Danny D. Shen

First-pass metabolism of midazolam by the human intestine

The in vivo intestinal metabolism of the CYP3A probe midazolam to its principal metabolite, 1′‐hydroxymidazolam, was investigated during surgery in 10 liver transplant recipients. After removal of the diseased liver, five subjects received 2 mg midazolam intraduodenally, and the other five received 1 mg midazolam intravenously. Simultaneous arterial and hepatic portal venous blood samples were collected during the anhepatic phase; collection of arterial samples continued after reperfusion of the donor liver. Midazolam, 1′‐hydroxymidazolam, and 1′‐hydroxymidazolam glucuronide were measured in plasma. A mass balance approach that considered the net change in midazolam (intravenously) or midazolam and 1′‐hydroxymidazolam (intraduodenally) concentrations across the splanchnic vascular bed during the anhepatic phase was used to quantitate the intestinal extraction of midazolam after each route of administration. For the intraduodenal group, the mean fraction of the absorbed midazolam dose that was metabolized on transit through the intestinal mucosa was 0.43 ± 0.18. For the intravenous group, the mean fraction of midazolam extracted from arterial blood and metabolized during each passage through the splanchnic vascular bed was 0.08 ± 0.11. Although there was significant intersubject variability, the mean intravenous and intraduodenal extraction fractions were statistically different (p = 0.009). Collectively, these results show that the small intestine contributes significantly to the first‐pass oxidative metabolism of midazolam catalyzed by mucosal CYP3A4 and suggest that significant first‐pass metabolism may be a general phenomenon for all high‐turnover CYP3A4 substrates.

Pharmacokinetics and pharmacodynamics of oral oxycodone in healthy human subjects: Role of circulating active metabolites

In vitro experiments suggest that circulating metabolites of oxycodone are opioid receptor agonists. Clinical and animal studies to date have failed to demonstrate a significant contribution of the O‐demethylated metabolite oxymorphone toward the clinical effects of the parent drug, but the role of other putative circulating active metabolites in oxycodone pharmacodynamics remains to be examined.

Clinical pharmacogenetics implementation consortium guidelines for cytochrome P450 2D6 genotype and codeine therapy: 2014 Update

Codeine is bioactivated to morphine, a strong opioid agonist, by the hepatic cytochrome P450 2D6 (CYP2D6); hence, the efficacy and safety of codeine are governed by CYP2D6 activity. Polymorphisms are a major cause of CYP2D6 variability. We summarize evidence from the literature supporting this association and provide therapeutic recommendations for codeine based on CYP2D6 genotype. This document is an update to the 2012 Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines for CYP2D6 genotype and codeine therapy.

Clinical Pharmacogenetics Implementation Consortium (CPIC) Guidelines for Codeine Therapy in the Context of Cytochrome P450 2D6 (CYP2D6) Genotype

Codeine is bioactivated to morphine, a strong opioid agonist, by the hepatic cytochrome P450 2D6 (CYP2D6); hence, the efficacy and safety of codeine as an analgesic are governed by CYP2D6 polymorphisms. Codeine has little therapeutic effect in patients who are CYP2D6 poor metabolizers, whereas the risk of morphine toxicity is higher in ultrarapid metabolizers. The purpose of this guideline (periodically updated at http://www.pharmgkb.org) is to provide information relating to the interpretation of CYP2D6 genotype test results to guide the dosing of codeine.

Enzyme-catalyzed processes of first-pass hepatic and intestinal drug extraction.

Oral bioavailability of pharmacologically effective drugs is often limited by first-pass biotransformation. In humans, both hepatic and intestinal enzymes can catalyze the metabolism of a drug as it transits between the gastrointestinal lumen and systemic blood for the first time. Although a spectrum of drug biotransformations can occur during first-pass, the most common are oxidations catalyzed by cytochromes P450. It is the isozymes CYP2D6, CYP3A4, CYP1A2, CYP2C9 and CYP2C19 that are most often implicated in first-pass drug elimination. For any given substrate, enzyme specificity, enzyme content, substrate binding affinity and sensitivity to irreversible catalytic events all play a role in determining the overall efficiency, or intrinsic clearance, of elimination. Several models have been proposed over the past twenty-five years that mathematically describe the process of drug extraction across the liver. The most widely used, the well-stirred model, has also been considered for depiction of first-pass drug elimination across the intestinal wall. With these models it has been possible to examine sources of interindividual variability in drug bioavailability including, variable constitutive enzyme expression (both genetic and environmentally determined), enzyme induction by drugs, disease and diet, and intrinsic or acquired differences in plasma protein binding and organ blood flow (food and drug effects). In recent years, the most common application of hepatic clearance models has been the determination of maximum organ availability of a drug from in vitro derived estimates of intrinsic metabolic clearance. The relative success of the in vitro-in vivo approach for both low and highly extracted drugs has led to a broader use by the drug industry for a priori predictions as part of the drug selection process. A considerable degree of effort has also been focused on gut wall first-pass metabolism. Important pathways of intestinal Phase II first-pass metabolism include the sulfation of terbutaline and isoproterenol and glucuronidation of morphine and labetalol. It is also clear that some of the substrates for CYP3A4 (e.g., cyclosporine, midazolam, nifedipine, verapamil and saquinavir) undergo significant metabolic extraction by the gut wall. For example, the first-pass extraction of midazolam by the intestinal mucosa appears, on average, to be comparable to extraction by the liver. However, many other CYP3A substrates do not appear susceptible to a gut wall first-pass, possibly because of enzyme saturation during first-pass or a limited intrinsic metabolic clearance. Both direct biochemical and indirect in vivo clearance data suggest significant inter-individual variability in gut wall CYP3A-dependent metabolism. The source of this constitutive variability is largely unknown. Because of their unique anatomical location, enzymes of the gut wall may represent an important and highly sensitive site of metabolically-based interactions for orally administered drugs. Again, interindividual variability may make it impossible to predict the likelihood of an interaction in any given patient. Hopefully, though, newer models for studying human gut wall metabolic extraction will provide the means to predict the average extraction ratio and maximum first-pass availability of a putative substrate, or the range of possible inhibitory or inductive changes for a putative inhibitor/inducer.

Comparison of Blood and Brain Mercury Levels in Infant Monkeys Exposed to Methylmercury or Vaccines Containing Thimerosal

Thimerosal is a preservative that has been used in manufacturing vaccines since the 1930s. Reports have indicated that infants can receive ethylmercury (in the form of thimerosal) at or above the U.S. Environmental Protection Agency guidelines for methylmercury exposure, depending on the exact vaccinations, schedule, and size of the infant. In this study we compared the systemic disposition and brain distribution of total and inorganic mercury in infant monkeys after thimerosal exposure with those exposed to MeHg. Monkeys were exposed to MeHg (via oral gavage) or vaccines containing thimerosal (via intramuscular injection) at birth and 1, 2, and 3 weeks of age. Total blood Hg levels were determined 2, 4, and 7 days after each exposure. Total and inorganic brain Hg levels were assessed 2, 4, 7, or 28 days after the last exposure. The initial and terminal half-life of Hg in blood after thimerosal exposure was 2.1 and 8.6 days, respectively, which are significantly shorter than the elimination half-life of Hg after MeHg exposure at 21.5 days. Brain concentrations of total Hg were significantly lower by approximately 3-fold for the thimerosal-exposed monkeys when compared with the MeHg infants, whereas the average brain-to-blood concentration ratio was slightly higher for the thimerosal-exposed monkeys (3.5 ± 0.5 vs. 2.5 ± 0.3). A higher percentage of the total Hg in the brain was in the form of inorganic Hg for the thimerosal-exposed monkeys (34% vs. 7%). The results indicate that MeHg is not a suitable reference for risk assessment from exposure to thimerosal-derived Hg. Knowledge of the toxicokinetics and developmental toxicity of thimerosal is needed to afford a meaningful assessment of the developmental effects of thimerosal-containing vaccines.

Comparative spinal distribution and clearance kinetics of intrathecally administered morphine, fentanyl, alfentanil, and sufentanil.

Background: Despite widespread use, little is known about the comparative pharmacokinetics of intrathecally administered opioids. The present study was designed to characterize the rate and extent of opioid distribution within cerebrospinal fluid, spinal cord, epidural space, and systemic circulation after intrathecal injection. Methods: Equal doses of morphine and alfentanil, fentanyl, or sufentanil were administered intrathecally (L3) to anesthetized pigs. Microdialysis probes were used to sample cerebrospinal fluid at L2, T11, T7, T3, and the epidural space at L2 every 5–10 min for 4 h. At the end of the experiment, spinal cord and epidural fat tissue were sampled, and each probe’s recovery was determined in vitro. Using SAAM II pharmacokinetic modeling software (SAAM Institute, University of Washington, Seattle, WA), the data were fit to a 16-compartment model that was divided into four spinal levels, each of which consisted of a caternary arrangement of four compartments representing the spinal cord, cerebrospinal fluid, epidural space, and epidural fat. Results: Model simulations revealed that the integral exposure (area under the curve divided by dose) of the spinal cord (i.e., effect compartment) to the opioids was highest for morphine because of its low spinal cord distribution volume and slow clearance into plasma. The integral exposure of the spinal cord to the other opioids was relatively low, but for different reasons: alfentanil has a high clearance from spinal cord into plasma, fentanyl distributes rapidly into the epidural space and fat, and sufentanil has a high spinal cord volume of distribution. Conclusions: The four opioids studied demonstrate markedly different pharmacokinetic behavior, which correlates well with their pharmacodynamic behavior.

EFFECT OF CYP3A5 POLYMORPHISM ON TACROLIMUS METABOLIC CLEARANCE IN VITRO

Previous investigations of solid organ transplant patients treated with tacrolimus showed that individuals carrying a CYP3A5*1 allele have lower dose-adjusted trough blood concentrations compared with homozygous CYP3A5*3 individuals. The objective of this investigation was to quantify the contribution of CYP3A5 to the hepatic and renal metabolic clearance of tacrolimus. Four primary tacrolimus metabolites, 13-O-desmethyl tacrolimus (13-DMT) (major), 15-O-desmethyl tacrolimus, 31-O-desmethyl tacrolimus (31-DMT), and 12-hydroxy tacrolimus (12-HT), were generated by human liver microsomes and heterologously expressed CYP3A4 and CYP3A5. The unbound tacrolimus concentration was low (4–15%) under all incubation conditions. For CYP3A4 and CYP3A5, Vmax was 8.0 and 17.0 nmol/min/nmol enzyme and Km,u was 0.21 and 0.21 μM, respectively. The intrinsic clearance of CYP3A5 was twice that of CYP3A4. The formation rates of 13-DMT, 31-DMT, and 12-HT were ≥1.7-fold higher, on average, in human liver microsomes with a CYP3A5*1/*3 genotype compared with those with a homozygous CYP3A5*3/*3 genotype. Tacrolimus disappearance clearances were 15.9 ± 9.8 ml/min/mg protein and 6.1 ± 3.6 ml/min/mg protein, respectively, for the two genotypes. In vitro to in vivo scaling using both liver microsomes and recombinant enzymes yielded higher predicted in vivo tacrolimus clearances for patients with a CYP3A5*1/*3 genotype compared with those with a CYP3A5*3/*3 genotype. In addition, formation of 13-DMT was 13.5-fold higher in human kidney microsomes with a CYP3A5*1/*3 genotype compared with those with a CYP3A5*3/*3 genotype. These data suggest that CYP3A5 contributes significantly to the metabolic clearance of tacrolimus in the liver and kidney.

THE HUMAN CYP3A SUBFAMILY: PRACTICAL CONSIDERATIONS*

STEVEN A. WRIGHTON,† ERIN G. SCHUETZ, KENNETH E. THUMMEL, DANNY D. SHEN, KENNETH R. KORZEKWA, and PAUL B. WATKINS Department of Drug Disposition Lilly Research Laboratories Lilly Corporate Center Mail Drop 0730 Indianapolis, Indiana 46285 Department of Pharmaceutical Sciences St. Jude Children’s Research Hospital Memphis, Tennessee 38105 Department of Pharmaceutics Box 357610 University of Washington Seattle, Washington 98195 Department of Pharmacy University of Washington Seattle, Washington 98195 Camitro Corporation 4040 Campbell Avenue Menlo Park, California 94025 University of North Carolina at Chapel Hill Chapel Hill, North Carolina 27599

In-vivo phenotyping for CYP3A by a single-point determination of midazolam plasma concentration

We investigated whether a single plasma midazolam concentration could serve as an accurate predictor of total midazolam clearance, an established in-vivo probe measure of cytochrome P450 3A (CYP3A) activity. In a retrospective analysis of data from 224 healthy volunteers, non-compartmental pharmacokinetic parameters were estimated from plasma concentration-time curves following intravenous (IV) and/or oral administration. Based on statistical moment theory, the concentration at the mean residence time (MRT) should be the best predictor of the total area under the curve (AUC). Following IV or oral midazolam administration, the average MRT was found to be approximately 3.5 h, suggesting that the optimal single sampling time to predict AUC was between 3 and 4 h. Since a 4-h data point was common to all studies incorporated into this analysis, we selected this time point for further investigation. The concentrations of midazolam measured 4 h after an IV or oral dose explained 80 and 91% of the constitutive interindividual variability in midazolam AUC, respectively. The 4-h midazolam measurement was also an excellent predictor of drug-drug interactions involving CYP3A induction and inhibition. Compared with baseline values, the direction and magnitude of change in midazolam AUC and the 4-h concentration were completely concordant for all study subjects. We conclude that a single 4-h midazolam concentration following IV or oral administration represents an accurate marker of CYP3A phenotype under constitutive and modified states. Moreover, the single-point approach offers an efficient means to phenotype and identify individuals with important genetic polymorphisms that affect CYP3A activity.