Ronald de Vries

Combination of ibrutinib with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for treatment-naive patients with CD20-positive B-cell non-Hodgkin lymphoma: a non-randomised, phase 1b study

BACKGROUND Present first-line therapy for diffuse large B-cell lymphoma, a subtype of non-Hodgkin lymphoma, is rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Ibrutinib, a novel oral Brutons tyrosine kinase inhibitor, has shown single-drug activity in relapsed or refractory B-cell malignancies. We investigated the safety and efficacy of ibrutinib in combination with R-CHOP for patients with previously untreated CD20-positive B-cell non-Hodgkin lymphoma. METHODS In this phase 1b, open-label, non-randomised study, patients were recruited across six centres in the USA and France. Eligibility was age 18 years or older and treatment-naive histopathologically confirmed CD20-positive B-cell non-Hodgkin lymphoma. In the dose-escalation phase (part 1), patients with diffuse large B-cell lymphoma, mantle-cell lymphoma, or follicular lymphoma were enrolled. The primary objective was to determine a recommended phase 2 dose of ibrutinib with a standard R-CHOP regimen, by assessing safety in all patients who received treatment. Patients received ibrutinib 280 mg, 420 mg, or 560 mg per day in combination with a standard R-CHOP regimen every 21 days. Safety of the recommended phase 2 dose was then assessed in a dose-expansion population, which consisted of patients with newly diagnosed diffuse large B-cell lymphoma (part 2). Secondary objectives included assessments of the proportion of patients who had an overall response, pharmacokinetics, and pharmacodynamics. This trial is registered with ClinicalTrials.gov, number NCT01569750. FINDINGS From June 22, 2012, to March 25, 2013, 33 patients were enrolled (part 1: 17; part 2: 16) and 32 received ibrutinib plus R-CHOP treatment (one patient in the part 2 cohort withdrew). The maximum tolerated dose was not reached and the recommended phase 2 dose for ibrutinib was 560 mg per day. The most common grade 3 or greater adverse events included neutropenia (73% [24 of 33 patients]), thrombocytopenia (21% [seven patients]), and febrile neutropenia and anaemia (18% each [six patients]). The most frequently reported serious adverse events were febrile neutropenia (18% [six patients]) and hypotension (6% [two patients]). 30 (94%) of 32 patients who received one or more doses of combination treatment achieved an overall response. All 18 patients with diffuse large B-cell lymphoma who received the recommended phase 2 dose had an overall response. For those subtyped and treated at the recommended phase 2 dose, five (71%) of seven patients with the germinal centre B-cell-like subtype and two (100%) patients with the non-germinal centre B-cell-like subtype had a complete response. R-CHOP did not affect pharmacokinetics of ibrutinib, and ibrutinib did not alter the pharmacokinetics of vincristine. Pharmacodynamic data showed Brutons tyrosine kinase was fully occupied (>90% occupancy) at the recommended phase 2 dose. INTERPRETATION Ibrutinib is well tolerated when added to R-CHOP, and could improve responses in patients with B-cell non-Hodgkin lymphoma, but our findings need confirmation in a phase 3 trial. FUNDING Janssen.

Which Human Metabolites Have We MIST? Retrospective Analysis, Practical Aspects, and Perspectives For Metabolite Identification and Quantification in Pharmaceutical Development

With the recent publication of the FDA guidance on metabolites in safety testing (MIST), a reflection is provided that describes the impact of this guidance on the processes of drug metabolite identification and quantification at various stages of drug development. First, a retrospective analysis is described that was conducted on 12 human absorption, metabolism, and excretion (AME) trials with the application of these MIST criteria. This analysis showed that the number of metabolites requiring identification, (semi)-quantification, and coverage in the toxicology species would substantially increase. However, a significant proportion of these metabolites were direct or indirect conjugates, a class of metabolites that was specifically addressed in the guidance as being largely innocuous. The nonconjugated metabolites were all covered in at least one toxicology animal species, with no need for additional safety evaluation. Second, analytical considerations pertaining to the efficient identification of metabolites are discussed. Topics include software-assisted detection and structural identification of metabolites, the emerging hyphenation of ultraperformance liquid chromatography (UPLC) with radioactivity detection, and the various ways to estimate metabolite abundance in the absence of an authentic standard. Technical aspects around the analysis of metabolite profiles are also presented, focusing on precautions to be taken in order not to introduce artifacts. Finally, a tiered approach for metabolite quantification is proposed, starting with quantification of metabolites prior to the multiple ascending dose study (MAD) in humans in only specific cases (Tier A). The following step is the identification and quantification of metabolites expected to be of pharmacological or toxicological relevance (based on MIST and other complementary criteria) in selected samples from the MAD study and preclinical studies in order to assess metabolite exposure coverage (Tier B). Finally, a metabolite quantification strategy for the studies after the MAD phase (Tier C) is proposed.

A device for dried blood microsampling in quantitative bioanalysis: overcoming the issues associated blood hematocrit

AIMS A cross-laboratory experiment has been performed on a novel dried blood sampler in order to investigate whether it overcomes issues associated with blood volume and hematocrit (HCT) that are observed when taking a subpunch from dried blood spot samples. MATERIALS & METHODS An average blood volume of 10.6 μl was absorbed by the samplers across the different HCTs investigated (20-65%). RESULTS No notable change of volume absorbed was noted across the HCT range. Furthermore, the variation in blood sample volumes across six different laboratories was within acceptable limits. CONCLUSION The novel volumetric absorptive microsampling device has the potential to deliver the advantages of dried blood spot sampling while overcoming some of the issues associated with the technology.

Comparison of triple quadrupole and high-resolution TOF-MS for quantification of peptides

BACKGROUND With an increased interest in peptides and proteins as potential new drug candidates, new approaches for sensitive and selective quantitative analysis are required. LC-MS/MS analysis provides a good alternative to immunoassays with reduced method development times and increased specificity. RESULTS We have evaluated two state-of-the-art triple quadrupole and high-resolution TOF mass spectrometers with respect to their performance for quantification of six peptides (glufibrinopeptide B, somatostatin, enfuvirtide, TRI1144, C34 and exenatide). The peptides were spiked into protein-precipitated plasma supernatant. Triple quadrupole quantification was performed in SRM mode, and in high-resolution, MS narrow-width extracted chromatograms were generated for quantification. Specificity, accuracy, reproducibility and robustness were found to be comparable between the two instruments. The triple quadrupole instrument is still the most sensitive instrument for quantification of peptides with a median factor of about four-times higher sensitivity (based on LLOQ evaluation). CONCLUSION Based on sensitivity, the newest generation triple quadrupole MS systems are still the preferred technology for quantification of peptides. Since the sensitivity difference between triple quadrupole instruments and the new-generation high-resolution TOF-MS instruments is minor, the latter offer a useful alternative whenever additional selectivity is preferred or the use of a generic approach not requiring method optimization is advantageous.

Update of the EBF recommendation for the use of DBS in regulated bioanalysis integrating the conclusions from the EBF DBS-microsampling consortium

The European Bioanalysis Forum dried blood spots/microsampling consortium is reporting back from the experiments they performed on further documenting the potential hurdles of the DBS technology. Their experiments focused on the impact of hematocrit changes, IS addition, spot homogeneity, aging of spots and stability of fresh blood and cards. Results from these experiments demonstrate that the issues of DBS in regulated bioanalysis are real and that the technology will need additional improvements to be ready for use as a general tool for regulated bioanalysis. In addition, results on fresh blood and card stability were shared at international meetings and will be reported at a later date.

Effects of various CYP2D6 genotypes on the steady-state plasma concentrations of risperidone and its active metabolite, 9-hydroxyrisperidone, in Japanese patients with schizophrenia.

The effects of various CYP2D6 genotypes on the steady-state plasma concentrations (Css) of risperidone and its active metabolite, 9-hydroxyrisperidone, were studied in 85 Japanese schizophrenic patients (27 men and 58 women) treated with 6 mg/d risperidone for at least 2 weeks. Plasma concentrations of risperidone and 9-hydroxyrisperidone were measured using liquid chromatography–tandem mass spectrometry. The patients had the following CYP2D6 genotypes: wild-type (wt) /wt (40 patients), CYP2D6 * 10 (* 10)/ wt (28), CYP2D6 * 5 (* 5)/ wt (8), * 10/ * 10 (5), * 5/ * 10 (3), and CYP2D6 * 4/CYP2D6 * 14 (1), respectively. The Css values of risperidone and 9-hydroxyrisperidone were corrected to the median body weight of 58 kg. The medians (ranges) of the Css of risperidone in the aforementioned genotype groups were 2.2 (0.37–35.7), 6.4 (2.1–26.5), 12.3 (4.7–39.5), 19.4 (13.4–26.4), 64.0 (41.6–68.8), and 91.8 nmol/L. Those values for risperidone-to-9-hydroxyrisperidone ratio were 0.03 (0.01–0.33), 0.06 (0.03–0.19), 0.14 (0.07–0.29), 0.28 (0.25–0.38), 0.48 (0.38–0.58), and 2.35, respectively. The Css of risperidone was significantly (P < 0.05 or P < 0.001) different among the four genotype groups (wt/wt, * 10/wt, * 5/wt, and * 10/ * 10), except between the * 5/wt and * 10/ * 10 groups. Also, the risperidone-to-9-hydroxyrisperidone ratio significantly (P < 0.005 or P < 0.001) differed among these genotype groups. No significant differences were found in the Css of 9-hydroxyrisperidone and the active moiety (the Css of risperidone plus 9-hydroxyrisperidone) among these genotype groups. This study confirms previous findings that the CYP2D6 status affects the Css of risperidone via its strong regulation of 9-hydroxylation of risperidone. However, similar active moiety of risperidone among different genotype groups suggests that the determination of the CYP2D6 genotype has little importance for clinical situations.

Effects of CYP2D6 Genotypes on Plasma Concentrations of Risperidone and Enantiomers of 9‐Hydroxyrisperidone in Japanese Patients with Schizophrenia

It has been shown that risperidone (+)‐9‐hydroxylation is enantioselectively catalyzed by the polymorphic CYP2D6 in human liver. This study aimed to examine the effect of CYP2D6 genotype on (+)‐9‐hydroxylation of risperidone in schizophrenic patients. Subjects were 38 Japanese schizophrenic inpatients receiving 6 mg/day of risperidone. Plasma concentrations of risperidone and (+)‐ and (−)‐9‐hydroxyrisperidone at steady state were quantified using LC/MS/MS and HPLC with α1 acid‐AGP chiral column, respectively. The CYP2D6*5(*5) and *10 alleles were identified using polymerase chain reaction (PCR) methods. Twenty patients had no mutated allele, 14 had one mutated allele, and 4 had two mutated alleles. There were significant differences in the steady‐state plasma concentrations of risperidone (ANOVA; p < 0.0001) among the three genotype groups, while the CYP2D6 genotype did not affect the steady‐state plasma concentrations of (+)‐9‐hydroxyrisperidone (p = 0.314) or (−)‐9‐hydroxyrisperidone (p = 0.957). The concentration ratio of risperidone to 9‐hydroxyrisperidone was strongly dependent on the CYP2D6 genotypes. This study suggests that CYP2D6 activity strongly influences the steady‐state plasma concentrations of risperidone and risperidone/9‐hydroxyrisperidone concentration ratios but is unlikely to determine enantioselectivity in the steady‐state plasma concentrations of 9‐hydroxyrisperidone in the clinical situation.

In-depth study of homogeneity in DBS using two different techniques: results from the EBF DBS-microsampling consortium

BACKGROUND At the start of their work, the European Bioanalysis Forum dried blood spots microsampling consortium did not form a dedicated team to investigate the spot homogeneity. However, two teams performed experiments that produced results relating to sample homogeneity. RESULTS The data, which were produced via two different approaches (a radiolabeled and a nonradiolabeled approach), are highly complementary and demonstrate clear effects on sample inhomogeneity due to the substrate type, compound and hematocrit levels. CONCLUSION The results demonstrate that sample inhomogeneity is a significant hurdle to the use of dried blood spots for regulated bioanalysis that should be investigated further in the method establishment phase if the whole spot is not sampled.

Stability: recommendation for best practices and harmonization from the Global Bioanalysis Consortium Harmonization Team.

This paper provides a comprehensive overview of stability-related aspects of quantitative bioanalysis and recommends science-based best practices, covering small and large molecules as well as chromatographic and ligand-binding assays. It addresses general aspects, such as the use of reference values, transferability and treatment of failing stability results, and also focuses on specific types of stability assessment: bench-top, freeze/thaw and long-term frozen stability, stock stability, extract stability, stability in whole blood, tissue and urine, and stability of endogenous analytes, in special matrix types and in incurred samples.

A phase I, open-label, single-dose, mass balance study of 14C-labeled abiraterone acetate in healthy male subjects

1. Metabolic disposition of 14C-abiraterone acetate (AA), a prodrug of abiraterone was assessed in a phase I, open-label, single-dose (1000 mg, approximately 100 μCi) study in healthy males (18–55 years, N = 8). Blood, urine, and faecal samples were obtained at specified timepoints for determination of abiraterone concentrations in the plasma, total radioactivity (TR), and the metabolite profile. 2. Most plasma AA concentrations were below the limit of quantification. The mean maximum plasma concentration (Cmax) of abiraterone was 10.4 ng/mL, mean area under the plasma concentration-time curve (AUC) from 0 to the last measurable plasma concentration (AUC0–last) was 74.8 ng·h/mL. The exposures for TR in plasma (Cmax = 3429 ng·eq/mL; AUC0–last = 26,683 ng eq·h/mL) and whole blood (Cmax = 1836 ng·eq/mL; AUC0–last = 12,162 ng·eq·h/mL) were >300-fold higher than abiraterone exposure in plasma. The majority of TR resided in the plasma compartment of blood. 3. Main circulating metabolites were abiraterone sulfate and N-oxide abiraterone sulfate. The main metabolite excreted in urine was N-oxide abiraterone sulfate (4.22% of TR). Major components of TR in faeces were unchanged AA (55.3% of TR) and abiraterone (22.3% of TR). Mean recovery of TR in faeces was 87.9%, indicating faeces as primary route of excretion.