolated a cDNA clone with 1710 bp encoding DPP II from a rat kidney cDNA library. The cDNA of rat DPP II contained an open reading frame of 1500 bp, coding for a protein of 500 amino acids. The first 10 residues of the purified enzyme matched the deduced protein sequence starting with residue 37, suggesting the presence of a signal peptide. The mature enzyme (464 residues) had a calculated molecular mass of 51400 Da, which was lower than the value (about 60000 Da) determined by SDS/PAGE; and the deduced amino acid sequence showed six potential N-glycosylation sites. The deduced amino acid sequence of rat DPP II shared high similarity with quiescent-cell proline dipeptidase (78% identity) and prolyl carboxypeptidase (38% identity) and bore the putative catalytic triad (Ser, Asp, His) conserved in serine peptidase families. We transiently transfected COS-7 cells with pcDNA3.1 containing the cloned cDNA and obtained the overexpression of an immunoreactive protein (of about 60000 Da). The transfected cells showed Lys-Ala-methylcoumarinamide-hydrolysing activity that was 50 times higher than the control cells.

Amino Acid Sequence↗

PubMed2001

Evaluation of absorption kinetics of orally administered theophylline in rats based on gastrointestinal transit monitoring by gamma scintigraphy.

The gastrointestinal (GI) transit and absorption of orally administered theophylline, a highly absorbable drug without presystemic elimination, were investigated under fasted and fed conditions using three rats in a crossover study. To evaluate the GI transit rate for each segment in vivo, a noninvasive technique, gamma scintigraphy, was employed using a nonabsorbable compound, (99m)Tc-labeled diethylenetriamine pentaacetic acid (DTPA). Using a gamma scintigraphic technique it is possible to simultaneously evaluate the GI transit and absorption of orally administered drug in the same individual. Theophylline was simultaneously administered along with [(99m)Tc]DTPA to animals in the fasted and fed states. Each GI transit pattern, simulated using the GI transit-kinetic model with a lag time factor, was well fitted to the experimental data. Gastric emptying rate varied in each study, even under the same experimental condition. The GI transit pattern for each segment was highly variable, especially in animals in the fed state. This inconsistency in transit pattern was mainly due to the variability in gastric emptying, which was much slower in animals in the fed compared with the fasted state. However, in spite of a large variability of GI transit kinetics, the plasma concentration-time curves of theophylline were well predicted by the GI transit-absorption model using the individual GI transit parameters obtained in the study. The absorption rate of theophylline was considerably reduced in animals in the fed state, because of the reduction of gastric emptying rate. Analysis using GI transit-absorption model and gamma scintigraphic technique made it possible to estimate the variable absorption kinetics regulated by GI transit with huge variability.

Administration, Oral↗

PubMed2001

Amino acids protect epithelial cells from local toxicity by absorption enhancer, sodium laurate.

To develop the safe absorption-enhancing formulation attenuating the local toxicity caused by an absorption enhancer, sodium laurate (C12), the effects of amino acids on the local toxicity by C12 were examined in rats. The absorption of phenol red, an unabsorbable marker drug, was significantly enhanced by 10 mM C12 in an in situ colon loop study and the addition of L-glutamine (L-Gln), L-arginine, or L-methionine at 10 mM did not change the promoting effect of C12. However, C12 significantly increased the elution of phospholipids, total protein, and lactate dehydrogenase, which are markers for local toxicity, from colon, but these amino acids attenuated the local toxicity caused by C12 significantly. Transport study using an Ussing-type chamber showed that the permeability of colonic membrane to phenol red was significantly enhanced by C12 and that L-Gln did not decrease the permeability enhanced by C12. Transmucosal electrical resistance was extensively decreased by C12, indicating that C12 could enhance the drug absorption at least partly by expanding the paracellular route. L-Gln significantly, but not completely, recovered resistance lowered by C12. Electrical potential difference was markedly reduced by C12, suggesting that C12 lowered the viability of mucosal cells, but 10 mM L-Gln significantly recovered potential difference almost to the control level. These results suggested the possibility that absorption-enhancing formulation with low local toxicity, which is low enough to be used practically, could be developed by using an amino acid like L-Gln as an ingredient attenuating the local toxicity caused by C12.

Amino Acids↗

PubMed2001

Progressive neuronal loss in the ventral posterior lateral and medial nuclei of thalamus in Niemann-Pick disease type C mouse brain.

Niemann-Pick disease type C (NP-C) disease is a progressive and fatal neurological disorder characterized by accumulation of cholesterol and glycosphingolipids in peripheral tissues and that of glycosphingolipids in the brain. A C57BL/KsJ-npc1(spm) mutant strain is a genetically authentic model of NP-C. This study investigated neuronal cell loss and lipid accumulation in the npc1(spm) mouse brain. Nissl-staining revealed abundant swollen neurons in the neocortex, piriform cortex, hippocampus and basal ganglia at 3-4 wk of age. In addition to loss of the Purkinje cells, we found a conspicuous cell loss in the ventral posterial lateral (VPL) and medial (VPM) nuclei of thalamus, which became apparent after 4-5 wk. Biochemical analyses revealed no increase of cholesterol in the lipid extracts whereas a substantial accumulation of cholesterol was detectable in most of the large neurons by filipin staining in the brain of homozygous mice. In contrast to the diffuse staining pattern in normal brains, the neuropils of the neurons in the brain of homozygous mice were stained in a punctate pattern. The ubiquitous accumulation excludes a direct role of cholesterol in the progressive neuronal loss in the Purkinje cell layer and in the VPL and VPM of the thalamus.

Aging↗

PubMed2001

Time-dependent oral absorption models.

The plasma concentration-time profiles following oral administration of drugs are often irregular and cannot be interpreted easily with conventional models based on first- or zero-order absorption kinetics and lag time. Six new models were developed using a time-dependent absorption rate coefficient, ka(t), wherein the time dependency was varied to account for the dynamic processes such as changes in fluid absorption or secretion, in absorption surface area, and in motility with time, in the gastrointestinal tract. In the present study, the plasma concentration profiles of propranolol obtained in human subjects following oral dosing were analyzed using the newly derived models based on mass balance and compared with the conventional models. Nonlinear regression analysis indicated that the conventional compartment model including lag time (CLAG model) could not predict the rapid initial increase in plasma concentration after dosing and the predicted Cmax values were much lower than that observed. On the other hand, all models with the time-dependent absorption rate coefficient, ka(t), were superior to the CLAG model in predicting plasma concentration profiles. Based on Akaike's Information Criterion (AIC), the fluid absorption model without lag time (FA model) exhibited the best overall fit to the data. The two-phase model including lag time, TPLAG model was also found to be a good model judging from the values of sum of squares. This model also described the irregular profiles of plasma concentration with time and frequently predicted Cmax values satisfactorily. A comparison of the absorption rate profiles also suggested that the TPLAG model is better at prediction of irregular absorption kinetics than the FA model. In conclusion, the incorporation of a time-dependent absorption rate coefficient ka(t) allows the prediction of nonlinear absorption characteristics in a more reliable manner.

Administration, Oral↗

PubMed2001

Isolation of NPC1-deficient Chinese hamster ovary cell mutants by gene trap mutagenesis.

Chinese hamster ovary cell mutants defective in the NPC1 gene (NPC1-trap) were generated by retrovirus-mediated gene trap mutagenesis from a parental cell line JP17 expressing an ecotropic retrovirus receptor. Insertion of the gene trap vector in the NPC1 gene and the absence of the gene product were verified by 5'RACE and immunological analyses, respectively. NPC1-trap cells showed intracellular accumulation of low-density lipoprotein (LDL)-derived cholesterol and had an increased level of unesterified cellular cholesterol. Cholesterol biosynthesis through the mevalonate pathway was upregulated in the mutant cells as assessed by [(14)C]acetate incorporation into cellular sterols. When JP17 cells were depleted of lipoproteins and then loaded with LDL, cell surface LDL receptors were promptly downregulated and the mature form of the sterol regulatory element-binding protein-1 disappeared from the nucleus. These responses to LDL were obviously retarded in NPC1-trap cells, suggesting an impaired response of the cholesterol-regulatory system to LDL. NPC1-trap cells will be a useful tool to study the regulation of cellular cholesterol homeostasis and the pathogenesis of Niemann-Pick disease type C.

Animals↗