Philip C. Smith

Quantification of human uridine-diphosphate glucuronosyl transferase 1A isoforms in liver, intestine, and kidney using nanobore liquid chromatography-tandem mass spectrometry.

Uridine-disphosphate glucuronosyl transferase (UGT) enzymes catalyze the formation of glucuronide conjugates of phase II metabolism. Methods for absolute quantification of UGT1A1 and UGT1A6 were previously established utilizing stable isotope peptide internal standards with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The current method expands upon this by quantifying eight UGT1A isoforms by nanobore high-performance liquid chromatography (HPLC) coupled with a linear ion trap time-of-flight mass spectrometer platform. Recombinant enzyme digests of each of the isoforms were used to determine assay linearity and detection limits. Enzyme expression level in human liver, kidney, and intestinal microsomal protein was determined by extrapolation from spiked stable isotope standards. Intraday and interday variability was <25% for each of the enzyme isoforms. Enzyme expression varied from 3 to 96 pmol/mg protein in liver and intestinal microsomal protein digests. Expression levels of UGT1A7, 1A8, and 1A10 were below detection limits (<1 pmol/mg protein) in human liver microsome (HLMs). In kidney microsomes the expression of UGT1A3 was below detection limits, but levels of UGT1A4, 1A7, 1A9, and 1A10 protein were higher relative to that of liver, suggesting that renal glucuronidation could be a significant factor in renal elimination of glucuronide conjugates. This novel method allows quantification of all nine UGT1A isoforms, many previously not amenable to measurement with traditional methods such as immunologically based assays. Quantitative measurement of proteins involved in drug disposition, such as the UGTs, significantly improves the ability to evaluate and interpret in vitro and in vivo studies in drug development.

Pharmacokinetics and metabolic profile of free, conjugated, and total silymarin flavonolignans in human plasma after oral administration of milk thistle extract.

Silymarin, a mixture of polyphenolic flavonoids extracted from milk thistle (Silybum marianum), is composed mainly of silychristin, silydianin, silybin A, silybin B (SBB), isosilybin A (ISBA), and isosilybin B. In this study, the plasma concentrations of free (unconjugated), conjugated (sulfated and glucuronidated), and total (free and conjugated) silymarin flavonolignans were measured using liquid chromatography-electrospray ionization-mass spectrometry, after a single oral dose of 600 mg of standardized milk thistle extracts to three healthy volunteers. Pharmacokinetic analysis indicated that silymarin flavonolignans were rapidly eliminated with short half-lives (1–3 and 3–8 h for free and conjugated, respectively). The AUC0→∞ values of the conjugated silymarin flavonolignans were 4- to 30-fold higher than those of their free fractions, with SBB (mean AUC0→∞ = 51 and 597 μg · h/l for free and conjugated, respectively) and ISBA (mean AUC0→∞ = 30 and 734 μg · h/l for free and conjugated, respectively) exhibiting higher AUC0→∞ values in comparison with other flavonolignans. Near the plasma peak times (1–3 h), the free, sulfated, and glucuronidated flavonolignans represented approximately 17, 28, and 55% of the total silymarin, respectively. In addition, the individual silymarin flavonolignans exhibited quite different plasma profiles for both the free and conjugated fractions. These data suggest that, after oral administration, silymarin flavonolignans are quickly metabolized to their conjugates, primarily forming glucuronides, and the conjugates are primary components present in human plasma.

Comparative pharmacokinetics of amphotericin B after administration of a novel colloidal delivery system, ABCD, and a conventional formulation to rats.

The pharmacokinetics and tissue distribution of two amphotericin B dosage forms were compared in rats. A novel lipid-based colloidal delivery system for amphotericin B (Amphotericin B Colloidal Dispersion [ABCD]) which reduces the toxicity of amphotericin B in animals was compared with a conventional micellar formulation. Male Sprague-Dawley rats received a single intravenous injection of 1.0 mg of ABCD, 5.0 mg of ABCD, or 1.0 mg of micellar amphotericin B per kg. Plasma and tissue samples were obtained at 0.5 to 96 h after dosing and analyzed for amphotericin B by high-pressure liquid chromatography. Animals receiving ABCD demonstrated reduced peak levels in plasma, a three- to sevenfold reduction in amphotericin B delivery to the kidneys (the major target organ for toxicity), and prolonged residence time compared with those receiving the micellar formulation. In contrast, amphotericin B concentrations in the liver were two- to threefold higher with ABCD than with the micellar formulation: nearly 100% of the amphotericin B administered as ABCD was recovered from the liver 30 min after dosing. These results suggest that the colloidal particles of ABCD are taken up by the liver, which then acts as a reservoir of amphotericin B.

Silymarin Ascending Multiple Oral Dosing Phase I Study in Noncirrhotic Patients With Chronic Hepatitis C

Silymarin, derived from the milk thistle plant Silybum marianum, is widely used for self‐treatment of liver diseases, including hepatitis C virus (HCV), and its antiviral activity has been demonstrated in vitro and in HCV patients administered an intravenous formulation of the major silymarin flavonolignans, silybin A and silybin B. The safety and dose‐exposure relationships of higher than customary oral doses of silymarin and its acute effects on serum HCV RNA were evaluated in noncirrhotic HCV patients. Four cohorts of 8 patients with well‐compensated, chronic noncirrhotic HCV who failed interferon‐based therapy were randomized 3:1 to silymarin or placebo. Oral doses of 140, 280, 560, or 700 mg silymarin were administered every 8 hours for 7 days. Steady‐state exposures for silybin A and silybin B increased 11‐fold and 38‐fold, respectively, with a 5‐fold increase in dose, suggesting nonlinear pharmacokinetics. No drug‐related adverse events were reported, and no clinically meaningful reductions from baseline serum transaminases or HCV RNA titer were observed. Oral doses of silymarin up to 2.1 g per day were safe and well tolerated. The nonlinear pharmacokinetics of silybin A and silybin B suggests low bioavailability associated with customary doses of silymarin may be overcome with doses above 700 mg.

Targeted Quantitative Proteomics for the Analysis of 14 UGT1As and -2Bs in Human Liver Using NanoUPLC–MS/MS with Selected Reaction Monitoring

Targeted quantitative proteomics using heavy isotope dilution techniques is increasingly being utilized to quantify proteins, including UGT enzymes, in biological matrices. Here we present a multiplexed method using nanoLC-MS/MS and multiple reaction monitoring (MRM) to quantify 14 UGT1As and UGT2Bs in liver matrices. Where feasible, we employ two or more proteotypic peptides per protein, with only four proteins quantified with only one proteotypic peptide. We apply the method to analysis of a library of 60 human liver microsome (HLM) and matching S9 samples. Ten of the UGT isoforms could be detected in liver, and the expression of each was consistent with mRNA expression reported in the literature. UGT2B17 was unusual in that ∼30% of liver microsomes had no or little (<0.5 pmol/mg protein) content, consistent with a known common polymorphism. Liver S9 UGT concentrations were approximately 10-15% those of microsomes. The method was robust, precise, and reproducible and provides novel UGT expression data in human liver that will benefit rational approaches to evaluate metabolism in drug development.

Effect of probenecid on the formation and elimination of acyl glucuronides: Studies with zomepirac

When 100 mg oral zomepirac was taken with 500 mg b.i.d. oral probenecid by six healthy subjects, the disposition of zomepirac was markedly altered. Probenecid decreased total plasma clearance of zomepirac by 64%, which resulted in an increase in bioavailability from 0.55 without probenecid to 0.84 when given concurrently. The apparent metabolic clearance of zomepirac to form zomepirac acyl glucuronide was reduced 71% and zomepirac renal clearance, a minor elimination route, was reduced by 79%. When assayed by a method that prevents degradation of the labile acyl glucuronide, zomepirac glucuronide concentrations in plasma were comparable to those of zomepirac. Probenecid decreased the renal clearance of zomepirac glucuronide by 72%, which, together with the increased zomepirac levels, resulted in a 2.8‐fold increase in the AUC of the conjugate. Urinary excretion of zomepirac glucuronide was reduced from 72% to 58% of the dose, but the excretion of free zomepirac was unchanged at 5% of the dose. The ratio of the total clearance/bioavailability of zomepirac in control subjects was 682 ± 246 ml/min, which is double the value reported in previous studies of zomepirac disposition. We believe that this difference is due to degradation of the unstable zomepirac acyl glucuronide in the previous analytic methodologies used. Qualitatively, the effects of probenecid on zomepirac disposition are similar to those previously reported for other drugs of this class that are metabolized to acyl glucuronides. However, zomepirac appears unusual in that significant levels of its acyl glucuronide metabolite are found in vivo.

Disposition and irreversible plasma protein binding of tolmetin in humans

The pharmacokinetics and irreversible plasma protein binding of tolmetin were studied in six healthy subjects after the administration of a single, 400 mg dose of tolmetin. With HPLC analysis, tolmetin, tolmetin glucuronide, and the isomers of tolmetin glucuronide, which result from intramolecular acyl migration in vivo, were detected in the plasma up to 4 hours after administration, whereas these conjugates were present in the urine up to 24 hours. Irreversible binding of tolmetin to plasma proteins occurred in all subjects. Irreversible binding exhibited a better correlation with exposure to tolmetin glucuronide (r = 0.5618) and the isomers of tolmetin glucuronide (r = 0.8200) than with exposure to tolmetin (−0.3635). This is consistent with the hypothesis that covalent binding occurs via the acyl glucuronide.

The Pharmacokinetics of Silymarin Is Altered in Patients with Hepatitis C Virus and Nonalcoholic Fatty Liver Disease and Correlates with Plasma Caspase-3/7 Activity

Silymarin, used by 30 to 40% of liver disease patients, is composed of six major flavonolignans, each of which may contribute to silymarins hepatoprotective properties. Previous studies have only described the pharmacokinetics for two flavonolignans, silybin A and silybin B, in healthy volunteers. The aim of this study was to determine the pharmacokinetics of the major silymarin flavonolignans in liver disease patients. Healthy volunteers and three patient cohorts were administered a single, 600-mg p.o. dose of milk thistle extract, and 14 blood samples were obtained over 24 h. Silybin A and B accounted for 43% of the exposure to the sum of total silymarin flavonolignans in healthy volunteers and only 31 to 38% in liver disease cohorts as a result of accumulation of silychristin (20–36%). Area under the curve (AUC0–24h) for the sum of total silymarin flavonolignans was 2.4-, 3.3-, and 4.7-fold higher for hepatitis C virus (HCV) noncirrhosis, nonalcoholic fatty liver disease (p ≤ 0.03), and HCV cirrhosis cohorts (p ≤ 0.03), respectively, compared with healthy volunteers (AUC0–24h = 2021 ng · h/ml). Caspase-3/7 activity correlated with the AUC0–24h for the sum of all silymarin conjugates among all subjects (R2 = 0.52) and was 5-fold higher in the HCV cirrhosis cohort (p ≤ 0.005 versus healthy). No correlation was observed with other measures of disease activity, including plasma alanine aminotransferase, interleukin 6, and 8-isoprostane F2α, a measure of oxidative stress. These findings suggest that the pharmacokinetics of silymarin is altered in patients with liver disease. Patients with cirrhosis had the highest plasma caspase-3/7 activity and also achieved the highest exposures for the major silymarin flavonolignans.

Safety and Pharmacokinetics of Bevirimat (PA-457), a Novel Inhibitor of Human Immunodeficiency Virus Maturation, in Healthy Volunteers

ABSTRACT Bevirimat (BVM; formerly known as PA-457) is a novel inhibitor of human immunodeficiency virus (HIV) maturation that is being developed for the treatment of HIV infection. The pharmacokinetics of this agent in healthy male volunteers were studied in a randomized, double-blind study in which the participants received single oral doses of placebo (n = 8) or escalating doses of BVM at 25, 50, 100, or 250 mg (n = 6 per dose); escalation was performed only after the pharmacokinetics and safety of the preceding dose had been evaluated. Plasma was collected over 480 h after dosing and urine was collected over 48 h after dosing for determination of the values of pharmacokinetic parameters. BVM was well absorbed after oral administration, with peak plasma concentrations being achieved 1 to 3 h after dosing. The half-life was 60 to 80 h. The exposure assessed by determination of the peak concentration and the area under the concentration-time curve was dose proportional. Single oral doses of BVM were well tolerated: there were no dose-limiting toxicities, and no serious adverse events were reported. These findings suggest that that BVM offers a favorable pharmacokinetic profile, with predictable pharmacokinetics following the oral administration of single doses. The long half-life of BVM may facilitate once-daily dosing.

The influence of norfloxacin and metronidazole on the disposition of mycophenolate mofetil.

The objective of this study was to investigate the effect of concurrent antibiotic administration on the disposition of mycophenolic acid (MPA) and mycophenolic acid glucuronide (MPAG) after oral administration of mycophenolate mofetil (MMF) in healthy subjects. Eleven healthy subjects were enrolled. The study was divided into 4 treatment periods. Subjects received MMF as a single oral 1‐g dose alone and were then randomized to 3 antibiotic treatment periods. The 3 periods included norfloxacin, metronidazole, and a combination of norfloxacin and metronidazole. Antibiotic treatment was started 3 days prior to each MMF pharmacokinetic study day and was given for a total of 5 days. On day 4 of each antibiotic phase, subjects received a single 1‐g oral dose of MMF. Plasma and urine samples were obtained over 48 hours after the MMF dose in all treatment periods and were quantitatively measured for MPA and MPAG. Pharmacokinetic parameters for MPA and MPAG were determined for all periods. Compared to MMF alone, the area under the plasma concentration versus time curve (AUC) of MPA was reduced by an average of 10%, 19%, and 33% when given with norfloxacin, metronidazole, and norfloxacin plus metronidazole, respectively. The AUC of MPAG was also reduced on average by 10%, 27%, and 41% in the corresponding periods. The combination of norfloxacin and metronidazole significantly reduced the AUC of MPA and MPAG in healthy subjects. This likely occurs as a result of reduced enterohepatic recirculation.