An Integrative Approach to Elucidate Mechanisms Underlying the Pharmacokinetic Goldenseal-Midazolam Interaction: Application of In Vitro Assays and Physiologically Based Pharmacokinetic Models to Understand Clinical Observations.

The natural product goldenseal is a clinical inhibitor of CYP3A activity, as evidenced by a 40%-60% increase in midazolam area under the plasma concentration versus time curve (AUC) after coadministration with goldenseal. The predominant goldenseal alkaloids berberine and (-)-ß-hydrastine were previously identified as time-dependent CYP3A inhibitors using human liver microsomes. Whether these alkaloids contribute to the clinical interaction, as well as the primary anatomic site (hepatic vs. intestinal) and mode of CYP3A inhibition (reversible vs. time-dependent), remain uncharacterized. The objective of this study was to mechanistically assess the pharmacokinetic goldenseal-midazolam interaction using an integrated in vitro-in vivo-in silico approach. Using human intestinal microsomes, (-)-ß-hydrastine was a more potent time-dependent inhibitor of midazolam 1'-hydroxylation than berberine (KI and kinact: 8.48 µM and 0.041 minutes-1, respectively, vs. >250 µM and ∼0.06 minutes-1, respectively). Both the AUC and Cmax of midazolam increased by 40%-60% after acute (single 3-g dose) and chronic (1 g thrice daily × 6 days) goldenseal administration to healthy adults. These increases, coupled with a modest or no increase (≤23%) in half-life, suggested that goldenseal primarily inhibited intestinal CYP3A. A physiologically based pharmacokinetic interaction model incorporating berberine and (-)-ß-hydrastine successfully predicted the goldenseal-midazolam interaction to within 20% of that observed after both chronic and acute goldenseal administration. Simulations implicated (-)-ß-hydrastine as the major alkaloid precipitating the interaction, primarily via time-dependent inhibition of intestinal CYP3A, after chronic and acute goldenseal exposure. Results highlight the potential interplay between time-dependent and reversible inhibition of intestinal CYP3A as the mechanism underlying natural product-drug interactions, even after acute exposure to the precipitant. SIGNIFICANCE STATEMENT: Natural products can alter the pharmacokinetics of an object drug, potentially resulting in increased off-target effects or decreased efficacy of the drug. The objective of this work was to evaluate fundamental mechanisms underlying the clinically observed goldenseal-midazolam interaction. Results support the use of an integrated approach involving established in vitro assays, clinical evaluation, and physiologically based pharmacokinetic modeling to elucidate the complex interplay between multiple phytoconstituents and various pharmacokinetic processes driving a drug interaction.

Assessing Transporter-Mediated Natural Product-Drug Interactions Via In vitro-In Vivo Extrapolation: Clinical Evaluation With a Probe Cocktail.

The botanical natural product goldenseal can precipitate clinical drug interactions by inhibiting cytochrome P450 (CYP) 3A and CYP2D6. Besides P-glycoprotein, effects of goldenseal on other clinically relevant transporters remain unknown. Established transporter-expressing cell systems were used to determine the inhibitory effects of a goldenseal extract, standardized to the major alkaloid berberine, on transporter activity. Using recommended basic models, the extract was predicted to inhibit the efflux transporter BCRP and uptake transporters OATP1B1/3. Using a cocktail approach, effects of the goldenseal product on BCRP, OATP1B1/3, OATs, OCTs, MATEs, and CYP3A were next evaluated in 16 healthy volunteers. As expected, goldenseal increased the area under the plasma concentration-time curve (AUC0-inf ) of midazolam (CYP3A; positive control), with a geometric mean ratio (GMR) (90% confidence interval (CI)) of 1.43 (1.35-1.53). However, goldenseal had no effects on the pharmacokinetics of rosuvastatin (BCRP and OATP1B1/3) and furosemide (OAT1/3); decreased metformin (OCT1/2, MATE1/2-K) AUC0-inf (GMR, 0.77 (0.71-0.83)); and had no effect on metformin half-life and renal clearance. Results indicated that goldenseal altered intestinal permeability, transport, and/or other processes involved in metformin absorption, which may have unfavorable effects on glucose control. Inconsistencies between model predictions and pharmacokinetic outcomes prompt further refinement of current basic models to include differential transporter expression in relevant organs and intestinal degradation/metabolism of the precipitant(s). Such refinement should improve in vitro-in vivo prediction accuracy, contributing to a standard approach for studying transporter-mediated natural product-drug interactions.

Simultaneous quantification of 25-hydroxyvitamin D3-3-sulfate and 25-hydroxyvitamin D3-3-glucuronide in human serum and plasma using liquid chromatography-tandem mass spectrometry coupled with DAPTAD-derivatization.

25-hydroxyvitamin D3-3-sulfate (25-OHD3-S) and 25-hydroxyvitamin D3-3-glucuronide (25-OHD3-G) are major conjugative metabolites of vitamin D3 found in the systemic circulation and potentially important reservoirs for 25-hydroxyvitamin D3. Simultaneous and accurate quantification of these metabolites could advance assessment of the impact of vitamin D3 on health and disease. In this study, a highly sensitive and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous quantification of 25-OHD3-S and 25-OHD3-G in human serum or plasma. Following protein precipitation, the analytes of interest were partially purified by solid-phase extraction and subjected to derivatization with 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD). Quantification of the analytes was based on multiple reaction monitoring (MRM) operated in the positive ion mode, and deuterated internal standards were used for each conjugative metabolite. Applying this method to the analysis of 25-OHD3-S and 25-OHD3-G concentrations in human serum or plasma samples achieved satisfactory reproducibility, accuracy and sensitivity. We subsequently used this method to simultaneously determine serum concentrations of the two metabolites in archived samples from a rifampin treatment study. Drug treatment had no effect on metabolite concentrations, but significantly increased the 25-OHD3-S/25-OHD3 concentration ratio (p=0.01). The availability of this new method should improve sample throughput and our ability to quantify and monitor circulating 25-OHD3-S and 25-OHD3-G concentrations.

Variability in Expression of CYP3A5 in Human Fetal Liver.

Members of the cytochrome P450 3A (CYP3A) subfamily of drug metabolizing enzymes exhibit developmental changes in expression in human liver characterized by a transition between CYP3A7 and CYP3A4 over the first few years of life. In contrast, the developmental expression of CYP3A5 is less well understood due to polymorphic expression of the enzyme in human tissues as a result of the prevalence of the CYP3A5*3 allele, which leads to alternative splicing. We further explored the expression of CYP3A5 and the impact of alternative splicing on the variability of CYP3A5 functional activity in a large bank of human prenatal liver samples (7 to 32 weeks of age postconception). The expression of normally spliced CYP3A5 mRNA in all human fetal liver samples varied 235-fold whereas CYP3A5 SV1 mRNA was only detected in fetal liver samples with at least one CYP3A5*3 allele. Formation of 1'-OH midazolam (MDZ) varied 79-fold, and the ratio of 1'-OH MDZ to 4-OH MDZ varied 8-fold and depended on the presence or absence of the CYP3A5*3 allele. Formation of 4-OH MDZ was significantly associated with 1'-OH MDZ (r(2) = 0.76, P < 0.0001) but varied (36-fold) independently of CYP3A5 genotype or expression. The substantial interindividual variability that remains even after stratification for CYP3A5 genotype suggests that factors such as environmental exposure and epigenetic alterations act in addition to genetic variation to contribute to the variability of CYP3A5 expression in human prenatal liver.

Identification of CYP3A7 for glyburide metabolism in human fetal livers.

Glyburide is commonly prescribed for the treatment of gestational diabetes mellitus; however, fetal exposure to glyburide is not well understood and may have short- and long-term consequences for the health of the child. Glyburide can cross the placenta; fetal concentrations at term are nearly comparable to maternal levels. Whether or not glyburide is metabolized in the fetus and by what mechanisms has yet to be determined. In this study, we determined the kinetic parameters for glyburide depletion by CYP3A isoenzymes; characterized glyburide metabolism by human fetal liver tissues collected during the first or early second trimester of pregnancy; and identified the major enzyme responsible for glyburide metabolism in human fetal livers. CYP3A4 had the highest metabolic capacity towards glyburide, followed by CYP3A7 and CYP3A5 (Clint,u=37.1, 13.0, and 8.7ml/min/nmol P450, respectively). M5 was the predominant metabolite generated by CYP3A7 and human fetal liver microsomes (HFLMs) with approximately 96% relative abundance. M5 was also the dominant metabolite generated by CYP3A4, CYP3A5, and adult liver microsomes; however, M1-M4 were also present, with up to 15% relative abundance. CYP3A7 protein levels in HFLMs were highly correlated with glyburide Clint, 16α-OH DHEA formation, and 4'-OH midazolam formation. Likewise, glyburide Clint was highly correlated with 16α-OH DHEA formation. Fetal demographics as well as CYP3A5 and CYP3A7 genotype did not alter CYP3A7 protein levels or glyburide Clint. These results indicate that human fetal livers metabolize glyburide predominantly to M5 and that CYP3A7 is the major enzyme responsible for glyburide metabolism in human fetal livers.

Tacrolimus placental transfer at delivery and neonatal exposure through breast milk.

AIM(S): The current investigation aims to provide new insights into fetal exposure to tacrolimus in utero by evaluating maternal and umbilical cord blood (venous and arterial), plasma and unbound concentrations at delivery. This study also presents a case report of tacrolimus excretion via breast milk. METHODS: Maternal and umbilical cord (venous and arterial) samples were obtained at delivery from eight solid organ allograft recipients to measure tacrolimus and metabolite bound and unbound concentrations in blood and plasma. Tacrolimus pharmacokinetics in breast milk were assessed in one subject. RESULTS: Mean (±SD) tacrolimus concentrations at the time of delivery in umbilical cord venous blood (6.6 ± 1.8 ng ml(-1)) were 71 ± 18% (range 45-99%) of maternal concentrations (9.0 ± 3.4 ng ml(-1)). The mean umbilical cord venous plasma (0.09 ± 0.04 ng ml(-1)) and unbound drug concentrations (0.003 ± 0.001 ng ml(-1)) were approximately one fifth of the respective maternal concentrations. Arterial umbilical cord blood concentrations of tacrolimus were 100 ± 12% of umbilical venous concentrations. In addition, infant exposure to tacrolimus through the breast milk was less than 0.3% of the mother's weight-adjusted dose. CONCLUSIONS: Differences between maternal and umbilical cord tacrolimus concentrations may be explained in part by placental P-gp function, greater red blood cell partitioning and higher haematocrit levels in venous cord blood. The neonatal drug exposure to tacrolimus via breast milk is very low and likely does not represent a health risk to the breastfeeding infant.

CYP3A5 gene variation influences cyclosporine A metabolite formation and renal cyclosporine disposition.

BACKGROUND: Higher concentrations of AM19 and AM1c9, secondary metabolites of cyclosporine A (CsA), have been associated with nephrotoxicity in organ transplant patients. The risk of renal toxicity may depend on the accumulation of CsA and its metabolites in the renal tissue. We evaluated the hypothesis that CYP3A5 genotype, and inferred enzyme expression, affects systemic CsA metabolite exposure and intrarenal CsA accumulation. METHODS: An oral dose of CsA was administered to 24 healthy volunteers who were selected based on their CYP3A5 genotype. CsA and its six main metabolites in whole blood and urine were measured by liquid chromatography-mass spectometry. In vitro incubations of CsA, AM1, AM9, and AM1c with recombinant CYP3A4 and CYP3A5 were performed to evaluate the formation pathways of AM19 and AM1c9. RESULTS: The mean CsA oral clearance was similar between CYP3A5 expressors and nonexpressors. However, compared with CYP3A5 nonexpressors, the average blood area under the concentration-time curve (AUC) for AM19 and AM1c9 was 47.4% and 51.3% higher in CYP3A5 expressors (P=0.040 and 0.011, respectively), corresponding to 30% higher AUCmetabolite/AUCCsA ratios for AM19 and AM1c9 in CYP3A5 expressors. The mean apparent urinary CsA clearance based on a 48-hr collection was 20.4% lower in CYP3A5 expressors compared with CYP3A5 nonexpressors (4.2±1.0 and 5.3±1.3 mL/min, respectively; P=0.037), which is suggestive of CYP3A5-dependent intrarenal CsA metabolism. CONCLUSIONS: At steady state, intrarenal accumulation of CsA and its secondary metabolites should depend on the CYP3A5 genotype of the liver and kidneys. This may contribute to interpatient variability in the risk of CsA-induced nephrotoxicity.

Pharmacokinetics of tacrolimus during pregnancy.

BACKGROUND: Information on the pharmacokinetics of tacrolimus during pregnancy is limited to case reports despite the increasing number of pregnant women being prescribed tacrolimus for immunosuppression. METHODS: Blood, plasma, and urine samples were collected over 1 steady-state dosing interval from women treated with oral tacrolimus during early to late pregnancy (n = 10) and postpartum (n = 5). Total and unbound tacrolimus as well as metabolite concentrations in blood and plasma were assayed by a validated liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) method. A mixed-effect linear model was used for comparison across gestational age and using postpartum as the reference group. RESULTS: The mean oral clearance (CL/F) based on whole-blood tacrolimus concentration was 39% higher during mid-pregnancy and late pregnancy compared with postpartum (47.4 ± 12.6 vs. 34.2 ± 14.8 L/h, P < 0.03). Tacrolimus-free fraction increased by 91% in plasma (f(P)) and by 100% in blood (f(B)) during pregnancy (P = 0.0007 and 0.002, respectively). Increased fP was inversely associated with serum albumin concentration (r = -0.7, P = 0.003), which decreased by 27% during pregnancy. Pregnancy-related changes in f(P) and f(B) contributed significantly to the observed gestational increase in tacrolimus whole-blood CL/F (r² = 0.36 and 0.47, respectively, P < 0.01). In addition, tacrolimus whole-blood CL/F was inversely correlated with both hematocrit and red blood cell counts, suggesting that binding of tacrolimus to erythrocytes restricts its availability for metabolism. Treating physicians increased tacrolimus dosages in study participants during pregnancy by an average of 45% to maintain tacrolimus whole-blood trough concentrations in the therapeutic range. This led to striking increases in unbound tacrolimus trough concentrations and unbound area under the concentration-time curve, by 112% and 173%, respectively, during pregnancy (P = 0.02 and 0.03, respectively). CONCLUSIONS: Tacrolimus pharmacokinetics are altered during pregnancy. Dose adjustment to maintain whole-blood tacrolimus concentration in the usual therapeutic range during pregnancy increases circulating free drug concentrations, which may impact clinical outcomes.

Evidence of significant contribution from CYP3A5 to hepatic drug metabolism.

CYP3A4 and CYP3A5 exhibit significant overlap in substrate specificity but can differ in product regioselectivity and formation activity. To further explore this issue, we compared the kinetics of product formation for eight different substrates, using heterologously expressed CYP3A4 and CYP3A5 and phenotyped human liver microsomes. Both enzymes displayed allosteric behavior toward six of the substrates. When it occurred, the "maximal" intrinsic clearance was used for quantitative comparisons. Based on this parameter, CYP3A5 was more active than CYP3A4 in catalyzing total midazolam hydroxylation (3-fold) and lidocaine demethylation (1.4-fold). CYP3A5 exhibited comparable metabolic activity as CYP3A4 (90-110%) toward dextromethorphan N-demethylation and carbamazepine epoxidation. CYP3A5-catalyzed erythromycin N-demethylation, total flunitrazepam hydroxylation, testosterone 6beta-hydroxylation, and terfenadine alcohol formation occurred with an intrinsic clearance that was less than 65% that of CYP3A4. Using two sets of human liver microsomes with equivalent CYP3A4-specific content but markedly different CYP3A5 content (group 1, predominantly CYP3A4; group 2, CYP3A4 + CYP3A5), we assessed the contribution of CYP3A5 to product formation rates determined at low substrate concentrations (< or = Km). Mean product formation rates for group 2 microsomes were 1.4- to 2.2-fold higher than those of group 1 (p < 0.05 for 5 of 8 substrates). After adjusting for CYP3A4 activity (itraconazole hydroxylation), mean product formation rates for group 2 microsomes were still significantly higher than those of group 1 (p < 0.05 for 3 substrates). We suggest that, under conditions when CYP3A5 content represents a significant fraction of the total hepatic CYP3A pool, the contribution of CYP3A5 to the clearance of some drugs may be an important source of interindividual variability.

Transiently altered acetaminophen metabolism after liver transplantation.

BACKGROUND AND OBJECTIVES: Acetaminophen (INN, paracetamol) is metabolized to N-acetyl-p-benzoquinone imine (NAPQI), a hepatotoxic metabolite, predominantly by cytochrome P450 (CYP) 2E1. Alterations in drug metabolism occur after organ transplantation. This study was designed to characterize acetaminophen disposition during the first 6 months after liver transplantation. METHODS: Thirteen liver transplant patients received an oral dose of acetaminophen (500 mg) on days 2, 10, 90, and 180 after transplantation. Serial blood samples were collected for 8 hours, and urine was collected for 24 hours. Liver biopsy specimens were obtained from the donor liver during transplantation (day 0) and on days 10, 90, and 180 after transplantation. RESULTS: There were significant time-dependent changes in acetaminophen metabolism after liver transplantation. When day 2 and day 10 were compared with day 180, the respective mean urinary recovery was 137% and 81% higher for thioether conjugates derived from NAPQI (P =.0002 and P =.01, respectively); 31% and 22% lower for acetaminophen sulfate (P =.0006 and P =.008, respectively); and 22% and 27% lower for acetaminophen glucuronide (P =.05 and P =.004, respectively). Metabolite formation clearances changed in concordance with the fractional urinary recovery. It was surprising that hepatic CYP2E1 content on day 10 after transplantation was only 20% higher, on average, than that found on day 180 (not significant). In contrast, hepatic CYP3A4 content was 984% higher, on average, when tissue from days 10 and 180 was compared after transplantation (P =.007). CONCLUSIONS: Increased recovery of acetaminophen thioether conjugates during the first 10 days after liver transplantation was a result of impaired glucuronidation and sulfation and enhanced NAPQI formation.