First-in-human study of CPL207280, a novel G-protein-coupled receptor 40/free fatty acid receptor 1 agonist, in healthy volunteers after single and multiple administration.

AIM: To assess the safety, tolerability and pharmacokinetic (PK) profile of single and multiple doses of CPL207280, a new G-protein-coupled receptor 40 agonist developed to treat type 2 diabetes (T2D). METHODS: The phase 1 study in healthy volunteers (White, age 18-55 years, body mass index 18.5-29.9 kg/m2 ) was performed after single (24 subjects, 5-480 mg) and multiple (32 subjects, 60-480 mg) once-daily administration of CPL207280.  The effect of food intake and interaction with metformin were evaluated in additional cohort (12 subjects, 120 mg). The primary objective was the safety and tolerability of CPL207280. Secondary objectives included PK and pharmacodynamic (PD) characteristics (glucose, insulin, C-peptide, proinsulin, glucagon levels) observed during the 14-day treatment period. RESULTS: No deaths or serious adverse events (AEs) were reported. All reported AEs were classified as unrelated to the study product. No clinically significant differences in safety parameters were observed between cohorts and no food or metformin effect on safety parameters was identified. The ascending dose of CPL207280 caused an increase in the PK parameters maximum observed plasma concentration (Cmax ) or area under the plasma concentration-time curve up to 24 h. However, dose-normalized Cmax decreased with ascending dose. There was no relationship between the CPL207280 dose or prandial state and terminal elimination half-life and terminal elimination rate constant. No clear relationship between CPL207280 dose and PD area under the effect curve values was observed. CONCLUSIONS: CPL207280 was found to be safe and well tolerated by healthy volunteers (with a low risk of hepatotoxicity) for up to 14 days of administration. The PK profile of CPL207280 supports single-daily administration and justifies further development of this therapy for patients with T2D.

How does the order of sample analysis influence the matrix effect during LC-MS bioanalysis?

Mass spectrometry coupled with liquid chromatography is a valuable tool for drug development and personalised drug therapy. The matrix effect is caused by enhancing or suppressing the analyte signal intensity by the interfering compounds of biological fluids. The matrix effect may influence the reliability of the quantitative results. Thus, its evaluation is a critical part of bioanalytical method validation. Identified factors affecting the matrix effect are the physicochemical properties of the analyte, type of biological material, analytical conditions, the ion source construction and calculation method. The order of analysis of test samples (pure solutions and post-extraction spiked samples) is another factor possibly affecting quantifying the matrix effect variability between sources. Our primary goal was to find which experimental design - interleaved or set of blocks - is more sensitive to detect matrix effect variability. Additionally, to better understand the reason of variability, we evaluated the influence of chromatographic elution and the type of plasma (normal, lipemic or hemolyzed), co-elution, and carry-over of phospholipids. We used chemometric methods: Principal Component Analysis and Partial Least-Squares Discriminant Analysis. Although a comparable (but statistically different) matrix effect (%RSDMF) is observed using the interleaved and block schemes, for some compounds, the order of the samples strongly influences the results. The interleaved scheme was generally more sensitive in detecting the matrix effect than the block scheme. Thus, reporting the order of samples is needed to ensure the repeatability of experiments. Chemometrics suggests that lipemic samples analyzed in isocratic conditions are most prone to the matrix effect. Different compositions of matrix lots of the same type - especially lipemic - may influence method reliability. Thus, evaluating more than one source of lipemic and hemolyzed plasma is recommended.

Replicates Number for Drug Stability Testing during Bioanalytical Method Validation-An Experimental and Retrospective Approach.

BACKGROUND: The stability of a drug or metabolites in biological matrices is an essential part of bioanalytical method validation, but the justification of its sample size (replicates number) is insufficient. The international guidelines differ in recommended sample size to study stability from no recommendation to at least three quality control samples. Testing of three samples may lead to results biased by a single outlier. We aimed to evaluate the optimal sample size for stability testing based on 90% confidence intervals. METHODS: We conducted the experimental, retrospective (264 confidence intervals for the stability of nine drugs during regulatory bioanalytical method validation), and theoretical (mathematical) studies. We generated experimental stability data (40 confidence intervals) for two analytes-tramadol and its major metabolite (O-desmethyl-tramadol)-in two concentrations, two storage conditions, and in five sample sizes (n = 3, 4, 5, 6, or 8). RESULTS: The 90% confidence intervals were wider for low than for high concentrations in 18 out of 20 cases. For n = 5 each stability test passed, and the width of the confidence intervals was below 20%. The results of the retrospective study and the theoretical analysis supported the experimental observations that five or six repetitions ensure that confidence intervals fall within 85-115% acceptance criteria. CONCLUSIONS: Five repetitions are optimal for the assessment of analyte stability. We hope to initiate discussion and stimulate further research on the sample size for stability testing.

Evaluation of tramadol human pharmacokinetics and safety after co-administration of magnesium ions in randomized, single- and multiple-dose studies.

BACKGROUND: Magnesium ions (Mg2+) increase and prolong opioid analgesia in chronic and acute pain. The nature of this synergistic analgesic interaction has not yet been explained. Our aim was to investigate whether Mg2+ alter tramadol pharmacokinetics. Our secondary goal was to assess the safety of the combination. METHODS: Tramadol was administered to healthy Caucasian subjects with and without Mg2+ as (1) single 100-mg and (2) multiple 50-mg oral doses. Mg2+ was administered orally at doses of 150 mg and 75 mg per tramadol dosing in a single- and multiple-dose study, respectively. Both studies were randomized, open label, laboratory-blinded, two-period, two-treatment, crossover trials. The plasma concentrations of tramadol and its active metabolite, O-desmethyltramadol, were measured. RESULTS: A total of 25 and 26 subjects completed the single- and multiple-dose study, respectively. Both primary and secondary pharmacokinetic parameters were similar. The 90% confidence intervals for Cmax and AUC0-t geometric mean ratios for tramadol were 91.95-102.40% and 93.22-102.76%. The 90% confidence intervals for Cmax,ss and AUC0-τ geometric mean ratios for tramadol were 93.85-103.31% and 99.04-105.27%. The 90% confidence intervals for primary pharmacokinetic parameters were within the acceptance range. ANOVA did not show any statistically significant contribution of the formulation factor (p > 0.05) in either study. Adverse events and clinical safety were similar in the presence and absence of Mg2+. CONCLUSIONS: The absence of Mg2+ interaction with tramadol pharmacokinetics and safety suggests that this combination may be used in the clinical practice for the pharmacotherapy of pain.

Low-oxidation-potential thiophene-carbazole monomers for electro-oxidative molecular imprinting: Selective chemosensing of aripiprazole.

New thiophene-carbazole functional and cross-linking monomers electropolymerizing at potentials sufficiently low for molecular imprinting of an electroactive aripiprazole antipsychotic drug were herein designed and synthesized. Numerous conducting molecularly imprinted polymer (MIP) films are deposited by electropolymerization at relatively low potentials by electro-oxidation of pyrrole, aniline, phenol, or 3,4-ethylenedioxythiophene (EDOT). However, their interactions with templates are not sufficiently strong. Hence, it is necessary to introduce additional recognizing sites in these cavities to increase their affinity to the target molecules. For that, functional monomers derivatized with substituents forming stable complexes with the templates are used. However, oxidation potentials of these derivatives are often, disadvantageously, higher than that of parent monomers. Therefore, we designed and synthesized new functional and cross-linking monomers, which are oxidized at sufficiently low potentials. The deposited MIP and non-imprinted polymer (NIP) films were characterized by PM-IRRAS and UV-vis spectroscopy and imaged with AFM. The structure of the aripiprazole pre-polymerization complex with functional monomers was optimized with density functional theory (DFT), and aripiprazole interactions with imprinted cavities were simulated with molecular mechanics (MM) and molecular dynamics (MD). MIP-aripiprazole film-coated electrodes were used as extended gates for selective determination of aripiprazole with the extended-gate field-effect transistor (EG-FET) chemosensor. The linear dynamic concentration range was 30-300 pM, and the limit of detection was 22 fM. An apparent imprinting factor of the MIP-1 was IF = 4.95. The devised chemosensor was highly selective to glucose, urea, and creatinine interferences. The chemosensor was successfully applied for aripiprazole determination in human plasma. The results obtained were compared to those of the validated HPLC-MS method.

10th Anniversary of a Two-Stage Design in Bioequivalence. Why Has it Still Not Been Implemented?

PURPOSE: In 2010 the European Medicines Agency allowed a two-stage design in bioequivalence studies. However, in the public domain there are mainly articles describing the theoretical and statistical base for the application of the two-stage design. One of the reasons seems to be the lack of practical guidance for the Sponsors on when and how the two-stage design can be beneficial in bioequivalence studies. METHODS: Different variants with positive and negative outcomes have been evaluated, including a pivotal study, pilot + pivotal study and two-stage study. The scientific perspective on the two-stage bioequivalence study has been confronted with the industrial one. RESULTS: Key information needed to conduct a bioequivalence study - such as in vitro data and pharmacokinetics - have been listed and organized into a decision scheme. Advantages and disadvantages of the two-stage design have been summarized. CONCLUSION: The use of the two-stage design in bioequivalence studies seems to be a beneficial alternative to the 2 × 2 crossover study. Basic information on the properties of the active substance and the characteristics of the drug form are needed to make an initial decision to carry out the two-stage study.

Incurred Sample Reanalysis: Time to Change the Sample Size Calculation?

Reliable results of pharmacokinetic and toxicokinetic studies are vital for correct decision making during drug discovery and development. Thus, ensuring high quality of bioanalytical methods is of critical importance. Incurred sample reanalysis (ISR)-one of the tools used to validate a method-is included in the bioanalytical regulatory recommendations. The methodology of this test is well established, but the estimation of the sample size is still commented on and contested. We have applied the hypergeometric distribution to evaluate ISR test passing rates in different clinical study sizes. We have tested both fixed rates of the clinical samples-as currently recommended by FDA and EMA-and a fixed number of ISRs. Our study revealed that the passing rate using the current sample size calculation is related to the clinical study size. However, the passing rate is much less dependent on the clinical study size when a fixed number of ISRs is used. Thus, we suggest using a fixed number of ISRs, e.g., 30 samples, for all studies. We found the hypergeometric distribution to be an adequate model for the assessment of similarities in original and repeated data. This model may be further used to optimize the sample size needed for the ISR test as well as to bridge data from different methods. This paper provides a basis to re-consider current ISR recommendations and implement a more statistically rationalized and risk-controlled approach.

Matrix effect screening for cloud-point extraction combined with liquid chromatography coupled to mass spectrometry: Bioanalysis of pharmaceuticals.

Matrix effects are one of the most challenging issues in the analysis of complex samples using liquid chromatography coupled to mass spectrometry (LC-MS). Apart from the instrumental origin, these effects are also related to sample preparation. Cloud-point extraction (CPE) is rarely combined with LC-MS as it requires the use of surfactants which might interfere with droplet evaporation. Thus, they are suspected to cause a significant matrix effect (signal suppression). In this paper, 73 model drugs with different physicochemical properties were screened to analyse how susceptible LC-MS is to the absolute and relative matrix effect (ME) when coupled with CPE for measurements in human plasma. Three combinations of the surfactant Triton X-114 concentration (1.5% or 6%) and extraction temperature (40 or 55 °C) in six pH values gave over 1300 analyte-sample preparation condition pairs. A new term - surfactant effect (calculated for the standard solution) - allowed us to distinguish between the surfactant effect and that related to interferences from human plasma. The screening revealed that CPE combined with LC-MS is not related to a significant ME in the optimal pH of extraction. A significant absolute ME (<85% or>115%) was observed only for 25% of the analytes. Data processing (principal component analysis, classification trees, partial least squares-discriminant analysis) based on the extraction conditions and molecular descriptors helped to identify compounds prone to the matrix effect and speed up method development. A low surfactant concentration and low temperature decreased both the absolute and relative ME. pH of the extraction influenced only the relative ME. Low retention time reduced the risk of relative ME, whereas high polarity and the possibility of hydrogen bond formation minimized the occurrence of the surfactant effect and absolute ME. A significant relative ME (>15%) was observed only for 11% of the compounds, thus CPE merged with LC-MS allowed to measure drug concentrations in a reliable manner for majority of compounds. The presented approach may be further applied to other analytes and matrices.

Bioanalytical method validation: new FDA guidance vs. EMA guideline. Better or worse?

Bioanalysis concerns the identification and quantification of analytes in various biological matrices. Validation of any analytical method helps to achieve reliable results that are necessary for proper decisions on drug dosing and patient safety. In the case of bioanalytical methods, validation additionally covers steps of pharmacokinetic and toxicological studies - such as sample collection, handling, shipment, storage, and preparation. We drew our attention to the difference of both the newest FDA Guidance and the EMA Guideline on bioanalytical method validation. We aimed to point out advantages of both documents from the laboratory perspective. The FDA and the EMA documents are similar, but not identical. The EMA describes the practical conduct of experiments more precisely, while the FDA presents reporting recommendations more comprehensively. There are also differences in recommended validation parameters. We hope that the International Council for Harmonisation will combine advantages of both documents to avoid confusing differences in terminology as well as the unnecessary effort of being compliant with two or more guidelines.

Quantitative evaluation of the matrix effect in bioanalytical methods based on LC-MS: A comparison of two approaches.

Liquid chromatography coupled to mass spectrometry (LC-MS) is a powerful tool for studying pharmacokinetics and toxicokinetics. Reliable bioanalysis requires the characterization of the matrix effect, i.e. influence of the endogenous or exogenous compounds on the analyte signal intensity. We have compared two methods for the quantitation of matrix effect. The CVs(%) of internal standard normalized matrix factors recommended by the European Medicines Agency were evaluated against internal standard normalized relative matrix effects derived from Matuszewski et al. (2003). Both methods use post-extraction spiked samples, but matrix factors require also neat solutions. We have tested both approaches using analytes of diverse chemical structures. The study did not reveal relevant differences in the results obtained with both calculation methods. After normalization with the internal standard, the CV(%) of the matrix factor was on average 0.5% higher than the corresponding relative matrix effect. The method adopted by the European Medicines Agency seems to be slightly more conservative in the analyzed datasets. Nine analytes of different structures enabled a general overview of the problem, still, further studies are encouraged to confirm our observations.

Extended 3D and 4D cumulative plots for evaluation of unmatched incurred sample reanalysis.

AIM: Incurred sample reanalysis (ISR) helps ensure the reliability of pharmacokinetic studies. An appropriate graph may facilitate the evaluation of an unmatched reanalyses or a failed ISR test. METHODS: We evaluated different ways of visualizing multidimensional ISR data using an extended cumulative ISR plot. RESULTS: 3D and 4D cumulative ISR plots enable comprehensive data analysis using a single plot. We propose to use color for percentage difference classes in bar and XY-scatter plots. For the latter the shape of symbols may represent analyte concentration class, study phase, analyst or subject. CONCLUSION: The extended 3D and 4D cumulative ISR plots facilitate in-study monitoring and post-study inspection of data. It helps find the root cause of unmatched ISR, thus increasing reliability of bioanalytical data.

Incurred sample reanalysis: adjusted procedure for sample size calculation.

AIM: The incurred sample reanalysis (ISR) helps to assure bioanalysis reliability. The regulatory guidelines recommend the reanalysis of up to 10% of the study samples for this test, but not all reanalyses are necessary to evaluate the test result. MATERIALS & METHODS: We have optimized ISR sample size calculation to eliminate negligible reanalyses. RESULTS: Adjusted procedure eliminates negligible reanalyses - up to 66% of currently analyzed ISRs - without affecting the test outcome. CONCLUSION: The procedure is universal as it may be applied in the studies compliant with EMA and US FDA requirements, for both small and large molecules. It may assist the evolution of bioanalytical method validation as the current ISR sample size recommendations seem to be ill-matched with the acceptance criteria.

Comprehensive graphical presentation of data from incurred sample reanalysis.

AIM: Incurred sample reanalysis (ISR) contributes to the reliability of pharmacokinetic studies. Despite regulatory guidelines having adopted ISR methodology, graphical presentation of data has been overlooked. MATERIALS & METHODS: Different graphs were tested for datasets including limited, standard and large numbers of ISR pairs. The datasets covered both passed and failed cases. RESULTS: We have developed a combination of complementary plots enabling the visual inspection of ISR data quality: %difference versus mean concentration and cumulative ISR plot. The former shows individual ISR datapoints and concentration-dependent trends, while the latter presents the contribution of individual pairs to the overall result as well as time-dependent trends. CONCLUSION: The proposed visualization of ISR data shows at a glance whether acceptance criteria for each sample and whole experiment are met or not. Standardized graphical presentation of ISR outcomes may increase quality of bioanalytical data.

Bioequivalence study of 2.5 mg film-coated bisoprolol tablets in healthy volunteers.

BACKGROUND: Bisoprolol is one of the most widely used beta-blockers characterised by cardioselectivity, and it has no intrinsic sympathomimetic activity. It is commonly used in the treatment of coronary heart disease and heart failure. AIM: The aim of study was to assess the bioequivalence of the film-coated tablets containing 2.5 mg of bisoprolol (Bisocard® - the medicinal product) to the original medicinal product (Concor Cor 2.5® - the reference). METHODS: A randomised, open-label, two-period, crossover, single-dose, relative bioavailability study was conducted in fasted healthy Caucasian volunteers. A single 10-mg oral dose (four tablets of 2.5 mg) of the test or reference product was followed by a 14-day wash-out period, after which the subjects received the alternative product. Blood was sampled within a period of 60 h post administration in pre-specified time points. Bisoprolol concentrations were determined by a validated LC-MS/MS method. The products were considered bioequivalent if the 90% confidence interval (CI) of the log-transformed geometric mean ratios (test vs. reference) for AUC(0-t), AUC(0-∞), and Cmax were within 80-125% limits. Adverse events were monitored during the study based on the subject claims and clinical parameters. RESULTS: Twenty-six healthy male and female volunteers (mean age ca. 29 years; body mass index 22.7 kg/m²) were in-cluded in the study, and 24 completed the clinical part. The geometric mean ratios (test/reference) for the log-transformed AUC(0-t), AUC(0-∞), and Cmax were 95.16% (90% CI 92.52-97.87%), 95.08% (90% CI 92.40-97.83%), and 100.00% (90% CI 94.83-105.45%), respectively. There were no significant differences in the pharmacokinetic parameters between the test and reference formulations. No serious adverse events were reported. CONCLUSIONS: The results of this single-dose study in healthy Caucasian volunteers indicate that Bisocard®; 2.5 mg film-coated tablets are bioequivalent to the reference product - Concor Cor 2.5®; 2.5 mg film-coated tablets. Both products had similar safety profile and have been well tolerated.

Is a deuterated internal standard appropriate for the reliable determination of olmesartan in human plasma?

A right choice of the internal standard is one of the most challenging tasks during bioanalytical method development. Surprisingly, among the HPLC-MS methods for the determination of a cardiovascular drug olmesartan in plasma only structural analogues or similar compounds were used as internal standards. We have tried to answer the question whether the stable isotope labelled (deuterated) internal standard, as recommended by regulatory agencies, can be used for the reliable determination of olmesartan in human plasma. An HPLC-MS method using this standard in a simplified liquid-liquid extraction procedure led to accurate and precise results in the linearity range of 5-2500ng/mL. The method is well suited for pharmacokinetic studies following a single 40mg oral dose of olmesartan medoxomil in humans. The method was fully validated according to international guidelines and successfully applied in a bioequivalence study in humans. The use of deuterated olmesartan as the internal standard afforded a reliable tool for regulatory bioanalysis that can indirectly contribute to therapy efficacy and improve the safety of patients treated with generic medicines.

ENVIRONMENTALLY FRIENDLY LC/MS DETERMINATION OF EPLERENONE IN HUMAN PLASMA.

Eplerenone (EPL), a selective aldosterone receptor antagonist, is indicated in the treatment of chronic heart failure and hypertension. It is hard to find a green assay among a few published methods for its determination in human plasma or serum. Following a liquid-liquid extraction with methyl t-butyl ether, eplerenone and isotope labelled eplerenone - used as an internal standard - were separated from the endogenous compounds on an Atlantis dCl8 column (150 x 3 mm, 3.0 [im). An isocratic elution with the mobile phase consisting of methanol and ammonium acetate (3 : 2, v/v) was used. A single quadrupole mass spectrometer was operated in positive electrospray ionization using the selected ion monitoring mode. The method is more environmentally-friendly than the previously reported assays. Acetonitrile in the mobile phase was replaced with methanol which is a removable solvent. Plasma sample volume was reduced to 250 pL which significantly decreased waste volume. Chlorinated solvents used previously for liquid-liquid extraction were eliminated and the safety of the laboratory staff was increased by eliminating diethyl ether. The method is characterized by a short analysis time, simple sample preparation and reduction of waste volume, which are important advantages when analyzing large numbers of samples. The method was validated according to international regulatory guidelines and may be applied to human pharmacokinetic studies following a single 25 or 50 mg oral dose.

HPLC-UV ASSAY OF IMATINIB IN HUMAN PLASMA OPTIMIZED FOR BIOEQUIVALENCE STUDIES.

lmatimb is an anticancer drug approved for the treatment of a number of cancers, mostly used in chronic myeloid leukemia. Numerous bioanalytical methods using high performance liquid chromatography coupled to ultraviolet detection point at the importance and necessity of the therapeutic drug monitoring of imatinib. Unfortunately, these methods are not optimized for single dose pharmacokinetic studies such as bioe- quivalence. In this study, attention was turned mostly to the analysis time, linearity range and interferences by endogenous components of the matrix and exogenous substances - especially metabolites. The method enables the quantification of imatinib in the presence of the main metabolite (N-desmethyl imatinib). Its potential back- conversion was examined during storage for 4 h at ambient temperature as well as for 239 days at -20°C. The sample preparation based on the liquid-liquid extraction was combined with a short analysis time of 7 min. Therefore, the method was suitable for analyzing large numbers of samples in a short time. The linearity range of 40-4000 ng/mL was optimized for human pharmacokinetic studies after a single 400 mg oral dose of ima- tinib. Successful application in a bioequivalence study confirmed the reliability of the method.

Cloud-point extraction is compatible with liquid chromatography coupled to electrospray ionization mass spectrometry for the determination of bisoprolol in human plasma.

Cloud-point extraction (CPE) draws increasing interest in a number of analytical fields including bioanalysis, but combining CPE and LC-MS with electrospray ionization (ESI) in the determination of drugs in biological fluids such as plasma, serum or blood has not been reported so far. Bisoprolol was determined in human plasma by CPE using Trition X-114 as a surfactant and metoprolol as the internal standard. NaOH concentration, temperature and Trition X-114 concentration were optimized. All analyses were performed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). All validation experiments met international acceptance criteria and no significant matrix effect was observed. The compatibility of CPE and LC-ESI-MS/MS was confirmed using clinical plasma samples and appropriate statistical tests. The determination of bisoprolol concentration in human plasma in the range 1.0-70ngmL(-1) by the CPE method leads to the results which are equivalent to those obtained by the widely used liquid-liquid extraction method. The results revealed that a structural analogue may be an appropriate internal standard when CPE is used as the extraction technique. CPE offers significant practical advantages over the classical extraction methods, including a positive impact on the environment, therefore its wider application in future pharmacokinetic studies is justifiable.

Simplified LC-MS/MS method enabling the determination of azithromycin in human plasma after a low 100mg dose administration.

A sensitive liquid chromatographic-tandem mass spectrometric method was developed and validated to study the pharmacokinetics of a low dose of azithromycin in human plasma. The sample preparation was based on liquid-liquid extraction with the low volume of methyl t-butyl ether. The chromatographic separation was performed on a Symmetry C18 column (50×2.1mm, 3.5µm). Gradient elution with ammonium acetate-acetonitrile and ammonium acetate-methanol was applied. Positive electrospray ionisation tandem mass spectrometry in the multiple reaction monitoring mode was used for the detection of azithromycin. The influence of a major metabolite, including the possibility of its back-conversion, on the quantification of azithromycin was evaluated. Isotope labelled azithromycin was used as the internal standard. The calibration curve was linear in the range of 0.5-250.0ng/mL. The new validated method was successfully applied to a pharmacokinetic study in humans following a single 100mg oral dose.