Olivier Kretz

Towards absolute quantification of therapeutic monoclonal antibody in serum by LC-MS/MS using isotope-labeled antibody standard and protein cleavage isotope dilution mass spectrometry.

Although LC-MS methods are increasingly used for the absolute quantification of proteins, the lack of appropriate internal standard (IS) hinders the development of rapid and standardized analytical methods for both in vitro and in vivo studies. Here, we have developed a novel method for the absolute quantification of a therapeutic protein, which is monoclonal antibody (mAb). The method combines liquid chromatography tandem mass spectrometry (LC-MS/MS) and protein cleavage isotope dilution mass spectrometry with the isotope-labeled mAb as IS. The latter was identical to the analyzed mAb with the exception that each threonine contains four (13)C atoms and one (15)N atom. Serum samples were spiked with IS prior to the overnight trypsin digestion and subsequent sample cleanup. Sample extracts were analyzed on a C18 ACE column (150 mm x 4.6 mm) using an LC gradient time of 11 min. Endogenous mAb concentrations were determined by calculating the peak height ratio of its signature peptide to the corresponding isotope-labeled peptide. The linear dynamic range was established between 5.00 and 1000 microg/mL mAb with accuracy and precision within +/-15% at all concentrations and below +/-20% at the LLOQ (lower limit of quantification). The overall method recovery in terms of mAb was 14%. The losses due to sample preparation (digestion and purification) were 72% from which about 32% was due to the first step of the method, the sample digestion. This huge loss during sample preparation strongly emphasizes the necessity to employ an IS right from the beginning. Our method was successfully applied to the mAb quantification in marmoset serum study samples, and the precision obtained on duplicate samples was, in most cases, below 20%. The comparison with enzyme-linked immunosorbent assay (ELISA) showed higher exposure in terms of AUC and Cmax with the LC-MS/MS method. Possible reasons for this discrepancy are discussed in this study. The results of this study indicate that our LC-MS/MS method is a simple, rapid, and precise approach for the therapeutic mAb quantification to support preclinical and clinical studies.

Antibiotic and Hemolytic Activity of a β2/β3 Peptide Capable of Folding into a 12/10‐Helical Secondary Structure

Antibiotic and Hemolytic Activity of a B2/B3 Peptide Capable of Folding into a 12/10-Helical Secondary Structure

New microfluidic-based sampling procedure for overcoming the hematocrit problem associated with dried blood spot analysis.

Hematocrit (Hct) is one of the most critical issues associated with the bioanalytical methods used for dried blood spot (DBS) sample analysis. Because Hct determines the viscosity of blood, it may affect the spreading of blood onto the filter paper. Hence, accurate quantitative data can only be obtained if the size of the paper filter extracted contains a fixed blood volume. We describe for the first time a microfluidic-based sampling procedure to enable accurate blood volume collection on commercially available DBS cards. The system allows the collection of a controlled volume of blood (e.g., 5 or 10 μL) within several seconds. Reproducibility of the sampling volume was examined in vivo on capillary blood by quantifying caffeine and paraxanthine on 5 different extracted DBS spots at two different time points and in vitro with a test compound, Mavoglurant, on 10 different spots at two Hct levels. Entire spots were extracted. In addition, the accuracy and precision (n = 3) data for the Mavoglurant quantitation in blood with Hct levels between 26% and 62% were evaluated. The interspot precision data were below 9.0%, which was equivalent to that of a manually spotted volume with a pipet. No Hct effect was observed in the quantitative results obtained for Hct levels from 26% to 62%. These data indicate that our microfluidic-based sampling procedure is accurate and precise and that the analysis of Mavoglurant is not affected by the Hct values. This provides a simple procedure for DBS sampling with a fixed volume of capillary blood, which could eliminate the recurrent Hct issue linked to DBS sample analysis.

Hepatic transport of PKI166, an epidermal growth factor receptor kinase inhibitor of the pyrrolo-pyrimidine class, and its main metabolite, ACU154.

PKI166, a specific inhibitor of the tyrosine kinase activity of two epidermal growth factor receptors, was under development for the treatment of cancer. In preclinical studies PKI166 was mainly cleared by metabolism, and its metabolites were eliminated by biliary excretion, emphasizing the role of liver transport processes for its disposition. Here the transport properties of [14C]PKI166 and its main metabolite [14C]ACU154, an O-glucuronide, were analyzed using 1) Madin-Darby canine kidney II (MDCKII) cells stably transfected with human multidrug resistance-associated protein 2 (MRP2) and/or human organic anion-transporting peptide 2 (OATP2) and 2) liver canalicular membrane vesicles (CMVs) prepared from Wistar and mrp2-deficient TR- rats. Analysis of transport through MDCKII cells revealed that [14C]ACU154 was a substrate of MRP2 and OATP2. Rat mrp2 was shown to transport [14C]ACU154 with a Km of approximately 1 μM. [14C]PKI166 efficiently crossed MDCKII cells, particularly toward the apical side, but expression of MRP2 and/or OATP2 did not increase the flux. The effect of PKI166 and ACU154 on transport of [3H]estradiol-17β-d-glucuronide (EG; via mrp2/MRP2 and OATP2) or [3H]taurocholic acid (TCA; via bile salt export pump (bsep) was analyzed. PKI166 inhibited the transport of [3H]EG by OATP2. ACU154 did strongly inhibit [3H]TCA uptake into CMVs from Wistar but not from TR- rats, demonstrating a dependence of bsep inhibition on mrp2 activity. ATP-dependent uptake of [3H]EG into CMVs from Wistar rats was inhibited by ACU154 but up to 4-fold increased by PKI166. In conclusion, OATP2 and MRP2/mrp2 were identified as transporters involved in ACU154 transport into bile. Both PKI166 and its O-glucuronide ACU154 affected mrp2/MRP2-, OATP2-, and/or bsep-mediated transport processes.

In vitro blood distribution and plasma protein binding of the iron chelator deferasirox (ICL670) and its iron complex Fe-[ICL670]2, for rat, marmoset, rabbit, mouse, dog and human

Deferasirox (Exjade, ICL670) is an orally active iron chelator. Two molecules of deferasirox can form a complex with ferric iron (Fe-[ICL670]2) that can be excreted, reducing body iron overload. The blood binding parameters across species and the interaction with human serum albumin were analyzed for deferasirox and its iron complex. Both molecules were very highly bound to plasma proteins in all the tested species with unbound fractions in plasma in the range of 0.4 to 1.8% and 0.2 to 1.2% for deferasirox and Fe-[ICL670]2, respectively; binding of the iron complex was either similar or higher in all the species. The high plasma protein binding was in line with a distribution mainly into the plasma fraction of blood; the fraction in plasma was around 100% for Fe-[ICL670]2 in all the species and 65 to 95% for deferasirox depending on the species. Investigations with isolated proteins pointed to serum albumin as the principal binding protein for deferasirox and its iron complex in human plasma. Competition binding experiments indicated that deferasirox at high concentrations displaced markers from the two main drug binding sites of human albumin, whereas Fe-[ICL670]2 displaced only warfarin. In the context of the pharmacokinetic properties of deferasirox and Fe-[ICL670]2, the data indicate the importance of plasma protein binding for their disposition and support a comparison of the pharmacokinetics of deferasirox and its iron complex across species. The low likelihood of clinically relevant drug displacement by deferasirox in plasma is discussed.

Intestinal Lymphatic Transport Enhances the Post-Prandial Oral Bioavailability of a Novel Cannabinoid Receptor Agonist Via Avoidance of First-Pass Metabolism

PurposeTo examine the effect of food on the oral bioavailability of a highly lipophilic, cannabinoid receptor agonist (CRA13) and to explore the basis for the food effect in lymph-cannulated and non-cannulated dogs.MethodsOral bioavailability was assessed in fasted and fed human volunteers and in lymph-cannulated dogs. In fasted dogs, the extent of absorption and oral bioavailability was also examined following administration of radiolabelled CRA13.ResultsFood had a substantial positive effect on the oral bioavailability of CRA13 in human volunteers (4.3–4.9 fold increase in

Laser diode thermal desorption-positive mode atmospheric pressure chemical ionization tandem mass spectrometry for the ultra-fast quantification of a pharmaceutical compound in human plasma.

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Fast simultaneous quantitative analysis of FTY720 and its metabolite FTY720-P in human blood by on-line solid phase extraction coupled with liquid chromatography-tandem mass spectrometry.

) and in dogs. The absolute bioavailability of parent drug was low in fasted dogs (8–20%), in spite of good absorption (72–75% of radiolabelled CRA13 recovered in the systemic circulation). In post-prandial lymph-cannulated dogs, bioavailability increased to 47.5% and the majority (43.7%) of the dose was absorbed via the intestinal lymphatic system.ConclusionsThe positive food effect for CRA13 does not appear to result from increased post-prandial absorption. Rather these data provide one of the first examples of a significant increase in bioavailability for a highly lipophilic drug, which is stimulated via almost complete post-prandial transport into the lymph, in turn resulting in a reduction in first-pass metabolism.