Search PubMed⌕ Search

Biomedical subjects

T Walle

Publications and source records attributed to T Walle.

At least 37 records · Page 2Linked to original sources
PubMed1999

Phase I and pharmacologic study of a 3-hour infusion of paclitaxel followed by cisplatinum and 5-fluorouracil in patients with advanced solid tumors.

A Phase I and pharmacological study of paclitaxel administered as an outpatient, 3-h i.v. infusion just before a 5-day regimen of daily cisplatinum (CP) and a continuous infusion of 5-fluorouracil (5-FU) was performed in patients with advanced solid tumors. A secondary objective was to determine the objective response rate to this regimen. Forty-two patients were enrolled and were evaluable for toxicities. Eighteen patients were previously untreated, whereas the rest had received prior treatment with radiation (J. H. Schiller et al., J. Clin. Oncol., 12: 241-248, 1994), chemotherapy (M. J. Kennedy et al., Clin. Cancer Res., 4: 349-356, 1998), or both modalities (J. H. Schiller et al., J. Clin. Oncol., 12: 241-248, 1994). The paclitaxel dose was escalated from 100-135-170-200-225 to 250 mg/m2, whereas i.v. 5-FU and CP doses were fixed at 1.0 g/m2/day continuous infusion and 20 mg/m2/day, respectively, daily for 5 days. Granulocyte colony-stimulating factor (G-CSF; 5 microg/kg/day) was administered s.c. from day 6, routinely after 250 mg/m2 dose of paclitaxel or after a lower dose of paclitaxel if ANC <500/microl or febrile neutropenia was observed. Patients were treated every 28 days. Plasma and urine samples were collected to determine the pharmacokinetics of paclitaxel. In previously untreated patients, the maximally tolerated dose of paclitaxel in the drug regimen was determined to be 170 mg/m2 without and 250 mg/m2 with G-CSF support. At the higher dose level, mucositis and thrombocytopenia were dose-limiting. In previously treated patients, these toxicities were observed at all dose levels of paclitaxel > or =135 mg/m2. With increasing doses of paclitaxel, a disproportionate increase in the peak concentrations, as well as the area under plasma concentration time-curve, was seen. This nonlinearity was due to saturable total body clearance and volume of distribution of paclitaxel (P < 0.001). The apparent plasma elimination half-life was unaffected by the dose of paclitaxel. CP and 5-FU had no apparent effect on the metabolism of paclitaxel. Among 32 patients evaluable for response, 22 demonstrated an objective response, including five complete remissions. Therefore, a regimen of 3-h infusion of 250 mg/m2 paclitaxel before CP and FU is tolerable with G-CSF (as above) support in previously untreated patients. The regimen also seems to be highly active against breast and esophageal cancers.

Adult↗
PubMed1999

Transport of genistein-7-glucoside by human intestinal CACO-2 cells: potential role for MRP2.

This study demonstrated rapid apical to basolateral absorption of genistein across Caco-2 cell monolayers with a P(app) of 20.0+/-0.8 x 10(-6)cm/sec. In contrast, genistein's main dietary source, genistein-7-glucoside (genistin), was not absorbed. In fact, genistin demonstrated basolateral to apical efflux of 1.28+/-0.10 x 10(-6) cm/sec, which exceeded the flux of the paracellular transport marker mannitol by 6-fold. Although genistin hydrolysis to genistein occurred in the preparation, it did not contribute significantly to these observations. In experiments trying to define the efflux mechanism for genistin, MK-571 reduced the efflux by 87% to 0.19+/-0.02 x 10(-6) cm/sec, implying transport by the polarized efflux pump MRP2.

Anion Transport Proteins↗
PubMed1998

Transport of quercetin and its glucosides across human intestinal epithelial Caco-2 cells.

There is mounting evidence from human epidemiological, animal in vivo, and in vitro studies to suggest beneficial effects related to the consumption of quercetin and its glucosides. However, there is limited knowledge on the oral bioavailability of these natural products. This study examined the intestinal epithelial membrane transport of quercetin, quercetin 4'-glucoside, and quercetin 3,4'-diglucoside, using the Caco-2 human colonic cell line, a model of human intestinal absorption. The apparent permeability (Papp) of each agent was measured in both apical to basal and basal to apical directions. The apical to basolateral flux of quercetin, Papp 5.8 +/- 1.1 x 10(-6) cm x sec(-1) (mean +/- SEM), was more than 10-fold higher than for the paracellular transport marker mannitol, 0.48 +/- 0.09 x 10(-6) cm x sec(-1) (P < 0.01). Under identical conditions, the Papp for the transcellular marker propranolol was about 5-fold higher than for quercetin (P < 0.001). Interestingly, the reverse, basolateral to apical, flux of quercetin (Papp 11.1 +/- 1.2 x 10(-6) cm x sec(-1)) was almost 2-fold higher than the apical to basolateral flux (P < 0.001). In similar experiments, quercetin 4'-glucoside demonstrated no absorption, Papp < 0.02 x 10(-6) cm x sec(-1) in the apical to basal direction, but did demonstrate basal to apical flux, Papp 1.6 +/- 0.2 x 10(-6) cm x sec(-1). Quercetin 3,4'-diglucoside showed a low apical to basolateral transport (Papp 0.09 +/- 0.03 x 10(-6) cm x sec(-1)); its reverse, basolateral to apical, transport was, however, 4-fold higher (P < 0.05). In these cells, glucose was actively transported with an apical to basolateral Papp of 36.8 +/- 1.1 x 10(-6) cm x sec(-1). These observations suggest facile absorption of quercetin through the human intestinal epithelium, but contrary to a previous proposal, they do not support an active transport process for quercetin glucosides.

Caco-2 Cells↗
PubMed1998

Enantioselective sulfation of beta 2-receptor agonists by the human intestine and the recombinant M-form phenolsulfotransferase.

The beta 2-receptor agonist class of drugs is metabolized in humans almost exclusively by sulfate conjugation. The objective of this investigation was to determine the influence of chemical structure on the stereoselectivity of the sulfoconjugation of these chiral drugs. The pure enantiomers of six beta 2-agonists, including those clinically most widely used, were all effectively sulfated both by the cytosol of the human intestine and the recombinant human M-form phenolsulfotransferase (PST). Whereas the apparent Km values (Km,app) for the sulfation of the individual drug enantiomers by the intestinal cytosol varied widely, ranging from 4.8 microM for (S)-isoproterenol to 889 microM for (S)-albuterol, these Km,app values were highly correlated with those obtained with M-PST (correlation coefficient 0.994). In contrast, the M-PST Vmax,app values were similar for all drug enantiomers, ranging from 276 to 914 pmol min-1 mg-1 protein, implying that substrate binding to M-PST by far is the main determinant of the sulfation activity. For isoproterenol, the Km,app for M-PST was 6.1 times higher for the active (R)- than for the inactive (S)-enantiomer. For other beta 2-agonists, the stereoselectivity decreased towards unity as the Km,app increased. However, for albuterol, containing a hydroxymethyl substituent at the aromatic ring, the stereoselectivity was dramatically reversed, with 10 times higher Km,app for the inactive (S)- than for the active (R)-enantiomer.

Adrenergic beta-2 Receptor Agonists↗
PubMed1998

Bioactivation of the cooked food mutagen N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by estrogen sulfotransferase in cultured human mammary epithelial cells.

Cooked food mutagens from fried meat and fish have recently been suggested to contribute to the etiology of breast cancer. Thus, the most prevalent of these compounds, i.e. 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, or rather its more mutagenic N-hydroxylated metabolite (N-OH-PhIP), forms DNA adducts in mammary cells, including human mammary epithelial (HME) cells. The objective of this study was to determine the involvement of estrogen sulfotransferase (EST), the only sulfotransferase identified in HME cells, in the further bioactivation of N-OH-PhIP. These studies were done in vitro using human recombinant EST and in intact HME cells. Human recombinant EST increased the covalent binding of [3H]N-OH-PhIP to calf thymus DNA approximately 3.5-fold in the presence of the sulfotransferase co-substrate 3'-phosphoadenosine-5'-phosphosulfate at each N-OH-PhIP concentration (1, 10 and 100 microM) (n = 6, P < 0.001). In contrast, EST did not catalyze the DNA binding of two other cooked food mutagens, N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline and N-hydroxy-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, which are mainly hepatocarcinogens. Cultured HME cells displayed high EST activity, which could be completely inhibited by 1 microM estrone. When the cells were incubated with [3H]N-OH-PhIP, binding to native DNA occurred at 60-240 pmol/mg DNA. This binding was inhibited to 55% of control by 1 microM estrone (P < 0.01, n = 8), suggesting that EST plays a significant role in carcinogen bioactivation in human breast tissue.

Biotransformation↗
PubMed1998

Extensive binding of the bioflavonoid quercetin to human plasma proteins.

Although the bioflavonoids, a large group of polyphenolic natural products, exert chemopreventive effects in cardiovascular disease and cancer, there is little information about the disposition of these dietary components in man. The objective of this study was to investigate the plasma-protein binding of the most abundant bioflavonoid, quercetin, using 14C-labelled quercetin. An ultracentrifugation assay (170,000 g for 16 h at 20 degrees C) was shown to sediment plasma proteins. Binding of quercetin to normal plasma was extensive (99.1+/-0.5%, mean +/- s.d., n = 5). The unbound fraction varied as much as 6-fold, 0.3-1.8%, between subjects. This high binding was independent of quercetin concentration over the range 1.5-15 microM (0.5-5 microg mL(-1)). Human serum albumin was the primary protein responsible for the binding of quercetin in plasma (99.4+/-0.1%). Binding by alpha1-acid glycoprotein (39.2+/-0.5%) and very-low-density lipoproteins (<0.5% of total quercetin) did not make substantial contributions to overall plasma binding. The equilibrium association constant for the binding of quercetin to serum albumin was 267+/-33 x 10(3) M(-1) (n = 15). Thermodynamic data for the binding of quercetin to serum albumin indicated spontaneous, endothermic association. Displacement studies suggested that in man the 'IIA' subdomain binding site of human serum albumin was the primary binding site for quercetin. Association of quercetin with erythrocytes was significantly (P < 0.001) reduced by plasma protein binding. These data indicate poor cellular availability of quercetin because of its extensive binding to plasma proteins.

Blood Proteins↗
PubMed1998

Taxol transport by human intestinal epithelial Caco-2 cells.

Taxol (paclitaxel) belongs to a new class of antimicrotubule anticancer drugs with clinical activity against common solid tumors and acute leukemias. Preclinical studies have suggested that taxol is not absorbed after oral doses. However, whether the observed low oral bioavailability is the result of poor absorption or extensive presystemic hepatic metabolism is not clear. For this reason, we studied the transepithelial flux of taxol, using the human colonic cell line Caco-2 as a model. The cells were grown to confluency on permeable polycarbonate membrane inserts, to permit flux experiments after loading of [3H]taxol on either the apical or basolateral side. The flux of taxol across the Caco-2 cell layer was linear with time for up to 3 hr. The flux from the basolateral to the apical side was 4-10 times greater than that from the apical to the basolateral side. Whereas the absorptive transport appeared linearly related to the taxol concentration (0.5-20 microM), the efflux was saturable. The apparent KM of the active efflux component was 16.5 microM. Verapamil (50 microM) significantly decreased the active transport component. These data support the conclusion that rapid passive diffusion of taxol through the intestinal epithelium is partially counteracted by the action of an outwardly directed efflux pump, presumably P-glycoprotein. However, the relatively high apparent permeability coefficient for the apical to basolateral taxol transport (4.4 +/- 0.4 x 10(-6) cm/s; N = 17) suggests that the drug may still be effectively absorbed in the intestinal tract.

Antineoplastic Agents, Phytogenic↗
PubMed1997

Stereoselective sulphate conjugation of fenoterol by human phenolsulphotransferases.

1. The objective of this study was to determine (1) the molecular site(s) of sulphoconjugation of fenoterol; (2) the human phenolsulphotransferase (PST) isoform(s) involved; and (3) the stereochemistry of the enzymatic reaction. 2. Using the human Hep G2 cell line, hplc isolation and FAB/ms/ms, it was determined that fenoterol is sulphated both in the 4'-hydroxyphenyl position and in one of the 3',5'-dihydroxyphenyl positions. 3. Recombinant human M-PST preferentially sulphated the 4'-hydroxyphenyl position. In contrast, recombinant P-PST exclusively sulphated the 3',5'-hydroxyphenyl position. 4. The M-PST-catalysed sulphation of the 4'-hydroxyphenyl position was highly selective for the active RR-enantiomer, whereas the sulphation of the 3',5'-dihydroxyphenyl position was slightly selective for the opposite SS-enantiomer. 5. The P-PST-catalysed sulphation of the 3',5'-hydroxyphenyl position was selective for the inactive SS-enantiomer.

Bronchodilator Agents↗
PubMed1996

Stimulatory as well as inhibitory effects of ethinyloestradiol on the metabolic clearances of propranolol in young women.

1. The metabolism of a single 80 mg oral dose of propranolol was determined in nine young women before and after administration of ethinyloestradiol alone (EE2) or in combination with norethindrone (OC). 2. Whereas the total clearance of propranolol (2713 +/- 404 ml min-1 (mean +/- s.e.mean)) was not significantly altered by either EE2 (3365 +/- 347 ml min-1) or the combined OC (2905 +/- 345 ml min-1), significant changes in all three primary metabolic pathways were observed. 3. The clearance through side-chain oxidation decreased from 345 +/- 55 ml min-1 to 262 +/- 33 ml min-1 after EE2 (P < 0.05). A similar reduction of cytochrome P450 metabolism by EE2 has been observed for other drugs. 4. The clearance through glucuronidation increased from 364 +/- 61 ml min-1 to 625 +/- 117 ml min-1 after EE2 (P < 0.01). Similar stimulation of glucuronic acid conjugation by EE2 has also been observed for other drugs. 5. The clearance through ring oxidation increased from 697 +/- 109 ml min-1 to 1280 +/- 162 ml min-1 after EE2 (P < 0.01). This observation appears to be a novel finding with EE2 and cytochrome P450 metabolism. 6. The treatment with OC produced changes in propranolol's metabolic clearances which were qualitatively similar to those generated by EE2.

Adrenergic beta-Agonists↗
PubMed1996

Stereoselective sulphate conjugation of salbutamol by human lung and bronchial epithelial cells.

1. The metabolism of (+)-, (-)- and (+/-)-salbutamol by sulphoconjugation was determined in vitro using human lung cytosol and bronchial epithelial BEAS-2B cell homogenate. 2. For the lungs the intrinsic clearance (Vmax/Km) value for the pharmacologically active (-)-salbutamol (0.49 +/- 0.32 ml min-1 g-1 protein) exceeded that of (+)-salbutamol (0.046 +/- 0.028 ml min-1 g-1 protein) by 11-fold. This was mainly due to a difference in Km value, which was 16 times higher for (+)-salbutamol (1300 +/- 170 microM) than for (-)-salbutamol (83 +/- 12 microM). 3. The stereoselectivity of sulphoconjugation of salbutamol was very similar in the BEAS-2B cells, although the absolute activity was considerably lower. 4. The enzyme catalyzing this reaction both in the lungs and in the BEAS-2B cells was the monoamine (M) form phenolsulphotransferase. 5. These observations emphasize that the smooth muscle of the bronchi most likely are exposed to considerably higher concentrations of the potentially toxic (+)-enantiomer than of the bronchodilating (-)-enantiomer during therapy with (+/-)-salbutamol.

Albuterol↗
PubMed1996

Flavonoids, potent inhibitors of the human P-form phenolsulfotransferase. Potential role in drug metabolism and chemoprevention.

The common dietary constituent quercetin was a potent inhibitor of sulfoconjugation of acetaminophen and minoxidil by human liver cytosol, partially purified P-form phenolsulfotransferase (PST), and recombinant P-form PST, with IC50 values of 0.025-0.095 microM. Quercetin inhibition of acetaminophen was noncompetitive with respect to acceptor substrate, with a Ki value of 0.067 microM. A number of other flavonoids, such as fisetin, galangin, myricetin, kaempferol, chrysin, and apigenin, were also potent inhibitors of P-form PST-mediated sulfation, with IC50 values < 1 microM. Studies of structural analogs indicated the flavonoid 7-hydroxyl group as particularly important for potent inhibition. Potential human metabolites of quercetin were poor inhibitors. Curcumin, genistein, and ellagic acid (other polyphenolic natural products) were also inhibitors of P-form PST, with IC50 values of 0.38-34.8 microM. Quercetin was also shown to inhibit sulfoconjugation by the human hepatoma cell line Hep G2. Although less potent in this intact cell system (IC50 2-5 microM), quercetin was still more potent than 2,6-dichloro-4-nitrophenol, the classical P-form PST inhibitor that has been shown to be an inhibitor also in vivo. These observations suggest the potential for clinically important drug interactions, as well as a possible role for flavonoids as chemopreventive agents in sulfation-induced carcinogenesis.

Arylsulfotransferase↗
PubMed1996

Improved bacterial expression of the human P form phenolsulfotransferase. Applications to drug metabolism.

Bacterial expression of human phenol phenolsulfotransferase (P-PST) has provided the opportunity to understand better the catalytic properties and biological role of this enzyme. However, as the yield of pure protein from the currently used expression system was low, we subcloned the P-PST c-DNA into pET-15b, a vector containing an oligohistidine domain, for improved expression. The fusion protein, His-P-PST, was isolated from the bacterial cytosol in a single affinity chromatography step, using a Ni2+ agarose column. The yield of His-P-PST from the pET-15b vector was improved 12-fold, compared with P-PST from the original vector. The purity was > 99%, as established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry scanning. The enzyme was stable for at least 3 weeks when stored in 20% glycerol at -80 degrees C. A very rapid deterioration of the enzyme during 37 degrees C incubations was effectively prevented by the addition of bovine serum albumin. The sulfonation of several substrates was very similar for His-P-PST and P-PST, with Vmax/KM values (first order rate constants) for the high-affinity substrate p-nitrophenol of 143 +/- 27 and 120 +/- 25 ml min-1 microgram-1 PST [mean +/- SE; not significant (NS)], respectively, and for the low-affinity substrate acetaminophen of 0.21 +/- 0.11 and 0.14 +/- 0.07 ml min-1 microgram-1 PST (NS). The Vmax/KM for the sulfonation of the isoproterenol enantiomers showed a (+)/(-)-enantiomer ratio of 6.2 for His-P-PST and 7.4 for P-PST. Interestingly, 3- to 10-fold higher apparent KM values were obtained for these substrates with the crude human liver cytosol, compared with the recombinant P-PSTs, suggested to be due to endogenous or dietary P-PST inhibitors in the liver. In addition, the inhibition of acetaminophen sulfonation by quercetin was very similar for His-P-PST and P-PST, with IC50 values of 0.10 +/- 0.03 and 0.05 +/- 0.01 microM (NS), respectively. The additional amino acid residues in the His-P-PST, compared with the recombinant P-PST, thus did not significantly alter the catalytic properties. This bacterial expression system should lend itself to routine use in studies of the metabolism of drugs and environmental chemicals.

Arylsulfotransferase↗
PubMed1995

Quercetin, a potent and specific inhibitor of the human P-form phenosulfotransferase.

The natural product quercetin was a potent inhibitor of the human P-form phenolsulfo-transferase with an IC50 value of 0.10 +/- 0.03 microM (mean +/- SEM; N = 5), which was three to four orders of magnitude more potent than its inhibition of other human sulfotransferases. The inhibition was noncompetitive with a Ki value of 0.10 microM. The potency and mechanism of this inhibition appear similar to those of the current standard P-form inhibitor, 2,6-dichloro-4-nitrophenol. Among other flavonoids examined, kaempferol was found to have an IC50 value of 0.39 +/- 0.07 microM, naringenin 10.6 +/- 1.6 microM and naringin 265 +/- 90 microM (N = 3). These observations suggest the potential for clinically important pharmacologic and toxicologic interactions by flavonoid-containing foods and beverages.

Arylsulfotransferase↗
PubMed1995

Extensive and saturable accumulation of paclitaxel by the human platelet.

Little is known about the cellular distribution of paclitaxel in humans. In the present study we examined the distribution of [3H]-paclitaxel in human blood. When 1 microM paclitaxel was incubated with fresh blood at 37 degrees C, the platelet/plasma concentration ratio was 240 +/- 17 (mean +/- SEM), whereas the red blood cell (RBC)/plasma concentration ratio was only 0.59 +/- 0.05. In kinetics experiments using platelet-rich plasma, we observed that the platelet accumulation of paclitaxel was highly temperature- and concentration-dependent. Scatchard analysis of the 37 degrees C uptake data demonstrated a dissociation constant (Kapp) of 0.80 +/- 0.10 microM and a maximal binding capacity of 672 +/- 102 pmol/10(9) platelets. It is proposed that the platelet accumulation of paclitaxel reflects binding to microtubules and may serve as a useful model for binding to less accessible cellular sites.

Adult↗
PubMed1995

Comparative in vitro cytotoxic effects of taxol and its major human metabolite 6 alpha-hydroxytaxol.

Taxol is metabolized by the liver microsomal cytochrome P450 enzyme system into its principal metabolite 6 alpha-hydroxytaxol (6HT). In the present in vitro studies 6HT was compared to taxol with respect to its effects on tubulin depolymerization, mitotic arrest, clonogenic survival and apoptosis in HL-60 cells. 6HT was generated by incubating taxol with human liver microsomes in a NADPH-generating system. HL-60 cells were incubated for 24 h with either taxol or 6HT, washed and placed in drug-free suspension or cultured for colony growth in agarose. For the suspension and colony culture growth of the cells, the IC50 concentrations of 6HT were 500 +/- 46 and 350 +/- 37 nM, while those of taxol were 3.2 +/- 0.2 and 2.8 +/- 0.5 nM, respectively. Immediately after a 24-h exposure of HL-60 cells to 50 nM taxol, electrophoresis of genomic DNA from HL-60 cells revealed an internucleosomal DNA fragmentation 'ladder'. In addition, 39% of the cells were arrested in mitosis and 16% showed the morphologic features of apoptosis. In contrast, an identical treatment with 6HT resulted in the mitotic arrest of only 2.8% of the cells, with 4.0% displaying apoptosis (P < 0.01); internucleosomal DNA fragmentation was not observed. 6HT was also significantly less effective than taxol in inhibiting the temperature-induced depolymerization of microtubules in a cell-free system. However, at equipotent concentrations, the effect of 6HT on tubulin depolymerization, mitotic arrest or apoptosis was similar to that of taxol. In addition, at concentrations of taxol or 6HT at or below their IC50, there was little tubulin depolymerization, mitotic arrest or apoptosis. The results presented here show that the biotransformation of taxol to 6HT substantially detoxifies taxol.

Apoptosis↗
PubMed1995

Taxol metabolism and disposition in cancer patients.

The objective of this study was to determine the metabolic fate and disposition of taxol in cancer patients. Five patients received 225 or 250 mg/m2 of taxol together with 100 microCi of [3H]taxol as a 3-hr infusion, followed by cisplatin and 5-fluorouracil. Urine, feces, and blood samples were collected for 120 hr and analyzed for total radioactivity, taxol, and metabolites by reversed-phase HPLC and tandem MS. Total urinary excretion was 14.3 +/- 1.4% (SE) of the dose, with unchanged taxol and an unknown polar metabolite as the main excretion products. Total fecal excretion was 71.1 +/- 8.2%, with 6 alpha-hydroxytaxol being the largest component by far. Unchanged taxol and four other metabolites could also be identified from fecal extracts. The plasma area under the curve for unchanged taxol was 20.5 +/- 2.3 microM.hr and that for total taxol metabolites was 14.2 +/- 4.5 microM.hr. The half-life of total metabolites (5.6 +/- 0.4 hr), however, greatly exceeded that of unchanged taxol (2.9 +/- 0.3 hr). Thus, at 5-hr posttaxol infusion, the plasma concentrations of the five metabolites together exceeded the taxol concentration by 2.4-fold. The findings from this study should be of importance as a guide to further therapeutic evaluation of this drug.

Adult↗