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1.
Anal Chem ; 93(46): 15236-15242, 2021 11 23.
Article in English | MEDLINE | ID: mdl-34762405

ABSTRACT

In multiple myeloma (MM) disease, malignant plasma cells produce excessive quantities of a monoclonal immunoglobulin (Ig), known as M-protein. M-protein levels are measured in the serum of patients with MM using electrophoresis techniques to determine the response to treatment. However, therapeutic monoclonal antibodies, such as isatuximab, may confound signals using electrophoresis assays. We developed a robust assay based on immunocapture and liquid chromatography coupled to high-resolution mass spectrometry (IC-HPLC-HRMS) in order to eliminate this interference. Following immunocapture of Ig and free light chains (LC) in serum, heavy chains (HC) and LC were dissociated using dithiothreitol, sorted by liquid chromatography and analyzed using HRMS (Q-Orbitrap). This method allowed the M-proteins to be characterized and the signals from isatuximab and M-proteins to be discriminated. As M-protein is specific to each patient, no standards were available for absolute quantification. We therefore used alemtuzumab (an IgG kappa mAb) as a surrogate analyte for the semiquantification of M-protein in serum. This assay was successfully validated in terms of selectivity/specificity, accuracy/precision, robustness, dilution linearity, and matrix variability from 10.0 to 200 µg/mL in human serum. This method was used for clinical assessment of samples and eliminated potential interference due to isatuximab when monitoring patients with MM.


Subject(s)
Antibodies, Monoclonal , Immunoglobulins/blood , Multiple Myeloma/diagnosis , Antibodies, Monoclonal, Humanized , Chromatography, High Pressure Liquid , Chromatography, Liquid , Humans , Mass Spectrometry
2.
Bioanalysis ; 11(6): 525-532, 2019 Mar.
Article in English | MEDLINE | ID: mdl-30973017

ABSTRACT

Following the completion of a detailed experimental protocol into the potential inhomogeneity of capillary liquid microsamples, which was performed at seven European Bioanalysis Forum member companies, the summary and conclusion on the data are reported here. It has been demonstrated that it is possible to generate homogeneous samples using these microsampling techniques; that the resultant microsamples can be accurate and precise and that capillary liquid microsampling data can be consistent with conventional larger volume plasma samples. However, the data contain some variability which is contributed to by the different range of experiences that each investigating site had with these techniques. Therefore, knowledge of the compounds, well-designed experiments and experience with these techniques are essential for the delivery of high quality data.


Subject(s)
Blood Chemical Analysis/methods , Blood Specimen Collection/methods , Blood Chemical Analysis/standards , Blood Specimen Collection/instrumentation , Blood Specimen Collection/standards , Europe , Humans , Pharmaceutical Preparations/blood , Reproducibility of Results
3.
Bioanalysis ; 11(6): 533-542, 2019 Mar.
Article in English | MEDLINE | ID: mdl-30973018

ABSTRACT

Aim: Microsampling in preclinical pharmacokinetics (PK) studies is currently widely adopted across the pharmaceutical industry. Materials & methods: The European Bioanalysis Forum liquid microsampling consortium member companies assessed the accuracy and precision of handheld pipettes and microcapillaries at volumes of less than 10 µl. The following key factors on pipetting performance were also evaluated: Pipette type (positive displacement, air displacement and microcapillary), experience of user and the liquid type. Water was selected as a best-case scenario for accuracy and precision determination and blood plasma as a 'real world' bioanalysis sample type. Conclusion: Accuracy and precision on the pipetted volume decreased at lower volumes and experienced laboratory technicians performed better compared with the infrequent users. With respect to the pipetting devices used, microcapillaries showed better or equivalent accuracy and precision compared with handheld pipettes across the volume range 1-8 µl independent of the matrix used.


Subject(s)
Blood Chemical Analysis/methods , Blood Specimen Collection/instrumentation , Blood Chemical Analysis/standards , Blood Specimen Collection/standards , Europe , Humans , Pharmaceutical Preparations/blood , Reproducibility of Results
4.
Eur J Pharm Sci ; 117: 68-79, 2018 May 30.
Article in English | MEDLINE | ID: mdl-29427702

ABSTRACT

Efficacy of drugs aimed at treating central nervous system (CNS) disorders rely partly on their ability to cross the cerebral endothelium, also called the blood-brain barrier (BBB), which constitutes the main interface modulating exchanges of compounds between the brain and blood. In this work, we used both, conventional pharmacokinetics (PK) approach and in situ brain perfusion technique to study the blood and brain PK of PKRinh, an inhibitor of the double-stranded RNA-dependent protein kinase (PKR) activation, in mice. PKRinh showed a supra dose-proportional blood exposure that was not observed in the brain, and a brain to blood AUC ratio of unbound drug smaller than 1 at all tested doses. These data suggested the implication of an active efflux at the BBB. Using in situ brain perfusion technique, we showed that PKRinh has a very high brain uptake clearance which saturates with increasing concentrations. Fitting the data to a Michaelis-Menten equation revealed that PKRinh transport through the BBB is composed of a passive unsaturable flux and an active saturable protein-mediated efflux with a km of ≅ 3 µM. We were able to show that the ATP-binding cassette (ABC) transporter P-gp (Abcb1), but not Bcrp (Abcg2), was involved in the brain to blood efflux of PKRinh. At the circulating PKRinh concentrations of this study, the P-gp was not saturated, in accordance with the linear brain PKRinh PK. Finally, PKRinh had high brain uptake clearance (14 µl/g/s) despite it is a good P-gp substrate (P-gp Efflux ratio ≅ 3.6), and reached similar values than the cerebral blood flow reference, diazepam, in P-gp saturation conditions. With its very unique brain transport properties, PKRinh improves our knowledge about P-gp-mediated efflux across the BBB for the development of new CNS directed drugs.


Subject(s)
Brain/metabolism , Central Nervous System Agents/pharmacokinetics , Protein Kinase Inhibitors/pharmacokinetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 2/genetics , Animals , Biological Transport , Central Nervous System Agents/blood , Female , Male , Mice, Inbred C57BL , Mice, Knockout , Protein Kinase Inhibitors/blood
5.
Bioanalysis ; 7(6): 653-9, 2015.
Article in English | MEDLINE | ID: mdl-25514576

ABSTRACT

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.


Subject(s)
Blood Specimen Collection/instrumentation , Dried Blood Spot Testing/instrumentation , Hematocrit , Absorption, Physicochemical , Animals , Artifacts , Humans , Rats
6.
J Mass Spectrom ; 38(8): 879-90, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12938109

ABSTRACT

The pharmacokinetics of intracellular drugs have recently aroused new interest because monitoring a drug's behaviour near the site of action can enhance knowledge of its efficacy and toxicity. Liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) is particularly attractive for intracellular analytes. Very few papers deal precisely with special features encountered in intracellular drug assay or with how closely the assay matches the actual recommendations. Particular problems are encountered mainly because the analytes are located intracellularly. This mainly concerns the handling of biological media, including provision of blank samples using Ficoll gradient separation, cell counts, optimisation of cell lysis, sample extraction, plotting standard curves using either fmol/10(6) cells or fmol/ml of extract or fmol/sample, the matrix effect as a function of the number of cells, stability before and during cell separation, as well as in storage conditions using clinical samples, biological matrix replacement and interference by endogenous compounds. This paper describes a strategy for the full validation and routine use of an LC/MS/MS assay applied to the simultaneous intracellular determination of the triphosphorylated anabolites of didanosine (2',3'-dideoxyadenosine triphosphate or ddA-TP) and stavudine (2',3'-didehydro-3'-deoxythymidine triphosphate or d4T-TP), two nucleoside reverse transcriptase inhibitors of HIV, in human peripheral blood mononuclear cells (PBMCs), as a guide for further LC/MS/MS assay of intracellular drugs.


Subject(s)
Anti-HIV Agents/pharmacokinetics , Chromatography, High Pressure Liquid/methods , Didanosine/pharmacokinetics , Leukocytes, Mononuclear/metabolism , Spectrometry, Mass, Electrospray Ionization/methods , Stavudine/pharmacokinetics , Didanosine/analysis , Dideoxyadenosine/analysis , Leukocytes, Mononuclear/chemistry , Stavudine/analysis
7.
Pharm Res ; 19(4): 367-76, 2002 Apr.
Article in English | MEDLINE | ID: mdl-12033366

ABSTRACT

PURPOSE: To compare the pharmacokinetics and bioavailability of an oligonucleotide delivered in a free form or using cationic or anionic synthetic carrier systems. METHODS: Whole body dynamic quantitative imaging and metabolism of a HIV antisense oligonucleotide intravenously administered either free or incorporated into synthetic carriers were compared in baboons. using non invasive positron emission tomography and an enzyme-based competitive hybridization assay, respectively. RESULTS: In its free form, the oligonucleotide showed high liver and kidney concentration, rapid plasmatic degradation and elimination from the body. Use of a cationic vector slightly protected the oligonucleotide against degradation and enhanced uptake by the reticulo-endothelial system. In contrast, the anionic vector dramatically enhanced the uptake in several organs, including the lungs, spleen and brain, with a prolonged accumulation of radioactivity in the brain. Using this vector, intact oligonucleotide was detected in plasma for up to two hours after injection. and the T 1/2beta and distribution volume increased by 4- and 7-fold, respectively. No evidence of toxicity was found after a single dose administration. CONCLUSIONS: The anionic vector improves significantly the bioavailability and the pharmacokinetics of the oligonucleotide, and is a promising delivery system for in vivo administration of therapeutic nucleic acids.


Subject(s)
Anions/pharmacokinetics , Drug Delivery Systems/methods , Genetic Vectors/administration & dosage , Genetic Vectors/chemical synthesis , Oligonucleotides, Antisense/pharmacokinetics , Tomography, Emission-Computed/methods , Animals , Anions/administration & dosage , Drug Carriers/administration & dosage , Drug Carriers/pharmacokinetics , Genetic Vectors/pharmacokinetics , Liposomes , Male , Oligonucleotides, Antisense/administration & dosage , Papio , Whole-Body Counting/methods
8.
Invest Ophthalmol Vis Sci ; 43(1): 253-9, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11773039

ABSTRACT

PURPOSE: The efficacy of sterically stabilized liposomes for delivering a model phosphodiester oligonucleotide intravitreally was investigated in the rabbit. METHODS: Ocular distribution and clearance from the vitreous humor of a model 16-mer oligothymidylate (pdT16) were evaluated in the rabbit by radioactivity measurements after intravitreal injection of either a solution or liposomes containing the [33P]pdT16 oligonucleotide. The integrity of pdT16 was investigated using a competitive hybridization assay. RESULTS: The residual concentration of the [33P]pdT16 oligonucleotide within the ocular tissues was significantly increased after intravitreal administration of the liposomal suspension compared with a simple solution. Administration of liposome-encapsulated pdT16 oligonucleotide resulted in sustained release into the vitreous and the retina-choroid compared with release from the solution and in a reduced distribution to nontarget tissues (sclera, lens). In addition, liposomes protected the phosphodiester oligonucleotide against degradation. This was not observed after administration of the free oligonucleotide. CONCLUSIONS: The intravitreal injection of a phosphodiester oligonucleotide encapsulated within liposomes is a new way of delivering intact oligonucleotide to the eye in a controlled manner. This offers interesting prospects for the treatment of retinal diseases.


Subject(s)
Drug Delivery Systems , Oligonucleotides/administration & dosage , Oligonucleotides/pharmacokinetics , Poly T/administration & dosage , Poly T/pharmacokinetics , Vitreous Body/metabolism , Animals , Choroid/metabolism , Choroid/pathology , Delayed-Action Preparations , Injections , Liposomes , Rabbits , Retina/metabolism , Retina/pathology , Thionucleotides/administration & dosage , Vitreous Body/pathology
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