James D. Hulse

Bioanalytical Method Validation—A Revisit with a Decade of Progress

This report is a synthesis of (1) the earlier conference on Analytical Methods Validation−Bioavailability, Bioequivalence and Pharmacokinetic Studies (Conference held in Arlington, VA, December 3–5, 1990 and the report published in Pharmaceutical Research, 9: 588-592, 1992) and (2) the workshop on “Bioanalytical Methods Validation—A Revisit with a Decade of Progress,” (Workshop held in Arlington, VA, January 12–14, 2000), sponsored by the American Association of Pharmaceutical Scientists and the U. S. Food and Drug Administration. The bioanalytical method validation workshop of January 12–14, 2000 was directed towards small molecules. A separate workshop was held in March 1–3, 2000 to discuss validation principles for macromolecules. The purpose of this report is to represent the progress in analytical methodologies over the last decade and assessment of the major agreements and issues discussed with regard to small molecules at both the conference and the workshop. The report is also intended to provide guiding principles for validation of bioanalytical methods employed in support of bioavailability, bioequivalence, and pharmacokinetic studies in man and in animals.

Analytical methods validation: Bioavailability, bioequivalence and pharmacokinetic studies: Sponsored by the American Association of Pharmaceutical Chemists, U.S. Food and Drug Administration, Fédération Internationale Pharmaceutique, Health Protection Branch (Canada) and Association of Official Analytical Chemists

Abstract This is a summary report of the conference on ‘Analytical Methods Validation: Bioavailability, Bioequivalence and Pharmacokinetic Studies.’ The conference was held from December 3 to 5, 1990, in the Washington, DC area and was sponsored by the American Association of Pharmaceutical Scientists, U.S. Food and Drug Administration, Federation Internationale Pharmaceutique, Health Protection Branch (Canada) and Association of Official Analytical Chemists. The purpose of the report is to represent our assessment of the major agreements and issues discussed at the conference. This report is also intended to provide guiding principles for validation of analytical methods employed in bioavailability, bioequivalence and pharmacokinetic studies in man and animals. The objectives of the conference were: (1) to reach a consensus on what should be required in analytical methods validation and the procedures to establish validation; (2) to determine processes of application of the validation procedures in the bioavailability, bioequivalence and pharmacokinetic studies; and (3) to develop a report on analytical methods validation (which may be referred to in developing future formal guidelines). Acceptable standards for documenting and validating analytical methods with regard to processes, parameters or data treatments were discussed because of their importance in assessment of pharmacokinetic. bioavailability, and bioequivalence studies. Other topics which were considered essential in the conduct of pharmacokinetic studies or in establishing bioequivalency criteria, including measurement of drug metabolites and stereoselectivc determinations, were also deliberated.

Pharmacokinetics of Hydroxyethyl Starch in Normal Subjects

Abstract: To determine the elimination of high‐molecular‐weight hydroxyethyl starch (HES, Mw 450,000) in normal subjects, ten volunteers were given 500 ml 6% HES solution by intravenous infusion, and serial blood and urine samples were collected for nonglucose total carbohydrate determination. On the average, 46 and 64 per cent of the dose was excreted in the urine within two and eight days, respectively. The plasma concentration declined rapidly during the first week after infusion. The average terminal half‐life was 17 days during the first 42 days, which accounted for elimination of about 90 per cent of the dose. The remainder was eliminated with a terminal half‐life of 48 days determined between days 42 and 83 of the study. As expected, the infusion of HES resulted in plasma volume expansion over a 48‐hour period during which time levels of nonglucose carbohydrates were above 3.5 mg/ml. HES is metabolized by α‐amylase in the body. During the first 48 hours after infusion of HES, plasma α‐amylase activity was significantly increased over control. Concomitantly, α‐amylase activity in urine was also elevated but not significantly so.

Hetastarch: An Overview of the Colloid and its Metabolism:

Hetastarch, ethoxylated amylopectin, has found clinical utility as a plasma volume expansion agent, a sedimenting agent during pheresis, and a pump priming fluid. Hetastarch is a complex mixture of derivatized amylopectin molecules of various molecular sizes. The derivatization causes resistance to enzymatic hydrolysis, therefore, allowing hetastarch sufficient vascular residence time to be an effective vascular osmotic agent. This has led to its use as a volume expander and to its consequent use as a pump priming fluid. The metabolism of hetastarch proceeds through α-amylase hydrolysis of glycosidic bonds, yielding molecules small enough for renal clearance, but does not result in complete hydrolysis. Hence, glucose is not a significant product of hetastarch metabolism. Metabolism proceeds at such a rate that volume expansion is seen for 24–36 hours with a maximum effect (100–172 percent of the infused volume) occurring shortly after infusion. Ninety percent of the dose is eliminated with a half-life of about 17 days.

Surrogate Biochemical Markers: Precise Measurement for Strategic Drug and Biologics Development

More efficient drug and biologics development is necessary for future success of pharmaceutical and biotechnology companies. One way to achieve this objective is to use rationally selected surrogate markers to improve the early decision‐making process. Using typical clinical chemistry methods to measure biochemical markers may not ensure adequate precision and reproducibility. In contrast, using analytical methods that meet good laboratory practices along with rational selection and validation of biochemical markers can give those who use them a competitive advantage over those who do not by providing meaningful data for earlier decision making.

Effect of High-Dose Oral Ganciclovir on Didanosine Disposition in Human Immunodeficiency Virus (HIV)-Positive Patients

This study was designed to investigate the interaction between high‐dose oral ganciclovir (6,000 mg/day) and didanosine at steady state in patients who were seropositive for human immunodeficiency virus (HIV) and cytomegalovirus (CMV) infection. The study was conducted as an open‐label, randomized, three‐period crossover study. Patients received (in random order) multiple oral doses of didanosine 200 mg every 12 hours alone, ganciclovir 2,000 mg every 8 hours alone, and ganciclovir 2,000 mg every 8 hours in combination with didanosine 200 mg every 12 hours. Blood and urine samples for determinations of drug concentrations were obtained on day 3 of each dose regimen. When ganciclovir was administered either before or 2 hours after didanosine, the mean increases in maximum concentration (Cmax), area under the concentration—time curve (AUC0–12), and percent excreted in urine of didanosine were 58.6% and 87.3%, 87.3% and 124%, and 100% and 153%, respectively. There were no statistically significant effects of didanosine on the steady‐state pharmacokinetics of ganciclovir in the presence of didanosine, irrespective of sequence of administration. There were no significant changes in renal clearance of didanosine, suggesting that the mechanism for the interaction does not involve competition for active renal tubular secretion. The mechanism responsible for increased didanosine concentrations and percent excreted in urine during concurrent ganciclovir therapy may be a result of increased bioavailability of didanosine. However, the mechanism appears to be saturated at oral ganciclovir doses of 3 g/day.

Pharmacokinetics of Esmolol in Hepatic Disease

Esmolol is an intravenous beta blocker with a short duration of action. The pharmacokinetics of esmolol and its acid metabolite, ASL‐8123, were studied in nine patients who had stable, biopsy‐proved Laennecs cirrhosis and in three normal volunteer controls. Kinetics were determined after a four‐hour continuous infusion of esmolol at a rate of 200 μg/kg/min. Blood samples were collected during the infusions and at frequent intervals thereafter. The parameters studied were the steady state concentration, the total body clearance, the elimination half‐life, the area under the curve, and the volume of distribution. No significant differences in any of these parameters were detected between control subjects and those with hepatic disease, for either esmolol or its acid metabolite. It is concluded from this study that Laennecs cirrhosis does not cause any change in the pharmacokinetics of esmolol or its major metabolite. Therefore, adjustments in dosage of esmolol are not required for patients with Laennecs cirrhosis.

Metabolism and Urinary Excretion of Esmolol in Humans

The urinary excretion patterns of esmolol, a short‐acting beta blocker, and its major metabolite were investigated in eight healthy men after intravenous infusion of 50, 100, 200, and 300 μg/kg/min of esmolol for six hours and 150 μg/kg/min for 24 hours. Esmolol and the metabolite concentrations in urine were determined by high‐performance liquid chromatography. The mean urinary recoveries of the unchanged drug were 0.64%, 0.67%, 0.69%, 0.77, and 0.98% after the 50, 100, 150, 200, and 300 μg/kg/min dose, respectively. Recovery of the metabolite was independent of dose, and the overall mean recovery accounted for 73% of administered dose. The results of this study indicate that esmolol is extensively metabolized, and the extent of the metabolism is not dose related in the dosage range used. The renal route plays a very minor role in the elimination of the drug but is important for the elimination of the metabolite.

An Evaluation of the Effect of Food on the Oral Bioavailability of Sustained‐Release Morphine Sulfate Tablets (ORAMORPH SR) After Multiple Doses

The effect of food on the oral bioavailability of sustained‐release morphine sulfate tablets (ORAMORPH SR; Roxane Laboratories, Inc., Columbus, OH; OSR) was examined in an open‐label, randomized, two‐period crossover study. Healthy male volunteers received a 30‐mg OSR tablet orally every 12 hours for seven doses during both the fasted and fed states. Dosing periods were separated by a 14‐day washout Volunteers in the fasted group received all doses either 2 hours before or after meals. Volunteers in the fed group received all doses immediately after meals. All meals were standardized. Serial blood samples were collected for analysis of plasma morphine concentration by radioimmunoassay. Pharmacokinetic parameters were calculated using plasma concentration data collected after the last dose at 72 hours of each dosing period. The two one‐sided t analysis indicated confidence intervals between 80% and 120% for maximum peak plasma concentration (Cmax), AUC72‐84hr, Cavg, and Cmin. The relative bioavailability of OSR administered after meals was 90.2% of that administered in the fasted state. As compared with the fasted condition, morphine bioavailability was essentially unchanged when multiple oral doses of 30‐mg OSR tablets were given after meals.

Tolerance and beta‐adrenergic blocking activity of flestolol, a short‐acting beta blocker

The tolerance and β‐adrenergic blocking activity of flestolol, a short‐acting β‐blocker, was investigated in 30 subjects. Flestolol infused intravenously at doses up to 100 μg/kg/min was found to be well tolerated. A dose‐dependent attenuation of isoproterenol‐induced tachycardia and increase in systolic blood pressure occurred with flestolol at doses ranging from 0.5 to 15.0 μg/kg/min. The average percent reduction in isoproterenol‐induced tachycardia (β‐blockade) at each dose of flestolol, 0.5, 2.5, 5.0, 15.0, and 50.0 μg/ kg/min, was 15.1%, 45.9%, 67.0%, 85.9%, and 90.3%, respectively. The onset of β‐blockade occurred within 30 minutes. After the end of flestolol infusion there was a marked reduction in β‐blockade within 6 minutes, with complete recovery from β‐blockade within 30 to 45 minutes. There was a statistically significant (P < 0.01) positive correlation between flestolol dosage and its blood levels (r = 0.91) as well as between the flestolol‐induced β‐blockade and its dosage (r = 0.62).