Erythromycin breath test.
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Anti-Anxiety Agents↗
Biomedical subjects
P B Watkins
Publications and source records attributed to P B Watkins.
The effect of an individual's cytochrome CYP3A4 activity on docetaxel clearance.
Docetaxel is a chemotherapeutic agent effective in the treatment of various solid tumors. Patients given a standard dose of docetaxel exhibit wide interpatient variation in clearance (CL) and toxic effects. Docetaxel undergoes metabolism by cytochrome CYP3A4. Thus, interpatient variability in CYP3A4 activity may account in part for differences in toxicity and CL. Twenty-one heavily pretreated patients with metastatic sarcomas received docetaxel (100 mg/m2). Hepatic CYP3A4 activity in each patient was measured by the [14C-N-methyl]erythromycin breath test (ERMBT). Blood samples were taken at selected times over the next 24 h for pharmacokinetic analysis. Phenotypic expression of hepatic CYP3A4 activity measured by the ERMBT varied over 20-fold (administered 14C exhaled in 1 h: mean, 2.53%; range, 0.25-5.35%), which is similar to a normal control population. CL of docetaxel varied nearly 6-fold (mean, 21.0 liters/h/m2; range, 5.4-29.1 liters/h/m2). The ERMBT was the best predictor of CL when compared with serum alanine aminotransferase, albumin, alkaline phosphatase, or serum alpha-1-acidic glycoprotein. The natural log of ERMBT accounted for 67% of the interpatient variation in CL. Multivariate analysis showed that the natural log of ERMBT and albumin together accounted for 72% of the interpatient variation in CL. The greatest toxicity was seen in patients with the lowest ERMBT. Hepatic CYP3A4 activity is the strongest predictor of docetaxel CL and accounts for the majority of interpatient differences in CL. Patients with low CYP3A4 activity are at risk for having decreased CL and may thus experience increased toxicity from docetaxel. Those with high activity may be receiving a suboptimal dose. By measuring CYP3A4 activity, the ERMBT may be clinically useful in tailoring doses of CYP3A4 substrates, such as docetaxel, in certain individuals.
Adult↗
Effects of a chargrilled meat diet on expression of CYP3A, CYP1A, and P-glycoprotein levels in healthy volunteers.
BACKGROUND & AIMS: Carcinogenic heterocyclic amines and polycyclic aromatic hydrocarbons present in chargrilled meat are substrates for inducible CYP1A and CYP3A enzymes and for P-glycoprotein. We examined whether consumption of a chargrilled meat diet results in induction of these proteins. METHODS: Ten healthy adults were fed a diet enriched with chargrilled meat for 7 days. Duodenal biopsy specimens were obtained on days 1, 5, and 12 and analyzed for CYP1A, CYP3A, and P-glycoprotein messenger RNA (mRNA) and protein. On days 5 and 12, hepatic CYP3A4 and CYP1A2 activities were measured and colon biopsies were performed. The levels of polycyclic aromatic hydrocarbon DNA adducts in peripheral blood mononuclear cells were measured on days 1, 4, 11, and 26. RESULTS: There was no detectable induction of CYP3A4, CYP3A5, or P-glycoprotein mRNAs or protein in small intestine or colon and no induction of hepatic CYP3A4 enzyme activity. In contrast, the chargrilled meat diet resulted in unequivocal induction of CYP1A enzymes in the liver and small intestine of each subject. There was an inverse correlation between the level of peripheral polycyclic aromatic hydrocarbon DNA adducts measured on day 11 and both liver CYP1A2 activity (P = 0.027) and enterocyte CYP1A1 protein concentration (P = 0.046). CONCLUSIONS: Ingestion of chargrilled meat results in induction of CYP1A enzymes but not CYP3A4 or P-glycoprotein. This observation, combined with the correlation between adduct levels and CYP1A expression, supports an adaptive role for CYP1A but not CYP3A4 or P-glycoprotein.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Cytochrome P-450 1A1 expression in human small bowel: interindividual variation and inhibition by ketoconazole.
Human cytochrome P-450 1A1 (CYP1A1) is located primarily in extrahepatic tissues. To begin the characterization of this enzyme in the small intestine, we screened a bank of 18 human small intestinal microsomal preparations for CYP1A1 catalytic [(7-ethoxyresorufin O-deethylase (EROD)] activity and protein content. Although EROD activity was below detectable limits in 12 of the preparations, 6 exhibited measurable activity (1.4-123.5 pmol/min/mg), some exceeding that for 2 human liver microsomal preparations (11.0 and 26.4 pmol/min/mg). This variation was not due to variable quality of the preparations because each sample displayed readily detectable CYP3A4 catalytic activity and immunoreactive protein. We inadvertently found that intestinal EROD activity was inhibitable by ketoconazole at a concentration commonly believed to selectively inhibit CYP3A4. The possibility that CYP3A4 metabolizes 7-ethoxyresorufin was excluded because there was no correlation between intestinal CYP3A4 catalytic and EROD activity, and cDNA-expressed human CYP3A4 exhibited no EROD activity. Moreover, CYP1A1 immunoreactive protein was most abundant in the three intestinal preparations with the highest EROD activities, and the mean apparent Ki of ketoconazole observed for these three preparations (40 nM) was essentially identical with that for cDNA-expressed human CYP1A1 (37 nM). In summary, there is large interindividual variation in CYP1A1 expression in human small bowel, and ketoconazole is not a selective CYP3A4 inhibitor in in vitro metabolism studies involving intestinal tissue obtained from some individuals. These observations raise the possibility that in vivo drug interactions involving ketoconazole could result from CYP1A1 inhibition in the intestine in some individuals.
Blotting, Western↗
First-pass midazolam metabolism catalyzed by 1alpha,25-dihydroxy vitamin D3-modified Caco-2 cell monolayers.
Cytochrome P-450 (CYP) 3A4 accounts for approximately 50% of all P-450s found in the small intestine (Paine et al., 1997) and contributes to the extensive and variable first-pass extraction of drugs such as cyclosporine and saquinavir. We recently demonstrated that CYP3A4 expression in a differentiated Caco-2 subclone is increased when cell monolayers are treated with 1alpha,25-dihydroxy-vitamin-D3 (Schmiedlin-Ren et al., 1997). This improved metabolic capacity permits the in vitro modeling of first-pass intestinal metabolic kinetics. Midazolam (MDZ) 1'-hydroxylation was used as a specific probe for CYP3A-mediated metabolism in modified Caco-2 monolayers. Caco-2 cells were grown to confluence on laminin-coated culture inserts, and then for two additional weeks in the presence of 1alpha,25-dihydroxy vitamin-D3. Cell monolayers were subsequently exposed to MDZ for varying lengths of time and concentrations. The amount of MDZ in the monolayer increased rapidly after apical drug administration, reaching a pseudo steady state within 6 min. The cellular uptake rate was considerably slower after a basolateral dose. By either route of administration, the rate of 1'-hydroxymidazolam formation was stable and linear for 2 h. Under basolateral sink conditions and low apical MDZ dosing concentration (1-8 microM), the first-pass extraction ratio was found to be approximately 15%. Higher dosing concentrations led to saturation of the hydroxylation reaction and reduction in the extraction ratio. The modified Caco-2 cell monolayer is an excellent model for studying drug absorption and first-pass intestinal metabolic kinetic processes. In this system, the selective CYP3A probe MDZ was rapidly absorbed, yet extensively metabolized, as is observed in vivo.
Biotransformation↗
Molecular and physical mechanisms of first-pass extraction.
This is a report of a symposium held at the March 1997 meeting of the American Society for Pharmacology and Therapeutics in San Diego. Our understanding of the events that control first-pass drug elimination in humans has increased tremendously by two sequential discoveries. First, cytochrome P-450s 3A4 and 5 are expressed at high concentrations in both hepatocytes and upper intestinal enterocytes, and therefore limit the systemic availability of many drugs. Second, P-glycoprotein is expressed at the lumenal surface of the intestinal epithelium and therefore also acts to oppose the absorption of unchanged drug. The following discussion brings together our current understandings of these interrelated phenomena to aid a more complete picture of how they may contribute both qualitatively and quantitatively to first-pass elimination.
Animals↗
Hepatic dysfunction associated with troglitazone.
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Alanine Transaminase↗
Caffeine based measures of CYP1A2 activity correlate with oral clearance of tacrine in patients with Alzheimer's disease.
AIMS: To study the potential utility of caffeine based probes of CYP1A2 enzyme activity in predicting the pharmokinetics of tacrine in patients with Alzheimer's disease. METHODS: The pharmokinetics of a single 40 mg oral dose of tacrine were measured in 19 patients with Alzheimer's disease. Each patient also received 2 mg kg(-1) [13C-3-methyl] caffeine orally and had breath and urine samples collected. RESULTS: Tacrine oral clearance (CL F(-1) kg(-1)), which varied 15-fold among the patients, correlated significantly with the 2 h total production of 13CO2 in breath (r=0.56, P=0.01), and with each of two commonly used urinary caffeine metabolite ratios: the 'paraxanthine/caffeine ratio' (1,7X + 1, 7U)/1,3,7X) (r=0.76, P=0.0002) and the 'caffeine metabolic ratio' (AFMU + 1X + 1U)/1, 7U)(r=0.76, P=0.0001). CONCLUSIONS: These observations support a central role for CYP1A2 in the in vivo disposition of tacrine and the potential for drug interactions when tacrine treated patients receive known inducers or inhibitors of this enzyme. The magnitude of the correlations we observed, however, are probably not sufficient to be clinically useful in individualizing tacrine therapy.
Administration, Oral↗
Sequences of intestinal and hepatic cytochrome P450 3A4 cDNAs are identical.
Cytochrome P450 (CYP or P450) 3A4 is known to be the major P450 expressed in the liver. More recently, CYP3A4 was also shown to be the major P450 in the intestine, where it plays an important role in the metabolism of some orally administered drugs. However, studies examining the catalytic properties of CYP3A4 have been largely based on the use of CYP3A4 enzyme obtained from liver or recombinant protein expressed from hepatic cDNA. To investigate whether intestinal and hepatic CYP3A4 enzymes are identical, we determined the sequences of intestinal CYP3A4 cDNAs. Compared with the published sequence for hepatic CYP3A4, we found a single base pair difference in the 3' untranslated region of intestinal cDNA from three individuals. We found this same base pair difference in cDNA obtained from the livers of three additional subjects. We conclude that hepatic and intestinal CYP3A4 cDNAs are identical and that the proteins expressed in these two tissues are therefore likely to be the same.
Cloning, Molecular↗
Grapefruit juice increases felodipine oral availability in humans by decreasing intestinal CYP3A protein expression.
The increase in oral availability of felodipine and other commonly used medications when taken with grapefruit juice has been assumed to be due to inhibition of CYP3A4, a cytochrome P450 that is present in liver and intestine. To evaluate the effect of repeated grapefruit juice ingestion on CYP3A4 expression, 10 healthy men were given 8 oz of grapefruit juice three times a day for 6 d. Before and after receiving grapefruit juice, small bowel and colon mucosal biopsies were obtained endoscopically, oral felodipine kinetics were determined, and liver CYP3A4 activity was measured with the [14C N-methyl] erythromycin breath test in each subject. Grapefruit juice did not alter liver CYP3A4 activity, colon levels of CYP3A5, or small bowel concentrations of P-glycoprotein, villin, CYP1A1, and CYP2D6. In contrast, the concentration of CYP3A4 in small bowel epithelia (enterocytes) fell 62% (P = 0.0006) with no corresponding change in CYP3A4 mRNA levels. In addition, enterocyte concentrations of CYP3A4 measured before grapefruit juice consumption correlated with the increase in Cmax when felodipine was taken with either the 1st or the 16th glass of grapefruit juice relative to water (r = 0. 67, P = 0.043, and r = 0.71, P = 0.022, respectively). We conclude that a mechanism for the effect of grapefruit juice on oral felodipine kinetics is its selective downregulation of CYP3A4 in the small intestine.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Role of intestinal P-glycoprotein (mdr1) in interpatient variation in the oral bioavailability of cyclosporine.
Interpatient differences in the oral clearance of cyclosporine (INN, ciclosporin) have been partially attributed to variation in the activity of a single liver enzyme termed CYP3A4. Recently it has been shown that small bowel also contains CYP3A4, as well as P-glycoprotein, a protein able to transport cyclosporine. To assess the importance of these intestinal proteins, the oral pharmacokinetics of cyclosporine were measured in 25 kidney transplant recipients who each had their liver CYP3A4 activity quantitated by the intravenous [14C-N-methyl]-erythromycin breath test and who underwent small bowel biopsy for measurement of CYP3A4 and P-glycoprotein. Forward multiple regression revealed that 56% (i.e., r2 = 0.56) and 17% of the variability in apparent oral clearance [log (dose/area under the curve)] were accounted for by variation in liver CYP3A4 activity (p < 0.0001) and intestinal P-glycoprotein concentration (p = 0.0059), respectively. For peak blood concentration, liver CYP3A4 activity accounted for 32% (p = 0.0002) and P-glycoprotein accounted for an additional 30% (p = 0.0024) of the variability. Intestinal levels of CYP3A4, which varied tenfold, did not appear to influence any cyclosporine pharmacokinetic parameter examined. We conclude that intestinal P-glycoprotein plays a significant role in the first-pass elimination of cyclosporine, presumably by being a rate-limiting step in absorption. Drug interactions with cyclosporine previously ascribed to intestinal CYP3A4 may instead be mediated by interactions with intestinal P-glycoprotein.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Steady-state pharmacokinetics of delavirdine in HIV-positive patients: effect on erythromycin breath test.
OBJECTIVE: The steady-state kinetics of delavirdine and desisopropyldelavirdine were evaluated in human immunodeficiency virus-positive patients after escalating oral doses and after repeated oral administrations at the same dose level. STUDY DESIGN: Patients (n = 8 males) were given escalating oral doses of delavirdine mesylate, in a sequential fashion, over 14 days for phases 1 (200 mg every 8 hours), 2 (300 mg every 8 hours), and 3 (400 mg every 8 hours). Control patients (n = 4 males) were given 300 mg oral doses of drug every 8 hours for all three phases. Hepatic CYP3A activity was evaluated with the erythromycin breath test (ERMBT). RESULTS: In the escalating-dose group, delavirdine displayed nonlinear kinetics as indicated by the decreasing oral clearance, maximum steady-state plasma concentration/minimum steady-state plasma concentration ratio, and log-linear terminal rate constant, as well as by increasing half-life at higher doses; the ratio of desisopropyl-delavirdine formation clearance to elimination clearance was also reduced. In the control group, the kinetics of delavirdine and desisopropyl-delavirdine were unchanged. Plasma protein binding was linear for delavirdine in the escalating-dose and control groups; on average, the fraction unbound was about 2.3% and 2.0%, respectively. Hepatic CYP3A activity was markedly reduced after short- and long-term exposure to all doses of delavirdine mesylate. Delavirdine could maximally inhibit 70% to 75% of predose ERMBT values, with an IC50 of about 0.9 mumol/L. CONCLUSION: Delavirdine is a potent and reversible inhibitor of hepatic CYP3A; it is also a substrate for this CYP450 isoform. It is likely that delavirdine will exhibit drug-drug interactions when coadministered with other CYP3A substrates.
Administration, Oral↗
Predicting drug interactions in vivo from experiments in vitro. Human studies with paclitaxel and ketoconazole.
This study was performed to evaluate whether concomitant treatment with ketoconazole could reduce the clearance of paclitaxel given to ovarian cancer patients. Paclitaxel, 175 mg/m2, was given as a 3-hour continuous intravenous infusion and repeated every 21 days. Initially, ketoconazole, 100 to 1600 mg, was given as a single oral dose 3 hours after paclitaxel. Later, ketoconazole, 200 mg, was given perorally 3 hours before paclitaxel. Plasma drug concentrations were measured by high-pressure liquid chromatography (HPLC), and cytochrome P450 3A (CYP3A) activity was measured with the erythromycin breath test (ERMBT). Ketoconazole did not alter plasma concentrations of paclitaxel or its principal metabolite, 6 alpha-hydroxypaclitaxel. Although there was marked inter- and intrapatient variability in ketoconazole pharmacokinetics, peak plasma concentrations in all but one course were below the 50% inhibitory concentration (IC50) point determined for inhibition of paclitaxel metabolism in vitro. Therefore, paclitaxel and ketoconazole can be coadministered safely without dose adjustments. There was no correlation between ERMBT measurements and serial plasma concentrations of paclitaxel. The erythromycin breath-test measurements did correlate with the corresponding ketoconazole plasma concentrations. The erythromycin breath test is a valuable tool for measuring instantaneous CYP3A activity in vivo. This clinical study confirms the results of prior studies with human-derived materials in vitro, reinforcing the notion that such studies are useful predictors of drug pharmacokinetics and interactions in vivo.
Antifungal Agents↗
Expression of enzymatically active CYP3A4 by Caco-2 cells grown on extracellular matrix-coated permeable supports in the presence of 1alpha,25-dihydroxyvitamin D3.
The human colon carcinoma cell line, Caco-2, is widely used as a model for oral absorption of xenobiotics. The usefulness of Caco-2 cells has been limited, however, because they do not express appreciable quantities of CYP3A4, the principle cytochrome P450 present in human small bowel epithelial cells. We report that treatment of Caco-2 cells with 1 alpha,25-dihydroxyvitamin D3, beginning at confluence, results in a dose- and duration-dependent increase in CYP3A4 mRNA and protein, with little apparent effect on the expression of CYP3A5 or CYP3A7. This treatment also results in increases in NADPH cytochrome P450 reductase and P-glycoprotein (the MDR1 gene product) but has no detectable effect on expression of CYP1A1, CYP2D6, cytochrome b5, liver or intestinal fatty acid binding proteins, or villin. Maximal expression of CYP3A4 requires an extracellular matrix on a permeable support and the presence of serum. In the treated cells, the intrinsic formation clearance of 1'-hydroxymidazolam (a reaction characteristically catalyzed by CYP3A enzymes) was estimated to be somewhat lower than that of human jejunal mucosa (1.14 and 3.67 ml/min/g of cells, respectively). The 1'-OH-midazolam/4-OH-midazolam product ratio produced by the cells (approximately 5.3) is comparable to, but somewhat lower than, that observed in human jejunal microsomes (7.4-15.4), which may reflect the presence of CYP3A7 in the Caco-2 cells. 25-Hydroxyvitamin D3 is less efficacious but reproduces the effects of the dihydroxy compound, whereas unhydroxylated vitamin D is without appreciable effect. These observations, together with the time course of response, suggest that the vitamin D receptor may be involved in CYP3A4 regulation. The culture model we describe should prove useful in defining the role of CYP3A4 in limiting the oral bioavailability of many xenobiotics.
Caco-2 Cells↗
Mechanisms of enhanced oral availability of CYP3A4 substrates by grapefruit constituents. Decreased enterocyte CYP3A4 concentration and mechanism-based inactivation by furanocoumarins.
Grapefruit juice increases the oral availability of a variety of CYP3A4 substrates. It has been shown that recurrent grapefruit juice ingestion results in a loss of CYP3A4 from the small bowel epithelium. We now show that the reduction in intestinal CYP3A4 concentration is rapid; a 47% decrease occurred in a healthy volunteer within 4 hr after consuming grapefruit juice. To identify the specific components of the juice responsible for this effect, we used a recently developed Caco-2 cell culture model of human intestinal epithelium that expresses catalytically active CYP3A4. We found that grapefruit oil and two furanocoumarin constituents (6', 7'-dihydroxybergamottin and a closely related dimer) caused a dose-dependent fall in CYP3A4 catalytic activity and immunoreactive CYP3A4 concentration. The effect was selective in that concentrations of CYP1A1 and CYP2D6 did not fall, consistent with previous results obtained in vivo. Assays of various juices confirmed that 6',7'-dihydroxybergamottin is the major furanocoumarin present and, although its concentration varies significantly among types and brands of grapefruit juice, it is consistently present in concentrations exceeding the IC50 (1 microM) for loss of midazolam 1'-hydroxylase activity determined in the Caco-2 cells. Studies with recombinant CYP3A4 revealed that 6', 7'-dihydroxybergamottin is a mechanism-based inactivator, which supports the idea that loss of CYP3A4 results from accelerated degradation of the enzyme. We conclude that the effect of grapefruit juice on oral availability of CYP3A4 substrates can be largely accounted for by the presence of 6',7'-dihydroxybergamottin although other furanocoumarins probably also contribute.
Biological Availability↗
The caffeine breath test does not identify patients susceptible to tacrine hepatotoxicity.
Therapy with tacrine, a promising new treatment for Alzheimer's disease, must be discontinued in up to 15% of patients because of hepatocellular toxicity. Recent studies using human liver microsomes have suggested that a single liver enzyme, cytochrome P450 1A2 (CYP1A2), catalyzes the major route of metabolism and elimination of tacrine, and also catalyzes the pathway(s) involved in the generation of reactive metabolites capable of covalent protein binding and cytotoxicity. Because CYP1A2 activity has been shown to vary up to 60-fold among patients, we proposed that a convenient measure of CYP1A2 activity, the [(13)C 3-methyl] caffeine breath test (CBT), might be clinically useful in identifying patients most susceptible to tacrine liver toxicity. To test this hypothesis, we administered the CBT to 37 patients with Alzheimer's disease before they began treatment with tacrine. Twenty patients received 2 mg/kg of [(13)C 3-methyl] caffeine. The remaining 17 patients received the commercially available CBT kit, which employs a constant 200-mg dose. The activities of two other major drug-metabolizing enzymes (cytochrome P450 3A4 and 2D6 [CYP3A4 and CYP2D6]) were also measured in these 17 patients. We found that the results obtained from the CBT protocol did not predict the peak serum alanine transaminase (ALT) observed in the patients. The measured CYP3A4 and CYP2D6 activities also failed to predict the susceptible patients. However, the result of the standardized-dose CBT correlated well with the logarithm of the steady-state plasma tacrine level obtained in 10 patients (R(2) = .69, P = .003). We conclude that the CBT will not be clinically useful in determining the subset of patients most susceptible to tacrine hepatotoxicity. However, the correlation we observed between CBT results and tacrine blood levels is the first evidence supporting a critical role for CYP1A2 activity in the disposition of the drug in vivo.
Aged↗
Erythromycin breath test and clinical transplantation.
The intravenous 14C-erythromycin breath test (ERMBT) appears to be a convenient and reproducible means of measuring the activity of a major drug-metabolizing enzyme present in the liver, termed CYP3A4. Interpatient differences in liver CYP3A4 activity, as measured by the ERMBT, seem to account, for the most part, for interindividual differences in the kinetics of cyclosporin A and FK506. The predictability of the test may be improved as an oral form of the test and improved oral formulations of transplant medications are developed. Although the ERMBT is a promising research tool, its role in clinical transplantation has yet to be defined.
Breath Tests↗
Selective expression of CYP3A5 and not CYP3A4 in human blood.
There is a marked variation between people in the activity of CYP3A4 in liver and intestine. We reasoned that if CYP3A4 was expressed in peripheral blood cells, a simple blood based test of CYP3A4 phenotype might be feasible. We prepared peripheral blood smears from healthy volunteers and performed immunostaining with a rabbit polyclonal antibody that selectively reacts with enzymes within the CYP3A subfamily. Staining was observed only within the cytoplasm of neutrophils (PMNs). cDNA prepared from isolated PMNs and mononuclear cells was subjected the polymerase chain reaction using as primers synthetic oligonucleotides that selectively amplify fragments of each known CYP3A cDNAs (CYP3A3, CYP3A4, CYP3A5, and CYP3A7). Amplification was only obtained with the CYP3A5 specific oligonucleotides, predominantly in PMNs, and the identity of the amplified fragment was confirmed by sequencing. Next, whole white cell homogenate prepared from human blood was reacted on immunoblots with a monoclonal antibody that recognizes all CYP3A proteins or an absorbed polyclonal antibody that recognizes only CYP3A5. Both antibodies recognized a protein in the white cells that comigrated with purified CYP3A5. However, metabolism of midazolam, a substrate of CYP3A, could not be detected in homogenates of isolated granulocytes, in homogenates of the whole WBC fractions, or in incubations with unlysed WBC preparations. We conclude that CYP3A4 is not expressed in peripheral blood and hence a blood phenotyping test for this enzyme will not be feasible. Our discovery that CYP3A5 is present may be important since this enzyme is also present in the liver intestine and kidney of many people.
Cytochrome P-450 CYP3A↗