Tailoring oral anticoagulant treatment in the era of multi-drug therapies for PAH and CTEPH.

The use of oral anticoagulants in the management of pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH) presents distinct therapeutic challenges and benefits. In PAH, the benefits of oral anticoagulation are uncertain, with studies yielding mixed results on their efficacy and safety. Conversely, oral anticoagulants are a cornerstone in the treatment of CTEPH, where their use is consistently recommended to prevent recurrent thromboembolic events. The choice between vitamin K antagonists (VKAs) and direct oral anticoagulants (DOACs) remains a significant clinical question, as each type presents advantages and potential drawbacks. Furthermore, drug-drug interactions (DDIs) with concomitant PAH and CTEPH treatments complicate anticoagulant management, necessitating careful consideration of individual patient regimens. This review examines the current evidence on oral anticoagulant use in PAH and CTEPH and discusses the implications of DDIs within a context of multi-drug treatments, including targeted drugs in PAH.

Full-dose rivaroxaban in patients with a history of bariatric surgery: bridging the knowledge gap through a phase 1 study.

BACKGROUND: Bariatric surgery (BS) induces significant changes in gastrointestinal anatomy, potentially influencing the pharmacokinetics of orally administered drugs such as rivaroxaban. OBJECTIVES: This phase 1 study aimed to assess the pharmacokinetics and safety of full-dose rivaroxaban in post-BS patients. METHODS: The ABSORB (Rivaroxaban Pharmacokinetics and Pharmacodynamics After Bariatric Surgery and in Morbid Obesity) study was a single-center, nonrandomized, multiple-dose, parallel-design bioequivalence trial. Adult patients with stable weight after Roux-en-Y gastric bypass (RYGB) or sleeve gastrectomy (SG) were compared with subjects with class III obesity and healthy controls. Participants received 20 mg of rivaroxaban daily for 8 days. RESULTS: Post-BS patients exhibited altered rivaroxaban pharmacokinetics, suggesting reduced absorption. Mean area under the concentration-time curve from time 0 to 24 hours after the first dose (RYGB, 1806.8 ng.h/mL; SG, 1648.9 ng.h/mL) was lower compared with that in controls (1893.5 ng.h/mL). At steady state, the area under the concentration-time curve values remained lower in BS groups (RYGB, 2129.9 ng.h/mL; SG, 1946.4 ng.h/mL) than in controls (2224.8 ng.h/mL). The maximum concentration after the first dose was lower in post-RYGB subjects (214.9 ng/mL) than in controls (264.1 ng/mL). This difference was less pronounced at steady state (RYGB, 256.9 ng/mL vs controls, 288.8 ng/mL). Neither BS group met bioequivalence criteria compared with controls, whereas the group with class III obesity met bioequivalence criteria compared with controls at steady state. CONCLUSION: Rivaroxaban displayed minor pharmacokinetic variations in post-BS patients. Given reported interindividual variability in the general population, these variations are unlikely to be of clinical significance. Our findings support rivaroxaban use in BS patients, emphasizing the need for further research in this area.

Tinzaparin, an alternative to subcutaneous unfractionated heparin, in patients with severe and end-stage renal impairment: a retrospective observational single-center study.

BACKGROUND: Tinzaparin could be easier to manage than unfractionated heparin in patients with severe renal impairment. However, clinical and pharmacologic data regarding its use in such patients are lacking. OBJECTIVES: The aims of this study were to determine, in patients with estimated glomerular filtration rate (eGFR) of <30 mL.min⁻1, tinzaparin pharmacokinetics (PK) parameters using a population PK approach and bleeding and thrombotic complications. METHODS: We performed a retrospective observational single-center study, including in-patients with eGFR of <30 mL.min⁻1 receiving prophylactic (4500 IU.d⁻1) or therapeutic (175 IU.kg⁻1.d⁻1) tinzaparin. Measured anti-Xa levels were analyzed using a nonlinear mixed-effects modeling approach. Individual predicted tinzaparin exposure markers at steady state were calculated for each patient and dosing regimen. The PK was also evaluated through Monte Carlo simulations based on the final covariate model parameter estimates. RESULTS: Over a 22-month period, 802 tinzaparin treatment periods in 623 patients were analyzed: two-thirds received a prophylactic dose, 66% had an eGFR of <20 mL.min⁻1, and 25% were on renal replacement therapy. In patients for whom anti-Xa measurements were performed (n = 199; 746 values), PK parameters, profiles, and maximum plasma concentrations were comparable with those in patients without renal impairment or in healthy volunteers. In the whole population, major bleeding occurred in 2.4% and 3.5% of patients receiving prophylactic and therapeutic doses over a median 9- and 7-day treatment period, respectively. No patients had thrombotic complications. CONCLUSION: Tinzaparin PK parameters and profiles were not affected by renal impairment. This suggests that tinzaparin, at therapeutic or prophylactic dose, could be an alternative to unfractionated heparin in hospitalized patients with severe renal impairment.

In Vitro/Ex Vivo Release Study of a Ground Umbilical Cord Matrix Loaded with Dexamethasone.

Eye drops containing steroids and antibiotics are widely used to treat a large range of ocular diseases of the ocular surface. They require frequent instillation or a high dosage, which can affect quality of life. We developed a biomaterial from human umbilical cord that can be loaded with drugs before being placed in the inferior conjunctival fornix. In the present work, this viro-inactivated, freeze-dried, and sterile foam was loaded with dexamethasone phosphate. We studied the release kinetic of 100 mg of biomaterial loaded with 100 µg of dexamethasone phosphate. Assays have shown that the product can be loaded with 100 µg of dexamethasone and allows a progressive release over time for at least 48 h. In addition, when compared with the instillation of the same dexamethasone quantity (100 µg), instilled regularly via eye-drop solution at 0.79 mg/mL, the drug penetration through corneal tissues was better with the dexamethasone-loaded biomaterial.

Genotype-Dependent Response to Desmopressin in Hemophilia A and Proposal of a Predictive Response Score.

BACKGROUND: Desmopressin (DDAVP) is used in patients with moderate/mild hemophilia A (PWMHs) to increase their factor VIII (FVIII) level and, if possible, normalize it. However, its effectiveness varies between individuals. The GIDEMHA study aims to investigate the influence of F8 gene variants. MATERIAL AND METHODS: The study collected the trajectory of FVIII levels from therapeutic intravenous DDAVP tests in four French hemophilia treatment centers. A pharmacological analysis was performed associated with efficacy scores according to F8 variants: absolute and relative responses, as well as new scores: absolute duration (based on duration with FVIII ≥ 0.50 IU.mL-1) and relative duration (based on half-life). RESULTS: From enrolled 439 PWMHs, 327 had a hot-spot F8 variant (with ≥5 PWMHs). For these, the median (min-max) basal and peak FVIII were 0.20 (0.02-0.040) and 0.74 (0.14-2.18) IU.mL-1 respectively, with FVIII recovery being 3.80 IU.ml-1 (1.15-14.75). The median FVIII half-life was 3.9 hours (0.7-15.9 hours). FVIII was normalized (≥0.50 IU.mL-1) in 224/327 PWMHs (69%) and the median time with normalized FVIII was 3.9 hours (0.0-54.1 hours). Following the response profiles to DDAVP defined by the four efficacy scores, four groups of F8 variants were isolated, and then compared using survival curves with normalized FVIII (p < 0.0001): "long-lastingly effective" [p.(Glu739Lys), p.(Ser2030Asn), p.(Arg2178His), p.(Gln2208Glu), and T-stretch deletion in intron 13]; "moderately effective" [p.(Ser112Phe), p.(Ala219Thr), p.(Thr2105Ile), p.Phe2146Ser), and p.(Asp2150Asn)]; "moderately ineffective" [p.Ala81Asp), p.(Gln324Pro), p.(Tyr492His), p.(Arg612Cys), p.(Met701Val), p.(Val2035Asn), and p.(Arg2178Cys)]; and "frequently ineffective" [c.-219C > T, p.(Cys2040Tyr), p.(Tyr2169His), p.(Pro2319Leu), and p.(Arg2326Gln)]. CONCLUSION: In view of our data, we propose indications for DDAVP use in PWMH based on F8 variants for minor and major invasive procedures.

Multicenter evaluation of the hemostatic activity of emicizumab in patients with severe hemophilia A.

BACKGROUND: Emicizumab has been approved for the prophylaxis of patients with hemophilia A with or without inhibitors. However, spontaneous and trauma-induced breakthrough bleeds have been reported in patients on emicizumab prophylaxis, and no laboratory assay has been validated to evaluate the hemostatic activity of emicizumab. OBJECTIVES: The thrombin generation assay (TGA) could be a surrogate marker of the hemostatic efficacy of emicizumab. The correlation between TGA and the methods used to measure emicizumab blood concentration was evaluated in this study. METHODS: TGA was modified by the use of a trigger reagent combining a very low concentration of tissue factor and activated factor (F)XI. Emicizumab quantification was performed by 3 methods: the modified 1-step FVIII assay and 2 methods based on liquid chromatography and mass spectrometry (LC-MS). RESULTS: Using tissue factor/activated FXI-triggered TGA and platelet-poor plasma, a relationship was observed between the area under the thrombin generation curve (endogenous thrombin potential [ETP]) and the clinical response of patients to emicizumab. The ultrastructure of fibrin clots was consistent with ETP results and showed that emicizumab had a hemostatic activity equivalent to 20 to 30 IU/dL of FVIII. Finally, pharmacokinetic/pharmacodynamic analyses showed no correlation between ETP and LC-MS nor with modified 1-stage FVIII assay, but a statistically significant correlation between the LC-MS methods and the time-to-peak results of the TGA. CONCLUSION: Using a modified TGA, this study showed that patients who experienced breakthrough bleeds while on emicizumab had a lower thrombin-generating capacity compared with others with good clinical response to emicizumab.

Latest advances in the reversal strategies for direct oral anticoagulants.

BACKGROUND: Since the late 2000s, Europe has granted approval for various thrombotic risk-related uses of direct oral anticoagulants (DOACs). Unlike traditional anticoagulants, DOACs do not necessitate routine coagulation monitoring. Nevertheless, clinical practice often encounters bleeding events associated with these medications, making the need for effective reversal strategies evident. OBJECTIVES: The study aims to take stock of current reversal strategies for DOACs, with a particular emphasis on the latest compounds that have been developed or are currently under development. METHODS: For obtaining information regarding the ongoing reversal strategies and the compounds under development, we referred to ClinicalTrials website, PubMed, and Google Scholar. RESULTS: In 2024, two specific antidotes to DOACs have already received approval when reversal of anticoagulation is needed owing to life-threatening or uncontrolled bleeding: idarucizumab that reverses the effects of dabigatran, and andexanet alfa, designed to counteract activated factor X inhibitors such as apixaban and rivaroxaban. Furthermore, ciraparantag, a potential universal reversal agent, is currently in advanced stages of clinical development. Concerns remain regarding the safety of specific reversal agents, especially concerning the risk of thrombosis. Additionally, the cost of these antidotes remains high. Consequently, nonspecific strategies to counteract anticoagulant medications, including activated charcoal, hemodialysis, and concentrates of coagulation factors, still have utility. CONCLUSION: With the validation of specific and nonspecific antidotes, DOACs could supplant traditional oral anticoagulants. This progress represents a significant advancement in anticoagulation therapy. However, ongoing research is crucial to address remaining safety concerns of the specific reversion agents of DOACs in clinical practice.

Design and validation of a custom-made system to measure transepithelial electrical impedance in human corneas preserved in active storage machine.

Corneal epithelial barrier represents one of the major limitations to ocular drug delivery and can be explored non-invasively through the evaluation of its electrical properties. Human corneas stored in active storage machine (ASM) could represent an interesting physiological model to explore transcorneal drug penetration. We designed a new system adapted to human corneas preserved in ASM to explore corneal epithelial barrier function ex-vivo. A bipolar set-up including Ag/AgCl electrodes adaptors to fit the corneal ASM and a dedicated software was designed and tested on freshly excised porcine corneas (n = 59) and human corneas stored 14 days in ASM (n = 6). Porcine corneas presented significant and proportional decrease in corneal impedance in response to increasing-size epithelial ulcerations and acute exposure to benzalkonium chloride (BAC) 0.01 and 0.05%. Human corneas stored 14 days in ASM presented a significant increase in corneal impedance associated with the restoration of a multi-layer epithelium and an enhanced expression of tight junctions markers zonula occludens 1, claudin 1 and occludin. These results support the relevance of the developed approach to pursue the exploration and development of human corneas stored in ASM as a physiological pharmacological model.

Factors Influencing Unfractionated Heparin Pharmacokinetics and Pharmacodynamics During a Cardiopulmonary Bypass.

BACKGROUND: Unfractionated heparin (UFH) is commonly used during cardiac surgery with a cardiopulmonary bypass to prevent blood clotting. However, empirical administration of UFH leads to variable responses. Pharmacokinetic and pharmacodynamic modeling can be used to optimize UFH dosing and perform real-time individualization. In previous studies, many factors that could influence UFH pharmacokinetics/pharmacodynamics had not been taken into account such as hemodilution or the type of UFH. Few covariates were identified probably owing to a lack of statistical power. This study aims to address these limitations through a meta-analysis of individual data from two studies. METHODS: An individual patient data meta-analysis was conducted using data from two single-center prospective observational studies, where different UFH types were used for anticoagulation. A pharmacodynamic/pharmacodynamic model of UFH was developed using a non-linear mixed-effects approach. Time-varying covariates such as hemodilution and fluid infusions during a cardiopulmonary bypass were considered. RESULTS: Activities of UFH's anti-activated factor/anti-thrombin were best described by a two-compartment model. Unfractionated heparin clearance was influenced by body weight and the specific UFH type. Volume of distribution was influenced by body weight and pre-operative fibrinogen levels. Pharmacodynamic data followed a log-linear model, accounting for the effect of hemodilution and the pre-operative fibrinogen level. Equations were derived from the model to personalize UFH dosing based on the targeted activated clotting time level and patient covariates. CONCLUSIONS: The population model effectively characterized UFH's pharmacokinetics/pharmacodynamics in cardiopulmonary bypass patients. This meta-analysis incorporated new covariates related to UFH's pharmacokinetics/pharmacodynamics, enabling personalized dosing regimens. The proposed model holds potential for individualization using a Bayesian estimation.

Comparisons between diluted thrombin time, ecarin chromogenic assays, and UPLC-MS for plasma level dabigatran quantification: Results from DRIVING study.

INTRODUCTION: The knowledge of dabigatran levels is helpful for decision-making in specific situations such as urgent surgery or when the question of reversal arises (uncontrolled bleeding, eligibility for thrombolysis). However, a limited number of observational studies are available regarding comparisons between quantification methods. The objective of the study was to compare dabigatran plasma levels using three assays including the reference method (high-performance liquid chromatography coupled with mass spectrometry), focusing on the agreement around the 30-50 ng/mL clinically relevant thresholds. METHODS: Sixty healthy volunteers from DRIVING trial (NCT01627665) were given a single 300-mg dabigatran etexilate dose. Serial blood samplings were performed at pre-defined time points (0 to 24 h). We analyzed plasma samples using ultra-performance-liquid chromatography coupled with tandem mass spectrometry (UPLC-MS) (dabigatran reference method); ii/diluted thrombin time (dTT) (Hemoclot-DTI-Hyphen-Biomed); iii/ecarin-based chromogenic assay (ECA-II-Stago). RESULTS: Nine hundred sixty samples were analyzed using the three assays (2759 values). dTT and ECA-II values were highly correlated with those of UPLC-MS (Deming regression). Most values >50 ng/mL were higher using dTT and ECA-II compared to UPLC-MS: biases were constant, +14% and +16% with dTT and ECA-II, respectively (Bland-Altman plots), suggesting that active metabolites accounted for ~15% of thrombin inhibition. Regarding values <30 ng/mL, 30-50 ng/mL, or ≥50 ng/mL, the agreement probability between dTT and ECA-II was of 90.6% [88.4-92.5] (Cohen's kappa coefficient 0.84). CONCLUSION: dTT and ECA-II assays rapidly provide accurate dabigatran-level results for clinical practice, both assays being suitable in emergency, taking into account the thrombin inhibitory effect of dabigatran metabolites.

Proteomic biomarkers for survival in systemic sclerosis-associated pulmonary hypertension.

BACKGROUND: Interstitial lung disease (ILD) and pulmonary hypertension (PH) represent the major causes of mortality in systemic sclerosis (SSc). Patients with systemic sclerosis and combined PH and ILD (SSc-PH-ILD) generally have a poor prognosis. Predictors of survival and of potential benefit of treatment are lacking in patients with SSc-PH-ILD. OBJECTIVE: To identify specific plasma protein expression patterns associated with survival in patients with SSc-PH-ILD. MATERIALS AND METHODS: Post-hoc analysis of a prospective multicenter French study in patients with PH-ILD. An untargeted proteomic analysis using mass spectrometry was performed to identify plasma protein changes associated with long-term overall survival in patients with SSc-PH-ILD. RESULTS: Thirty two patients were included in the analysis, of whom 13 died during follow-up (median survival: 76.5 months). At baseline, survivors had less severe hemodynamic impairment [pulmonary vascular resistance of 4.4 Wood Units (IQR 3-5.2) vs. 6.2 Wood Units (IQR 4.2-10.7)] and higher carbon monoxide diffusing capacity [median 39% (IQR 35-44%) vs. 25% (IQR 22-30.5%)], than the 13 patients who died. Seven proteins, associated with haemostasis and fibrosis, were differentially expressed according to patients' survival. In the survivor group, two proteins were increased (ADAMTS13, SERPIND1) and five were decreased (PTGDS, OLFM1, C7, IGFBP7, FBN1) compared to the non-survivor groups. CONCLUSION: The prognosis of SSc-PH-ILD patients is poor. This proteomic approach found 7 plasma proteins (involved in haemostasis and fibrosis pathways) associated with survival. These potential biomarkers may be good candidates to prognostic enrichment.

Characterization of a recombinant factor IX molecule fused to coagulation factor XIII-B subunit.

INTRODUCTION AND AIM: Severe haemophilia B (HB) is characterized by spontaneous bleeding episodes, mostly into joints. Recurrent bleeds lead to progressive joint destruction called haemophilic arthropathy. The current concept of prophylaxis aims at maintaining the FIX level >3-5 IU/dL, which is effective at reducing the incidence of haemophilic arthropathy. Extended half-life FIX molecules make it easier to achieve these target trough levels compared to standard FIX concentrates. We previously reported that the fusion of a recombinant FIX (rFIX) to factor XIII-B (FXIIIB) subunit prolonged the half-life of the rFIX-LXa-FXIIIB fusion molecule in mice and rats 3.9- and 2.2-fold, respectively, compared with rFIX-WT. However, the mechanism behind the extended half-life was not known. MATERIALS AND METHODS: Mass spectrometry and ITC were used to study interactions of rFIX-LXa-FXIIIB with albumin. Pharmacokinetic analyses in fibrinogen-KO and FcRn-KO mice were performed to evaluate the effect of albumin and fibrinogen on in-vivo half-life of rFIX-LXa-FXIIIB. Finally saphenous vein bleeding model was used to assess in-vivo haemostatic activity of rFIX-LXa-FXIIIB. RESULTS AND CONCLUSION: We report here the key interactions that rFIX-LXa-FXIIIB may have in plasma are with fibrinogen and albumin which may mediate its prolonged half-life. In addition, using the saphenous vein bleeding model, we demonstrate that rFIX-FXIIIB elicits functional clot formation that is indistinguishable from that of rFIX-WT.

Plasma lipidomic analysis to investigate putative biomarkers of P-glycoprotein activity in healthy volunteers.

P-glycoprotein (P-gp) is an efflux transporter involved in the bioavailability of many drugs currently on the market. P-gp is responsible for several drug-drug interactions encountered in clinical practice leading to iatrogenic hospital admissions, especially in polypharmacy situations. ABCB1 genotyping only reflects an indirect estimate of P-gp activity. Therefore, it would be useful to identify endogenous biomarkers to determine the P-gp phenotype to predict in vivo activity prior to the initiation of treatment and to assess the effects of drugs on P-gp activity. The objective of this study was to assess changes in plasma lipidome composition among healthy volunteers selected on the basis of their ABCB1 genotype and who received clarithromycin, a known inhibitor of P-gp. Untargeted lipidomic analysis based on liquid chromatography-tandem mass spectrometry was performed before and after clarithromycin administration. Our results revealed changes in plasma levels of some ceramides (Cers) {Cer(d18:1/22:0), Cer(d18:1/22:1), and Cer(d18:1/20:0) by ~38% (p < 0.0001), 13% (p < 0.0001), and 13% (p < 0.0001), respectively} and phosphatidylcholines (PCs) {PC(17:0/14:1), PC(16:0/18:3), and PC(14:0/18:3) by ~24% (p < 0.001), 10% (p < 0.001), and 23.6% (p < 0.001)} associated with both ABCB1 genotype and clarithromycin intake. Through the examination of plasma lipids, our results highlight the relevance of untargeted lipidomics for studying in vivo P-gp activity and, more generally, to safely phenotyping transporters.

Proteomics analyses of human plasma reveal triosephosphate isomerase as a potential blood marker of methotrexate resistance in rheumatoid arthritis.

OBJECTIVES: To assess differentially expressed blood proteins between patients with active rheumatoid arthritis (RA) and patients in remission after methotrexate (MTX) treatment, with the aim of identifying a biomarker of methotrexate resistance (MTXR). METHODS: Two populations of RA patients treated with a stable dose of subcutaneous MTX for at least 3 months were constituted according to the DAS28: remission (DAS28 < 2.6; n = 24) and active disease (DAS28 > 3.2; n = 32). The two groups of RA patients were homogeneous regarding their epidemiological characteristics, except for the duration of treatment which was longer in the remission group. After collection of a blood sample, plasma protein digestion was performed, followed by untargeted proteomics analysis. Then, a targeted analysis was performed to confirm the results of the untargeted approach. RESULTS: Untargeted proteomics analysis revealed 8 plasma proteins differentially expressed between the two groups of patients. Among them, triosephosphate isomerase (TPI-1) and glucose-6-phosphate isomerase (GPI), which are main actors of glycolysis, were found down-regulated in the active group. This result was confirmed for TPI-1 in the targeted proteomics analysis. CONCLUSIONS: A first step was achieved in the search for biomarker of MTXR with identification of two actors of glycolysis (TPI-1 and GPI). The next step will be to confirm these results in a larger cohort, including samples from treatment-naive patients, to assess the predictive potential of these protein markers.

Acoustic Aerosol Delivery: Assessing of Various Nasal Delivery Techniques and Medical Devices on Intrasinus Drug Deposition.

This study aims to evaluate the impact of the nasal delivery technique and nebulizing technologies (using different frequencies of oscillating airflow) for acoustic aerosol targeting of maxillary sinuses. Sodium fluoride (chemical used as a marker), tobramycin (drug used as a marker) and 99mTc-DTPA (radiolabel aerosol) were used to assess the intrasinus aerosol deposition on a nasal cast. Two commercial medical devices (PARI SINUS nebulizer and NL11SN ATOMISOR nebulizer) and various nasal delivery techniques (one or two nostrils connected to the aerosol inlet, the patient with the soft palate closed or open during the acoustic administration of the drug, the presence or not of flow resistance in the nostril opposite to the one allowing the aerosol to be administered) were evaluated. The closed soft palate condition showed a significant increase in drug deposition even though no significant difference in the rest of the nasal fossae was noticed. Our results clearly demonstrated a higher intrasinus aerosol deposition (by a factor 2-3; respectively 0.03 ± 0.007% vs. 0.003 ± 0.0002% in the right maxillary sinus and 0.027 ± 0.006% vs. 0.013 ± 0.004% in the left maxillary sinus) using the acoustic airflow generated by the PARI SINUS compared to the NL11SN ATOMISOR. The results clearly demonstrated that the optimal conditions for aerosol deposition in the maxillary sinuses were obtained with a closed soft palate. Thus, the choice of the nebulizing technology (and mainly the frequency of the pulsating aerosol generated) and also the recommendation of the best nasal delivery technique are key factors to improve intrasinus aerosol deposition.

Impact of age on in vitro metabolism of clopidogrel: a potential explanation for high on-treatment platelet reactivity in the elderly?

Background: High on-treatment platelet reactivity has been reported in 30% of patients on clopidogrel and 50% in elderly patients; however, little is known about the mechanisms of this biological resistance. One hypothesis is an age-related impaired hepatic metabolism of the prodrug clopidogrel, leading to a lower formation of its active metabolite (clopidogrel-AM). Objectives: To compare the levels of clopidogrel-AM formed in vitro using "old" and "young" human liver microsomes (HLMs) and their consequences on platelet functions. Methods: We developed an in vitro model using "old" (73.6 ± 2.3 years) and "young" (51.2 ± 8.5 years) HLMs, added to platelet-rich plasma from 21 healthy donors with or without clopidogrel (50 µM) and incubated at 37 °C for 30 (T30) and 45 minutes (T45). Clopidogrel-AM was quantified by liquid chromatography-mass spectrometry/mass spectrometry method. Platelet aggregation was performed by light transmission aggregometry. Results: The generation of clopidogrel-AM increased over time and reached concentrations comparable with those reported in treated patients. At T30, mean clopidogrel-AM concentrations were significantly higher with "young" (8.56 µg/L; 95% CI, 5.87-11.24) than with "old" HLMs (7.64 µg/L; 95% CI, 5.14-10.14; P = .002); and at T45, 11.40 µg/L; 95% CI (7.57-15.22) vs 10.63 µg/L, 95% CI (7.10-14.15), P = .02 (n = 21). Despite a significant inhibition of platelet aggregation, no significant difference was found in light transmission aggregometry (adenosine diphosphate, 10 µM) after clopidogrel metabolism by "old" or "young" HLMs, probably because of low sensitivity of the method to small variations of clopidogrel-AM. Conclusion: In this original model combining metabolic and functional approaches, less clopidogrel-AM was produced with HLMs from older patients. This provides support for a decreased CYP450 activity that may contribute to high on-treatment platelet reactivity in elderly patients.

A French Real-World Evidence Study Evaluating the Efficacy, Safety, and Pharmacokinetic Parameters of rVIII-SingleChain in Patients with Hemophilia A Receiving Prophylaxis.

BACKGROUND: rVIII-SingleChain is a recombinant factor VIII (FVIII) with increased binding affinity to von Willebrand factor compared with other FVIII products. rVIII-SingleChain is indicated for the treatment and prevention of bleeding episodes in patients with hemophilia A. OBJECTIVES: To collect real-world evidence data from patients treated with rVIII-SingleChain to confirm the efficacy and safety established in the clinical trial program and carry out a population pharmacokinetic (PK) analysis. METHODS: This interim analysis includes data, collected between January 2018 - September 2021, from patients treated with rVIII-SingleChain prophylaxis at French Hemophilia Treatment centers. Data on annualized bleeding rates, dosing frequency, and consumption before and after switching to rVIII-SingleChain were recorded. A population PK analysis was also conducted to estimate PK parameters. RESULTS: Overall, 43 patients switched to prophylaxis with rVIII-SingleChain either from a previous prophylaxis regimen or from on-demand treatment. Following the switch to rVIII-SingleChain, patients maintained excellent bleed control. After switching to rVIII-SingleChain, most patients maintained or reduced their regimen. Interestingly, a majority of patients treated >2 ×/weekly with a standard half-life FVIII reduced both injection frequency and FVIII consumption with rVIII-SingleChain. A PK analysis revealed a lower clearance of rVIII-SingleChain (1.9 vs. 2.1 dL/h) and a longer half-life both in adolescents/adults (n = 28) and pediatric (n = 6) patients (15.5 and 11.9 hours, respectively vs. 14.5 and 10.3 hours) than previously reported. CONCLUSIONS: Patients who switched to rVIII-SingleChain prophylaxis demonstrated excellent bleed control and a reduction in infusion frequency. A population PK analysis revealed improved PK parameters compared with those reported in the clinical trial.

Development of simple and rapid method for Emicizumab quantification by LC-MS/MS in human plasma.

Emicizumab is a new therapeutic monoclonal antibody indicated for prophylaxis in severe haemophilia A patients. Pharmacokinetic variability has been reported in clinical studies, thus dose optimisation based on quantification of plasma drug concentration could be considered to reduce this variability. Therefore, a reliable and accurate quantification of emicizumab is required. In this study, we developed a method for absolute quantification of emicizumab using liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS). Sample preparation was based on organic solvent precipitation of proteins followed by trypsin digestion. A signature peptide of emicizumab was used for quantification by LC-MS/MS. A stable isotope-labelled peptide was used as an internal standard. Finally, 6 samples from patients treated with emicizumab were quantified by LC-MS/MS and compared with those obtained with the modified one-stage activated partial prothrombin time technique (aPTT) based FVIII activity. The LC-MS/MS method was validated according to FDA recommendations. Good linearity of the calibration curves was observed over the range 5-150 µg/mL. The cross-validation showed an acceptable correlation of the developed LC-MS/MS method with the modified aPTT-based FVIII activity assay, and the Bland-Altman analysis did not show any significant bias.

Relationship Between Plasmatic Metformin Concentration and Renal Replacement Therapy: A Multicenter Cohort Study.

BACKGROUND: Metformin is the first-line treatment used for type 2 diabetes mellitus for more than 60 years. Metformin-associated lactic acidosis is the most serious adverse effect of metformin and is most widely defined as metabolic acidosis with elevated lactate levels in the presence of metformin. However, there is no consensus regarding the role of metformin in metformin-associated lactic acidosis onset. This study aimed to determine the metformin toxicity threshold (the metformin plasma concentration that predicts the occurrence of lactic acidosis) and the metformin dialysis threshold (the metformin plasma concentration strongly correlated with dialysis introduction). METHODS: This was a retrospective multicenter cohort study conducted from January 1, 2013, to December 31, 2020. All consecutive adult patients with at least one metformin-detectable blood concentration measurement were included. RESULTS: In total, 169 patients (92 men; mean age, 70 ± 11 years) were included in this study. A receiver operating characteristic analysis using Youden index showed that a metformin plasma concentration threshold of 17.9 mg/L was associated with lactic acidosis (sensitivity: 43.8%; specificity: 90.5%). Another receiver operating characteristic analysis using Youden index showed that a metformin plasma concentration threshold of 17.5 mg/L was associated with dialysis (sensitivity, 53.0%; specificity: 94.2%). CONCLUSIONS: The retrospective study design, lack of clinical data, and selection bias (patients in whom metformin was prescribed owing to pathological conditions) were major limitations, resulting in only preliminary findings. However, this study could serve as a basis for future prospective clinical studies to evaluate the use of these clinical threshold values as therapeutic guides.

TNF-α and IL-1ß Exposure Modulates the Expression and Functionality of P-Glycoprotein in Intestinal and Renal Barriers.

Inflammation is characterized by an increased secretion of proinflammatory cytokines known to alter the expression and functionality of drug transporters. Since P-glycoprotein (P-gp) plays a key role in the pharmacokinetics of several drugs, these modulations could further affect drug exposure. In this context, this study aims to investigate the impact of in vitro cytokine exposure on the expression and activity of P-gp using the intestinal model Caco-2 and the human renal cells RPTEC/TERT1. Cells were exposed to various concentrations of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß for 24 or 72 h. Gene expression was then assessed by RT-qPCR followed by absolute quantification of P-gp using liquid chromatography coupled with mass spectrometry. Then, the activity of P-gp was assessed by the intracellular accumulation of rhodamine 123. TNF-α increased both the gene expression and P-gp activity by 15-40% in each model. Minor modulations were observed at the protein level with increases of up to 8% for RPTEC/TERT1 cells and 24% for Caco-2 cells. Conversely, IL-1ß led to a downregulation of gene, protein, and functionality by 48 and 25% in intestinal and renal cells, respectively. Taken together, these data highlighted that gene expression levels and functional activity of P-gp are altered by the pro-inflammatory cytokines in intestinal and renal cells. Such pronounced changes in human P-gp could result in altered exposure to drug substrates. Further in vivo studies are needed to confirm the impact of inflammation on drug pharmacokinetics.