Establishing a systematic framework to characterise in vitro methods for human hepatic metabolic clearance.

Hepatic metabolic clearance is one of the most important factors driving the overall kinetics of chemicals including substances used in various product categories such as pesticides, biocides, pharmaceuticals, and cosmetics. A large number of in vitro systems from purified isozymes and subcellular organelles to hepatocytes in simple cultures and in complex scaffold setups are available for measuring hepatic metabolic clearance for different applications. However, there is currently no approach for systematically characterising and comparing these in vitro methods in terms of their design, applicability and performance. To address this, existing knowledge in the field of in vitro human hepatic metabolic clearance methods was gathered and analysed in order to establish a framework to systematically characterise methods based on a set of relevant components. An analogous framework would be also applicable for non-human in vitro systems. The components are associated with the biological test systems used (e.g. subcellular or cells), the in vitro method (e.g. number of cells, test item solubility), related analytical techniques, data interpretation methods (based on substrate depletion/metabolite formation), and performance assessments (precision and accuracy of clearance measurements). To facilitate the regulatory acceptance of this class of methods, it is intended that the framework provide the basis of harmonisation work within the OECD.

Direct and quantitative evaluation of the human CYP3A4 contribution (fm) to drug clearance using the in vitro SILENSOMES model.

1. Among the different in vitro studies recommended by the regulatory agencies, no gold-standard model can easily and directly measure the quantitative CYP450 contributions to drug biotransformation. In this article, we propose an original strategy, called SilensomesTM, to produce human liver microsomes silenced for one specific CYP450, thanks to specific mechanism-based inhibitors (MBI). 2. Using azamulin as a specific CYP3A4 MBI, we demonstrated the proof of concept that CYP3A4 can be totally, specifically (even against 3A5) and permanently (at least for six years) inhibited by our process. Thus, comparing clearance in control and CYP3A4-SilensomesTM, CYP3A4 contributions were determined for 11 CYP3A4 substrates which correlated with known in vivo contributions and revealed accuracy with less than 10% error. In comparison, contributions determined using recombinant human CYP450 (rhCYP450s) were less accurate (more than 10% error for 30% of the tested CYP3A4 substrates). 3. This easy and ready-to-use in vitro method combines the advantages of existing models (specificity of rhCYP450s and representativeness of HLM) without their drawbacks. The same strategy could be used to silence other major CYP450s one-by-one to provide a complete direct CYP450 quantitative phenotyping kit.

Characterization of the comparative drug binding to intra- (liver fatty acid binding protein) and extra- (human serum albumin) cellular proteins.

1. This study compared the extent, affinity, and kinetics of drug binding to human serum albumin (HSA) and liver fatty acid binding protein (LFABP) using ultrafiltration and surface plasmon resonance (SPR). 2. Binding of basic and neutral drugs to both HSA and LFABP was typically negligible. Binding of acidic drugs ranged from minor (fu > 0.8) to extensive (fu < 0.1). Of the compounds screened, the highest binding to both HSA and LFABP was observed for the acidic drugs torsemide and sulfinpyrazone, and for ß-estradiol (a polar, neutral compound). 3. The extent of binding of acidic drugs to HSA was up to 40% greater than binding to LFABP. SPR experiments demonstrated comparable kinetics and affinity for the binding of representative acidic drugs (naproxen, sulfinpyrazone, and torsemide) to HSA and LFABP. 4. Simulations based on in vitro kinetic constants derived from SPR experiments and a rapid equilibrium model were undertaken to examine the impact of binding characteristics on compartmental drug distribution. Simulations provided mechanistic confirmation that equilibration of intracellular unbound drug with the extracellular unbound drug is attained rapidly in the absence of active transport mechanisms for drugs bound moderately or extensively to HSA and LFABP.

Comparative pharmacokinetics between a microdose and therapeutic dose for clarithromycin, sumatriptan, propafenone, paracetamol (acetaminophen), and phenobarbital in human volunteers.

A clinical study was conducted to assess the ability of a microdose (100 µg) to predict the human pharmacokinetics (PK) following a therapeutic dose of clarithromycin, sumatriptan, propafenone, paracetamol (acetaminophen) and phenobarbital, both within the study and by reference to the existing literature on these compounds and to explore the source of any nonlinearity if seen. For each drug, 6 healthy male volunteers were dosed with 100 µg (14)C-labelled compound. For clarithromycin, sumatriptan, and propafenone this labelled dose was administered alone, i.e. as a microdose, orally and intravenously (iv) and as an iv tracer dose concomitantly with an oral non-labelled therapeutic dose, in a 3-way cross over design. The oral therapeutic doses were 250, 50, and 150 mg, respectively. Paracetamol was given as the labelled microdose orally and iv using a 2-way cross over design, whereas phenobarbital was given only as the microdose orally. Plasma concentrations of total (14)C and parent drug were measured using accelerator mass spectrometry (AMS) or HPLC followed by AMS. Plasma concentrations following non-(14)C-labelled oral therapeutic doses were measured using either HPLC-electrochemical detection (clarithromycin) or HPLC-UV (sumatriptan, propafenone). For all five drugs an oral microdose predicted reasonably well the PK, including the shape of the plasma profile, following an oral therapeutic dose. For clarithromycin, sumatriptan, and propafenone, one parameter, oral bioavailability, was marginally outside of the normally acceptable 2-fold prediction interval around the mean therapeutic dose value. For clarithromycin, sumatriptan and propafenone, data obtained from an oral and iv microdose were compared within the same cohort of subjects used in the study, as well as those reported in the literature. For paracetamol (oral and iv) and phenobarbital (oral), microdose data were compared with those reported in the literature only. Where 100 µg iv (14)C-doses were given alone and with an oral non-labelled therapeutic dose, excellent accord between the PK parameters was observed indicating that the disposition kinetics of the drugs tested were unaffected by the presence of therapeutic concentrations. This observation implies that any deviation from linearity following the oral therapeutic doses occurs during the absorption process.