Therapeutic drug monitoring and virological response at week 48 in a cohort of HIV-1-infected patients switching to dolutegravir/rilpivirine dual maintenance therapy (ANRS-MIE-BIRIDER study).

AIMS: Dolutegravir (DTG) and rilpivirine (RPV) dual therapy is now recommended as a switch option in virologically suppressed HIV patients. Literature suggests that virological failure with dual therapy could possibly relate to subtherapeutic drug concentrations. In this study, we aimed at describing the DTG and RPV trough plasma concentrations (Cmin) and plasma HIV-1 RNA viral load (VL) during maintenance dual therapy. METHODS: We performed a retrospective analysis of DTG and RPV therapeutic drug monitoring in people living with HIV/AIDS (PLWHA) with dual therapy in 9 French centres. DTG and RPV trough plasma concentrations were estimated using a Bayesian approach to predict Cmin. The relationship between the pharmacokinetics of DTG and RPV and VL > 50 copies (cp)/mL was explored using joint nonlinear mixed models. The frequency of subtherapeutic threshold (DTG Cmin below 640 ng/mL and RPV Cmin below 50 ng/mL) were compared between PLWHA presenting VL > 50 cp/mL or not during the study. RESULTS: At baseline, 209 PLWHA were enrolled in the study. At week 48, 19 people living with HIV/AIDS (9.1%) discontinued their treatment and 15 PLWHA (7.1%) exhibited VL > 50 cp/mL. Six PLWHA out of 15 (40.0%) with VL > 50 cp/mL during the follow-up had at least 1 Cmin below the respective thresholds while only 26/194 patients (13.4%) without virological replication had at least 1 concentration below the threshold (P = .015). CONCLUSION: A majority of PLWHA receiving DTG/RPV maintenance dual therapy demonstrated VL < 50 cp/mL but virological replication was more frequent in people living with HIV/AIDS with subtherapeutic Cmin.

Proposals for a standardized procedure of validation of DNA extraction and allelic discrimination assays in pharmacogenomics according to ISO15189 requirements.

OBJECTIVES: In the era of quality management in clinical laboratories, method validation can be a challenge without appropriate guidelines, such as in the field of pharmacogenetics. The present work describes a method validation for DNA extraction and CYP3A5*3 genotyping, which would meet ISO15189:2012 requirements. METHODS: DNA extraction was performed using a QIAamp DSP DNA Blood kit, DNA purity and concentration were determined using a Nanodrop, and the genotyping assay was a real-rime PCR using TaqMan reagents. Validation criteria were similar to those usually verified when validating methods in the analytical field: specificity, sensitivity, cross-over contamination, stability of reagents, robustness, lower and upper limits of detection, and between-run and within-run precisions. A comparison to alternate or reference methods was also performed (i.e. QiAamp kit versus DNA extractor and TaqMan genotyping versus Sanger sequencing). Each validation step is described from the pharmacogenetic point of view, as well as acceptance criteria for both DNA extraction [i.e. concentration relative SD (RSD) below 25%, verified purity, and no DNA in blank samples] and genotyping assay (i.e. specificity and diagnostic sensitivity, RSD of mean threshold cycle below 15%, no amplification in blank samples). RESULTS: Concerning CYP3A5 genotyping following a DNA extraction described as an example, validation criteria were met, allowing routine use of this analytical process. Cost estimation of the overall validation procedure was approximately 290 euros, concerning reagents and consumables. CONCLUSION: This work aims to provide a reference for method validation for pharmacogenetic analysis using real-time PCR to detect single nucleotide polymorphisms, in accordance with ISO15189:2012.

Influence of Antiplatelet Agents on the Lipid Composition of Platelet Plasma Membrane: A Lipidomics Approach with Ticagrelor and Its Active Metabolite.

Lipids contained in the plasma membrane of platelets play an important role in platelet function. Modifications in the lipid composition can fluidify or rigidify the environment around embedded receptors, in order to facilitate the access of the receptor by the drug. However, data concerning the lipid composition of platelet plasma membrane need to be updated. In addition, data on the impact of drugs on plasma membrane composition, in particular antiplatelet agents, remain sparse. After isolation of platelet plasma membrane, we assessed, using lipidomics, the effect of ticagrelor, a P2Y12 antagonist, and its active metabolite on the lipid composition of these plasma membranes. We describe the exact lipid composition of plasma membrane, including all sub-species. Ticagrelor and its active metabolite significantly increased cholesterol and phosphatidylcholine ether with short saturated acyl chains 16:0/16:0, and decreased phosphatidylcholine, suggesting overall rigidification of the membrane. Furthermore, ticagrelor and its active metabolite decreased some arachidonylated plasmalogens, suggesting a decrease in availability of arachidonic acid from the membrane phospholipids for synthesis of biologically active mediators. To conclude, ticagrelor and its active metabolite seem to influence the lipid environment of receptors embedded in the lipid bilayer and modify the behavior of the plasma membrane.

Validation of an HPLC-MS/MS Method for the Determination of Plasma Ticagrelor and Its Active Metabolite Useful for Research and Clinical Practice.

Ticagrelor is an antiplatelet agent which is extensively metabolized in an active metabolite: AR-C124910XX. Ticagrelor antagonizes P2Y12 receptors, but recently, this effect on the central nervous system has been linked to the development of dyspnea. Ticagrelor-related dyspnea has been linked to persistently high plasma concentrations of ticagrelor. Therefore, there is a need to develop a simple, rapid, and sensitive method for simultaneous determination of ticagrelor and its active metabolite in human plasma to further investigate the link between concentrations of ticagrelor, its active metabolite, and side effects in routine practice. We present here a new method of quantifying both molecules, suitable for routine practice, validated according to the latest Food and Drug Administration (FDA) guidelines, with a good accuracy and precision (<15% respectively), except for the lower limit of quantification (<20%). We further describe its successful application to plasma samples for a population pharmacokinetics study. The simplicity and rapidity, the wide range of the calibration curve (2-5000 µg/L for ticagrelor and its metabolite), and high throughput make a broad spectrum of applications possible for our method, which can easily be implemented for research, or in daily routine practice such as therapeutic drug monitoring to prevent overdosage and occurrence of adverse events in patients.

Cholesterol-Rich Microdomains Contribute to PAR1 Signaling in Platelets Despite a Weak Localization of the Receptor in These Microdomains.

Platelet protease-activated receptor 1 (PAR1) is a cell surface G-protein-coupled receptor (GPCR) that acts as a thrombin receptor promoting platelet aggregation. Targeting the PAR1 pathway by vorapaxar, a PAR1 antagonist, leads to a reduction in ischemic events in cardiovascular patients with a history of myocardial infarction or with peripheral arterial disease. In platelets, specialized microdomains highly enriched in cholesterol act as modulators of the activity of several GPCRs and play a pivotal role in the signaling pathway. However, their involvement in platelet PAR1 function remains incompletely characterized. In this context, we aimed to investigate whether activation of PAR1 in human platelets requires its localization in the membrane cholesterol-rich microdomains. Using confocal microscopy, biochemical isolation, and proteomics approaches, we found that PAR1 was not localized in cholesterol-rich microdomains in resting platelets, and only a small fraction of the receptor relocated to the microdomains following its activation. Vorapaxar treatment increased the level of PAR1 at the platelet surface, possibly by reducing its endocytosis, while its colocalization with cholesterol-rich microdomains remained weak. Consistent with a cholesterol-dependent activation of Akt and p38 MAP kinase in thrombin receptor-activating peptide (TRAP)-activated platelets, the proteomic data of cholesterol-rich microdomains isolated from TRAP-activated platelets showed the recruitment of proteins contributing to these signaling pathways. In conclusion, contrary to endothelial cells, we found that PAR1 was only weakly present in cholesterol-rich microdomains in human platelets but used these microdomains for efficient activation of downstream signaling pathways following TRAP activation.

Ticagrelor increases its own potency at the P2Y12 receptor by directly changing the plasma membrane lipid order in platelets.

BACKGROUND AND PURPOSE: Although the amphiphilic nature of the widely used antithrombotic drug Ticagrelor is well known, it was never considered as a membranotropic agent capable of interacting with the lipid bilayer in a receptor-independent way. In this study, we investigated the influence of Ticagrelor on plasma membrane lipid order in platelets and if this modulates the potency of Ticagrelor at the P2Y12 receptor. EXPERIMENTAL APPROACH: We combined fluorescent in situ, in vitro and in silico approaches to probe the interactions between the plasma membrane of platelets and Ticagrelor. The influence of Ticagrelor on the lipid order of the platelet plasma membrane and large unilamellar vesicles was studied using the advanced fluorescent probe NR12S. Furthermore, the properties of model lipid bilayers in the presence of Ticagrelor were characterized by molecular dynamics simulations. Finally, the influence of an increased lipid order on the dose-response of platelets to Ticagrelor was studied. KEY RESULTS: Ticagrelor incorporates spontaneously into lipid bilayers and affects the lipid order of the membranes of model vesicles and isolated platelets, in a nontrivial composition and concentration-dependent manner. We showed that higher plasma membrane lipid order in platelets leads to a lower IC50 value for Ticagrelor. It is shown that membrane incorporation of Ticagrelor increases its potency at the P2Y12 receptor, by increasing the order of the platelet plasma membrane. CONCLUSION AND IMPLICATIONS: A novel dual mechanism of Ticagrelor action is suggested that combines direct binding to P2Y12 receptor with simultaneous modulation of receptor-lipid microenvironment.

Molecular modeling in cardiovascular pharmacology: Current state of the art and perspectives.

Molecular modeling in pharmacology is a promising emerging tool for exploring drug interactions with cellular components. Recent advances in molecular simulations, big data analysis, and artificial intelligence (AI) have opened new opportunities for rationalizing drug interactions with their pharmacological targets. Despite the obvious utility and increasing impact of computational approaches, their development is not progressing at the same speed in different fields of pharmacology. Here, we review current in silico techniques used in cardiovascular diseases (CVDs), cardiological drug discovery, and assessment of cardiotoxicity. In silico techniques are paving the way to a new era in cardiovascular medicine, but their use somewhat lags behind that in other fields.

Bictegravir/emtricitabine/tenofovir alafenamide combination in the management of kidney transplant patients with HIV receiving immunosuppressants.

We report here a drug-drug interaction with tacrolimus in a HIV-positive patient with renal transplant, after switch from highly active antiretroviral therapy with boosted protease inhibitors to the combination bictegravir/emtricitabine/tenofovir alafenamide. Although the tacrolimus doses were adapted to take account of the pharmacokinetic interactions with protease inhibitors, a tacrolimus overdosage occurred in the patient nonetheless. Through this case report, we highlight the need to consider a sufficient timeframe of withdrawal of protease inhibitors, which induce a prolonged drug-drug interaction with tacrolimus. To conclude, we purport that the combination bictegravir/emtricitabine/tenofovir alafenamide could be an attractive alternative in the context of transplantation provided a discontinuation of boosted protease inhibitors for more than 48 hours before introducing tacrolimus.

ß-lactam dosing at the early phase of sepsis: Performance of a pragmatic protocol for target concentration achievement in a prospective cohort study.

PURPOSE: We hypothesized that a protocol of standardized fixed dose using prolonged infusion during the early phase of sepsis may avoid insufficient ß-lactam concentrations. METHODS: In this single center prospective study, patients with sepsis and vasopressors were enrolled if they were treated by either piperacillin-tazobactam, meropenem or cefepime. Βeta-lactams were administered at fixed dose by prolonged infusion. Targeted plasma concentrations for piperacillin, meropenem and cefepime were above 80 mg/L, 8 mg/L and 38 mg/L respectively. Three blood samples were collected per patient over the first 48 h of treatment. Primary endpoint was target concentration achievement during the 48 first hours, defined as all plasma concentrations above the targeted threshold. RESULTS: Among the 89 patients completing the three samples, target concentrations were achieved for 61 (69%). Target concentrations were achieved in 20 (53%), 32 (89%), and 9 (60%) of the patients treated with piperacillin, meropenem and cefepime, respectively. By multivariate analysis, lower APACHE 2 score, higher baseline MDRD creatinine clearance, and piperacillin use were independently associated with insufficient ß-lactam concentrations. CONCLUSION: Despite a fixed dose antibiotic administration protocol with prolonged infusion insufficient ß-lactam concentration was frequent at the early phase of sepsis, especially in less severe patients, without renal failure, and treated with piperacillin. In septic patients with vasopressors, piperacillin dosing higher than 16 g may be needed to achieve the recommended target concentration. TRIAL REGISTRATION: NCT02820987.

Real-Life Impact on Lipid Profile of a Switch from Tenofovir Disoproxil Fumarate to Tenofovir Alafenamide in HIV-Infected Patients.

BACKGROUND: Tenofovir disoproxil fumarate is a prodrug of tenofovir diphosphate that exposes patients to renal toxicity over the long term. Tenofovir alafenamide, a new prodrug, now makes it possible to reduce toxicity, but at the cost of an alteration in lipid profile. There is currently no recommendation for follow-up of lipid profile when switching from tenofovir disoproxil fumarate to tenofovir alafenamide. OBJECTIVE: Our study aimed to evaluate the effects on renal function and lipid profile of a switch from tenofovir disoproxil fumarate to tenofovir alafenamide, and the consequences for patient management. METHODS: Demographic, clinical and biological data was recorded from a retrospective clinical cohort study in real-life, including patients who switched from tenofovir disoproxil fumarate to tenofovir alafenamide. A descriptive analysis of the study population, with a comparison of biological parameters using the paired Student t test for paired data was performed. RESULTS: From January 2016 to January 2019, a total of 103 patients were included. There was no significant difference in renal function before vs after the switch in therapy (p=0.29 for creatinine, p=0.30 for phosphoremia). We observed a change in lipid profile, with a significant increase in total cholesterol (p=0.0006), HDL cholesterol (p=0.0055) and triglycerides (p=0.0242). Four patients received lipid-lowering therapy after switching. CONCLUSION: In patients who switch from tenofovir disoproxil fumarate to tenofovir alafenamide, lipid profile is altered, and may require initiation of lipid-lowering therapy. It seems necessary to monitor lipid parameters after this switch, despite the absence of an official recommendation.

A simple and fast HPLC-MS/MS method for simultaneous determination of direct oral anticoagulants apixaban, dabigatran, rivaroxaban in human plasma.

Direct Oral Anticoagulants (DOACs) available for the treatment and prevention of thromboembolic diseases include dabigatran, a direct thrombin (IIa) inhibitor, and apixaban, edoxaban and rivaroxaban, which are direct inhibitors of Stuart factor (Xa). DOACs have a different pharmacokinetic and pharmacodynamics profiles, with less probable drug-drug interactions than vitamin K antagonists. They do not require systematic therapeutic monitoring except in specific clinical situations such as emergency procedures or drug non-compliance. Furthermore, anticoagulant effects of DOACs could be impacted by renal impairment, drug-drug interactions, food interactions, or pharmacogenetic variability. In this context, we developed a method for simultaneous determination of dabigatran, rivaroxaban and apixaban in human plasma using high performance liquid chromatography coupled with a mass spectrometry assay and applied it to 26 patient samples. Our method presents a total run time of 5 min and extends from 25 to 1000 µg/L for apixaban and dabigatran; and from 5 to 1000 µg/L for rivaroxaban. Intra- and inter-assay accuracy were between -22.3 and 25.4%; and - 23.7 and 3.8%, respectively. Precision at low and high concentrations were below 17.5%. Frozen samples were stable up to 3 months. No significant cross-contamination was observed. In conclusion, our assay can be used during clinical studies and in daily routine practice for the management of specific clinical situations at reasonable cost.

Lethal Neonatal Progression of Fetal Cardiomegaly Associated to ACAD9 Deficiency.

ACAD9 (acyl-CoA dehydrogenase 9) is an essential factor for the mitochondrial respiratory chain complex I assembly. ACAD9, a member of acyl-CoA dehydrogenase family, has high homology with VLCAD (very long-chain acyl-CoA dehydrogenase) and harbors a homodimer structure. Recently, patients with ACAD9 deficiency have been described with a wide clinical spectrum ranging from severe lethal form to moderate form with exercise intolerance.We report here a prenatal presentation with intrauterine growth retardation and cardiomegaly, with a fatal outcome shortly after birth. Compound heterozygous mutations, a splice-site mutation - c.1030-1G>T and a missense mutation - c.1249C>T; p.Arg417Cys, were identified in the ACAD9 gene. Their effect on protein structure and expression level was investigated. Protein modeling suggested a functional effect of the c.1030-1G>T mutation generating a non-degraded truncated protein and the p.Arg417Cys, creating an aberrant dimer. Our results underscore the crucial role of ACAD9 protein for cardiac function.