Microsampling, a reduced volume sampling method, has successfully gained attention at the International Conference on Harmonization (ICH) level and established benefits support its use in Toxicokinetic (TK) studies. These improved sampling techniques are less invasive and in large animal species improve animal welfare (refinement). To evaluate if the plasma concentrations of drugs were influenced by the blood sampling method, the traditional method from femoral vein and microsampling from tail vein in Cynomolgus monkeys were compared. The pharmacokinetic parameters (Cmax, Tmax and AUC) of four drugs (selected based on acid-base and volume of distribution properties) in non-human primate were correlated. The plasma samples were quantified using standard LC-MS/MS methods, qualified to evaluate the precision and accuracy before the analysis of real samples. The results reported in this work demonstrated the suitability of microsampling in supporting PK/TK studies in non-human primates. The data show that the exposure of drugs tested after blood collection using standard procedure from femoral vein and microsampling from tail vein is correlated and is not influenced by acid-base characteristics and volume of distribution.

LNA-i-miR-221, a 13-mer oligonucleotide, has proved favorable efficacy and safety profiles in the preclinical studies, leading to being approved for use in clinical trials by regulatory authorities. The objective of this study was to develop and validate LC-MS/MS methods to quantify LNA-i-miR-221 in human plasma and urine. Chromatographic separation was performed with a gradient system on HALO C18 column using hexafluoro-2-propanol/triethylamine buffer and methanol as mobile phase. LNA-i-miR-221 was detected on tandem mass spectrometer with electrospray ionization source in negative ion mode. The methods showed good linearity within the calibration range of 50-25000 ng/mL and 50-50000 ng/mL for human plasma and urine, respectively. The methods proved to be accurate, precise and selective in both human matrices. These validated methods are reliable and are currently in use to support a first-inhuman clinical trial of LNA-i-miR-221 in patients affected by refractory multiple myeloma and advanced solid tumors. (C) 2020 Elsevier B.V. All rights reserved.

AIM Development of a high-sensitivity chiral LC-MS/MS method was required to evaluate a combination of pramipexole (S-PPX) and its enantiomer dexpramipexole (R-PPX) in a proposed clinical trial. The previously available methods suffered from low sensitivity for the (S)-enantiomer in the presence of the more abundant (R)-enantiomer. Based on the projected dosing regimen in the clinical trial, a 5000-fold improvement in sensitivity was required for the (S)-enantiomer. METHODOLOGY Spiked human plasma samples were extracted by liquid-liquid extraction using ethyl acetate and injected onto a CHIRALPAK ID column under pH gradient conditions. CONCLUSION An improved analytical method was developed and validated with a final LLQ for (S)-PPX of 0.1 ng/ml in the presence of 2000 ng/ml of (R)-PPX.

Abstract miRNAs therapeutics is based on the use of oligonucleotide engineered to efficiently replace or sequester endogenous miRNAs. miR-221 is upregulated in several tumors, including multiple myeloma (MM), where it promotes cancer cell proliferation mainly via targeting the negative regulators of G1/S cell cycle progression p27 and/or p57. We previously demonstrated that direct miR-221 inhibition by the use of an original LNA inhibitor, LNA-i-miR-221, induces significant anti-MM activity and upregulates canonical targets in vitro and in vivo.To assess pharmacokinetics (PK)/pharmacodynamics of LNA-i-miR-221, we set-up, and report here, novel assays for oligo quantification in plasma, urine and tissues of NOD.SCID mice and Cynomolgus monkeys. We investigated the LNA-i-miR-221 PKs by UHPLC-MS/MS, following solid-phase extraction, in plasma and urine, and tissue uptake by in-situ hybridization (ISH). In particular, PK profile was calculated by UHPLC-MS/MS after single dose i.p injection of LNA-i-miR-221 (25 mg/kg) in mouse and the Tmax was identified at 1.5 hours with Cmax of 1110 ng/mL. Three hours after bolus injection, the plasma concentration was about 60% reduced and the molecule was not detectable at later time-points. The plasma exposure of the oligo showed an AUClast of 2330 h*ng/mL. Similarly, in monkeys the LNA-i-miR-221 plasma concentration following a single i.v. bolus injection (8.75 mg/kg) peaked at a Tmax of ½ hour, with a Cmax of 23600 ng/mL. The plasma exposure of the oligo showed an AUClast of 49300h*ng/mL, C0 of 61400 ng/mL and the t1/2 was 12.8 hours. Urine PK evaluation was performed in monkey on samples collected from 2 up to 48 hours. The excreted LNA-i-miR-221 was quantified equal to 0.68 mg with a total urinary recovery of approximately 2.68% of the amount injected (25,375 mg). Specifically, the urinary concentration/time-points plot shows the excretion of 9394,5 ng/mL (1.63% of recovery) in the interval of 2-6 hours. In addition, murine tissues analyzed by ISH revealed strong signals and long-lasting accumulation of the oligo in mice vital organs together with upregulation of the main canonical miR-221 target p27, as assessed by IHC. In addition, ISH analysis excluded that the LNA-i-miR-221 crosses the blood-brain barrier. All together PK results demonstrated that LNA-i-miR-221 has a short serum half-life with a rapid tissue uptake and minimum urinary excretion of the intact molecule, with a very long tissue half-life and biological activity. Moreover, we report preliminary toxicity analysis in non-human primates. No changes in the monkeys’ behavior, body weight or food consumption were observed in treated animals.In conclusion our results suggest that LNA-i-miR-221 is a promising anti-MM agent associated with a favorable PK, long-lasting tissue bioavailability and good safety profile in mice and in monkeys, providing the rationale for translation of this novel miR-221 inhibitor in early clinical investigation Citation Format: Maria Teresa Di Martino, Maria Eugenia Gallo Cantafio, Boye S. Nielsen, Mariamena Arbitrio, Annamaria Gullà, Chiara Mignogna, Massimo Breda, Niels M. Frandsen, Pierosandro Tagliaferri, Pierfrancesco Tassone. Pharmacokinetics and biodistribution of LNA-i-miR-221 in NOD.SCID mice and Cynomolgus monkeys. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1079.

CHF5074 has been shown to inhibit brain &bgr;-amyloid deposition and attenuate memory deficits in different transgenic mice models of Alzheimer disease. We evaluated the safety, pharmacokinetics, and pharmacodynamics of 3 ascending dose regimens of CHF5074 (200, 400, and 600 mg/d for 14 d) in a double-blind, placebo-controlled, parallel group study involving 48 healthy subjects. Plasma, urine, and cerebrospinal fluid (CSF) samples were collected for measuring drug and main metabolite concentrations and potential biomarkers of pharmacodynamic activity (&bgr;-amyloid1-40, &bgr;-amyloid1-42, soluble CD40 ligand, and tumor necrosis factor-&agr;). All subjects completed the study, and no serious or severe adverse events were reported. The maximum tolerated dose was close to 600 mg/d with mild diarrhea being the most frequent adverse event at this dose. CHF5074 reached peak plasma levels 2 to 3 hours after drug administration and then was slowly eliminated (t1/2z=30 h) in the urine as glucoronide. Systemic exposure to the drug appeared to be dose-proportional with a 2-fold accumulation ratio at steady state. Metabolite plasma levels peaked at 4 to 5 hours and accounted for about 25% of the parent compound. Drug levels in the CSF were dose-proportional. The drug dose-dependently lowered the levels of the soluble CD40 ligand, a marker of microglia activation, in both plasma and CSF samples.

BioanalysisVol. 4, No. 14 White PaperFree AccessRecommendations on ISR in multi analyte assays, QA/bioanalytical consultants and GCP by Global CRO Council for Bioanalysis (GCC)Timothy Sangster, John Maltas, Petra Struwe, Jim Hillier, Mark Boterman, Mira Doig, Massimo Breda, Fabio Garofolo, Maria Cruz Caturla, Philippe Couerbe, Christine Schiebl, Colin Pattison, Lee Goodwin, Rudi Segers, Wei Garofolo, Lois Folguera, Dieter Zimmer, Thomas Zimmerman, Maria Pawula, Daniel Tang, Chris Cox, Chiara Bigogno, Dick Schoutsen, Theo de Boer, Rachel Green, Richard Houghton, Romuald Sable, Christoff Siethoff, Tammy Harter & Stuart BestTimothy SangsterCharles River, Edinburgh, UK, John MaltasBASi, Kenilworth, Warwickshire, UK, Petra StruweCelerion, Fehraltorf, Switzerland, Jim HillierGen-Probe Life Sciences, Manchester, UK, Mark BotermanABL, Assen, The Netherlands, Mira DoigABS Laboratories, Welwyn Garden City, UK, Massimo BredaAccelera, Nerviano, Italy, Fabio GarofoloAlgorithme Pharma/Simbec Research, Laval, QC, Canada/Merthyr Tydfil, UK, Maria Cruz CaturlaAnapharm Europe, Barcelona, Spain, Philippe CouerbeATLANBIO, Saint-Nazaire, France, Christine SchieblCelerion, Fehraltorf, Switzerland, Colin PattisonCharles River, Edinburgh, UK, Lee GoodwinCovance Laboratories, Harrogate, UK, Rudi SegersEurofins, Breda, The Netherlands, Wei GarofoloGlobal CRO Council: Global CRO Council (GCC), 15 Sunview Dr, Toronto, Ontario, L4H 1Y3, Canada. ; www.global-cro-council.org, Lois FolgueraHarlan Laboratories, Breda, The Netherlands, Dieter ZimmerHarlan Laboratories, Itingen, Switzerland, Thomas ZimmermanHarlan Laboratories, Itingen, Switzerland, Maria PawulaHuntingdon Life Sciences, Huntingdon, UK, Daniel TangICON, Shanghai, China, Chris CoxMillipore BioPharma Services, Abingdon, UK, Chiara BigognoNiKem Research, Baranzate, Italy, Dick SchoutsenNOTOX, 's-Hertogenbosch, The Netherlands, Theo de BoerQPS Netherlands BV, Groningen, The Netherlands, Rachel GreenQuotient Bioresearch, Fordham, UK, Richard HoughtonQuotient Bioresearch, Fordham, UK, Romuald SableSGS, Wavre, Belgium, Christoff SiethoffSwiss BioQuant AG, Reinach, Switzerland, Tammy HarterUnilabs York Bioanalytical Solutions, York, UK; presently at Covance Laboratories, North Yorkshire, UK & Stuart BestXceleron, York, UKPublished Online:9 Aug 2012https://doi.org/10.4155/bio.12.172AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail Keywords: consultantEMAGCCGCPThe Global CRO Council for Bioanalysis (GCC) was formed in an effort to bring together many senior-level representatives of CROs to openly discuss bioanalysis and the regulatory challenges, many of them unique to the outsourcing industry. CROs are unique in that they work with many different pharmaceutical companies and agencies, which results in a distinctive and comprehensive perspective on scientific approaches in relation to regulatory requirements. More information on GCC unique structure can be found in the publication titled 'Formation of a Global Contract Research Organization Council for Bioanalysis' [1] and its scientific activities have been subsequently published in articles and White Papers [2–6].IntroductionThe 5th GCC meeting was held on 13 November 2011 in Barcelona, Spain. In attendance were 29 senior-level representatives from 24 CROs on behalf of nine countries, mainly from Europe. The topics selected for discussion, suggested at the 3rd GCC meeting held in Guildford, UK, on 3–4 July 2011 were: ▪ Assay qualification and validation: attaining clear definitions in relation to biomarker assays;▪ European Medicines Agency (EMA) Bioanalytical Method Validation guideline;▪ Incurred sample reproducibility (ISR) in multi-analyte assays: aim of reaching a consensus on the method of sample selection and the number of samples to be assessed;▪ Regulation of quality assurance (QA)/bioanalytical consultants and provide a list of recommendations for hiring a 'qualified consultant';▪ Regulatory requirements for good clinical practice (GCP).EMA Bioanalytical Method Validation guidelineFollowing its publication in July 2011, the new EMA bioanalytical guideline [7] was to be the main topic on the agenda.The guideline on Bioanalytical Method Validation was thoroughly evaluated and discussed via an extensive survey and during both the 4th and 5th GCC Closed Forums. CRO leaders were able to openly share opinions and perspectives and to agree on unified bioanalytical recommendations specifically in relation to this new EMA guideline. The following topics were discussed: ▪ Reference standards: certificates of analysis and internal standards (IS) (section 4.1);▪ Calibration curve and accuracy (sections 4.1.4 and 4.1.5);▪ IS stability, processed sample stability and matrix effect (sections 4.1.8 and 4.1.9);▪ Analysis of study samples and incurred samples reanalysis (sections 5, 5.1, 5.2, 5.5 and 6);▪ Ligand binding assays (LBA) (section 7).The GCC recommendations are presented in the White Paper, 'Global CRO Council (GCC) Recommendations on the Interpretation of the new EMA Guideline on Bioanalytical Method Validation' [5].Assay qualification & validation for biomarker assaysDue to the magnitude of this topic, the GCC recommendations on biomarker validation will be presented in an independent White Paper [6].▪ Recommendation #1: ISR, including multi-analyte assaysIn 2006, at the 3rd AAPS/US FDA Bioanalytical Workshop (Crystal City III) [8], it was decided that ISR was to be conducted for both non-clinical and clinical studies. At the AAPS Workshop on ISR held in February 2008, specific recommendations on ISR, including aspects to be considered in implementing a robust ISR program, were presented and discussed [9]. Several 'non-consensus' topics were highlighted, including multi-analyte methods. Between 2008 and 2011, ISR has been discussed at subsequent conferences and forums (WRIB, EBF, GCC), but no consensus has been reached with respect to multi-analyte methods. The new EMA bioanalytical guideline addresses ISR but not in detail regarding multi-analyte assays.A survey was circulated to the GCC European members posing questions on the approach to ISR, with particular reference to multi-analyte assays.From the survey and subsequent discussions, the majority of CROs consider that ISR is conducted to demonstrate both the validity of incurred sample analysis and the performance/robustness of the analytical method in the laboratory. It was the general opinion that ISR should be independent (i.e., kept separate from study sample analysis) and that reanalyzing a few samples within a batch alongside study samples may also not be adequate to assess reproducibility of the method. It was considered that performing ISR in separate batches would also make any investigations of trends or failures less complicated. Additionally, it was reiterated that ISR should be performed as soon as practical after the original analysis to limit any influence of incurred sample stability.From the survey results, ISR on tissue analysis is not always performed. For tissue analysis, qualified methods have been recommended within the bioanalytical community and following discussions it was recommended that ISR should only be performed when the tissue analysis is a primary end point of the study and is performed using a validated assay.For ISR in general, three options were discussed to determine how many of the samples analyzed were to be reanalyzed: ▪ For bioequivalence studies reanalyze 10% of samples and for non-bioequivalence studies reanalyze 5% of samples, regardless of size;▪ For studies with up to 1000 samples, reanalyze 10% of samples and for studies with over 1000 samples reanalyze 5% of samples, but at least 100 samples;▪ For studies up to 1000 samples reanalyze 10% of samples and for studies with over 1000 samples reanalyze 10% of the first 1000 samples and 5% of any remaining samples.The third option matches the EMA guideline and was the one preferred by attendees. It is also agreed that a minimum number of ISR samples should be specified even if it is not regulated by the FDA or the EMA; for example, 20 samples.It was agreed that typically two samples from a subject would be selected for ISR, one around the tmax and one close to the elimination phase. For toxicology studies it was discussed whether to select more samples from fewer groups (e.g., the highest and lowest concentration dose groups only) or fewer samples from more groups. The general opinion was that performing ISR on the highest and lowest concentration dose groups may not be sufficient as there could be differences, for example in metabolite profile, gender and dose group. It was also agreed that for composite profiles, sample selection should mimic profiles with an equal number of samples from the tmax and the elimination phase.Only samples with concentrations at least three-times the LLOQ concentration should be selected. Samples that had undergone dilution should be included, and where possible the same dilution factor should be used for both analyses. The setting of criteria for the selection of late elimination phase samples (e.g., number of half lives from the tmax) was discussed but the consensus was to leave the selection of late elimination phase samples as it is currently – samples should be selected 'by visual inspection' rather than by calculation.The use of split samples, for example, two aliquots originating from the same sample stored in similar containers under similar conditions, was discussed and it was agreed that each split is considered equivalent and the use of different splits for ISR should be treated the same as per sample analysis. It was agreed that if there is insufficient sample volume remaining of the initial aliquot or it has reached the limit of the established freeze–thaw cycles then another sample split should be used for the ISR investigation. Additionally, how to apply ISR to dried blood spot assays was discussed and it was agreed that equivalence between spots is established during validation and, therefore, a different spot could be used for ISR.Almost all ISR results are calculated by comparing the difference between the original and ISR concentrations with the mean of the two concentrations (as per Crystal City recommendation and EMA guideline), though occasionally a CRO may be requested to calculate ISR using different criteria.Further discussions covered extreme outliers (e.g., >50% bias from the original value) and trends, and when an investigation and/or reanalysis should be performed. In summary, for extreme outliers it was agreed that if the ISR acceptance criteria were met then extreme outliers were not required to be investigated unless they formed a trend, and for trends (a series of results that show commonality, i.e., in terms of runs, populations, subjects or animals, time-points, dilutions and so on) an investigation should be performed even if ISR acceptance criteria were met.From the survey and subsequent discussions, the majority of members have performed ISR on multi-analyte assays. Currently ISR investigations are conducted for each analyte with sample selection based on all analytes, applying the same rules used for selecting samples for single analyte ISR. As each analyte may have a different concentration–time profile, this may result in an increase in the number of samples per subject. However, all samples selected should be used for ISR for all analytes, with the exception of samples where results are less than three-times the LLOQ concentration. If the ISR analysis is performed using a sample diluted applying a validated dilution but the original value was obtained from an undiluted sample for that analyte (or vice versa), as long as the measured concentration is greater than three-times the LLOQ, the result with a different dilution regime to the original analysis should be reported, rather than reanalyzing again with the dilution factor applied during the original analysis.For multi-analyte assays, where one or more analytes are not being assessed (e.g., a batch has been rejected for one analyte and is being reanalyzed for that analyte alone), as data could be generated for all analytes, it was discussed whether or not the data from the previously accepted analytes could be used for ISR. Although it was agreed to not routinely use these additional data, it was recognized that this could be useful, for example where sample volume is limited and only one more reanalysis is possible. It is recommended that if these additional data are to be used then this should be stated a priori in the study plan or in an amendment.The GCC recommendation on ISR is shown in Box 1.▪ Recommendation #2: regulation of QA/bioanalytical consultants & provide a list of recommendations for hiring a 'qualified consultant'Findings from a mock FDA inspection conducted by an independent QA consultant were presented at the 3rd GCC Closed Forum and the validity of some of these findings was debated. The overall consensus was that some of the potential findings raised by the QA consultant were out of date or even incorrect. Therefore, it was decided that the hiring of QA/bioanalytical consultants should be discussed in more depth, and a survey was circulated to the GCC European members posing questions on the hiring of consultants – 16 completed surveys were returned.From the survey, members consider that a consultant should have state-of-the-art knowledge of bioanalytical activities, understand quality practices with focus on GLP and/or GCP and bring in specific expertise that may not be available or may not be in adequate abundance in-house.GCC members sometimes hire consultants themselves but often a consultant has been hired by a client to act on their behalf. Members generally accept a consultant representing a client company without restriction, although some members do have a formalized process and request details, references and so on. Almost half of members check the expertise of the consultant on arrival at their site.Although some feel that consultants mandated by a client think they need to find something relevant to justify their mission, from experience, members have found that consultants hired by clients are mostly fair and complementary. It is general opinion that although there are some consultants who do not appear to be up to date with current practices and thinking or are not relevantly qualified, there are consultants who are able to manage the regulatory and technical aspects of bioanalytical activities.Consultants hired by the CRO often work as an independent workforce whilst following the CRO's standard operating procedures (SOPs), and depending on the length of the assignment they may have their own training documentation (CV/expertise summary) and may be listed on the organization chart. However, these consultants do not sign regulatory documents and few members have confidence that a consultant could be hired to implement a new topic without support from the organization.The main objectives for CROs hiring consultants are: as a teacher to fill in a technical or regulatory knowledge gap (e.g., IT/computer system validation); as an investigator to conduct mock inspections and gap analysis; as a partner by assisting with excess internal workload (e.g., peaks in QA, implementation of new hardware or software); or to help maintain independence of the QA function. None of the members who contributed had ever hired a consultant to act as a Study Director or Principal Investigator.Although most members do not have a specific procedure in place, recommendation and reputation is often the starting point for hiring a consultant, although there was general agreement that a member may not feel comfortable hiring a consultant who was recently or currently working for a competitor. The current process of hiring a consultant is mainly via interviews in combination with a file review, including assessment of CV and expertise. In general, it is a QA representative or the test facility management that approves that the consultant is qualified.The GCC recommendation on consultants is shown in Box 2.▪ Recommendation #3: regulatory requirements for GCP (contracts, study conduct & training)At the 3rd GCC Closed Forum, the implementation of GCP was discussed and it was determined that knowledge and experience of GCP varied widely across European countries. With the aim of ensuring all members had the knowledge to be able to implement GCP, it was suggested that sharing of experiences between members would facilitate this. A survey was circulated to the GCC European members posing questions on GCP experiences to date.GCP is a collection of ethical and scientific quality systems for designing, conducting and reporting of clinical trials that involve participation of human subjects. GCP is required to ensure the rights, safety and well-being of trial subjects are protected and to ensure the credibility of the clinical trial data. GCP has developed into formal guidelines as a result of instances of abuse of human rights, harm to subjects and serious fraud.The European Clinical Trials Directive 2001/20/EC (EU CTD) was introduced to establish standardization of research activity in clinical trials throughout the European community [101]. It provides a framework that sets out how clinical trials investigating the safety or efficacy of a medicinal product in humans must be conducted, including medicinal trials with healthy volunteers and small-scale or pilot studies. The aims of the EU CTD are to provide greater protection to subjects participating in clinical trials, ensure quality of conduct and harmonize regulation and conduct of clinical trials throughout Europe.The International Conference on Harmonisation Guidance on Good Clinical Practice (CPMP/ICH/135/95) is an international standard for GCP [102]. The Good Clinical Practice Directive 2005/28/EC supplements the EU CTD, strengthening the legal basis for requiring Member States to comply with the principles and guidelines of GCP, as set out in the ICH-GCP guidelines [101]. However, these guidelines offer no direct guidance on how laboratory analysis should be conducted. Therefore, the UK GLP monitoring authority, the Medicines and Healthcare Products Regulatory Agency (MHRA), produced a guidance on the maintenance of regulatory compliance in laboratories that perform the analysis or evaluation of clinical trial samples [10]. The EMA has now issued a reflection paper for laboratories that perform the analysis or evaluation of clinical trial samples [11].For laboratories undertaking clinical sample analysis, the accepted standards for GCP are similar to those of GLP with respect to: ▪ Method validation, ISR;▪ Repeat analysis;▪ Data recording, reporting and retention of data;▪ Facilities and equipment maintenance;▪ Computerized systems;▪ QA and QC, SOPs and policies.However, there are some important differences between GCP and GLP:▪ In GCP, the sponsor or sponsor representative is the Management Authority, that is, at the bioanalytical CRO there is no equivalent role to the GLP Study Director. Therefore, unlike GLP, the sponsor has legal responsibilities regarding integrity of data and conduct of the study. The responsible scientist for bioanalysis reports directly to the sponsor and must inform the sponsor without delay of any serious breach, which could affect either patient safety or the scientific value/integrity of the trial. Examples of such breaches include unscheduled breaking of the blinding code, thus risking patient confidentiality, unexpected test results or deviations from the clinical protocol;▪ In GCP, the Principal Investigator is a healthcare professional and not the responsible bioanalytical scientist.From the survey, a variety of regulatory guidelines are followed and quoted, including MHRA, EMA, OECD, GLP, GCP, ISO17025, ICH and Declaration of Helsinki. Most members have some form of procedure for GCP in place, many encompassing training, job descriptions, individuals' roles and competency requirements with respect to GCP, and requests for additional work. Approximately half of the members have specific SOPs for GCP in place, with some incorporating GCP into their current SOPs.The majority of members have only a bioanalytical function, and few have the ability to run a preclinical and/or clinical trial. Therefore, only a small minority of members accept all of the sponsors trial-related duties, including initiation and administration of the Clinical Trial Protocol. However, it was widely acknowledged that the quality of clinical protocols have noticeably improved following GCP inspections.Approximately two-thirds of members had already undergone an inspection for GCP, and most would like certification for GCP, similar to GLP certification. Those that also had clients audits generally found no significant differences from regulatory inspections (FDA, MHRA and so on). The primary focus of GCP inspections in practice has been concerned with contract composition, training records, documentation and data integrity, audit trails, traceability of sample data, sample chain of custody, patient confidentiality, patient safety, patient consent, patient blinding and anonymity and reporting of adverse reactions and potential breaches.During the discussions one member presented a finding from a GCP inspection where it was claimed that the method was not well defined (i.e., inappropriate range, as more than 3% of samples were reassayed diluted). In this situation it was felt that the inspector evaluated under their own personal approach as there are no parameters or acceptance criteria described for the number of diluted samples in any bioanalytical guidelines or official paper. No other attendee had encountered this during an inspection and it was considered that this was an isolated incident.The GCC recommendation on GCP is shown in Box 3.Future perspectiveThe GCC will continue to provide recommendations on hot topics in bioanalysis of global interest and expand its membership by coordinating its activities with the regional and international meetings held by the pharmaceutical industry. Please contact the GCC for the dates and times of future GCC Closed Meetings, and for all membership information [103].Box 1. The GCC recommendation on incurred sample reproducibility.▪ The GCC recommends that incurred sample reproducibility (ISR) should be performed in discrete batches separated from the study sample analysis within a short time period decided a priori based on knowledge of the stability of the analytes and matrix involved. ISR should be calculated by comparing the difference between the original and ISR results with the mean of the two results.▪ ISR is not required to be performed for tissue analysis unless the tissue analysis is a primary end point of the study and is performed using a validated assay.▪ For studies where up to 1000 samples are analyzed, reanalyze 10% of samples; for studies where more than 1000 samples are analyzed, reanalyze 10% of the first 1000 samples with an additional 5% of samples above 1000 samples. At least 20 samples should be analyzed. Typically two samples from a subject should be selected for ISR, one around the tmax and one close to the elimination phase, selected by visual inspection, and with concentrations at least three-times the LLOQ concentration.▪ For toxicology studies, it is recommended that ISR should be performed in all dose groups covering both male and females and for composite profiles sample selection should mimic profiles with an equal number of samples from the tmax and elimination phase.▪ For samples that had undergone dilution, the same dilution factor should be used for both analyses where possible. Additionally, different sample splits/aliquots or dried matrix spots can be used for ISR analysis.▪ For extreme outliers (>50%) the GCC recommends that if the ISR acceptance criteria are met then extreme outliers are not required to be investigated unless they form a trend, and for trends an investigation should be performed even if ISR acceptance criteria are met.▪ For multi-analyte assays, ISR should be conducted for each analyte and sample selection should be based on all analytes, applying the same rules used for selecting samples for single analyte ISR. This may result in an increase in the number of samples per subject with all samples selected to be used for ISR for all analytes, with the exception of samples where results are less than three-times the LLOQ concentration. If the ISR analysis is performed using a sample diluted applying a validated dilution but the original value was obtained from an undiluted sample for that analyte (or vice versa), as long as the measured concentration is greater than three-times the LLOQ, the result with different dilution regimen to the original analysis should be reported.▪ For multi-analyte assays, where a sample is being reanalyzed for a limited number of analytes, the additional data for the other analyte(s) should not normally be used for ISR. If these data are, for example, in cases where sample volume is limited and only one more reanalysis is possible then this should be stated a priori in the study plan or in an amendment.Box 2. The GCC recommendation on consultants.▪ The GCC recommends that an organization has defined procedures for hiring consultants, including the initial selection, assessment of qualifications and experience, approval process, integration within the organization and contractual arrangements, and for acceptance of consultants acting on behalf of clients.▪ The process for hiring consultants should be similar to hiring a new employee. The review of CVs should lead to a selection of candidate(s) for interview, where the contents of the CV should be interrogated further, including qualifications, experience, testimonials and continued relevant training (e.g., supplier's training for hardware/software, attendance at regulatory and scientific meetings). References should also be requested and followed up. A consultant should be fit-for-purpose, that is, they should be GLP, good clinical practice as required, knowledgeable (up to date with technology/regulations) and have relevant consultancy experience.▪ The process for accepting consultants acting on behalf of clients should be agreed between the CRO and client. The GCC recommends that the client conducts the more detailed process for hiring consultants, whilst the CRO performs an additional brief CV review prior to the visit and a brief interview on arrival.▪ The GCC also suggests that CROs could, and often do, act as consultant for their clients; for example, CRO quality assurance departments could perform facility audits for clients, as they have breadth and depth of knowledge and experience due to the wide variety of clients that they support. CROs are also experts in their particular field, as their focus is bioanalysis rather than drug development as a whole and, as such, are up to date with technology and regulatory requirements.▪ The consensus of the GCC members was that working with qualified consultants could be an opportunity to decrease internal costs by managing workflows and increasing knowledge and skills more efficiently. However, if consultants are to be more widely used, it may be advisable for them to become regulated or affiliated to a set code or standard to ensure a consistent, high standard for all consultants.Box 3. The GCC recommendation on good clinical practice.▪ The GCC recommends consideration of the following points concerning contracts: ▪ There should be a contract in place agreeing good clinical practice (GCP) compliance that identifies the responsibilities of all parties, that is, sponsor, CRO, clinic and so on;▪ A signed copy of the Clinical Trial Protocol (CTP) should be made available to the analytical laboratory;▪ Other trial associated documentation should be available, for example laboratory manuals, written assurance that informed consent has been obtained and so on;▪ Relevant CTP amendments should be provided in a timely manner to the analytical laboratory via a mechanism agreed between the sponsor and the analytical laboratory;▪ The CTP should only include work covered by the informed consent given by the trial subjects;▪ Consent change or withdrawal should be agreed to be communicated from the sponsor and the procedure identified in the CTP;▪ The CTP should be understood – specifically aspects relating to the analysis to be performed at the analytical laboratory;▪ An Analytical Phase Plan/Protocol (APP) describing the analytical work to be done, including a designated responsible scientist who will manage the analytical phase and claim GCP compliance, should be agreed and signed by the sponsor;▪ Additional work and subcontracting should have sponsor approval, be included in the CTP or CTP amendment and be covered by informed consent;▪ There should be no contradictions between any parts of the contract, for example, master service agreement, preferred provider agreement, quote, CTP, APP and so on.▪ The GCC recommends consideration of the following additional points concerning study conduct at the CRO: ▪ Management, quality assurance and analyst activity expectations similar to GLP;▪ No confidential information should be evidenced or inferred by the sample label or accompanying documentation; a procedure should be described at the analytical laboratory on how to deal with breaches of this nature;▪ Policies for dealing with missing, unexpected or poorly labeled samples should be documented;▪ Samples should not be analyzed until their identity is confirmed and approved by the sponsor;▪ Potential serious breaches should be immediately reported to the sponsor so that the potential health and safety risks can be asses

An open letter written by the Global CRO Council for Bioanalysis (GCC) describing the GCC survey results on stability data from co-administered and co-formulated drugs was sent to multiple regulatory authorities on 14 December 2011. This letter and further discussions at different GCC meetings led to subsequent recommendations on this topic of widespread interest within the bioanalytical community over the past 2 years.

) Guideline on Bioanalytical Method Validation (BMV), during the 4th GCC (23 October 2011, Washington DC, USA) and 5th GCC (14 November 2011, Barcelona, Spain) Closed Forums. These North American and European events provided a unique opportunity for CRO leaders to openly share opinions and perspectives and to agree on unified bioanalytical recommendations specifically in relation with the new EMA guideline.The Global CRO Council for Bioanalysis (GCC)

CHF5074, a new gamma-secretase modulator, has been shown to inhibit brain plaque deposition and attenuate or reverse memory deficits in different human amyloid precursor protein transgenic mice models of Alzheimer's disease (JPET 2007; 323: 822-30, Br J Pharmacol 2009; 156: 982-93; J Alzheimers Dis 2010; 20: 159-73; J Alzheimers Dis 2011, in press). We evaluated the safety, tolerability and pharmacokinetics of CHF5074 after multiple oral administrations to healthy male young volunteers. Three ascending oral dose regimens of CHF5074 (200, 400 and 600 mg/day for 14 days) were evaluated in a double-blind, placebo-controlled, parallel groups study involving 48 subjects. In each of the three study sessions, subjects were randomized to CHF5074 (n = 12) or placebo (n = 4). Overall, 36 subjects received CHF5074 and 12 placebo. An independent Data & Safety Monitoring Board (DSMB) reviewed safety and tolerability data derived from each treatment session before recommending advancement to subsequent treatment sessions. During each session, subjects were closely monitored for 18 days and vital signs, electrocardiographic activity, neuropsychological tests and laboratory safety tests were measured. Plasma samples were collected daily during drug administration and up to 96 hours after the last drug administration for measuring drug and main metabolite (CHF5074-glucuronide) concentrations using a validated LC-MS/MS analytical method. The study has been completed but the data are still blinded. All subjects completed the study. There were no serious adverse events. The most frequent adverse event was mild-to-moderate diarrhea. The DSMB concluded that maximum tolerated dose was not reached but that 600 mg/day was nearing the threshold. Preliminary pharmacokinetic analyses indicated that CHF5074 reached peak levels 2-3 hours after drug administration and was slowly eliminated from plasma (terminal half-life of about 30 hours). Systemic exposure to the drug appeared proportional to dose with a two-fold accumulation ratio at steady-state. Metabolite plasma levels peaked at 4-5 hours and accounted for about 25% of the parent compound. This study indicated that CHF5074 is well tolerated by healthy male subjects after multiple oral administrations of doses up to and including 600 mg/day. The pharmacokinetics of CHF5074 appears linear in the studied dose range with plasma exposure increasing in a predictable dose-proportional manner.

The 3rd Global CRO Council Closed Forum was held on the 3rd and 4th July 2011 in Guildford, United Kingdom, in conjunction with the 19th International Reid Bioanalytical Forum. In attendance were 21 senior-level representatives from 19 CROs on behalf of nine European countries and, for many of the attendees, this occasion was the first time that they had participated in a GCC meeting. Therefore, this closed forum was an opportunity to increase awareness of the aim of the GCC and how it works, share information about bioanalytical regulations and audit findings from different agencies, their policies and procedures and also to discuss some topics of interest and aim to develop ideas and provide recommendations for bioanalytical practices at future GCC meetings in Europe.

tor for Alzheimer disease. A single copy of this variant leads to a 3 fold increased risk compared with the most common APOE E3 allele whereas inheritance of the rare APOE E2 reduces this risk by about half. However, despite the discovery of these strong genetic clues, the mechanism whereby apoE confers risk remains uncertain. Methods: In order to decipher how the different ApoE isoforms (ApoEE2, -E3 and -E4) impact the formation and stability of amyloid plaques, AAV4 vectors coding for each ApoE isoform were injected into the ventricle of 7 month-old APP/PS mice. Using in vivo multiphoton imaging, we tracked populations of amyloid deposits at baseline and after exposure to ApoE over a two-month interval, thus allowing us to have a dynamic view of amyloidosis progression in a living animal. Results: We observed that the kinetic of amyloid deposits progression varies according to each isoform, so that ApoE4 injected mice have a 36% increase of senile plaques (p < 0.05) whereas mice treated with ApoE2 present a 25% decrease of the number of amyloid deposits (p < 0.05) compared to ApoE3 after 2 months. The post-mortem analysis revealed the presence of human ApoE proteins in the epithelial lining of the ventricle but it also decorates plaques in the cortex, reflecting a large diffusion of the protein and its accumulation where Aß peptides are deposited. In some of the injected mice, the amount of ApoE protein detected by ELISA reached the level of ApoE measured in transgenic animals, confirming that this approach may be efficient to increase the global content of a secreted protein throughout the entire brain. Conclusions: Overall, our results suggest that a single injection of an AAV4 vector into the ventricle leads to a diffuse and sustained overexpression of the different ApoE isoforms in the mouse brain. Moreover, the ApoE4 overexpression accelerates the progression of amyloid deposition whereas ApoE2 alleviates the course of the disease. These results demonstrated that manipulation of the ApoE status in the brain may be an interesting approach to modulate amyloidosis.

5-Fluorouracil (5-FU) is a cytostatic agent that has been widely used in the treatment of various solid tumours for more than 20 years, and is still considered to be among the most active antineoplastic agents in advanced colorectal cancer and malignancies of the head and neck. A large number of non-chromatographic and chromatographic methods for the quantitation of 5-FU, related prodrugs and their metabolites in biological matrices have been developed in the last 30 years to support preclinical and clinical studies. However, 5-FU monitoring has not been widely used, at least not in the USA, and certainly not outside the clinical research setting, given the absence of simple, fast and inexpensive testing methods for 5-FU monitoring. Recent developments with testing based on liquid chromatography-tandem mass spectrometry and a nanoparticle antibody-based immunoassay may facilitate routine monitoring of 5-FU in daily clinical practice. In this review the advantages and disadvantages of the bioanalytical methods developed and used for 5-FU, its metabolites and related prodrugs are discussed.

CHF5074, a new gamma-secretase modulator, has been shown to inhibit brain plaque deposition and attenuate or reverse associated memory deficits in different human amyloid precursor protein transgenic mice models of Alzheimer's disease (JPET 2007; 323: 822-30, Br J Pharmacol 2009; 156: 982-93; J Alzheimers Dis 2010, in press). We evaluated for the first time the safety, tolerability and pharmacokinetics of CHF5074 after single oral administration to healthy male volunteers. Six ascending oral doses of CHF5074 (25, 50, 100, 200, 400 and 600 mg) were evaluated in a double-blind, placebo-controlled, parallel groups study involving 60 healthy volunteers. In each of six study sessions, subjects were randomized to CHF5074 or placebo. Overall, 48 subjects received CHF5074 and 12 placebo. An independent Data & Safety Monitoring Board reviewed safety and tolerability data derived from each treatment session before recommending advancement to subsequent treatment sessions. During each session, subjects were closely monitored for 72 hours and vital signs, electrocardiographic activity, neuropsychological tests and laboratory safety tests were measured. Plasma samples were collected up to 72 hours after drug administration for measuring drug and main metabolite (CHF5074-glucoronide) concentrations. The study has been completed but the data are still blinded. The maximum tolerated dose was not reached since all adverse events were mild in intensity and were judged as not clinically significant. Preliminary pharmacokinetic analyses indicated that CHF5074 reached peak levels 2-3 hours after drug administration and was slowly eliminated from plasma (terminal half-life of about 25 hours). Systemic exposure to the drug appeared proportional to dose. Metabolite plasma levels peaked at 4-5 hours and accounted for about 30% of the parent compound. This first-in-man study with CHF5074 indicated that the drug is well tolerated after single oral administration to healthy subjects of doses up to and including 600 mg. The pharmacokinetics of CHF5074 appear linear in the studied dose range with plasma exposure increasing in a predictable dose-proportional manner.

A highly sensitive inductively coupled plasma mass spectrometry (ICP-MS) method with microwave-assisted sample digestion for the determination of total platinum in rat whole and ultrafiltrate plasma was developed and validated. A first step of this study concerned the optimization of the mineralization procedure, in order to obtain good extraction recovery (higher than 90%) and repeatability (less than 6%) and the absence of matrix effect. ICP-MS analysis was then performed using the “hot plasma/protective ion extraction” mode, achieving high sensitivity and very high signal/noise ratio. Iridium was added as internal standard. The method was then submitted to validation, performed according to the FDA Bioanalytical Validation Methods guidelines and to the Eurachem guide. Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision, accuracy and stability. An instrumental LOQ of 1.9 ng L−1, corresponding to a concentration of 955 ng L−1 in matrix under the adopted conditions, was obtained, allowing the quantitative analysis of Pt ultratraces. Instrumental linearity was verified in the range 1.9–14,000 ng L−1, corresponding to a concentration range from 955 ng L−1 to 6825 μg L−1 in matrix. Accuracy was evaluated by analyzing control samples for both matrices at different concentration levels; a good agreement (<15%) was obtained. Sample stability was tested by analyzing control samples maintained for 4 h at room temperature or submitted to three freezing–thawing cycles. Finally, the developed method was applied to the analysis of plasma and ultrafiltrate plasma of rats treated with oxaliplatin-base drug, thus demonstrating its reliability in pharmacokinetic studies.

Liquid chromatography-tandem mass spectrometry (LC/MS/MS) has become the technology of choice for bioanalysis, due to its high selectivity and high sample throughput. However, concerns have grown that this technique may be subject to errors due to "invisible" interferences, in particular ion-suppression. Investigations on ion-suppression from formulation agents have only been published to a limited extent. Such effects can be of particular importance in pre-clinical discovery studies where drugs may be formulated with large amount of solubilisers and bioanalysis may use fast generic methods. In a preliminary pharmacokinetic study we observed strong ion-suppression from a polysorbate co-solvent, which, if undetected, would have given highly erroneous pharmacokinetic results and possibly could have led to the inappropriate elimination of a promising drug candidate. Different chromatographic methods were tested indicating that the separation step was essential in controlling these effects. A method based on matrix dilution is proposed to check for these effects during the use of discovery support methods, where full validation is not practical. Some excipients commonly used in formulations are polydispersed polymers, for which very limited pharmacokinetic information is available. Further investigation is needed to better understand the mechanisms of ion-suppression and the kinetics of the suppressing species to allow the development of new LC/MS/MS based analytical strategies, which will not be subject to such ionisation interferences.

Estramustine phosphate has been used for over 20 years in the treatment of advanced prostate cancer. Recent reports of promising results from clinical combination studies, and neoadjuvant studies, have caused a renewed interest in this relatively old drug. After oral or intravenous administration, estramustine phosphate is metabolised to four main metabolites: estramustine, estromustine, estradiol and estrone. Due to the potential biological activity of all these estramustine phosphate metabolites, it is necessary to analyse the parent compound and all four metabolites in plasma, tissues and tumours. Consequently, various methods have been developed for the bioanalysis of these compounds. In this review various methods are described for the analysis of estramustine phosphate and its metabolites in biological fluids and tissues. Methods described included RIA, TLC, HPLC, HPLC-MS, GC and GC-MS.

A simple and fast analytical method using hydrophilic interaction liquid chromatography (HILIC) coupled with mass spectrometry was developed to analyse 5-fluorouracil (5-FU) in plasma and tissues. The HILIC system overcomes problems reported in obtaining satisfactory retention of 5-FU with other types of HPLC systems. After addition of internal standard (IS) (5-Chlorouracil (5-CU)), plasma proteins were precipitated with acetonitrile, and tissue samples homogenised with a micro-dismembrator. The analysis was performed using a polymer-based column (Ashaipak NH2) and the compounds were eluted under gradient conditions at 1 ml/min using a mobile phase containing a mixture of ammonium formate and acetonitrile. MS detection used a API 4000 mass spectrometry with heated nebulizer source and multiple reaction monitoring operated in the negative ion mode. The mass transitions of 5-FU and its internal standard were 129 m/z-->42m/z and 145 m/z-->42 m/z, respectively. The lower limits of quantitation in plasma and tissues were about 5 ng/ml and 10 ng/g, respectively, using 25 microl of plasma and 50mg of tissue. Good linearity, accuracy and precision were obtained in all matrices tested. The suitability and robustness of the method for in vivo samples were confirmed by analysis of mouse plasma, muscle and tumour from animals dosed with 5-FU.