Eric Fluhler

Interspecies prediction of human drug clearance based on scaling data from one or two animal species.

A data-driven approach was adopted to derive new one- and two-species-based methods for predicting human drug clearance (CL) using CL data from rat, dog, or monkey (n = 102). The new one-species methods were developed as CLhuman/kg = 0.152 · CLrat/kg, CLhuman/kg = 0.410 · CLdog/kg, and CLhuman/kg = 0.407 · CLmonkey/kg, referred to as the rat, dog, and monkey methods, respectively. The coefficient of the monkey method (0.407) was similar to that of the monkey liver blood flow (LBF) method (0.467), whereas the coefficients of the rat method (0.152) and dog method (0.410) were considerably different from those of the LBF methods (rat, 0.247; dog, 0.700). The new rat and dog methods appeared to perform better than the corresponding LBF methods, whereas the monkey method and the monkey LBF method showed improved predictability compared with the rat and dog one-species-based methods and the allometrically based “rule of exponents” (ROE). The new two-species methods were developed as CLhuman = arat-dog · W human0.628 (referred to as rat-dog method) and CLhuman = arat-monkey · W human0.650 (referred to as rat-monkey method), where arat-dog and arat-monkey are the coefficients obtained allometrically from the corresponding two species. The predictive performance of the two-species methods was comparable with that of the three-species-based ROE. Twenty-six Wyeth compounds having data from mouse, rat, dog, monkey, and human were used to test these methods. The results showed that the rat, dog, monkey, rat-dog, and rat-monkey methods provided improved predictions for the majority of the compounds compared with those for the ROE, suggesting that the use of three or more species in an allometrically based approach may not be necessary for the prediction of human exposure.

Workshop Report: Crystal City V—Quantitative Bioanalytical Method Validation and Implementation: The 2013 Revised FDA Guidance

In September 2013, the FDA released a draft revision of the Bioanalytical Method Validation (BMV) Guidance, which included a number of changes to the expectations for bioanalysis, most notably the inclusion of biomarker assays and data. To provide a forum for an open, inclusive discussion of the revised draft BMV Guidance, the AAPS and FDA once again collaborated to convene a two-and-a-half day workshop during early December 2013 in Baltimore, MD, USA. The resulting format embodied extensive open discussion and each thematic session included only brief, concise descriptions by Agency and industry representatives prior to opening the floor discussion. The Workshop was built around four thematic sessions (Common Topics, Chromatographic, Ligand-Binding Assays, and Biomarkers) and a final session with international regulators, concluding with a review of the outcomes and recommendations from the thematic sessions. This Workshop report summarizes the outcomes and includes topics of agreement, those where the FDA will consider the Industry’s perspective, and those where the workshop provided a first open dialogue. This article will be available to the bioanalytical community at http://www.aaps.org/BMV13.

A sensitive human bone assay for quantitation of tigecycline using LC/MS/MS

Tigecycline (Tygacil,Wyeth) is a first-in-class, broad spectrum antibiotic with activity against multiple-resistant organisms. In order to address the unexpectedly low tigecycline concentrations in human bone samples analyzed using a LC/MS/MS method developed elsewhere, we have developed and validated a new and sensitive human bone assay for the quantitation of tigecycline using LC/MS/MS. The new method utilizes the addition of a stabilizing agent to the human bone sample, homogenization of human bone in a strong acidic-methanol extraction solvent, centrifugation of the bone suspension, separation by liquid chromatography, and detection of tigecycline by mass spectrometry. Linearity was demonstrated over the concentration range from 50 to 20,000 ng/g using a 0.1g human bone sample. The intra- and inter-day accuracy of the assay was within 100+/-15%, and the corresponding precision (CV) was <15%. The stability of tigecycline was evaluated and shown to be acceptable under the assay conditions. The extraction recovery of tigecycline with the current method was 79% when using radio-labeled rat bone samples as a substitute for human bone samples. Twenty-four human bone samples collected previously from volunteers without infections who had elective orthopedic surgery after receiving a single dose of tigecycline were re-analyzed using the current validated method. Tigecycline concentrations in these samples ranged from 238 to 794 ng/g with a mean value 9 times higher than the mean concentration previously reported. The data demonstrated that the current method has significantly higher extraction efficiency than the previously reported method.

Repeat Analysis and Incurred Sample Reanalysis: Recommendation for Best Practices and Harmonization from the Global Bioanalysis Consortium Harmonization Team

The A7 harmonization team (A7 HT), a part of the Global Bioanalysis Consortium (GBC), focused on reviewing best practices for repeat analysis and incurred sample reanalysis (ISR) as applied during regulated bioanalysis. With international representation from Europe, Latin America, North America, and the Asia Pacific region, the team first collated common practices and guidance recommendations and assessed their suitability from both a scientific and logistical perspective. Subsequently, team members developed best practice recommendations and refined them through discussions and presentations with industry experts at scientific meetings. This review summarizes the team findings and best practice recommendations. The few topics where no consensus could be reached are also discussed. The A7 HT recommendations, together with those from the other GBC teams, provide the basis for future international harmonization of regulated bioanalytical practices.

PK of immunoconjugate anticancer agent CMD-193 in rats: ligand-binding assay approach to determine in vivo immunoconjugate stability

BACKGROUND Antibody-drug conjugates (ADCs) are a new generation of anticancer therapeutics. The objective of this manuscript is to propose a methodology that can be used to assess the stability of the ADCs by using the PK data obtained by ligand-binding assays that measure various components of ADCs. RESULTS The ligand-binding assays format of different components of ADCs provided unique valuable PK information. The mathematical manipulation of the bioanalytical data provided an insight into the in vivo integrity, indicating that the loading of the calicheamicin on the G193 antibody declines in an apparent slow first-order process. CONCLUSION This report demonstrates the value of analyzing various components of the ADC and their PK profiles to better understand the disposition and in vivo stability of ADCs.

Determination of tigecycline in human skin using a novel validated LC–MS/MS method

BACKGROUND To develop and validate a sensitive and novel bioanalytical method for measuring tigecycline concentrations in human skin using LC-MS/MS. RESULTS The method utilizes addition of a stabilizing agent to the human skin or surrogate (human liver or rat skin), homogenization of human skin in a strong acidic-methanol extraction solvent, centrifugation of the skin suspension, filtration of the skin suspension supernatant, separation by LC (Polaris™ C18-A 50 × 2.0 mm), and detection of tigecycline by MS/MS. Linearity was 50-20,000 ng/g, using a sample size of 100 mg. The intra-and inter-day accuracy and precision of the assay met acceptance criteria. CONCLUSION This method has been successfully applied to 17 incurred human skin samples from volunteers with surgical infections who received intravenous doses of tigecycline (100 mg initial loading dose and 50 mg every 12 h for at least 2 days). Tigecycline concentrations in these samples ranged from 185 to 2853 ng/g.

Understanding Bioanalysis Regulations

Bioanalytical data submitted to global health authorities in support of drug approval applications is expected to meet certain regulatory standards and requirements. Bioanalytical assay validation and the conduct of study sample analysis is addressed with a multitude of regulatory guidance documents. This chapter provides an overview of the regulations from a historical perspective to current day developments. Originally written around bioanalysis to support pharmacokinetic studies, bioanalysis regulatory language is now being applied to endogenous biomarker assays and evaluating immunogenicity of large molecule therapeutics. Quality management and documentation are also discussed in this chapter along with the process of how modern bioanalysis regulations are continuing to develop.

Novel Application of the Two‐Period Microtracer Approach to Determine Absolute Oral Bioavailability and Fraction Absorbed of Ertugliflozin

Ertugliflozin, a sodium glucose cotransporter‐2 inhibitor, is approved in the United States for treatment of type 2 diabetes mellitus. A novel two‐period study design with 14C microtracer dosing in each period was used to determine absolute oral bioavailability (F) and fraction absorbed (Fa) of ertugliflozin. Eight healthy adult men received 100‐μg i.v. 14C‐ertugliflozin (400 nCi) dose 1 h after a 15‐mg oral unlabeled ertugliflozin dose (period 1), followed by 100 μg 14C‐ertugliflozin orally along with 15 mg oral unlabeled ertugliflozin (period 2). Unlabeled ertugliflozin plasma concentrations were determined using high‐performance liquid‐chromatography tandem mass spectrometry (HPLC‐MS/MS). 14C‐ertugliflozin plasma concentrations were determined using HPLC‐accelerator mass spectrometry (AMS) and 14C urine concentrations were determined using AMS. F ((area under the curve (AUC)p.o./14C‐AUCi.v.)*(14C‐Dosei.v./Dosep.o.)) and Fa ((14C_Total_Urinep.o./14C_Total_Urinei.v.)* (14C‐Dosei.v./14C‐Dosep.o.)) were estimated. Estimates of F and Fa were 105% and 111%, respectively. Oral absorption of ertugliflozin was complete under fasted conditions and F was ∼100%. Ertugliflozin was well tolerated.

Conference Report: New setting for the Land O’Lakes bioanalytical conference

The University of Wisconsin bioanalytical conference is presented each year by the Extension Services in Pharmacy, the professional development department within the School of Pharmacy. The purpose of this 3-day conference was to provide an educational forum to discuss issues and applications associated with the analysis of xenobiotics, metabolites, biologics and biomarkers in biological matrices. The conference was designed to include and encourage an open exchange of scientific and methodological applications for bioanalysis. To increase the interactive nature of the conference the program was composed of a mixture of lectures, interactive discussions, poster sessions and roundtables. This paper summarizes the presentations at the Fourteenth Annual Conference, offered in a new venue.

Conference Report: Emerging technology for bioanalysis in the next decade

This University of Wisconsin School of Pharmacy bioanalytical conference is presented each year by the Extension Services in Pharmacy, the professional development department within the School. The purpose of this 4-day conference is to provide an educational forum to discuss issues and applications associated with the analysis of xenobiotics, metabolites, biologics and biomarkers in biological matrices. The conference is designed to include and encourage an open exchange of scientific and methodological applications for bioanalysis. To increase the interactive nature of the conference, the program was a mixture of lectures, poster sessions, round table discussions and workshops. This article summarizes the presentations at the 13th Annual Conference.