Carol Gleason

Pharmacokinetics of cefepime after single and multiple intravenous administrations in healthy subjects.

The pharmacokinetics of cefepime in 31 young, healthy volunteers were assessed after the administration of single and multiple 250-, 500-, 1,000-, or 2,000-mg intravenous doses. Each subject received a single dose of cefepime via a 30-min intravenous infusion on day 1 of the study. Starting from day 2, subjects received multiple doses of cefepime every 8 h for 9 days, and on the morning of day 11, they received the last dose. Serial blood and urine samples were collected after administration of the first dose and on days 1, 6, and 11. Cefepime concentrations in plasma and urine were assayed by using reverse-phase high-performance liquid chromatography with UV detection. Data were evaluated by noncompartmental methods to determine pharmacokinetic parameters. The mean half-life of cefepime was approximately 2 h and did not vary with the dose or duration of dosing. The regression analyses of peak levels (Cmax) in plasma at the end of the 30-min intravenous infusion and the area under the plasma concentration-versus-time curve (AUCo-infinity) showed a dose-proportional response. The steady-state volume of distribution (Vss) was approximately 18 liters and was independent of the administered dose. The multiple-dose pharmacokinetic data are suggestive of a lack of accumulation or change in clearance of cefepime on repeated dosing. Cefepime was excreted primarily unchanged in urine. The recovery of intact cefepime in urine was invariant with respect to the dose and accounted for over 80% of the dose. The values for renal clearance ranged from 99 to 132 ml/min and were suggestive of glomerular filtration as the primary excretion mechanism. It is concluded that cefepime linear pharmacokinetics in healthy subjects.

International Pig-a gene mutation assay trial: Evaluation of transferability across 14 laboratories†‡

A collaborative international trial was conducted to evaluate the reproducibility and transferability of an in vivo mutation assay based on the enumeration of CD59‐negative rat erythrocytes, a phenotype that is indicative of Pig‐a gene mutation. Fourteen laboratories participated in this study, where anti‐CD59‐PE, SYTO 13 dye, and flow cytometry were used to determine the frequency of CD59‐negative erythrocytes (RBCCD59−) and CD59‐negative reticulocytes (RETCD59−). To provide samples with a range of mutant phenotype cell frequencies, male rats were exposed to N‐ethyl‐N‐nitrosourea (ENU) via oral gavage for three consecutive days (Days 1–3). Each laboratory studied 0, 20, and 40 mg ENU/kg/day (n = 5 per group). Three sites also evaluated 4 mg/kg/day. At a minimum, blood samples were collected three times: predosing and on Days 15 and 30. Blood samples were processed according to a standardized sample processing and data acquisition protocol, and three endpoints were measured: %reticulocytes, frequency of RETCD59−, and frequency of RBCCD59−. The methodology was found to be reproducible, as the analysis of technical replicates resulted in experimental coefficients of variation that approached theoretical values. Good transferability was evident from the similar kinetics and magnitude of the dose‐related responses that were observed among different laboratories. Concordance correlation coefficients showed a high level of agreement between the reference site and the test sites (range: 0.87–0.99). Collectively, these data demonstrate that with adequate training of personnel, flow cytometric analysis is capable of reliably enumerating mutant phenotype erythrocytes, thereby providing a robust in vivo mutation assay that is readily transferable across laboratories. Environ. Mol. Mutagen. 2011.

Safety, tolerance, and pharmacokinetic evaluation of cefepime after administration of single intravenous doses.

In this double-blind, single-dose phase I study, the safety and tolerance of cefepime were assessed in 24 healthy male subjects, with ceftazidime as the control drug. Four subjects in each of the six dose groups (62.5, 125, 250, 500, 1,000, or 2,000 mg as a 30-min intravenous infusion) received each antibiotic, according to a crossover design, with a 2-day washout period between treatments. Blood and urine samples were obtained to characterize the pharmacokinetics of cefepime. Plasma and urine samples were assayed for intact cefepime. Samples containing ceftazidime were discarded. The adverse effects observed in the study were mild and infrequent, with prompt recovery from adverse experiences and abnormal laboratory values. The cefepime pharmacokinetic parameters for the therapeutically significant doses of 250 to 2,000 mg appeared to be proportional to dose and similar to literature values for ceftazidime. The elimination half-life of about 2 h was independent of the dose. Urinary recovery of intact cefepime was invariant with respect to dose; an overall mean value of 82% of dose was obtained for the four highest levels. Mean renal clearance was 105 ml/min and suggestive of glomerular filtration as the primary excretion mechanism. In normal humans, the safety and pharmacokinetic profiles of cefepime are very similar to those of ceftazidime.

International Pig‐a gene mutation assay trial (Stage III): Results with N‐methyl‐N‐nitrosourea

N‐methyl‐N‐nitrosourea (MNU) was evaluated in the in vivo Pig‐a mutation assay as part of an International Collaborative Trial to investigate laboratory reproducibility, 28‐day study integration, and comparative analysis with micronucleus (MN), comet, and clinical pathology endpoints. Male Sprague Dawley rats were treated for 28 days with doses of 0, 2.5, 5, and 10 mg MNU/kg/day in two independent laboratories, GlaxoSmithKline (GSK) and Bristol Myers Squibb (BMS). Additional studies investigated the low‐dose region (<2.5 mg/kg/day). Reticulocytes were evaluated for Pig‐a phenotypic mutation, CD59‐negative reticulocytes/erythrocytes (RETsCD59−/ RBCsCD59−) on Days 1, 4, 15, 29, 43, and 57, and for micronucleated reticulocytes (MN‐RETs) on Days 4 and 29. Comet analysis was conducted for liver and whole blood, and hematology and clinical chemistry was investigated. Dose‐dependent increases in the frequency of RETsCD59− and RBCsCD59− were observed by Day 15 or 29, respectively. Dose‐dependent increases were observed in %MN‐RET on Days 4 and 29, and in mean %tail intensity in liver and in blood. Hematology/clinical chemistry data demonstrated bone marrow toxicity. Data comparison between GSK and BMS indicated a high degree of concordance with the Pig‐a mutation assay results, consistent with previous observations with MNU and N‐ethyl‐N‐nitrosourea. These data confirm that complementary genotoxicity endpoints can be effectively incorporated into routine toxicology studies, a strategy that can provide information on gene mutation, chromosome damage, and DNA strand breaks in a single repeat dose rodent study. Collectively, this would reduce animal usage while providing valuable genetic toxicity information within the context of other toxicological endpoints. Environ. Mol. Mutagen., 2011.

Pharmacokinetics of the novel cephalosporin cefepime (BMY-28142) in rats and monkeys.

The disposition of the novel cephalosporin cefepime (BMY-28142) was characterized for intravenous administration of single doses to rats and cynomolgus monkeys, the species used most extensively for safety evaluation of the compound. Serial blood samples were collected from individual animals, and plasma was analyzed for intact cefepime by a high-pressure liquid chromatography-UV method. Assay results were evaluated by compartmental and noncompartmental methods to characterize pharmacokinetics for each species and dosage regimen. For intravenous (i.v.) bolus administration of 28 to 386 mg/kg (body weight) to rats, total body clearance (CL; 11.0 ml/min per kg) was essentially invariant with the dose; however, the terminal half-life (t1/2) and the steady-state distribution volume (Vss) increased with increasing dose level. After administration of 87 to 1,502 mg/kg by i.v. infusion, CL (12.5 ml/min per kg) was again similar for all dose groups. Mean t1/2 values (1.3 to 4.6 h) appeared unusually long for a cephalosporin in rats, and inordinately variable. No consistent differences among dose group mean Vss values were found. The maximal concentration of drug in plasma at the end of infusion was not a linear function of dose. For the cynomolgus monkey, kinetic parameters for 5-min i.v. infusions were linearly related to dose over the range of 10 to 600 mg/kg. Mean parameter values were t1/2 = 1.7 h, CL = 1.6 ml/min per kg, and Vss = 0.21 liters/kg. The pharmacokinetic results indicate substantive differences between the two species with respect to their response to toxicologic doses of cefepime.

Recommendations for Use and Fit-for-Purpose Validation of Biomarker Multiplex Ligand Binding Assays in Drug Development

Multiplex ligand binding assays (LBAs) are increasingly being used to support many stages of drug development. The complexity of multiplex assays creates many unique challenges in comparison to single-plexed assays leading to various adjustments for validation and potentially during sample analysis to accommodate all of the analytes being measured. This often requires a compromise in decision making with respect to choosing final assay conditions and acceptance criteria of some key assay parameters, depending on the intended use of the assay. The critical parameters that are impacted due to the added challenges associated with multiplexing include the minimum required dilution (MRD), quality control samples that span the range of all analytes being measured, quantitative ranges which can be compromised for certain targets, achieving parallelism for all analytes of interest, cross-talk across assays, freeze-thaw stability across analytes, among many others. Thus, these challenges also increase the complexity of validating the performance of the assay for its intended use. This paper describes the challenges encountered with multiplex LBAs, discusses the underlying causes, and provides solutions to help overcome these challenges. Finally, we provide recommendations on how to perform a fit-for-purpose-based validation, emphasizing issues that are unique to multiplex kit assays.

Comparison of the effects of food on the pharmacokinetics of cefprozil and cefaclor.

The objective of this study was to assess the effects of food on the pharmacokinetics of cefprozil and cefaclor. A group of 12 healthy male volunteers received a single 250-mg dose of cefprozil or cefaclor under fasting conditions as well as after the intake of food. There was a 1-week washout period between each treatment. Serial blood samples were collected and assayed for cefprozil or cefaclor by specific high-pressure liquid chromatographic methods. The mean +/- standard deviation peak concentration (Cmax) of cefprozil in plasma was 6.13 +/- 1.22 micrograms/ml under the fasting condition and 5.27 +/- 1.06 micrograms/ml after breakfast, and these values were not significantly different from each other. The corresponding median time to reach Cmax was prolonged after food intake, but this difference was not significant. The mean Cmax values of cefaclor decreased significantly from 8.70 +/- 2.72 micrograms/ml under the fasting condition to 4.29 +/- 1.52 micrograms/ml after breakfast, and the corresponding median times to reach Cmax were significantly prolonged. The mean half-lives of cefprozil and cefaclor were nearly identical for the two treatments, suggesting that the elimination kinetics of these cephalosporins remained unaltered when the drugs were administered with food. The area under the plasma-concentration-versus-time curves for fasted and fed conditions were not significantly different for both drugs. The results of this study indicate that the extent of absorption and rate of elimination of both cephalosporins remain unaltered in the presence of food. However, the absorption rate of cefaclor is significantly reduced in the presence of food, while that of cefprozil remains unaltered. As a result, the Cmax of cefaclor is significantly reduced in the presence of food, whereas that of cefprozil is not significantly affected. Cefprozil can be administered with a meal without markedly affecting levels in blood.

Phase I study of multiple-dose cefprozil and comparison with cefaclor.

The objectives of this study were to assess the safety and tolerance of cefprozil, to characterize the pharmacokinetics of cefprozil after administration of multiple doses of the drug, and to compare these pharmacokinetic parameters with those obtained with cefaclor. The volunteers received 28 doses of 250, 500, or 1,000 mg of cefprozil or 500 mg of cefaclor every 8 h for 10 days. Serial blood samples and the total volume of urine voided by each individual were collected for pharmacokinetic evaluation on days 1, 5, and 10. Both cephalosporins were well tolerated after multiple oral dosing. The peak levels in plasma (Cmax) of cefprozil ranged from 5.7 to 18.3 micrograms/ml after oral administration of 250- to 1,000-mg doses. The regression analysis of Cmax on cefprozil dose showed a dose-linear response. The mean Cmax of cefaclor ranged from 15.2 to 16.7 micrograms/ml and did not change significantly on multiple dosing. The overall mean terminal half-life of cefprozil was 1.2 h and was invariant with respect to dose or duration of dosing. The area under the plasma-concentration-versus-time curve from 0 h to infinity (AUC0-infinity) of cefprozil increased in a dose-proportional manner with an increase in dose. The overall urinary recovery (61% of dose) and renal clearance values of cefprozil were generally invariant with respect to dose and duration of dosing. While cefprozil was apparently absorbed less rapidly and achieved lower Cmax values than cefaclor, the AUC0-infinity of cefprozil was nearly twofold greater than that of cefaclor. The half-life of cefprozil was also twofold longer than that observed for cefaclor. Although the urinary recovery of cefaclor (75% of dose) was significantly higher than that of cefprozil (61% of dose), the concentrations of cefprozil in urine remained significantly higher than those of cefaclor from 2 to 8 h postdosing. If the therapeutic concept is maintained that levels of beta-lactam antibiotics in plasma should exceed the MIC for the offending organisms over a period that approximates the dosing interval, then cefprozil would appear to be suitable for twice-daily administration, whereas cefaclor should probably be administered three or even four times a day.

Development and validation of an LC–MS/MS assay for the quantitation of a PEGylated anti-CD28 domain antibody in human serum: overcoming interference from antidrug antibodies and soluble target

AIM To support drug development of a PEGylated anti-CD28 domain antibody, a sensitive and robust LC-MS/MS assay was developed for the first in-human multiple ascending dose study. MATERIALS & METHODS The procedure consists of a protein precipitation with acidified acetonitrile, followed by trypsin digestion of the supernatant. A surrogate peptide from the complementarity determining region was quantified with an LC-MS/MS assay using a stable isotope-labeled internal standard with flanking amino acids. An acid dissociation step was found to be essential to achieve full analyte recovery in the presence of antidrug antibodies and soluble target CD28. RESULTS & CONCLUSION The fully validated LC-MS/MS assay demonstrates good accuracy (% deviation ≤6.3) and precision (%CV ≤5.2) with an lower limit of quantitation of 10 ng/ml.

Fit-for-purpose bioanalytical cross-validation for LC–MS/MS assays in clinical studies

The paradigm shift of globalized research and conducting clinical studies at different geographic locations worldwide to access broader patient populations has resulted in increased need of correlating bioanalytical results generated in multiple laboratories, often across national borders. Cross-validations of bioanalytical methods are often implemented to assure the equivalency of the bioanalytical results is demonstrated. Regulatory agencies, such as the US FDA and European Medicines Agency, have included the requirement of cross-validations in their respective bioanalytical validation guidance and guidelines. While those documents provide high-level expectations, the detailed implementation is at the discretion of each individual organization. At Bristol-Myers Squibb, we practice a fit-for-purpose approach for conducting cross-validations for small-molecule bioanalytical methods using LC-MS/MS. A step-by-step proposal on the overall strategy, procedures and technical details for conducting a successful cross-validation is presented herein. A case study utilizing the proposed cross-validation approach to rule out method variability as the potential cause for high variance observed in PK studies is also presented.