Xevinapant Combined with Pembrolizumab in Patients with Advanced, Pretreated, Colorectal and Pancreatic Cancer: Results of the Phase Ib/II CATRIPCA Trial.

PURPOSE: Xevinapant is an orally available inhibitor of apoptosis proteins (IAP) inhibitor. Preclinical data suggest that IAP antagonism may synergize with immune checkpoint blockers by modulating the NFκB pathway in immune cells. PATIENTS AND METHODS: Adult patients with non-high microsatellite instability advanced/metastatic pancreatic ductal adenocarcinoma (PDAC) or colorectal cancer were enrolled in this phase Ib/II study and received pembrolizumab 200 mg every 3 weeks intravenously, and ascending doses of oral xevinapant (100, 150, and 200 mg daily for 14 days on/7 days off). Dose escalation followed a 3+3 design with a 21-day dose-limiting toxicity (DLT) evaluation period. Following the determination of the recommended phase II dose (RP2D), 14 patients with PDAC and 14 patients with colorectal cancer were enrolled in expansion cohorts to assess preliminary efficacy. RESULTS: Forty-one patients (26 males) with a median age of 64 years were enrolled: 13 in the dose escalation and 28 in the two expansion cohorts. No DLT was observed during dose escalation. The RP2D was identified as xevinapant 200 mg/day + pembrolizumab 200 mg every 3 weeks. The most common adverse events (AE) were fatigue (37%), gastrointestinal AE (decreased appetite in 37%, nausea in 24%, stomatitis in 12%, and diarrhea and vomiting in 10% each), and cutaneous AE (pruritus, dry skin, and rash seen in 20%, 15%, and 15% of patients, respectively). The best overall response according to RECIST1.1 was partial response (confirmed) in 1 (3%), stable disease in 4 (10%), and progressive disease in 35 (88%). CONCLUSIONS: Xevinapant combined with pembrolizumab was well tolerated with no unexpected AEs. However, antitumor activity was low.

Anticancer Activities of Novel Nicotinamide Phosphoribosyltransferase Inhibitors in Hematological Malignancies.

Targeting cancer cells that are highly dependent on the nicotinamide adenine dinucleotide (NAD+) metabolite is a promising therapeutic strategy. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme catalyzing NAD+ production. Despite the high efficacy of several developed NAMPT inhibitors (i.e., FK866 (APO866)) in preclinical studies, their clinical activity was proven to be limited. Here, we report the synthesis of new NAMPT Inhibitors, JJ08, FEI191 and FEI199, which exhibit a broad anticancer activity in vitro. Results show that these compounds are potent NAMPT inhibitors that deplete NAD+ and NADP(H) after 24 h of drug treatment, followed by an increase in reactive oxygen species (ROS) accumulation. The latter event leads to ATP loss and mitochondrial depolarization with induction of apoptosis and necrosis. Supplementation with exogenous NAD+ precursors or catalase (ROS scavenger) abrogates the cell death induced by the new compounds. Finally, in vivo administration of the new NAMPT inhibitors in a mouse xenograft model of human Burkitt lymphoma delays tumor growth and significantly prolongs mouse survival. The most promising results are collected with JJ08, which completely eradicates tumor growth. Collectively, our findings demonstrate the efficient anticancer activity of the new NAMPT inhibitor JJ08 and highlight a strong interest for further evaluation of this compound in hematological malignancies.

Inhibition of vascular calcification by inositol phosphates derivatized with ethylene glycol oligomers.

Myo-inositol hexakisphosphate (IP6) is a natural product known to inhibit vascular calcification (VC), but with limited potency and low plasma exposure following bolus administration. Here we report the design of a series of inositol phosphate analogs as crystallization inhibitors, among which 4,6-di-O-(methoxy-diethyleneglycol)-myo-inositol-1,2,3,5-tetrakis(phosphate), (OEG2)2-IP4, displays increased in vitro activity, as well as more favorable pharmacokinetic and safety profiles than IP6 after subcutaneous injection. (OEG2)2-IP4 potently stabilizes calciprotein particle (CPP) growth, consistently demonstrates low micromolar activity in different in vitro models of VC (i.e., human serum, primary cell cultures, and tissue explants), and largely abolishes the development of VC in rodent models, while not causing toxicity related to serum calcium chelation. The data suggest a mechanism of action independent of the etiology of VC, whereby (OEG2)2-IP4 disrupts the nucleation and growth of pathological calcification.

UHPLC-MS/MS assay for simultaneous determination of amlodipine, metoprolol, pravastatin, rosuvastatin, atorvastatin with its active metabolites in human plasma, for population-scale drug-drug interactions studies in people living with HIV.

Thanks to highly active antiretroviral treatments, HIV infection is now considered as a chronic condition. Consequently, people living with HIV (PLWH) live longer and encounter more age-related chronic co-morbidities, notably cardiovascular diseases, leading to polypharmacy. As the management of drug-drug interactions (DDIs) constitutes a key aspect of the care of PLWH, the magnitude of pharmacokinetic DDIs between cardiovascular and anti-HIV drugs needs to be more thoroughly characterized. To that endeavour, an UHPLC-MS/MS bioanalytical method has been developed for the simultaneous determination in human plasma of amlodipine, metoprolol, pravastatin, rosuvastatin, atorvastatin and its active metabolites. Plasma samples were subjected to protein precipitation with methanol, followed by evaporation at room temperature under nitrogen of the supernatant, allowing to attain measurable plasma concentrations down to sub-nanogram per milliliter levels. Stable isotope-labelled analytes were used as internal standards. The five drugs and two metabolites were analyzed using a 6-min liquid chromatographic run coupled to electrospray triple quadrupole mass spectrometry detection. The method was validated over the clinically relevant concentrations ranging from 0.3 to 480 ng/mL for amlodipine, atorvastatin and p-OH-atorvastatin, and 0.4 to 480 ng/mL for pravastatin, 0.5 to 480 ng/mL for rosuvastatin and o-OH-atorvastatin, and 3 to 4800 ng/mL for metoprolol. Validation performances such as trueness (95.4-110.8%), repeatability (1.5-13.4%) and intermediate precision (3.6-14.5%) were in agreement with current international recommendations. Accuracy profiles (total error approach) were lying within the limits of ±30% accepted in bioanalysis. This rapid and robust UHPLC-MS/MS assay allows the simultaneous quantification in plasma of the major currently used cardiovascular drugs and offers an efficient analytical tool for clinical pharmacokinetics as well as DDIs studies.

Development and validation of a multiplex UHPLC-MS/MS method for the determination of the investigational antibiotic against multi-resistant tuberculosis macozinone (PBTZ169) and five active metabolites in human plasma.

The emergence of Mycobacterium tuberculosis strains resistant to current first-line antibiotic regimens constitutes a major global health threat. New treatments against multidrug-resistant tuberculosis (MDR-TB) are thus eagerly needed in particular in countries with a high MDR-TB prevalence. In this context, macozinone (PBTZ169), a promising drug candidate with an unique mode of action and highly potent in vitro tuberculocidal properties against MDR Mycobacterium strains, has now reached the clinical phase and has been notably tested in healthy male volunteers in Switzerland. To that endeavor, a multiplex UHPLC-MS/MS method has been developed for the sensitive and accurate human plasma levels determination of PBTZ169 along with five metabolites retaining in vitro anti-TB activity. Plasma protein precipitation with methanol was carried out as a simplified sample clean-up procedure followed by direct injection of the undiluted supernatant for the bioanalysis of the six analytes within 5 min, using 1.8 µm reversed-phase chromatography coupled to triple quadrupole mass spectrometry employing electrospray ionization in the positive mode. Stable isotopically-labelled PBTZ169 was used as internal standard (ISTD), while metabolites could be reliably quantified using two unlabeled chemical analogues selected as ISTD from a large in-house analogous compounds library. The overall methodology was fully validated according to current recommendations (FDA, EMEA) for bioanalytical methods, which include selectivity, carryover, qualitative and quantitative matrix effect, extraction recovery, process efficiency, trueness, precision, accuracy profiles, method and instrument detection limits, integrity to dilution, anticoagulant comparison and short- and long-term stabilities. Stability studies on the reduced metabolite H2-PBTZ169 have shown no significant impact on the actual PBTZ169 concentrations determined with the proposed assay. This simplified, rapid, sensitive and robust methodology has been applied to the bioanalysis of human plasma samples collected within the frame of a phase I clinical study in healthy volunteers receiving PBTZ169.

LC-MS/MS method for the simultaneous analysis of seven antimalarials and two active metabolites in dried blood spots for applications in field trials: Analytical and clinical validation.

In epidemiological studies, antimalarials measurements in blood represent the best available marker of drugs exposure at population level, an important driver for the emergence of drug resistance. We have developed a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) for the simultaneous quantification of 7 frequently used antimalarials (amodiaquine, chloroquine, quinine, sulfadoxine, pyrimethamine, mefloquine, lumefantrine) and 2 active metabolites (N-desethyl-amodiaquine, desbutyl-lumefantrine) in 10-µl dried blood spots (DBS). This sampling approach is suitable for field studies wherein blood samples processing, transportation and storage are problematic. Sample preparation included extraction from a 3 mm-disk punched out of the DBS with 100-µl of methanol + 1% formic acid containing deuterated internal standards for all drugs. Good performances were achieved in terms of trueness (-12.1 to +11.1%), precision (1.4-15.0%) and sensitivity, with lower limits of quantification comprised between 2 ng/ml (sulfadoxine) and 20 ng/ml (chloroquine, quinine, pyrimethamine, mefloquine, lumefantrine and desbutyl-lumefantrine). All analytes were stable in DBS kept for 24 h at room temperature and at 37 °C. The developed assay was applied within the frame of a pharmacokinetic study including 16 healthy volunteers who received a single dose of artemether-lumefantrine. Lumefantrine concentrations in plasma and in DBS were highly correlated (R = 0.97) at all time points, confirming the assumption that lumefantrine concentrations determined in DBS confidently reflect blood concentrations. The blood/plasma ratio of 0.56 obtained using the Bland-Altman approach (and corresponding to the slope of the linear regression) is in line with very low penetration of lumefantrine into red blood cells. This sensitive multiplex LC-MS/MS assay enabling the simultaneous analysis of antimalarials in DBS is suitable for epidemiological studies in field conditions.

ERE-dependent transcription and cell proliferation: Independency of these two processes mediated by the introduction of a sulfone function into the weak estrogen estrothiazine.

The synthetic coumestrol derivative 6,12-dihydro-3-methoxy-1-benzopyrano[3,4-b][1,4]benzothiazin-6-one (estrothiazine, ESTZ) has been identified as a weak estrogen receptor α (ERα) ligand unable to compete with tritiated estradiol. The biological activity of this compound, supported by a methoxy group in position 3, seems mainly to result from its capacity to activate ERα dimerization without any participation of coactivators. In support of this view and referring to conventional estrogens, an ESTZ metabolism study conducted with hepatic human microsomes failed to provide any argument in favour of an estrogenic activity dependent on a metabolic conversion of the compound into hydroxylated metabolites with strong receptor activation ability. Interestingly, we failed to detect any oxidation of the sulfur atom of the compound. In the light of pharmacological literature data concerning sulfonylation, we assessed ERα-mediated activities generated by two sulfonylated ESTZ derivatives in which the methoxy group that plays a key role in its mechanism of action was maintained or removed. Sulfonylated ESTZ, even in its demethoxylated form, induced ERE-mediated transcriptions in MCF-7 breast cancer cells, without affecting the ERα turnover rate. In contrast to ESTZ, this compound failed to enhance the proliferation of ERα-positive breast cancer cells, suggesting that its sulfone function confers upon the receptor a capacity to elicit some of the known characteristics associated with estrogenic responses. Moreover, we demonstrated that this sulfone may contribute to ERα dimerization without any requirement of the methoxy group. Nevertheless, it seems to cooperate with this group, as reflected by a weak ability of the sulfonylated form of ESTZ to compete with tritiated estradiol for ERα-binding. Assessment of the docking of this compound within the ligand-binding domain of the receptor by molecular dynamics provided an explanation for this observation since the sulfone is engulfed in a small hydrophobic pocket involving the residues Leu-346, Leu-349, Ala-350 and Leu-384, also known to recruit coactivators. This work not only reports the sulfone functional group as a pharmacophore for estrogenic activity, but also opens new perspectives for the development of estrogenic molecules with therapeutic purpose and devoid of proliferative side effects.

Characterization of oxycodone in vitro metabolism by human cytochromes P450 and UDP-glucuronosyltransferases.

The hepatic metabolism of oxycodone by cytochromes P450 (CYP) and the UDP-glucuronosyltransferases (UGT), the main metabolic enzymes of phase I and phase II, respectively, was assessed in vitro. The N-demethylation by CYP3A4/5 and the O-demethylation by CYP2D6 in human liver microsomes (HLM) followed Michaelis-Menten kinetics, with intrinsic clearances of 1.46µL/min/mg and 0.35µL/min/mg, respectively. Although noroxycodone and oxymorphone mainly contribute to the elimination of oxycodone, the simulated total in vivo clearance using in vitro phase I metabolism was underestimated. For the first time, metabolism of oxycodone by UGT was deeply investigated using HLM, recombinant enzymes and selective inhibitors. Oxycodone-glucuronide was mainly produced by UGT2B7 (Km=762±153µM, Vmax=344±20 peak area/min/mg) and to a lesser extent by UGT2B4 (Km=2454±497µM, Vmax=201±19 peak area/min/mg). Finally, the kinetics of the drug-drug interactions were assessed using two CYP and UGT cocktail approaches. Incubations of HLM with phase I and phase II drug probes showed that oxycodone mainly decreased the in vitro activities of CYP2D6, CYP3A4/5, UGT1A3, UGT1A6 and UGT2B subfamily with an important impact on UGT2B7.

An extensive cocktail approach for rapid risk assessment of in vitro CYP450 direct reversible inhibition by xenobiotic exposure.

Acute exposure to environmental factors strongly affects the metabolic activity of cytochrome P450 (P450). As a consequence, the risk of interaction could be increased, modifying the clinical outcomes of a medication. Because toxic agents cannot be administered to humans for ethical reasons, in vitro approaches are therefore essential to evaluate their impact on P450 activities. In this work, an extensive cocktail mixture was developed and validated for in vitro P450 inhibition studies using human liver microsomes (HLM). The cocktail comprised eleven P450-specific probe substrates to simultaneously assess the activities of the following isoforms: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2 and subfamily 3A. The high selectivity and sensitivity of the developed UHPLC-MS/MS method were critical for the success of this methodology, whose main advantages are: (i) the use of eleven probe substrates with minimized interactions, (ii) a low HLM concentration, (iii) fast incubation (5min) and (iv) the use of metabolic ratios as microsomal P450 activities markers. This cocktail approach was successfully validated by comparing the obtained IC50 values for model inhibitors with those generated with the conventional single probe methods. Accordingly, reliable inhibition values could be generated 10-fold faster using a 10-fold smaller amount of HLM compared to individual assays. This approach was applied to assess the P450 inhibition potential of widespread insecticides, namely, chlorpyrifos, fenitrothion, methylparathion and profenofos. In all cases, P450 2B6 was the most affected with IC50 values in the nanomolar range. For the first time, mixtures of these four insecticides incubated at low concentrations showed a cumulative inhibitory in vitro effect on P450 2B6.

Inhibition screening method of microsomal UGTs using the cocktail approach.

A rapid method for the simultaneous determination of the in vitro activity of the 10 major human liver UDP-glucuronosyltransferase (UGT) enzymes was developed based on the cocktail approach. Specific substrates were first selected for each UGT: etoposide for UGT1A1, chenodeoxycholic acid for UGT1A3, trifluoperazine for UGT1A4, serotonin for UGT 1A6, isoferulic acid for UGT1A9, codeine for UGT2B4, azidothymidine for UGT2B7, levomedetomidine for UGT2B10, 4-hydroxy-3-methoxymethamphetamine for UGT2B15 and testosterone for UGT2B17. Optimal incubation conditions, including time-based experiments on cocktail metabolism in pooled HLMs that had been performed, were then investigated. A 45-min incubation period was found to be a favorable compromise for all the substrates in the cocktail. Ultra-high pressure liquid chromatography coupled to an electrospray ionization time-of-flight mass spectrometer was used to separate the 10 substrates and their UGT-specific glucuronides in less than 6 min. The ability of the cocktail to highlight the UGT inhibitory potential of xenobiotics was initially proven by using well-known UGT inhibitors (selective and nonselective) and then by relating some of the screening results obtained by using the cocktail approach with morphine glucuronidation (substrate highly glucuronidated by UGT 2B7). All the results were in agreement with both the literature and with the expected effect on morphine glucuronidation.

Comparison of liquid chromatography and supercritical fluid chromatography coupled to compact single quadrupole mass spectrometer for targeted in vitro metabolism assay.

The goal of this study was to evaluate the combination of powerful chromatographic methods and compact single quadrupole MS device for simple in vitro cytochrome P450 (CYP) inhibition assay, instead of more expensive triple quadrupole MS/MS detectors. For this purpose, two modern chromatographic approaches (ultra-high pressure liquid chromatography (UHPLC) and ultra-high performance supercritical fluid chromatography (UHPSFC)) were tested in combination with simple MS detector. To show the applicability for an in vitro CYP-mediated metabolism assay using the cocktail approach, a method was first developed in UHPLC-MS to separate a mixture of 8 probe substrates and 8 CYP-specific metabolites. A screening procedure was initially applied to determine the best combination of a column, an organic modifier and a mobile-phase pH, followed by fine tuning of the conditions (i.e., gradient profile, temperature and pH) using HPLC modelling software. A similar sequential method development procedure was also evaluated for UHPSFC-MS. For method development, where peak tracking is necessary, the use of single quadrupole MS was found to be extremely valuable for following the investigated analytes. Ultimately, a baseline separation of the 16 compounds was achieved in both UHPLC-MS and UHPSFC-MS with an analysis time of less than 7 min. In a second series of experiments, sensitivity was evaluated, and LOQ values were between 2 and 100 ng/mL in UHPLC-MS, while they ranged from 2 to 200 ng/mL in UHPSFC-MS. Based on the concentrations employed for the current in vitro phase I metabolism assay, these LOQ values were appropriate for this type of application. Finally, the two analytical methods were applied to in vitro CYP-dependent metabolism testing. Two well-known phytochemical inhibitors, yohimbine and resveratrol, were investigated, and reliable conclusions were drawn from these experiments with both UHPLC-MS and UHPSFC-MS. At the end, the proposed strategy of optimized chromatography combined with simple MS device has been shown to potentially compete with the widely used combination of generic chromatography and highly selective MS/MS device for simple in vitro CYP inhibition assays. In addition, our analytical method may be easier to use in a routine environment; the instrument cost is significantly reduced and the two developed methods fit for purpose.

Phenotyping of CYP450 in human liver microsomes using the cocktail approach.

The cocktail approach is an advantageous strategy used to monitor the activities of several cytochromes P450 (CYPs) in a single test to increase the throughput of in vitro phenotyping studies. In this study, a cocktail mixture was developed with eight CYP-specific probe substrates to simultaneously evaluate the activity of the most important CYPs, namely, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and the CYP3A subfamily. After cocktail incubation in the presence of human liver microsomes (HLMs), the eight selected substrates and their specific metabolites were analyzed by ultra-high-pressure liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry. Qualitative and quantitative data were simultaneously acquired to produce an overview of the extended phase I biotransformation routes for each probe substrate in the HLMs and to generate phenotypic profiles of various HLMs. A comparison of the cocktail strategy with an individual substrate assay for each CYP produced similar results. Moreover, the cocktail was tested on HLMs with different allelic variants and/or in the presence of selective inhibitors. The results were in agreement with the genetic polymorphisms of the CYPs and the expected effect of the alterations. All of these experiments confirmed the reliability of this cocktail assay for phenotyping of the microsomal CYPs.

A cocktail approach for assessing the in vitro activity of human cytochrome P450s: an overview of current methodologies.

An assessment of cytochrome P450 (CYP) enzyme activity is essential for characterizing the phase I metabolism of biological systems or to evaluate the inhibition/induction properties of xenobiotics. CYPs have generally been investigated individually by single probes, and metabolite formation has been monitored by liquid chromatography-mass spectrometry (LC-MS). To increase the throughput, many probes have been applied to assess multiple CYP activities simultaneously within a single experiment. This strategy is called the cocktail approach, and it has already been reviewed for in vivo applications, but never for in vitro ones. This review focuses for the first time on an in vitro cocktail approach, and it references the most notable articles on this topic. The advantages and limitations of applying cocktails for the in vitro activity assessment of major human CYPs, namely, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and subfamily CYP3A, are discussed. This article considers the probe reaction selections for each CYP according to regulatory recommendations, probe metabolic properties (i.e., specificity and turnover), probe concentrations and analytical sensitivity, but it also highlights a challenge specific to cocktail design, which is probe-probe interaction. The last part of the review reports some methodologies for incubating these cocktails and discusses some important issues regarding the incubation time, enzyme concentrations and sample preparation.

Contribution of various types of liquid chromatography-mass spectrometry instruments to band broadening in fast analysis.

When performing fast LC with 50mm narrow-bore columns packed with small particles, the LC instrumentation can give rise to non-negligible band broadening. In the present study, the loss in chromatographic efficiency attributed to nine different mass spectrometers of various brands, ionization source geometries and types of analyzers was assessed. In their standard configurations, the extra-column variance of these UHPLC-MS systems was estimated to vary from 20 to >100 µL(2). However, it was demonstrated that these differences arise exclusively from the chromatographic system (i.e., injector, tubing, valves, heater) and from the tubing employed to interface the UHPLC instrument with the MS device. By minimizing the tubing used for each UHPLC system, the extra-column variance was reduced to approximately 17-19 µL(2) at 600 µL/min, for all types of configurations. To achieve optimal chromatographic performance, it is therefore of prime importance to optimize the UHPLC configuration prior to conducting MS. The tubing located between the UHPLC system and the ionization source entrance was found to be particularly critical, as it contributes to band broadening even in the gradient mode. Using an optimized UHPLC-MS configuration, the loss in efficiency with a 50 × 2.1mm I.D. column was negligible for k>7. However, the efficiency loss with 1mm I.D. columns remained non-negligible for all current instrumentation, even for solutes with a value of k>20. Indeed, for a mixture of isobaric substrates and metabolites analyzed in gradient mode, the peak widths decreased by approximately 50% between a standard and optimized UHPLC-MS configuration, considering a 50 × 2.1mm, 1.7 µm column. The peak broadening was changed by 230% on a 50 × 1 mm, 1.7 µm stationary phase, for the same system configurations.