Discovery and Preclinical Pharmacology of INE963, a Potent and Fast-Acting Blood-Stage Antimalarial with a High Barrier to Resistance and Potential for Single-Dose Cures in Uncomplicated Malaria.

A series of 5-aryl-2-amino-imidazothiadiazole (ITD) derivatives were identified by a phenotype-based high-throughput screening using a blood stage Plasmodium falciparum (Pf) growth inhibition assay. A lead optimization program focused on improving antiplasmodium potency, selectivity against human kinases, and absorption, distribution, metabolism, excretion, and toxicity properties and extended pharmacological profiles culminated in the identification of INE963 (1), which demonstrates potent cellular activity against Pf 3D7 (EC50 = 0.006 μM) and achieves artemisinin-like kill kinetics in vitro with a parasite clearance time of \u3c24 h. A single dose of 30 mg/kg is fully curative in the Pf-humanized severe combined immunodeficient mouse model. INE963 (1) also exhibits a high barrier to resistance in drug selection studies and a long half-life (T1/2) across species. These properties suggest the significant potential for INE963 (1) to provide a curative therapy for uncomplicated malaria with short dosing regimens. For these reasons, INE963 (1) was progressed through GLP toxicology studies and is now undergoing Ph1 clinical trials

Développement de nouvelles approches génériques de spectrométrie de masse pour la quantification de protéines thérapeutiques dans des études précliniques

This PhD thesis focused on the development of generic mass spectrometry (MS)-based workflows for monoclonal antibody (mAb)-related therapeutic protein quantification in pre-clinical species. First, the development of bottom-up sample preparation protocols either based on direct serum digestion or immuno-capture allowed mAb-related therapeutic protein quantification over five orders of magnitude whereas the employment of peptides from the constant region of the mAb demonstrated the versatility of such generic liquid chromatography tandem MS (LC-MS/MS)-based approaches. Second, high-resolution MS (HRMS) instruments were evaluated as an alternative to triple quadrupole mass analyzers, traditionally utilized for bottom-up mAb quantification by LC-MS/MS. The major benefit of HRMS incorporation into the workflow was associated with the possibility to quantify simultaneously mAb-related therapeutic proteins directly at an intact level, providing an information level far beyond the one obtained with bottom-up LC-MS/MS methodologies. Hence, the pivotal role of HRMS for the qualitative and quantitative analyses of mAb-related therapeutic proteins was further outlined throughout this doctoral work.Ce travail de thèse s’est focalisé sur le développement des approches génériques de spectrométrie de masse (MS) pour la quantification des anticorps monoclonaux (mAbs) et de leurs produits dérivés dans des études précliniques. Premièrement, le développement des protocoles de préparation d’échantillons basée sur la digestion directe à partir de sérum ou comportant une étape d’immuno-précipitation spécifique par anticorps a permis la quantification des mAbs couvrant une large gamme d'étalonnage de cinq ordres de grandeur. En outre, l'emploi de peptides provenant de la région constante du mAb a démontré la polyvalence de telles approches génériques de chromatographie liquide en tandem MS (LC-MS/MS). Deuxièmement, les instruments de MS à haute résolution (HRMS) ont étés évalués dans le cadre de cette thèse en tant qu'alternative aux spectromètres de masse de type triple quadripôle traditionnellement utilisés pour l’analyse bottom-up quantitative. L’avantage majeur de l’intégration des analyseurs de HRMS a été associé à la possibilité de l’analyse quantitative simultanée des mAbs et leurs produits associés directement au niveau de la protéine fournissant un niveau d'informations bien au-delà de celui obtenu avec des approches bottom-up. Par conséquent, l’apport essential de la HRMS pour les analyses qualitative et quantitative des protéines thérapeutiques de type mAbs et produits associés a été démontré dans cette thèse

Use of predicted versus measured CCS values from different instrument platforms, and isomer separation on the SELECT SERIES Cyclic IMS

Biotransformation activities require the comparison of metabolites across species and studies. In general, chromatographic retention time, accurate mass measurement and mass spectral data are used to align metabolites. Isomeric metabolite comparison may be more challenging particularly when retention times may differ depending on the analytical conditions used. Additionally, the elemental formulae as well as MS/MS spectra can be identical which significantly increases the complexity of the data interpretation and localization of the biotransformation. The use of collision cross section (CCS) values to compare metabolites analyzed using the SELECT SERIES Cyclic IMS and the SYNAPT G2-Si Q-Tof instruments located in different facilities has been shown here and demonstrates the benefit of such analyte-specific physiochemical property to align metabolites across studies.Moreover, computational prediction of CCS values may provide an additional data asset, allowing the comparison of predicted with measured CCS values. This can further provide additional insights to differentiate between isomers. The prediction can also be used to suggest when additional cyclic ion mobility separation (cIMS) would be beneficial in the separation of isomers and increase confidence in any assignment with the use of higher ion mobility resolution. Examples are given here where cIMS has been used to separate oxygenated metabolites of ranitidine and imipramine. This alternative separation mechanism adds to the separating power of UPLC and is of benefit when isomers co-elute

SMART Digest™ compared with pellet digestion for analysis of human immunoglobulin G1 in rat serum by liquid chromatography tandem mass spectrometry

The newly developed SMART Digest™ kit was applied for the sample preparation of human immunoglobulin G1 (hIgG1) in rat serum prior to qualitative and quantitative analyses by liquid chromatography tandem mass spectrometry (LC–MS/MS). The sequence coverages obtained for the light and heavy chains of hIgG1A were 50 and 76%, respectively. The calibration curve was linear from 1.00 to 1000 μg/ml for three of four generic peptides. Overall, the SMART Digest™ kit resulted in similar quantitative data (linearity, sensitivity, accuracy, and precision) compared with the pellet digestion protocol. However, the SMART Digest™ required only 2 h of sample preparation with fewer reagents

Generic Hybrid Ligand Binding Assay Liquid Chromatography High-Resolution Mass Spectrometry-Based Workflow for Multiplexed Human Immunoglobulin G1 Quantification at the Intact Protein Level: Application to Preclinical Pharmacokinetic Studies

The quantitative analysis of human immunoglobulin G1 (hIgG1) by mass spectrometry is commonly performed using surrogate peptides after enzymatic digestion. Since some limitations are associated with this approach, a novel workflow is presented by hybridizing ligand binding assay (LBA) with liquid chromatography–high-resolution mass spectrometry (LC–HRMS) for hIgG1 quantification directly at the intact protein level. Different hIgG1s, including a [¹³C]-labeled version used as internal standard, were immuno-enriched from rat serum with a fully automated platform based on streptavidin coated tips and a biotinylated mouse anti-hIgG capture antibody targeting the fragment crystallizable region followed by overnight deglycosylation prior to LC–HRMS analysis. The proposed quantitative workflow utilized extracted ion chromatograms (XICs) from the nondeconvoluted full-scan MS spectrum. The assay was validated in terms of selectivity, sensitivity, accuracy/precision, carry-over, dilution linearity, and reproducibility. Consistent data between the conventional approach based on surrogate peptide analysis and our proposed workflow were obtained <i>in vitro</i> and <i>in vivo</i> with the advantage of a less extensive sample pretreatment. Multiplexing capabilities for simultaneous quantification of different hIgG1s within the same spiked sample were also exemplified. Altogether our results pave the way not only for the thorough application of intact hIgG1 quantification by LBA-LC–HRMS but also as a generic quantitative analytical method for other hIgG isotypes or next generation biotherapeutics

Potential of measured relative shifts in collision cross section values for biotransformation studies.

Ion mobility spectrometry-mass spectrometry (IMS-MS) separates gas phase ions due to differences in drift time from which reproducible and analyte-specific collision cross section (CCS) values can be derived. Internally conducted in vitro and in vivo metabolism (biotransformation) studies indicated repetitive shifts in measured CCS values (CCSmeas) between parent drugs and their metabolites. Hence, the purpose of the present article was (i) to investigate if such relative shifts in CCSmeas were biotransformation-specific and (ii) to highlight their potential benefits for biotransformation studies. First, mean CCSmeas values of 165 compounds were determined (up to n = 3) using a travelling wave IMS-MS device with nitrogen as drift gas (TWCCSN2, meas). Further comparison with their predicted values (TWCCSN2, pred, Waters CCSonDemand) resulted in a mean absolute error of 5.1%. Second, a reduced data set (n = 139) was utilized to create compound pairs (n = 86) covering eight common types of phase I and II biotransformations. Constant, discriminative, and almost non-overlapping relative shifts in mean TWCCSN2, meas were obtained for demethylation (- 6.5 ± 2.1 Å2), oxygenation (hydroxylation + 3.8 ± 1.4 Å2, N-oxidation + 3.4 ± 3.3 Å2), acetylation (+ 13.5 ± 1.9 Å2), sulfation (+ 17.9 ± 4.4 Å2), glucuronidation (N-linked: + 41.7 ± 7.5 Å2, O-linked: + 38.1 ± 8.9 Å2), and glutathione conjugation (+ 49.2 ± 13.2 Å2). Consequently, we propose to consider such relative shifts in TWCCSN2, meas (rather than absolute values) as well for metabolite assignment/confirmation complementing the conventional approach to associate changes in mass-to-charge (m/z) values between a parent drug and its metabolite(s). Moreover, the comparison of relative shifts in TWCCSN2, meas significantly simplifies the mapping of metabolites into metabolic pathways as demonstrated

Quantitative analysis of hIgG1 in monkey serum by LC-MS/MS using mass spectrometric immunoassay

Background: A sensitive LC-MS/MS method for human immunoglobulin G1 (hIgG1) quantification in monkey serum using mass spectrometric immunoassay disposable automation research tips (MSIA-D.A.R.T.s) is reported. Results: The hIgG1 was captured with biotinylated mouse anti-hIGg1 antibody targeting the fragment crystallizable (Fc) region (50.0 µg/ml) using 1000 cycles. Elution from the streptavidin coated MSIA-D.A.R.T.s was conducted with 0.4% trifluoroacetic acid in water. The method was selective and linear from 10.0-1000 ng/ml using 100 µl of serum. The method was evaluated regarding accuracy, precision, carry-over, dilution, auto-sampler stability and applied for the determination of hIgG1 concentration in monkey serum after intravitreal administration. Conclusions: The present assay is suitable for quantitative analysis of hIgG1 based therapeutic (fusion-) proteins in monkey serum at low levels

Ultra-fast quantitative mass spectrometry based method for ceritinib analysis in human plasma and its application for clinical use

Background: We report an ultra-fast method for the quantitative analysis in human plasma of ceritinib, a drug recently approved in the USA for the treatment of non-small-cell lung cancer (NSCLC). Results: Plasma samples were precipitated with acetonitrile containing [13C6]-ceritinib as internal standard and analyzed in 10 seconds each using Laser Diode Thermal Desorption-positive mode Atmospheric Chemical Pressure Ionization tandem mass spectrometry (LDTD-APCI-MS/MS). The method was selective and found to be linear from 5.00-1000 ng/ml using 100 µL plasma. The intra- and inter-days precision and accuracy data met acceptance criteria. The normalized recovery was 69%, carry over and matrix effects were not observed. The method was successfully used to measure clinical samples and consistent data were obtained with the reference LC-ESI-MS/MS method. Conclusions: The developed ultra-fast assay is suitable for quantitative analysis of ceritinib in plasma from clinical trials

Quantitative analysis of hIgG1 in monkey serum by LC–MS/MS using mass spectrometric immunoassay

Background: A sensitive LC-MS/MS method for human immunoglobulin G1 (hIgG1) quantification in monkey serum using mass spectrometric immunoassay disposable automation research tips (MSIA-D.A.R.T.s) is reported. Results: The hIgG1 was captured with biotinylated mouse anti-hIGg1 antibody targeting the fragment crystallizable (Fc) region (50.0 µg/ml) using 1000 cycles. Elution from the streptavidin coated MSIA-D.A.R.T.s was conducted with 0.4% trifluoroacetic acid in water. The method was selective and linear from 10.0-1000 ng/ml using 100 µl of serum. The method was evaluated regarding accuracy, precision, carry-over, dilution, auto-sampler stability and applied for the determination of hIgG1 concentration in monkey serum after intravitreal administration. Conclusions: The present assay is suitable for quantitative analysis of hIgG1 based therapeutic (fusion-) proteins in monkey serum at low levels

Laser Diode Thermal Desorption-positive mode Atmospheric Pressure Chemical Ionization Tandem Mass Spectrometry applied for the ultra-fast quantitative analysis of BKM120 in human plasma: method validation and its application to clinical studies

A sensitive and ultra-fast method utilizing the LDTD-APCI ion source coupled to tandem mass spectrometry was developed and validated for the quantitative analysis of BKM120 in human plasma. Samples originating from protein precipitation (PP) followed by salting-out assisted liquid-liquid extraction (SALLE) were spotted on the LazWell™ plate prior their thermal desorption and analysis by tandem mass spectrometry. Two MRM transitions (m/z 411.4367.4 and m/z 411.4307.4) were used to quantify BKM120. The present method was validated in terms of selectivity, linearity, accuracy, precision, stability, extraction recovery and matrix effect. The linearity of the method was found to be within the concentration range of 5.00 and 2000 ng mL-1. The sample analysis run time was 10 seconds as compared to 4.5 min for the current validated LC-ESI-MS/MS method. The LDTD-APCI-MS/MS method was successfully applied for the analysis of BKM120 in clinical samples obtained from 3 clinical studies. The resultant data was consistent with the results obtained with the validated LC-ESI-MS/MS assay. This work demonstrates that LDTD-APCI-MS/MS is a reliable method for the ultra-fast quantitative analysis of BKM120 and can be used to support its bioanalysis in the frame of the clinical trials