Biomedical subjects

J Borlak

Publications and source records attributed to J Borlak.

31 records · Page 2Linked to original sources

PubMed2000

Molecular diagnosis of a familial nonhemolytic hyperbilirubinemia (Gilbert's syndrome) in healthy subjects.

Recent research has shown that congenital nonhemolytic low grade hyperbilirubinemias in patients with Gilbert's syndrome (GS) are linked to mutations in the TATA box upstream of the uridine 5'-diphosphoglucose glucuronosyltransferase (UGT1A1) gene leading to an impaired bilirubin glucuronidation. In routine clinical practice GS patients can, however, only be suspected by exclusion of other causes of hyperbilirubinemia or substantial liver diseases. We developed a new, sensitive, convenient, and economic polymerase chain reaction (PCR) method for the rapid and reliable identification of gene polymorphisms in the TATA box of the UGT1A1 gene using fluorescence resonance energy transfer (FRET). With this procedure the genotype frequency in a cohort of 266 unrelated individuals from Southern Germany was investigated and the allelic distribution for individual genotypes was 43:45:12 for the (TA)(6)TAA:(TA)(6)TAA/(TA)(7)TAA:(TA)(7)TAA alleles, respectively. The homozygous (TA)(7)TAA genotype was strongly associated with suspected Gilbert's patients and its prevalence in our cohort of 266 Southern German individuals was 12.4%. In this cohort total mean serum bilirubin levels ranged from 5 micromol/L (wild-type 6/6 allele) to 57 micromol/L serum total bilirubin (mutant 7/7 homozygous allele). Median (interquartile range) serum total bilirubin levels were 12 (6) and 21 (13) for the homozygous wild-type and mutant allele, respectively. Our assay enables individual guidance for dose adjustment in suspected GS patients undergoing long-term drug therapies, especially if glucuronidation via UGT1A1 is a major metabolic pathway.

Adult↗

PubMed2000

Isolation and cultivation of Ca2+ tolerant cardiomyocytes from the adult rat: improvements and applications.

1. Primary cultures of cardiomyocytes provide a valuable tool for the study of the pharmacological and toxicological properties of drugs and chemicals, but for several technical reasons cardiomyocytes from adult animals are not routinely used in long-term culture. Because of significant advances in cardiovascular research, tissue engineering and cell transplantation, the need to isolate primary cells from adult animal and/or human tissue is likely to increase in the future. 2. The most common protocols for the isolation and cultivation of cardiomyocytes have been reviewed and the various approaches have been compared. The recent advances in cell culture techniques and the use of the cytoprotective agent, e.g. 2,3-butanedione monoxime greatly increases cell yield and cell viability of isolated and cultured cardiomyocytes. New concepts emerge that enabled an assessment of cellular differentiation in cultured cardiomyocytes and certain specific nuclear transcription factors may play a pivotal role in this process.

Animals↗

PubMed2000

Cytochrome P450 enzyme activity and protein expression in primary porcine enterocyte and hepatocyte cultures.

1. A method for the isolation and cultivation of porcine hepatocytes and porcine duodenal enterocytes for the investigation of drug oxidation reactions has been established. 2. Hepatocytes as well as enterocytes metabolized ethoxyresorufin (EROD) and ethoxycoumarin (ECOD) effectively, the rate being 31+/-17 pmol/h x dish (EROD) and 9530+/-4062 pmol/h x dish (ECOD) in the case of hepatocytes, and 9+/-4 pmol/h x dish (EROD) and 510+/-467 pmol/h x dish (ECOD) in the case of enterocytes. Diazepam, another CYP monooxygenase substrate, was also metabolized by porcine hepatocytes but not with porcine enterocytes, thus indicating differences in the metabolic competence of the liver and the gut. 3. The ability to induce enzymes responsible for the metabolism of ethoxyresorufin and ethoxycoumarin was investigated in vitro on treatment of the cell cultures with either 50 microM 3-methylcholanthrene (3-MC) or 50 microM beta-naphthoflavone (beta-NF). With enterocyte cultures, ECOD activity was inducible up to 20-fold, whereas EROD remained unchanged following treatment with either 3-MC or beta-NF. 4. Western blotting provided additional evidence for the expression of CYP1A1 and CYP3A4 at the protein level and treatment of cultured enterocytes with 30 microM Aroclor 1254 or 50 microM beta-NF resulted in enhanced expression of the CYP1A protein, and CYP3A4 protein expression was induced following treatment with 50 microM DEX, 2 mM PB, 30 microM Aroclor 1254 or 50 microM beta-NF. 5. The metabolism of diazepam was also investigated with baculovirus-expressed human CYP enzymes (2C8, 2C9-ARG, 2C9-CYS, 2C19, 3A4, 3A4+cytochrome b5 and 3A5) and evidence was obtained to suggest the formation of temazepam and oxazepam by enzymes of the CYP3A subfamily. Small amounts (32+/-12 ng/ml) of desmethyldiazepam were additionally recovered in microsomal preparations of all CYP-transfected cell lines. 6. In conclusion, porcine duodenal enterocytes can successfully be cultured for a short period and may be used as a tool for studying intestinal metabolism, whereas porcine hepatocytes can be cultured for prolonged periods (>10 days) reliably to investigate hepatic drug oxidation reactions.

Albumins↗

PubMed2000

Cellular dedifferentiation of endothelium is linked to activation and silencing of certain nuclear transcription factors: implications for endothelial dysfunction and vascular biology.

We investigated the gene expression of the nuclear transcription factors c/EBPalpha, GATA-2, and the silencer Oct-1 in conjunction with the gene expression of all major cytochrome P450 genes and of eNOS in cultures of endothelial cells of the rat. The purity of cultured endothelial cells was also confirmed by flow cytometry measurements of PECAM-1, a surface antigen of endothelial cells. Taken collectively, the gene expression and flow cytometry studies provide strong evidence for c/EBPalpha, GATA-2, and Oct-1 to play a key role in the cellular dedifferentiation of endothelial cells; gene expression of eight individual CYP genes in conjunction with protein activity could be significantly increased upon treatment with Aroclor 1254, a well-documented chemical inducer of a battery of genes. Nevertheless, the gene expression of c/EBPalpha, GATA-2, and most of the CYP genes was dramatically reduced (up to 90%) in cell cultures lacking PECAM-1 expression; in strong contrast, expression of the silencer Oct-1 was massively increased (approximately 14-fold). We thus conclude activation of the silencer Oct-1 to be strongly correlated with loss of PECAM-1 and eNOS gene expression, e.g., loss of cellular differentiation and endothelial function; in conjunction, gene expression of all major P450 isoforms was dramatically reduced in cultures of dedifferentiated endothelial cells. This process of cellular dedifferentiation and endothelial dysfunction was accompanied by down-regulation of endothelial specific transcription factors.

Animals↗

PubMed1998

Effects of intraduodenal administration of tarazepide on pancreatic secretion and duodenal EMG in neonatal calves.

The influence of CCK-A receptor antagonism on pancreatic exocrine secretion and duodenal EMG, and the mechanism(s) involved in CCK-induced pancreatic secretion were studied in conscious calves. Seven 1-week-old calves were fitted with a pancreatic duct catheter, duodenal cannula and duodenal electrodes. Pancreatic exocrine secretion and duodenal EMG were studied following intraduodenal CCK-A receptor antagonist (Tarazepide), intravenous atropine, and intravenous or intraduodenal CCK-8 administrations. Tarazepide decreased duodenal electric activity, reduced interdigestive pancreatic secretion, especially protein; reduced cephalic and early postprandial (milk) induced secretion of bicarbonate and protein. Pancreatic protein secretion to intravenous CCK-8 was little affected by atropine, but was significantly reduced by Tarazepide+/-atropine; in contrast, protein secretion to intraduodenal CCK-8 was abolished by Tarazepide or atropine. We conclude that pre- and especially early postprandial pancreatic secretion are partly controlled via CCK-A (mainly mucosal) mediated mechanisms.

Animals↗

PubMed1998

Sensitive method for the determination of KC 12291 in rat plasma and urine by high-performance liquid chromatography.

A sensitive and selective ion-pair reversed-phase HPLC method has been developed for the quantitative determination of KC 12291 and its major metabolite, KC 13194, in rat plasma and urine. An Ultrasphere ODS column constructed by using a mobile phase of 1 mM 1-octanesulfonic acid containing acetonitrile-0.1 M triethylamine phosphate buffer, pH 2.2 (29:71, v/v in plasma and 27:73, v/v in urine), an internal standard and a fluorescent detector (excitation 265 nm, emission 370 nm) were used for the separation and the quantitative determination, respectively. The plasma samples were made alkaline and both compounds were cleaned up by the use of liquid-liquid extraction. The limit of quantification was 10 ng/ml for KC 12291 in plasma and urine and for KC 13194 and 100 ng/ml in plasma, respectively. The assay has been validated with respect to system suitability, accuracy, precision, recovery, stability and ruggedness. All validated parameters were found to be within the necessary limits.

Animals↗

PubMed1998

Antifibrillatory efficacy of long-term tedisamil administration in a postinfarcted canine model of ischemic ventricular fibrillation.

The electrophysiologic and antifibrillatory properties of tedisamil (KC-8857) were studied in vivo in a conscious canine model of sudden cardiac death. Male mongrel dogs were anesthetized, and surgical anterior myocardial infarction was induced by a 2-h occlusion, with reperfusion of the left anterior descending coronary artery. Three to five days after infarction, dogs were subjected to programmed electrical stimulation (PES) to identify those at risk for ischemia-induced ventricular fibrillation. Previous studies documented that dogs with a significant anterior-wall infarction develop ventricular tachycardia in response to PES and are at an increased risk for sudden cardiac death on imposition of a transient ischemic event in a region remote from the infarct-related artery. PES-inducible animals were randomized to either oral placebo or oral tedisamil treatment (3 mg/kg, b.i.d for 4 days, Group 1, n = 8). Control animals received empty gelatin capsules (Group 2, n = 8). The effective refractory period and QTc interval were unchanged after 3 days of oral placebo or tedisamil dosing. Arrhythmic activity after drug administration was not observed in dogs treated with tedisamil. PES induction of ventricular tachycardia was reduced significantly in the tedisamil-treated group (100% inducible before drug vs. 9% inducible after drug; p < 0.05). In the sudden-cardiac-death protocol, tedisamil reduced the incidence of lethal ischemic arrhythmias developing in response to acute posterolateral myocardial ischemia. Tedisamil-treated animals exhibited a 100% compared with a 25% survival rate in the control group (p < 0.05). Anterior-wall infarct size, expressed as a percentage of the left ventricle, did not differ between groups: Group 1 = 20 +/- 1%; Group 2 = 22 +/- 1%. Our findings suggest that tedisamil might be useful in the prevention of malignant ventricular arrhythmias in myocardial ischemic injury.

Administration, Oral↗

PubMed1996

Tedisamil Attenuates Ventricular Fibrillation in a Conscious Canine Model of Sudden Cardiac Death.

BACKGROUND: The electrophysiologic and antifibrillatory properties of tedisamil (KC-8857;3,7-di-(cyclopropylmethyl)-9,9-tetramethylene-3,7-diazabicyclo[3.3.1]-nonane dihydrochloride) were studied in a conscious canine model of sudden cardiac death. METHODS AND RESULTS: Three to five days after surgically induced myocardial infarction (2-hour occlusion of the left anterior descending coronary artery), animals were subjected to programmed electrical stimulation to identify those at risk for ischemia-induced ventricular fibrillation. Sixty minutes after tedisamil (10 mg/kg, administered orally) PES was repeated. Tedisamil increased the ventricular effective refractory period from 106 +/- 6 to 134 +/- 7 ms (P <.05) compared to placebo treatment, which did not alter the ERP (123 +/- 6 to 116 +/- 5 ms). Tedisamil prolonged the QTc interval, from a predrug value of 308 +/- 14 to 327 +/- 14 ms, postdrug. The extent of the surgically induced anterior wall myocardial infarct did not differ between groups, tedisamil, 29 +/- 2%, and placebo, 28 +/- 2% of the left ventricle. CONCLUSIONS: Tedisamil conferred protection against ischemia induced ventricular fibrillation; 7 of 10 tedisamil-treated dogs survived, compared to 4 of 14 surviving in the vehicle treated group (P <.05). Although we observed instances of vomiting and/or diarrhea in several dogs after a single oral administration of tedisamil, the data indicate that oral administration of tedisamil provides protection from ischemia-induced ventricular fibrillation in the postinfarcted conscious canine. The mechanism by which tedisamil achieves its antifibrillatory effect may be related to its ability to prolong the ERP of the ventricular myocardium without altering ventricular conduction velocity.

Journal Article↗

PubMed1996

Topics in xenobiochemistry--application of microdialysis techniques in pharmacokinetic studies.

The principle of the microdialysis technique, the microdialysis system and its application to pharmacokinetic studies is discussed. By applying the microdialysis technique to pharmacokinetic studies the number of animals needed can be substantially reduced, because the technique involves a sampling technique which does not withdraw body fluid and so does not disturb blood homeostasis. Therefore, there are no limiting factors to the amount of samples taken from one animal. The utility offered by the microdialysis technique will have a bearing on future pharmacokinetic studies and it is to be expected that the importance of microdialysis in pharmacokinetic studies will grow in the future.

Animals↗

PubMed1991

Effects of a standardized meal on the pharmacokinetics of the new cardiotonic agent piroximone.

The influence of food on the pharmacokinetics of piroximone (MDL 19.205, CAS 84490-12-0) was evaluated in two groups of 6 healthy male volunteers receiving either 25 or 50 mg of the drug. Single doses were administered intravenously and orally under fasting conditions or orally with a standard breakfast on 3 different days with a washout period of at least 3 days in-between doses, according to an open, 3-way crossover, randomized design. Pharmacokinetic parameters (Cmax, tmax, AUC, t1/2, Cl, aVd, UEx) were not affected by food administration, but significant differences were found in t1/2 calculated from the decay of plasma concentrations in response to oral administration of 25 mg and 50 mg treatment doses. The urinary excretion of piroximone was significantly reduced after oral administration, when compared with the values obtained after intravenous application. In addition, extra-renal clearance was significantly reduced in the 50 mg treatment group, when compared with the values obtained in response to 25 mg. Bioavailability of piroximone calculated from AUC data compared favorably with data obtained from urinary recovery results.

Adolescent↗

PubMedDate not supplied

The role of Gilbert's syndrome and frequent NAT2 slow acetylation polymorphisms in the pharmacokinetics of retigabine.

Retigabine (RGB) is an investigational antiepileptic drug, which undergoes extensive UGT1A1, 1A9 and 1A4-mediated N-glucuronidation and N-acetylation. The mono-acetylated metabolite of RGB has some pharmacological activity and is denoted AWD21-360. We investigated whether the pharmacokinetics (PK) of RGB and AWD21-360 are altered in subjects with Gilbert's syndrome (GS) and/or with frequent N-acetyltransferase 2 (NAT2) slow acetylator (SA) polymorphisms. Based on consistent genotyping and phenotyping screening results, 37 Caucasian subjects (21-46 years; 31 men, six women) were assigned to one of the following groups: (1) absence of GS (non-GS)/rapid acetylator (RA) (N=11); (2) GS/RA (N=8); (3) non-GS/SA (N=11); (4) GS/SA (N=7). Subjects received single and multiple (b.i.d.) 200-mg oral RGB doses over 5 days. Blood samples were collected up to 60 h after dosing for plasma PK of RGB and AWD21-360. Group comparisons were performed by ANOVA. Single-dose PK of RGB and AWD21-360 and multiple-dose PK of RGB did not differ significantly between groups. After multiple dose treatment, RA subjects showed a significantly higher total exposure to AWD21-360 of about 32% (95% CI 101.9-172.5) relative to SA subjects (P=0.0362). The UGT1A1 metabolic capacity (i.e. presence or absence of GS), however, did not significantly affect the overall exposure to AWD21-360. The results indicate that the PK of RGB is unaltered in individuals with GS, in subjects with NAT2 SA status, and in carriers of both variants, whereas the total exposure to AWD21-360 is significantly related to the RA or SA status of subjects. Results further suggest that metabolic switching to the mono-acetylated metabolite AWD21-360 may partially compensate for the impaired glucuronidation capacity in GS subjects. RGB treatment showed no significant differences in tolerability and safety between groups.

Acetylation↗

PubMedDate not supplied

Expression of basolateral and canalicular transporters in rat liver and cultures of primary hepatocytes.

Basolateral and canalicular proteins are expressed in the liver and besides their role in the transport of bilirubin, glutathione, hormones and various glucuronides, they also function as transporters of a wide range of drugs. Despite their frequent use in drug research, little is known about the expression of genes coding for transporters in cultures of primary sandwiched hepatocytes. The kinetics of gene expression of canalicular and basolateral membrane transporters in cultures of primary rat sandwiched hepatocytes were investigated, and the expression of cMOAT, spgp, mdrla and mdr2 were shown to be comparable with transcript levels observed in vivo. Strikingly, expression of the basolateral membrane transporter ntcp and oatpl and 2 were dramatically reduced, the level being < 10 and < 5%, respectively, of those found in vivo in rat liver tissue. Notably, mRNA expression of the canalicular membrane transporter mdrlb was increased up to 13-fold. The findings point to a dramatic change in the expression of basolateral and canalicular transporters in cultured hepatocytes, and this should be considered when hepatocytes are used for drug profiling studies.

ATP Binding Cassette Transporter, Subfamily B↗

PubMedDate not supplied

Isolation and characterization of metabolically competent pulmonary epithelial cells from pig lung tissue.

Administration of drugs by inhalation opens new possibilities for entry into the systemic circulation and cultures of porcine pulmonary epithelial cells (PECs) may prove to be valuable in the prediction of pulmonary metabolism of drugs in humans. This paper, therefore, reports a method for the routine isolation and cultivation of PECs from slaughterhouse animals. On average 1.5x10(6) cells g-1 tissue were isolated by discontinuous density-gradient centrifugation. Cells were subsequently cultivated on collagen-coated plates and characterized by staining for alkaline phosphatase, by tannic acid staining of lamellar bodies and by surfactant protein (SP) expression at days 0, 3 and 6 in culture. Over 70% of purified cells were positive for SP-C and tannic acid staining and thus defined as epithelial cells of alveolar origin (AECs). The AEC phenotype was also confirmed by specific binding of marker lectins (Maclura pomifera and Helix pomatia) and by studying gene expression and activity of cytochrome P450 monooxygenases. Testosterone, ethoxyresorufin, benzyloxyresorufin and verapamil were used as substrates for cytochrome P450-catalysed oxidations and cultured cells were found to be differentiated as well as metabolically competent during cultivation. Therefore, this culture system enables in depth pulmonary biotransformation and toxicity studies.

Animals↗