Search

PDF file - 112K, Supplementary Figure 2 - in vivo study body weights

PDF file - 316K, Supplementary Figure 1 - in vivo study body weights

PDF file - 72K

The COVID-19 pandemic challenged pharmaceutical and bioanalytical communities at large, in the development of vaccines and therapeutics as well as supporting ongoing drug development efforts. Existing processes were challenged to manage loss of staffing at facilities along with added workloads for COVID-19-related study support including conducting preclinical testing, initiating clinical trials, conducting bioanalysis and interactions with regulatory agencies, all in an ultra-rapid timeframes. A key factor of success was creative rethinking of processes and removing barriers – some of which hitherto had been considered immovable. This article describes how bioanalysis was crippled at the onset of the pandemic but how innovative and highly collaborative efforts across teams within and outside of both pharma, bioanalytical labs and regulatory agencies worked together remarkably well.

Aim: Bioanalytical methods undergo many revisions and modifications throughout drug development to meet the objectives of the study and development program. Results: Validated LC–MS/MS methodology used to quantify abemaciclib and four metabolites in human plasma is described. The method, initially validated to support the first-in-human study, was successfully modified to include additional metabolites as in vitro and in vivo information about the activity and abundance of human metabolites became available. Consistent performance of the method over time was demonstrated by an incurred sample reanalysis passing rate exceeding 95%, across clinical studies. An overview of the numerous methods involved during the development of abemaciclib, including the quantification of drugs evaluated as combination regimens and used as substrates during drug–drug interaction studies, is presented. Conclusion: Robust bioanalytical methods need to be designed with the flexibility required to support the evolving study objectives associated with registration and post-registration trials.

The disposition and metabolism of prexasertib, a CHK-1 inhibitor was characterised over a 120 h period following a single 170-mg intravenous dose of [14C]prexasertib (50 µCi) to 6 patients with adv...

There is growing interest in the implementation of microsampling approaches for the quantitation of circulating concentrations of analytes in biological samples derived from nonclinical and clinical studies involved in drug development. This interest is partly due to the ethical advantages of taking smaller blood volumes, particularly for studies in rodents, children and the critically ill. In addition, these technologies facilitate sampling to be performed in previously intractable locations and occasions. Further, they enable the collection of samples for additional purposes (extra time points, biomarkers, sampling during a clinical event, etc). This article gives a comprehensive insight to the utilization of these approaches in drug discovery and development, and provides recommendations for best practice for nonclinical, clinical and bioanalytical aspects.

There is continuing interest in the development and application of various microsampling technologies for drug development. The AAPS bioanalytical community microsampling subgroup and the European Bioanalysis Forum conducted a survey of their members (39 individual organizations). This gives a snapshot of current practices and demonstrates that implementation of microsampling approaches is becoming increasingly commonplace, but not universal. Greater adoption was observed for nonclinical studies, particularly nonregulatory. A number of respondents reported that they have included microsampling data in regulatory submissions. Another important observation was that where microsampling is employed for clinical studies, dried blood approaches predominate, reflecting the interest in their use where they enable sample collection which is not feasible with standard approaches or to derive richer data sets.

The use of quality control (QC) samples in bioanalysis is well established and consistent with regulatory guidance. However, a systematic evaluation of whether QC samples serve the intended purpose of improving data quality has not been undertaken. The Translational and ADME Sciences Leadership Group (TALG) of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) conducted an evaluation to assess whether closer agreement is observed when comparing pharmacokinetic data from two passed runs, than when comparing data from failed and passed (retest) runs. Analysis of data collected across organizations, molecular types and analytical platforms, revealed that bioanalytical methods are very reproducible; and that QC samples improve the overall quality of pharmacokinetic concentration data and justifies their continued use.

The microsampling workshop generated recommendations pertaining to blood sampling site (venous blood versus capillary blood), when to conduct a bridging study, statistical approaches to establish correlation/concordance and deciding on sample size, opportunities and challenges with patient-centric sampling, and how microsampling technology can enrich clinical drug development. Overall, the goal was to provide clarity and recommendations and enable the broader adoption of microsampling supporting patients’ needs, convenience, and the transformation from clinic-centric to patient-centric drug development. The need and adoption of away-from-clinic sampling techniques has become critical to maintain patient safety during the current COVID-19 pandemic.

[ ]of writing, it is unclear whether this will result in a permanent change in how blood sampling for clinical trials and routine healthcare are performed, although it seems unlikely that the progression of these sampling technologies will be slowed by the current situation Many of the early publications in this area had a primary focus on the development of blood sampling technologies, validation of bioanalytical methods and were limited to applications supporting nonclinical and clinical studies for pharmaceutical drug development Bioanalysis 7(16) (2015) https://www future-science com/toc/bio/7/16 Neil Spooner: Spooner Bioanalytical Solutions Ltd, Hertford, UK and School of Life and Medical Sciences, University of Hertfordshire, UK Melanie Anderson: Merck and Co , Inc , West Point, PA 19486, USA Enaksha R Wickremsinhe: Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA

Historically, ligand-binding assays for pharmacokinetic samples employed duplicate rather than singlet-based analysis. Herein, the Translational and absorption, distribution, metabolism and excretion (ADME) Sciences Leadership Group of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) presents a study aiming to determine the value of duplicate versus singlet-based testing. Based on analysis of data collected from eight organizations for 20 drug candidates representing seven molecular types and four analytical platforms, statistical comparisons of validation and in-study quality controls and study unknown samples demonstrated good agreement across duplicate sets. Simulation models were also used to assess the impact of sample duplicate characteristics on bioequivalence outcomes. Results show that testing in singlet is acceptable for assays with % CV ≤15% between duplicates. Singlet-based approach is proposed as the default for ligand-binding assays while a duplicate-based approach is needed where imprecision and/or inaccuracy impede the validation of the assay.

Bioanalytical methods evolve throughout clinical development timelines, resulting in the need for establishing equivalency or correlation between different methods to enable comparison of data across different studies. This is accomplished by the conduct of cross validations and correlative studies to compare and describe the relationship. The incurred sample reanalysis acceptance criterion seems to be adopted universally for cross validations and correlative studies; however, this does not identify any trends or biases between the two methods (datasets) being compared. Presented here are graphing approaches suitable for comparing two methods and describing equivalence or correlation. This article aims to generate awareness on graphing techniques that can be adopted during cross validations and correlative studies.

Aim: Design and execution of a dried blood spot (DBS-LC–MS/MS) assay for pharmacokinetic analyses in oncology patients. Results & discussion: The methodology was validated to collect and store DBS samples from multiple clinical sites, and analyze blood with diverse hematocrit ranges (25–55) to match the potential patient population. Bridging data comparing DBS and plasma showed high degree of concordance with DBS:plasma ratios of 0.81, demonstrating no preferential uptake or association with cellular components of the blood. Pharmacokinetic analysis supporting clinical development was performed using 20 μl of blood collected as DBS. Incurred sample reanalysis showed high correlation. Conclusion: Successful validation of a DBS method and implementation in the clinic enabled pharmacokinetic analysis during the clinical development of a novel oncolytic agent in oncology patients.

The disposition and metabolism of prexasertib, a CHK-1 inhibitor was characterised over a 120 h period following a single 170-mg intravenous dose of [

Sampling blood for toxicokinetic (TK) evaluation in rodents is typically performed using a satellite group of animals to avoid depleting the blood volume and inducing an additional stressor in the main study animals. This practice does not allow for direct comparison of individual animal toxicity to exposure. These studies evaluated serial collection of twelve, 40-µl blood samples from each rat from either a tail clip or a saphenous vein bleed and its impact on toxicologic parameters over 4- and 14-day periods. The results show the feasibility of successfully collecting TK samples from main study animals, using either of the two techniques. Both procedures were amenable to execution by a single technician using dried blood spot sampling. Any changes observed in the primary markers of erythroid mass between the nonbled control rats and repeat sampled rats were minimal and the range of values often overlapped. This technique would improve the quality of data generated from toxicology studies by allowing a direct comparison of systemic exposure to toxicity while at the same time reducing the number of rats by obviating the need for satellite groups.

Incurred sample reanalysis (ISR) is used to ensure the validity and reliability of bioanalytical data. Additionally, ISR results also help identify issues that could influence or bias the data. Overall, based on a decade of experimental data generated at Eli Lilly and Company, ISR failures are few with less than 5% of ISR samples failing to meet acceptance criteria. In a majority of situations, the cause for ISR failures has been ‘human-error.’ However, there are examples where ISR has helped identify issues related to the stability of the analyte or the ruggedness of the method. As a strategy, it is beneficial to conduct ISR following the completion of a few sample runs, so any potential issues impacting the validity and reliability of the data can be identified and rectified early.

Background: Execution of experiments to introduce dried blood spot (DBS) sampling for preclinical GLP studies and subsequent clinical studies. Results: Bridging data showed high concordance with DBS:plasma ratios of 0.9 in rats and 1.1 in dogs, demonstrating no preferential uptake or association with cellular components of the blood. The DBS methodology was fully validated incorporating additional experiments pertinent to DBS sampling, storage and analysis. Individual hematocrit (Hct) values in the test animals (rats and dogs) were within the validated Hct range. DBS concentration data and the resulting TK profiles were not impacted by an Hct bias. Incurred sample reanalysis showed high correlation in dogs (97%) and rats (100%) meeting acceptance criteria. Conclusion: Successfully validated and adopted DBS for preclinical GLP studies.

Purpose: The PI3K/mTOR pathway is frequently aberrated in cancer. LY3023414 is a potent and selective ATP-competitive inhibitor of class I PI3K isoforms, mTOR, and DNA-PK. Here we report the dose-escalation results of the first-in-human phase I study of LY3023414. Patients and Methods: A 3+3 dose escalation for once-daily and twice-daily oral dosing of LY3023414 was followed by an expansion cohort for CYP3A4 drug–drug interaction (DDI) assessment. The primary objective was to determine the recommended phase 2 dose (RP2D). Additional objectives included safety, pharmacokinetics/pharmacodynamics, and antitumor activity. Results: Forty-seven patients with solid tumors received LY3023414 at once-daily (20–450 mg) or twice-daily dosing (150–250 mg). Dose-limiting toxicities were observed at 450 mg once-daily (thrombocytopenia, hypotension, hyperkalemia) in three of three patients, 250-mg twice-daily dosing (hypophosphatemia, fatigue, mucositis) in three of four patients, and in one of 15 patients at 200 mg twice-daily (nausea). Common related AEs included nausea (38%), fatigue (34%), and vomiting (32%) and were mostly mild or moderate. LY3023414 pharmacokinetics demonstrated dose-dependent increase in exposure with ≥ 90% target inhibition at doses ≥150 mg. DDI analysis demonstrated LY3023414 to be a weak inhibitor of CYP3A4. Durable partial response was observed in a patient with endometrial cancer harboring PIK3R1 and PTEN truncating mutations, and 13 additional patients (28%) had a decrease in their target lesions by up to 30%. Conclusions: LY3023414 has a tolerable safety profile and single-agent activity in patients with advanced cancers. The RP2D of LY3023414 monotherapy is 200 mg twice daily based on safety, tolerability, and pharmacokinetic/pharmacodynamic data. Clin Cancer Res; 24(14); 3253–62. ©2018 AACR.

LY2603618 is a selective inhibitor of deoxyribonucleic acid damage checkpoint kinase 1 (CHK1) and has been in development for the enhancement of chemotherapeutic agents. The study described was to assess the potential interaction between LY2603618 and cytochrome P450 isoform 2D6 (CYP2D6) substrate desipramine in patients with cancer. Before clinical investigation, in silico simulations (using Simcyp®) were conducted. An open-label, two-period, fixed-sequence study was planned in 30 patients with advanced or metastatic cancers, in which a 50 mg oral dose of desipramine was administered alone and in combination with 275 mg of LY2603618 (i.v. infusion). An interim analysis was planned after 15 patients completed both periods. Ratios of geometric least squares means (LSMs) of primary pharmacokinetic (PK) parameters and 90% repeated confidence intervals (RCIs) between desipramine plus LY2603618 and desipramine alone were calculated. Lack of an interaction was declared if the 90% RCI fell between 0.8 and 1.25. The LSM ratios (90% RCI) for areas under the plasma concentration–time curve from time zero to tlast (AUC[0-tlast]) and to infinity (AUC[0-∞]) and maximum plasma concentration (Cmax) were 1.14 (1.04, 1.25), 1.09 (0.99, 1.21) and 1.16 (1.05, 1.29). In silico simulations were predictive of clinical results. Single doses of 275 mg LY2603618 administered with 50 mg desipramine were generally well tolerated. In conclusion, no clinically significant interaction was observed between LY2603618 and desipramine in patients with cancer. In silico predictions of clinical results demonstrated that mechanistic and physiologically based PK approaches may inform clinical study design in cancer patients. Copyright © 2014 John Wiley & Sons, Ltd.

RATIONALE Pharmacokinetic data to support clinical development of pharmaceuticals are routinely obtained from liquid plasma samples. The plasma samples require frozen shipment and storage and are extracted off-line from the liquid chromatography/tandem mass spectrometry (LC/MS/MS) systems. In contrast, the use of dried blood spot (DBS) sampling is an attractive alternative in part due to its benefits in microsampling as well as simpler sample storage and transport. However, from a practical aspect, sample extraction from DBS cards can be challenging as currently performed. The goal of this report was to integrate automated serial extraction of large numbers of DBS cards with on-line liquid chromatography/high-resolution accurate mass spectrometry (LC/HRAMS) bioanalysis. METHODS An automated system for direct DBS extraction coupled to a LC/HRAMS was employed for the quantification of midazolam (MDZ) and α-hydroxymidazolam (α-OHMDZ) in human blood. The target analytes were directly extracted from the DBS cards onto an on-line chromatographic guard column followed by HRAMS detection. No additional sample treatment was required. The automated DBS LC/HRAMS method was developed and validated, based on the measurement at the accurate mass-to-charge ratio of the target analytes to ensure specificity for the assay. RESULTS The automated DBS LC/HRAMS method analyzed a DBS sample within 2 min without the need for punching or additional off-line sample treatment. The fully automated analytical method was shown to be sensitive and selective over the concentration range of 5 to 2000 ng/mL. Intra- and inter-day precision and accuracy was less than 15% (less than 20% at the LLOQ). The validated method was successfully applied to measure MDZ and α-OHMDZ in an incurred human sample after a single 7.5 mg dose of MDZ. CONCLUSIONS The direct DBS LC/HRAMS method demonstrated successful implementation of automated DBS extraction and bioanalysis for MDZ and α-OHMDZ. This approach has the potential to promote workload reduction and sample throughput increase. Copyright © 2014 John Wiley & Sons, Ltd.

Background: Online automated extraction of dried blood spots (DBS) via direct extraction to a solid-phase extraction (SPE) cartridge and bioanalysis by high-resolution accurate mass spectrometry was examined. The methodology was validated and used to investigate the effect of hematocrit on assay bias using partial and whole spot extractions from accurately dispensed blood samples. Results: The completed analysis of a DBS sample was accomplished within 2 to 3 min using the online DBS-SPE platform. Hematocrit related bias was observed (>15%) for the partial DBS extractions, but not when the whole DBS was eluted. Conclusion: Results demonstrate successful implementation of automated online DBS-SPE high-resolution accurate mass spectrometry analysis and the remediation of hematocrit bias using a capillary micro dispenser for accurate spotting of blood samples.

An integrated automated approach has been developed for the direct determination of drug concentrations using a SCAP DBS system for online extraction and analysis of dried blood spots (DBS) from DBS paper cards to a multidimensional liquid chromatography system coupled to a high-resolution QTOF mass spectrometry (LC-HRMS). An accurate, precise, selective, and sensitive two-dimensional liquid chromatography-high-resolution mass spectrometry (2D LC-HRMS) assay was developed and validated using small volumes of rat blood (approximately 1.25 μL) from a 2 mm DBS punch. The methodology was validated according to internationally accepted regulated bioanalysis acceptance criteria in order to establish the validity of the combination of online DBS assay and use of HRMS for quantitative bioanalysis. The fully automated procedure exhibited acceptable linearity (r(2)0.997) over the concentration range of 5 to 1000 ng/mL. Intra- and interday precision and accuracy runs indicated relative errors less than 20% at the LLOQ level and less than 15% at all other levels. The direct extraction and analysis of DBS samples resulted in a 5-fold improvement in assay sensitivity compared to conventional off-line extraction of punched DBS samples. In addition, the impact of blood hematocrit (Hct) on accurate quantification of the studied drugs also was evaluated, comparing Hct values of 30% and 60% against standards prepared at 45%. Hematocrit experiments show that Hct can influence the accuracy of drugs quantified by DBS and needs to be thoroughly evaluated prior to committing to validating a DBS assay. The online DBS system coupled to the LC-HRMS was then successfully applied to a pharmacokinetic study performed on male Sprague-Dawley rats after administration of a single dose of 5 and 10 mg/kg for midazolam and desipramine, respectively.

Purpose: Current practices applied to mouse pharmacokinetic (PK) studies often use large numbers of animals with sporadic or composite sampling that inadequately describe PK profiles. The purpose of this work was to evaluate and optimize blood microsampling techniques coupled with dried blood spot (DBS) and LC-MS/MS analysis to generate reliable PK data in mice. In addition, the feasibility of cross-over designs was assessed and recommendations are presented. Methods: The work describes a comprehensive evaluation of five blood microsampling techniques (tail clip, tail vein with needle hub, submandibular, retro-orbital, and saphenous bleeding) in CD-1 mice. The feasibility of blood sampling was evaluated based on animal observations, ease of bleeding, and ability to collect serial samples. Methotrexate, gemfibrozil and glipizide were used as test compounds and were dosed either orally or intravenously, followed by DBS collection and LC-MS/MS analysis to compare PK with various bleeding methods. Results: Submandibular and retro-orbital methods that required non-serial blood collections did not allow for inter-animal variability assessments and resulted in poorly described absorption and distribution kinetics. The submandibular and tail vein with needle-hub methods were the least favorable from a technical feasibility perspective. Serial bleeding was possible with cannulated animals or saphenous bleeding in non-cannulated animals. Conclusions: Of the methods that allowed serial sampling, the saphenous method when executed as described in this report, was most practical, reproducible and provided for assessment of inter-animal variability. It enabled the collection of complete exposure profiles from a single mouse and the conduct of an intravenous/oral cross-over study design. This methodology can be used routinely, it promotes the 3Rs principles by achieving reductions in the number of animals used, decreased restraints and animal stress, and improved the quality of data obtained in mouse PK studies. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page

Background: Understanding the distribution of the analyte between the cellular and noncellular (plasma) components of the blood is important, especially in situations where dried blood spot (DBS) data need to be compared with plasma data, or vice versa. Results: Pearson’s coefficient, Lin’s coefficient and the Bland–Altman analysis are appropriate to evaluate the concordance between DBS and plasma data from bridging studies. Percent recovery plots generated using the ex vivo blood:plasma ratio and the regression equations demonstrate the best approach for predicting plasma concentrations from DBS. Conclusion: Statistical analysis of bridging study data is needed to characterize the relationship or concordance between blood (DBS) and plasma. The outcomes also provide guidance on selecting the most appropriate approach to transform DBS data to plasma, or vice versa. However, the biological and statistical evidence must be weighed together when deciding if DBS is suitable for preclinical and/or clinical development.

Objective: LY2603618, a selective inhibitor of checkpoint kinase 1 (CHK1) and key regulator of the DNA damage checkpoint, may enhance the effects of antimetabolites. This phase I study defined the recommended phase II dose of LY2603618 combined with gemcitabine. Patients and Methods: Patients with advanced/metastatic disease were administered doses of LY2603618 (70-250 mg/m2 or flat-fixed doses of 200 or 230 mg) after gemcitabine (1,000 mg/m2). Safety and pharmacokinetics (PK) were assessed. Results: Among the 50 patients enrolled, frequent adverse events possibly related to study drug treatment included fatigue (44%), decreased platelets (42%), decreased neutrophils (32%), nausea (26%), and decreased hemoglobin (20%). Systemic exposure of LY2603618 increased dose dependently, while clearance was relatively dose independent. The mean LY2603618 half-life varied; however, the durations were still suitable for maintaining human exposures while minimizing accumulation. LY2603618 PK were not altered by gemcitabine administration. Plasma exposures that correlate with the maximal pharmacodynamic effect in nonclinical models were achieved for all doses. One patient with non-small cell lung cancer carcinoma achieved a partial response; 22 patients had stable disease. Conclusions: The maximum tolerated dose of LY2603618 combined with gemcitabine was 200 mg/m2, but a fixed LY2603618 dose of 230 mg combined with gemcitabine was selected as the recommended phase II dose.

Gemcitabine (dFdC, 2',2'-difluorodeoxycytidine) is metabolized by cytidine deaminase (CDA) and deoxycytidine kinase (DCK), but the contribution of genetic variation in these enzymes to the variability in systemic exposure and response observed in cancer patients is unclear. Wild-type enzymes and variants of CDA (Lys27Gln and Ala70Thr) and DCK (Ile24Val, Ala119Gly, and Pro122Ser) were expressed in and purified from Escherichia coli, and enzyme kinetic parameters were estimated for cytarabine (Ara-C), dFdC, and its metabolite 2',2'-difluorodeoxyuridine (dFdU) as substrates. All three CDA proteins showed similar K(m) and V(max) for Ara-C and dFdC deamination, except for CDA70Thr, which had a 2.5-fold lower K(m) and 6-fold lower V(max) for Ara-C deamination. All four DCK proteins yielded comparable metabolic activity for Ara-C and dFdC monophosphorylation, except for DCK24Val, which demonstrated an approximately 2-fold increase (P < 0.05) in the intrinsic clearance of dFdC monophosphorylation due to a 40% decrease in K(m) (P < 0.05). DCK did not significantly contribute to dFdU monophosphorylation. In conclusion, the Lys27Gln substitution does not significantly modulate CDA activity toward dFdC, and therefore would not contribute to interindividual variability in response to gemcitabine. The higher in vitro catalytic efficiency of DCK24Val toward dFdC monophosphorylation may be relevant to dFdC clinical response. The substrate-dependent alterations in activities of CDA70Thr and DCK24Val in vitro were observed for the first time, and demonstrate that the in vivo consequences of these genetic variations should not be extrapolated from one substrate of these enzymes to another.

Background: The adoption of dried blood spot (DBS) sampling and analysis to support drug discovery and development requires the understanding of its bioanalytical feasibility as well as the distribution of the analyte in blood. Results: Demonstrated the feasibility of adopting DBS for four test analytes representing diverse physico-chemical as well as pharmacokinetic parameters. The key findings include the use of a single extraction procedure across all four analytes, assay range of 1 to 5000 ng/ml, stability in whole blood as well as on-card, and the non-impact of blood volume. In vivo data were used to calculate the blood-to-plasma ratio (using both AUC and average of individual time points), which was then used to predict plasma concentration from DBS data. The predicted data showed an excellent correlation with actual plasma data. Conclusion: Transition from plasma to DBS can be supported for preclinical studies by conducting a few well-defined bioanalytical experiments followed by an in vivo bridging study. Blood:plasma ratio derived from the bridging study can be used to predict plasma concentrations from DBS data.

Representing the first industry-led workshop on this topic, the Drug Information Association (DIA) and Pharmaceutical Research and Manufacturers of America (PhRMA) cosponsored a workshop on dried blood spots (DBS) sampling in the pharmaceutical industry. Sampling from DBS on specialty papers is a novel methodology in the area of pharmacokinetics and toxicokinetics that serves to replace conventional plasma analysis for pharmaceutical development. As the awareness around this technology has increased exponentially in recent years, this 1-day symposium aimed to share current best practices for DBS use and implementation, sample collection, handling and associated bioanalysis. The event drew over 150 industry and agency participants; an impressive attendance for a relatively new technology within the industry. Overall, the day was divided into six separate technical sessions plus poster sessions, and included concurrent breakout sessions dedicated to three key areas surrounding DBS implementation: bioanalytical, toxicology/safety assessment and clinical/pharmacokinetics.

In this study, we report a method for direct determination of gemcitabine incorporation into human DNA. Gemcitabine (dFdC), a structural analog of the nucleoside deoxycytidine (dC), derives its primary antitumor activity through interruption of DNA synthesis. Unlike other surrogate measures, DNA incorporation provides a mechanistic end point useful for dose optimization. DNA samples (ca. 25 microg) were hydrolyzed using a two-step enzymatic procedure to release dFdC which was subsequently quantified by LC-ESI-MS/MS using stable isotope labeled internal standards and selected reaction monitoring (SRM). dFdC was quantitated and reported relative to deoxyguanosine (dG) since dG is the complementary base for both dFdC and dC. The SRM channel for dG was detuned using collision energy as the attenuating parameter in order to accommodate the difference in relative abundance for these two analytes (104) and enable simultaneous quantification from the same injection. The assay was shown to be independent of the amount of DNA analyzed. The method was validated for clinical use using a 3 day procedure assessing precision, accuracy, stability, selectivity, and robustness. The validated ranges for dFdC and dG were 5-7500 pg/mL and 0.1-150 microg/mL, respectively. Results are presented which confirm that the ratio of dFdC to dG in DNA isolated from tumor cells incubated with dFdC increases with increased exposure to the drug and that dFdC can also be quantified from DNA extracted from blood.

The 2015 9th Workshop on Recent Issues in Bioanalysis (9th WRIB) took place in Miami, Florida with participation of over 600 professionals from pharmaceutical and biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. It is once again a 5-day week long event – a full immersion bioanalytical week – specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches including the focus on biomarkers and immunogenicity. This 2015 White Paper encompasses recommendations that emerged from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to advance scientific excellence, improve quality and deliver better regulatory compliance. Due to its length, the 2015 edition of this comprehensive White Paper has been divided into three parts. Part 1 covers the recommendations for small molecule bioanalysis using LCMS. Part 2 (hybrid LBA/LCMS and regulatory agencies’ inputs) and Part 3 (large molecule bioanalysis using LBA, biomarkers and immunogenicity) will also be published in volume 7 of Bioanalysis, issues 23 and 24, respectively.

Background This Phase-I-study aimed to determine the recommended Phase-II-dosing-schedule of LY2334737, an oral gemcitabine prodrug, in patients with advanced/metastatic solid tumors. Pharmacokinetics, cytokeratin-18 (CK18) levels, genetic polymorphisms, and antitumor activity were additionally evaluated. Methods Patients received escalating doses of LY2334737 either every other day for 21 days (d) followed by 7 days-drug-free period (QoD-arm) or once daily for 7 days every other week (QD-arm). The 28 days-cycles were repeated until disease progression or unacceptable toxicity. Standard 3 + 3 dose-escalation was succeeded by a dose-confirmation phase (12 additional patients to be enrolled on the maximum tolerated dose [MTD]). Results Forty-one patients received QoD- (40–100 mg) and 32 QD-dosing (40-90 mg). On QoD, 3/9 patients experienced dose-limiting toxicities (DLTs) on the 100 mg dose (2 × G3 diarrhea, 1 × G3 transaminase increase); 1 additional DLT (G3 diarrhea) occurred during dose confirmation at 90 mg (12 patients). On QD, 1 patient each experienced DLTs on 60 mg (G3 transaminase increase) and 80 mg (G3 prolonged QTcF-interval); 2/7 patients had 3 DLTs on the 90 mg dose (diarrhea, edema, liver-failure; all G3). The MTD was established at 90 mg for the QoD-arm. Seven patients on QoD and 4 on QD achieved SD (no CR + PR). Pharmacokinetics showed a dose-proportional increase in exposure of LY2334737 and dFdC without accumulation after repeated dosing. Significant increases in CK18 levels were observed. Genetic polymorphism of the cytidine deaminase gene (rs818202) could be associated with ≥ G3 hepatotoxicity. Conclusions Both schedules displayed linear pharmacokinetics and acceptable safety profiles. The recommended dose and schedule of LY2334737 for subsequent Phase-II-studies is 90 mg given QoD for 21 day.

Traditional pharmacokinetic analysis in nonclinical studies is based on the concentration of a test compound in plasma and requires approximately 100 to 200 µL blood collected per time point. However, the total blood volume of mice limits the number of samples that can be collected from an individual animal—often to a single collection per mouse—thus necessitating dosing multiple mice to generate a pharmacokinetic profile in a sparse-sampling design. Compared with traditional methods, dried blood spot (DBS) analysis requires smaller volumes of blood (15 to 20 µL), thus supporting serial blood sampling and the generation of a complete pharmacokinetic profile from a single mouse. Here we compare plasma-derived data with DBS-derived data, explain how to adopt DBS sampling to support discovery mouse studies, and describe how to generate pharmacokinetic and pharmacodynamic data from a single mouse. Executing novel study designs that use DBS enhances the ability to identify and streamline better drug candidates during drug discovery. Implementing DBS sampling can reduce the number of mice needed in a drug discovery program. In addition, the simplicity of DBS sampling and the smaller numbers of mice needed translate to decreased study costs. Overall, DBS sampling is consistent with 3Rs principles by achieving reductions in the number of animals used, decreased restraint-associated stress, improved data quality, direct comparison of interanimal variability, and the generation of multiple endpoints from a single study.

Although traditionally reserved for proteomic analysis, nanoESI has found increased use for small molecule applications related to drug metabolism/pharmacokinetics (DMPK). NanoESI, which refers to ESI performed at flow rates in the range of 200 to 1000 nL/min using smaller diameter emitters (10 to 100 microm id), produces smaller droplets than conventional ESI resulting in more efficient ionization. Benefits include greater sensitivity, enhanced dynamic range, and a reduced competition for ionization. These advantages may now be harnessed largely due to the introduction of a commercial system for automated nanoESI infusion. This development in turn has allowed ADME (absorption, distribution, metabolism, and excretion) scientists to consider novel approaches to mass spectrometric analysis without direct LC interfacing. While it is freely acknowledged that nanoESI infusion is not likely to supplant LC-MS as the primary analytical platform for ADME, nanoESI infusion has been successfully applied to both quantitative (bioanalysis) and qualitative (metabolite identification) applications. This review summarizes published applications of this technology and offers a perspective on where it fits best into the DMPK laboratory.

This paper was developed with the support of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ). IQ is a not-for-profit organization of pharmaceutical and biotechnology companies with a mission of advancing science-based and scientifically driven standards and regulations for pharmaceutical and biotechnology products worldwide. Within the IQ, various working groups (WG) have been formed, where the microsampling WG is committed to providing a scientific forum for the advancement of both wet and dry microsampling techniques within the pharmaceutical industry. This first output from the microsampling WG is to summarize and reflect on the current knowledge and opinions on DBS sampling, to stimulate discussion, and to encourage future creative applications of DBS sampling. Dried blood spot (DBS) sampling has established itself as an innovative sampling technique where wet blood is spotted onto absorbent paper or other paper materials and allowed to dry (1–4). DBS offers several potential benefits inherent to the technique, namely a low blood volume, simplified blood sample collection (5), and convenient sample storage and transfer. In certain applications, DBS sampling has been shown to stabilize certain analytes or metabolites without the addition of chemical modifiers (6–9). DBS has been routinely applied for decades in neonatal screening for phenylketonuria and other congenital metabolic disorders (10). The utility of DBS sampling has also been demonstrated for therapeutic drug monitoring (11) and for epidemiological studies (e.g., HIV and HBV detection/monitoring) (12) due to the practical advantages along with simplified sample collection and handling procedures. Finally, DBS can also be used for quantitative biomarker (PD) assessment from blood, where appropriate. However, the technique is relatively new to the pharmaceutical industry and to government regulators overseeing new drug applications. Nevertheless, over the past 5 to 7 years, the technique has been extensively evaluated for quantifying drug exposure in nonclinical and/or clinical studies in various stages of drug discovery and development. The ease to collect, transfer, store, and process small volumes of blood samples has generated considerable interest in providing utility in volume-limited situations (e.g., small rodent, human pediatric studies) for toxicokinetic (TK), pharmacokinetic (PK), or pharmacodynamic (PD) sampling. Discovery and nonclinical studies Rodent animal models are typically employed in these studies. The reduced blood volumes required for DBS can enable serial bleeding and, consequently, elimination of satellite animal groups and reduction of compound use. The ability to eliminate the satellite animal groups enables the assessment of exposure and toxic effects within the same animal. Studies involving expensive animal models (i.e., transgenic mice, knock-out mice, humanized mice, etc.) further highlight a persuasive scientific and economic case for DBS sampling since a complete pharmacokinetic profile can be obtained from a single study animal without the need for extra rodents merely for generating exposure data. These are perfectly in line with the principles of the 3Rs: reduction, refinement, and replacement of humane animal research (13–15). With greater emphasis from the regulatory authorities to study new drugs for infants, neonates, and pediatric populations, the requirement to conduct associated nonclinical juvenile rodent toxicity studies serves as an ideal scenario where the advantage of low blood volume in DBS sampling is undeniable. Although the advantages of DBS heavily favor rodent studies, it can also be used to refine non-rodent studies.

Pharmacokinetic data to support clinical development of pharmaceuticals are routinely obtained from liquid plasma samples. The plasma samples require frozen shipment and storage and are extracted off-line from the liquid chromatography/tandem mass spectrometry (LC/MS/MS) systems. In contrast, the use of dried blood spot (DBS) sampling is an attractive alternative in part due to its benefits in microsampling as well as simpler sample storage and transport. However, from a practical aspect, sample extraction from DBS cards can be challenging as currently performed. The goal of this report was to integrate automated serial extraction of large numbers of DBS cards with on-line liquid chromatography/high-resolution accurate mass spectrometry (LC/HRAMS) bioanalysis.An automated system for direct DBS extraction coupled to a LC/HRAMS was employed for the quantification of midazolam (MDZ) and α-hydroxymidazolam (α-OHMDZ) in human blood. The target analytes were directly extracted from the DBS cards onto an on-line chromatographic guard column followed by HRAMS detection. No additional sample treatment was required. The automated DBS LC/HRAMS method was developed and validated, based on the measurement at the accurate mass-to-charge ratio of the target analytes to ensure specificity for the assay.The automated DBS LC/HRAMS method analyzed a DBS sample within 2 min without the need for punching or additional off-line sample treatment. The fully automated analytical method was shown to be sensitive and selective over the concentration range of 5 to 2000 ng/mL. Intra- and inter-day precision and accuracy was less than 15% (less than 20% at the LLOQ). The validated method was successfully applied to measure MDZ and α-OHMDZ in an incurred human sample after a single 7.5 mg dose of MDZ.The direct DBS LC/HRAMS method demonstrated successful implementation of automated DBS extraction and bioanalysis for MDZ and α-OHMDZ. This approach has the potential to promote workload reduction and sample throughput increase.

The disposition and metabolism of a Chk-1 inhibitor (LY2603618) was characterized following a 1-h intravenous administration of a single 250-mg dose of [14C]LY2603618 (50 µCi) to patients with advanced or metastatic solid tumors. LY2603618 was well tolerated with no clinically significant adverse events. Study was limited to three patients due to challenges of conducting ADME studies in patients with advanced cancer. Plasma, urine and feces were analyzed for radioactivity, LY2603618 and metabolites. LY2603618 had a half-life of 10.5 h and was the most abundant entity in plasma, accounting for approximately 69% of total plasma radioactivity. The second most abundant metabolites, H2 and H5, accounted for10% of total circulating radioactivity. The major route of clearance was via CYP450 metabolism. The mean total recovery of radioactivity was 83%, with approximately 72% of the radioactivity recovered in the feces and approximately 11% in the urine. LY2603618 represented approximately 6% and 3% of the administered dose in feces and urine, respectively. A total of 12 metabolites were identified. In vitro phenotyping indicated that CYP3A4 was predominantly responsible for the metabolic clearance of LY2603618. Additionally, aldehyde oxidase was involved in the formation of a unique human and non-human primate metabolite, H5.

This Phase 1 study aimed to determine the recommended Phase 2 dose of LY2334737, an oral gemcitabine prodrug, when combined with standard dose docetaxel treatment in patients with advanced solid tumors. Pharmacokinetics (PK) and antitumor activity were additionally evaluated.Patients with advanced/metastatic solid tumors received escalating doses of LY2334737 once daily (QD) for 14 days, followed by a 7-day drug-free period. Docetaxel was given at 75 mg/m(2) every 3 weeks (q3w). Cycles were repeated until progressive disease (PD) or unacceptable toxicity.Of 22 patients recruited, all Caucasian, 7 received an LY2334737 dose of 10 mg/day, 10 received 20 mg/day, 5 received 30 mg/day. Nineteen patients discontinued due to PD, 2 due to adverse events, 1 due to investigator decision. Dose-limiting toxicities: 2× febrile neutropenia (G3), 2× fatigue (1× G2, 1× G3), 1× neutropenia (G4). The maximum tolerated dose (MTD) was identified to be 10 mg/day. Two patients achieved partial response, 10 patients stable disease. Enrollment was stopped after unexpected hepatic toxicities were observed with LY2334737 QD for 14 days per cycle in another study of Japanese patients. PK data were consistent with the first-in-man study of LY2334737 and did not reveal any drug-drug interaction between LY2334737 and docetaxel.Combination of LY2334737 at doses up to 30 mg/day QD for 14 days per cycle with docetaxel 75 mg/m(2) q3w resulted in an undesirable toxicity profile and a low MTD of 10 mg/day. Alternative treatment schedules of LY2334737 should be explored.

Gemcitabine is an intravenously administered nucleoside analog chemotherapeutic agent. The ability to deliver this agent as an oral drug would allow greater flexibility of administration and patient convenience, however, attempts have been fraught with high first-pass metabolism and potential intestinal toxicity. Alternatively, an amide prodrug of gemcitabine (LY2334737) was discovered, which is able to avoid the extensive first-pass metabolism that occurs following administration of gemcitabine. Preclinical in vitro and in vivo experiments were conducted to evaluate the hydrolysis and pharmacokinetics of LY2334737 and its downstream metabolites. In mice, rats, and dogs, the prodrug is absorbed largely intact across the intestinal epithelium and delivers LY2334737 to systemic circulation. The hydrolysis of LY2334737 is relatively slow, resulting in sustained release of gemcitabine in vivo. In vitro experiments identified carboxylesterase 2 (CES2) as a major enzyme involved in the hydrolysis of LY2334737, but with relatively low intrinsic clearance. Following hydrolysis of the prodrug, gemcitabine is cleared predominantly via the formation of its inactive metabolite dFdU. Both biliary and renal excretion was responsible for the elimination of LY2334737 and its metabolites in both mice and dogs.

In this study we report a high sensitive method for the simultaneous analysis of LY2334737 (2'-deoxy-2',2'-difluoro-N-(1-oxo-2-propylpentyl)-cytidine), an amide prodrug of gemcitabine (2', 2'-difluoro-deoxycytidine), along with its active drug gemcitabine and its major metabolite dFdU (2',2'-difluoro-deoxyuridine) by LC-MS/MS. Quantification of all three analytes within a single analysis was challenging because the physio-chemical properties of LY2334737 were significantly different from gemcitabine and dFdU and was accomplished by incorporating column-switching. The assay was fully validated to quantify LY2334737 from 0.1 to 100ng/mL, gemcitabine from 0.25 to 100ng/mL and dFdU from 1 to 1000ng/mL in order to cover the diverse concentration ranges expected in clinical samples. A 25-fold dilution was also validated to accommodate any samples outside this range. Overall, the assay had good accuracy (ranging from -7.0 to 1.2% relative error) and precision (ranging from 2.1 to 8.4% relative standard deviation). Extraction efficiency was greater than 80% for all three analytes and there were no matrix effects. Plasma samples were stable for 24h at room temperature, 660 days in frozen storage, and at least 4 freeze-thaw cycles, at both -20 and -70°C. Data from clinical trials showed that plasma concentrations for LY2334737, gemcitabine, and dFdU were successfully quantified from a single LC-MS/MS analysis and that the assay ranges selected for the three analytes were appropriate and minimized the need for reanalysis.

Definitive plasma protein binding (PB) studies in drug development are routinely conducted with radiolabeled material, where the radiochemical purity limits quantitative PB measurement. Recent and emerging regulatory guidances increasingly expect quantitative determination of the fraction unbound (Fu) for key decision making. In the present study, PB of 11 structurally- and therapeutically-diverse drugs spanning the full range of plasma binding was determined by equilibrium dialysis of non-radiolabeled compound and was validated against the respective definitive values obtained by accepted radiolabeled protocols. The extent of plasma binding was in agreement with the radiolabeled studies; however, the methodology reported herein enables reliable quantification of Fu values for highly-bound drugs and is not limited by the radiochemical purity. In order to meet the rigor of a development study, equilibrium dialysis of unlabeled drug must be supported by an appropriately validated bioanalytical method along with studies to determine compound solubility and stability in matrix and dialysis buffer, nonspecific binding to the dialysis device, and ability to achieve equilibrium in the absence of protein. The presented methodology establishes an experimental protocol for definitive PB measurement, which enables quantitative determination of low Fu values, necessary for navigation of new regulatory guidances in clinical drug development.

A liquid chromatography-tandem mass spectrometry method was developed to chromatographically separate the four stereoisomers of the active metabolite of prasugrel, R-138727, in human plasma after derivatization with bromomethoxyacetophenone to stabilize the molecule. This technique was designed to determine the relative contribution of each stereoisomer, based on statistical analyses of each stereoisomer's chromatographic peak areas. The methodology was validated and used for the analysis of clinical samples in which R-138727 had been derivatized at the time of blood collection. This technique can be useful to determine the ratios of stereoisomers in biological samples (e.g., plasma) especially in situations in which authentic standards of each individual stereoisomer are scarce or unavailable. In humans, the metabolic formation of R-138727 from prasugrel was found to be stereoselective, where 84% of R-138727 was present as RS and RR, the two most pharmacologically potent isomers, whereas the SR and SS enantiomers accounted for approximately 16%. The ratios of the R-138727 stereoisomers were consistent among subjects, regardless of the dose or time of sample collection or whether the blood was sampled after the first dose or after 4 weeks of therapy.