In detail

Bruxelles Woluwe

The expertise is thus mainly centered on a theme; the study of the fate of xenobiotics in the organism and the factors affecting it. Our projects cover together multiple fields of pharmacotherapy. These areas include mainly:  - Immunossupressants - Lipid lowering drugs - Anti-HIV - Anticoagulants - Antibiotic

Patient survival and graft outcome after kidney transplantation have drastically improved in recent decades, mainly because of major improvements in immuno-suppressive therapy. However, optimal immunosuppression is difficult to achieve in an individual patient. Indeed, the use of immunosuppressive drugs such as tacrolimus (Tac) is complicated by a high toxicity profile combined with a narrow therapeutic window. An important part of the variability observed in drug response is thought to be the consequence of substantial inter- but also intra-individual differences in drug PK. Some patients have relatively fast drug clearance; others exhibit a slower drug elimination rate, while some depict varying drug levels despite no dosage change. This variation in drug clearance is of importance, since it might be related to an increased risk of under- or overexposure, which can ultimately lead to a higher frequency of acute graft rejection or adverse events.

Although our previous discoveries in humans have led to personalize the initial Tac dose through new genotype-based dosage guidelines (see below, human studies), the residual unexplained PK variability is still substantial (>50%).

The importance of the gut microbiota for explaining the fate of Tac in the organism has been largely understudied. Not only gut microorganisms express numerous enzymes able to directly metabolize xenobiotics but also, they are able to control the host Absorption, Distribution, Metabolism and Excretion (ADME) phenotype through different processes.

One of our current projects in collaboration with Laure Bindels (MNUT), combines in vitro, in vivo and clinical investigations aiming at characterizing how Tac PK and host microbiota are interrelated. The aim is to shed light on the mechanisms linking the gut microbiota to the Tac inter but also intra-individual PK variability. In our first animal experiment, mice (n=10 per group) were treated with Tac (3mg/kg body weight) +/- an oral non-resorbable antibiotic (ATB) cocktail that ensures gut microbiota depletion (2,6 log10 reduction). Our results demonstrate that effective ATB-mediated microbiota depletion decreases Tac systemic exposure. This observation suggests that the gut microbiota influences TAC metabolism.

Tacrolimus Area under the concentration curve (AUC) at day 7 post-exposure in mice treated qith Tac alone or with AB cocktail.

 

Further experiments in mice are currently performed to investigate the etiology behind this important observation.

During the last decade, we demontrated that carriership of genetic variants in the Cytochrome P450-mediated drug metabolism is associated with a rough 30% reduction in in vivo metabolic activity and led to 50% lower tac dose requirements in patients. This observation led us to propose new dosage guidelines based on a validated popPK model in adult renal transplant, which can be useful in the frame of pre-emptive genotyping and dosage adjustment prior to transplantation.

New dosing guidelines for tacrolimus therapy in renal transplant recipients according to CYP3A genotype.

 

In collaboration with the CUSL (Prof. Michel Mourad), We are currently conducting a clinical study investigating the potential influence of a patient’s microbiome on the tac dose requirement and the response to IS treatment to try to unravel the reason for the PK variability unexplained by host factors. We aim at recruiting 100 patients under Tac-based IS therapy (at present, n=50). We expect that this project will lead us to validate new biomarkers of Tac (and other IS drugs) PK variabilities and help refining our dosage recommendations. Moreover, important microbial biomarker identification might also become an asset for improving drug therapy, with the possible inclusion of e.g. antimicrobials for decreasing certain species, or probiotics in order to promote the most useful ones.

Since cardiovascular diseases are a real public health problem, lipid lowering medication are widely used to decrease cholesterol and triglyceride levels in the general population. There is, however, a great interindividual variation in response to therapy that is not mastered. Again, data suggest that a part of this variability might be attributed to PK differences. Atorvastatin is the world’s bestselling drug of all time. However, despite this clinical success, and although doses are titrated according to cholesterol measurements, many individuals are unable to reach their respective targeted cholesterol levels. In addition, many patients suffer from side effects, and up to 10% of patients taking atorvastatin experienced muscle-related adverse drug reactions (ADRs) 

 

In vitro investigations

The pathophysiology of statin-induced myopathy is fairly understood and local PK mechanisms determining drug cellular accumulation remain largely unexplored. To get into myocytes atorvastatin undergoes passive diffusion but also active transport. The influx protein OATP2B1 and the efflux proteins MRP1, MRP4 and are expressed at the sarcolemmal membrane of skeletal muscle fibres. We have developed recombinant HEK293 cellular models overexpressing either OATP2B1 or MRP1 or both transporters. 

(a)HEK293 non-transfected cells (b) stable double transfectants overexpressing OATP2B1 (OFP spark-red fluorescent tag) and MRP1 (Green Fluorescent protein [GFP] tag) and (c) stable single transfectant overexpressing an OATP2B1 variant.

In collaboration with the group of Giulio Muccioli (BPBL), we have demonstrated that Atorvastatin is a good substrate of these 2 efflux pumps as we observed a significant increase in Atorvastatin intracellular concentrations in OATP2B1 (influx) overexpressing cells whereas overexpression of MRP1 (efflux) was associated with a significantly decreased accumulation (data not shown).

Intracellular accumulation of atorvastatin in HEK293 (black) and in recombinant overexpressing OATP2B1 (red) after 2h of incubation with increasing concentrations of atorvastatin.

As both OATP and MRP transporters generate opposite drug transport, we have also assessed the combined impact of these transporters when they are co-expressed. In those double transfectants, we were able to show that ATV MRP1-efflux counteract OATP2B1 influx.

Intracellular accumulation of atorvastatin in HEK293 (black), in HEK293 overexpressing OATP2B1 (red) and HEK overexpressing both OATP2B1 and MRP1 (green) after 2h of incubation with increasing concentrations of atorvastatin.

Our next move was to introduce natural genetic variations in the cDNA of those proteins (OATP2B1 or MRP1) and to analyze the functional consequences of these SNPs on the intracellular PK of atorvastatin. With those mutagenetic experiments, we have pinpointed two natural genetic variations significantly affecting either MRP1 or OATP2B1 activity towards Atorvastatin.

Intracellular accumulation of atorvastatin in HEK293 (black), in HEK293 overexpressing WT OATP2B1+MRP1 (green), HEK overexpressing WT OATP2B1 but MRP1 variant (rs45511401) (purple) and HEK overexpressing WT MRP1 but OATP2B1 variant (rs12422149) after 2h of incubation with increasing concentrations of atorvastatin.

Finally, we plan to transpose and develop recombinant cultured primary differentiated human skeletal muscle myoblasts (HSMM) characterized by physiological expression of drug transporters in order to weight the consequences of transporter modulation (DDI, SNPs…) on drug accumulation and myocyte toxicity with specific biomarkers.

 

Human studies

To pursue these investigations a step further, the group has collaborated with Prof. Dr JL Balligand (FATH, IREC) to unravel the reasons for PK variability of atorvastatin in clinics. In this projetc, we also work with Prof Giulio Muccioli (BPBL) for the analytical part of the project and with Prof Vincent Haufroid (LTAP, IREC) for the pharmacogenetic aspect. 
Our collaborative project aims at deciphering the potential of popPK for optimization of statin therapy. Our prospective study now includes 83 patients treated with atorvastatin for hypercholesterolemia. In those patients, drug and metabolites measurements were performed at up ty 3 visits and patients were genotyped for some important biotransformation and transporter protein genes (e.g. CYP3A, ABCs, SLCOs…). PK results show a high variability in both atorvastatin and its metabolites concentrations.

Plasma concentrations of Atorvastatin (red), atorvastatin lactone (purple), 4- (light green) and 2- (blue) hydroxy-Atorvastatin in 83 patients.

By combining those data with a more rich-sampled cohort (collaboration, Anders Asberg, Oslo University Hospital), we have developed a two-compartmental PopPK model and tested the influence of covariates on PK parameters such as apparent clearance (CL/F). Our primary analysis indicates that CL/F was reduced in carriers of the SLCO1B1 521C allele compared to carriers of the T allele. The next step will be to model the PK-PD relationship and test whether genetic polymorphisms influence the clinical response to the drug.

Human studies

In close collaboration with the infectious disease unit of CUSL, in 2016, in a pilot study involving 135 patients treated with Darunavir, a potent protease inhibitor, we have demonstrated that significant PK drug-drug interaction exists between Darunavir and Etravirine, another coadministrated anti-HIV drug. We have also highlighted that this interaction is partly mediated by genetic polymorphisms in CYP3A5. Aside, in this study, we have shown that anti-HIV drugs accumulates differentially in circulating lymphocytes and that, for instance, Etravirine accumulates more efficiently in PBMCs compared to Darunavir. This is particularly important as lymphocytes represent the site where the drug exerts his therapeutic action. In a recent study, we have characterized darunavir PK by developing a population model based on data collected in a large cohort of 140 Darunavir treated HIV-infected patients. Alpha-1 acid glycoprotein level, sex, and genetic polymorphisms in the CYP3A5 and SLCO3A1 genes were found to be significant predictors of darunavir PK. The model was thoroughly evaluated using internal and external validation techniques.

The model also allowed us to simulate the effect of alternative dose regimens in populations representative of clinical practice. A reduction of the standard 800 mg once-daily dosage to 600 or 400 mg once-daily was found to be safe in a large proportion of patients. On the other hand, intermittent therapy (five out of seven days) constituted an unsafe option in most subjects. Whether individual patients could benefit from these alternative regimens could be predicted by our model. Additionally, optimal sampling strategies for darunavir were derived, showing how to best design future studies or how to optimize therapeutic monitoring for this drug. The following step would be to evaluate the appropriateness of these new recommendations in a prospective randomized study.