Irena Loryan

Influence of sex on propofol metabolism, a pilot study: implications for propofol anesthesia

PurposeThe basis of high intersubject variability of propofol metabolism is unclear. Therefore, we examined the influence of genetic polymorphisms of the key metabolizing enzymes cytochrome P450 2B6 (CYP2B6) and uridine diphosphate (UDP)-glucuronosyltransferase 1A9 (UGT1A9), age, and sex on propofol biotransformation in vitro and in vivo.MethodsPlasma concentrations of propofol, 4-hydroxypropofol, and their glucuronides were measured over 20 min in 105 patients after a single intravenous bolus of propofol. Propofol 4-hydroxylation activity, genotypes, and content of CYP2B6 protein in 68 human livers were determined. The common single nucleotide polymorphisms (SNPs) for the CYP2B6 and UGT1A9 genes were analyzed by polymerase chain reaction (PCR).ResultsPlasma levels of propofol metabolites showed high interindividual variability (range of coefficient of variation 89–128%). This was supported by in vitro data showing similar variability of propofol 4-hydroxylation in liver microsomes and 1.9-fold higher CYP2B6 protein content in the livers from women. No significant relationships were revealed between the SNPs studied and propofol metabolism. However, patients’ sex had a pronounced effect on propofol metabolism. Thus, women had higher amounts of propofol glucuronide (1.25-fold; p = 0.03), 4-hydroxypropofol-1-glucuronide (2.1-fold; p = 0.0009), and 4-hydroxypropofol-4-glucuronide (1.7-fold; p = 0.02) as shown by the weight-corrected area under the time–plasma concentration curve of metabolites. Additionally, the sexual dimorphism in 4-hydroxypropofol glucuronidation was prominent in the 35- to 64-year-old subgroup.ConclusionsNo significant effects of CYP2B6 and UGT1A9 SNPs or age on propofol metabolism were revealed in this pilot study, but there was a pronounced effect of sex, a finding that indicates an important factor for the previously described sex difference in systemic clearance of propofol seen.

Mechanistic Understanding of Brain Drug Disposition to Optimize the Selection of Potential Neurotherapeutics in Drug Discovery

ABSTRACTPurposeThe current project was undertaken with the aim to propose and test an in-depth integrative analysis of neuropharmacokinetic (neuroPK) properties of new chemical entities (NCEs), thereby optimizing the routine of evaluation and selection of novel neurotherapeutics.MethodsForty compounds covering a wide range of physicochemical properties and various CNS targets were investigated. The combinatory mapping approach was used for the assessment of the extent of blood-brain and cellular barriers transport via estimation of unbound-compound brain (Kp,uu,brain) and cell (Kp,uu,cell) partitioning coefficients. Intra-brain distribution was evaluated using the brain slice method. Intra- and sub-cellular distribution was estimated via calculation of unbound-drug cytosolic and lysosomal partitioning coefficients.ResultsAssessment of Kp,uu,brain revealed extensive variability in the brain penetration properties across compounds, with a prevalence of compounds actively effluxed at the blood-brain barrier. Kp,uu,cell was valuable for identification of compounds with a tendency to accumulate intracellularly. Prediction of cytosolic and lysosomal partitioning provided insight into the subcellular accumulation. Integration of the neuroPK parameters with pharmacodynamic readouts demonstrated the value of the proposed approach in the evaluation of target engagement and NCE selection.ConclusionsWith the rather easily-performed combinatory mapping approach, it was possible to provide quantitative information supporting the decision making in the drug discovery setting.

Molecular properties determining unbound intracellular and extracellular brain exposure of CNS drug candidates

In the present work we sought to gain a mechanistic understanding of the physicochemical properties that influence the transport of unbound drug across the blood-brain barrier (BBB) as well as the intra- and extracellular drug exposure in the brain. Interpretable molecular descriptors that significantly contribute to the three key neuropharmacokinetic properties related to BBB drug transport (Kp,uu,brain), intracellular accumulation (Kp,uu,cell), and binding and distribution in the brain (Vu,brain) for a set of 40 compounds were identified using partial least-squares (PLS) analysis. The tailoring of drug properties for improved brain exposure includes decreasing the polarity and/or hydrogen bonding capacity. The design of CNS drug candidates with intracellular targets may benefit from an increase in basicity and/or the number of hydrogen bond donors. Applying this knowledge in drug discovery chemistry programs will allow designing compounds with more desirable CNS pharmacokinetic properties.

Sex difference in formation of propofol metabolites : a replication study

Women recover faster from propofol anaesthesia and have been described to have a higher incidence of awareness during surgery, compared to men – an effect that may be inherent in sex differences in propofol metabolism. In an observational study, 98 ASA I‐II patients treated with continuous propofol infusion were recruited. The associations between sex and CYP2B6 and UGT1A9 polymorphisms with dose‐ and weight‐adjusted area under the total plasma level time curves (AUC) for propofol, and its metabolites propofol glucuronide (PG), 4‐hydroxypropofol (OHP) and hydroxyl glucuronide metabolites 4‐hydroxypropofol‐1‐O‐β‐D‐glucuronide (Q1G) and 4‐hydroxypropofol‐4‐O‐β‐D‐glucuronide (Q4G), were analysed. Significantly higher AUC of PG (1.3 times, p = 0.03), Q1G (2.9 times, p < 0.001), Q4G (2.4 times, p < 0.01) and OHP (4.6 times, p = 0.01) were found in women (n = 53) than in men (n = 45) after intravenous infusion of propofol using target‐controlled infusion system. There was, however, no significant impact of gene polymorphisms on propofol biotransformation. The results, which are supported by a previous pilot study using a propofol bolus dose, suggest that, compared to men, more rapid propofol metabolism may occur in women – a factor that may contribute to the mentioned differences in the efficacy of propofol anaesthesia between male and female patients.

Effect of transporter inhibition on the distribution of cefadroxil in rat brain

BackgroundCefadroxil, a cephalosporin antibiotic, is a substrate for several membrane transporters including peptide transporter 2 (PEPT2), organic anion transporters (OATs), multidrug resistance-associated proteins (MRPs), and organic anion transporting polypeptides (OATPs). These transporters are expressed at the blood–brain barrier (BBB), blood-cerebrospinal fluid barrier (BCSFB), and/or brain cells. The effect of these transporters on cefadroxil distribution in brain is unknown, especially in the extracellular and intracellular fluids within brain.MethodsIntracerebral microdialysis was used to measure unbound concentrations of cefadroxil in rat blood, striatum extracellular fluid (ECF) and lateral ventricle cerebrospinal fluid (CSF). The distribution of cefadroxil in brain was compared in the absence and presence of probenecid, an inhibitor of OATs, MRPs and OATPs, where both drugs were administered intravenously. The effect of PEPT2 inhibition by intracerebroventricular (icv) infusion of Ala-Ala, a substrate of PEPT2, on cefadroxil levels in brain was also evaluated. In addition, using an in vitro brain slice method, the distribution of cefadroxil in brain intracellular fluid (ICF) was studied in the absence and presence of transport inhibitors (probenecid for OATs, MRPs and OATPs; Ala-Ala and glycylsarcosine for PEPT2).ResultsThe ratio of unbound cefadroxil AUC in brain ECF to blood (Kp,uu,ECF) was ~2.5-fold greater during probenecid treatment. In contrast, the ratio of cefadroxil AUC in CSF to blood (Kp,uu,CSF) did not change significantly during probenecid infusion. Icv infusion of Ala-Ala did not change cefadroxil levels in brain ECF, CSF or blood. In the brain slice study, Ala-Ala and glycylsarcosine decreased the unbound volume of distribution of cefadroxil in brain (Vu,brain), indicating a reduction in cefadroxil accumulation in brain cells. In contrast, probenecid increased cefadroxil accumulation in brain cells, as indicated by a greater value for Vu,brain.ConclusionsTransporters (OATs, MRPs, and perhaps OATPs) that can be inhibited by probenecid play an important role in mediating the brain-to-blood efflux of cefadroxil at the BBB. The uptake of cefadroxil in brain cells involves both the influx transporter PEPT2 and efflux transporters (probenecid-inhibitable). These findings demonstrate that drug-drug interactions via relevant transporters may affect the distribution of cephalosporins in both brain ECF and ICF.

In-depth neuropharmacokinetic analysis of antipsychotics based on a novel approach to estimate unbound target-site concentration in CNS regions: link to spatial receptor occupancy

The current study provides a novel in-depth assessment of the extent of antipsychotic drugs transport across the blood–brain barrier (BBB) into various brain regions, as well as across the blood–spinal cord barrier (BSCB) and the blood–cerebrospinal fluid barrier (BCSFB). This is combined with an estimation of cellular barrier transport and a systematic evaluation of nonspecific brain tissue binding. The study is based on the new Combinatory Mapping Approach (CMA), here further developed for the assessment of unbound drug neuropharmacokinetics in regions of interest (ROI), referred as CMA-ROI. We show that differences exist between regions in both BBB transport and in brain tissue binding. The most dramatic spatial differences in BBB transport were found for the P-glycoprotein substrates risperidone (5.4-fold) and paliperidone (4-fold). A higher level of transporter-mediated protection was observed in the cerebellum compared with other brain regions with a more pronounced efflux for quetiapine, risperidone and paliperidone. The highest BBB penetration was documented in the frontal cortex, striatum and hippocampus (haloperidol, olanzapine), indicating potential influx mechanisms. BSCB transport was in general characterized by more efficient efflux compared with the brain regions. Regional tissue binding was significantly different for haloperidol, clozapine, risperidone and quetiapine (maximally 1.9-fold). Spatial differences in local unbound concentrations were found to significantly influence cortical 5-HT2A receptor occupancy for risperidone and olanzapine. In conclusion, the observed regional differences in BBB penetration may potentially be important factors contributing to variations in therapeutic effect and side effect profiles among antipsychotic drugs.

Drug Discovery Methods for Studying Brain Drug Delivery and Distribution

Methods used in drug discovery laboratories for assessing the delivery of small molecules to the brain have changed significantly in recent years. There is now more focus on measuring or estimating target unbound drug concentrations in the brain and evaluating the quantitative aspects of drug transport across the blood–brain barrier (BBB). The techniques for investigation of the rate and extent of BBB transport of new chemical entities (NCEs) are discussed in this chapter. Combinatory methodology for rapid mapping of the extent of brain drug delivery via assessment of the unbound drug brain partitioning coefficient is presented. The chapter also explains the procedures for approximation of subcellular distribution of NCEs, particularly into the lysosomes. The principles, technical issues, advantages, and potential applications of techniques for evaluation of intra-brain distribution, i.e., equilibrium dialysis-based brain homogenate and brain slice methods, are described. The assessment of extent of BBB transport and intracellular distribution of NCEs, the identification of intra-brain distribution patterns, and their integration with pharmacodynamic measurements are valuable implements for candidate evaluation and selection in drug discovery and development.

Rate and extent of mitragynine and 7-hydroxymitragynine blood-brain barrier transport and their intra-brain distribution: the missing link in pharmacodynamic studies: MG and 7-OHMG CNS exposure

Mitragyna speciosa is reported to be beneficial for the management of chronic pain and opioid withdrawal in the evolving opioid epidemic. Data on the blood–brain barrier (BBB) transport of mitragynine and 7‐hydroxymitragynine, the active compounds of the plant, are still lacking and inconclusive. Here, we present for the first time the rate and the extent of mitragynine and 7‐hydroxymitragynine transport across the BBB, with an investigation of their post‐BBB intra‐brain distribution. We utilized an in vitro BBB model to study the rate of BBB permeation of the compounds and their interaction with efflux transporter P‐glycoprotein (P‐gp). Mitragynine showed higher apical‐to‐basolateral (A‐B, i.e. blood‐to‐brain side) permeability than 7‐hydroxymitragynine. 7‐Hydroxymitragynine showed a tendency to efflux, with efflux ratio (B‐A/A‐B) of 1.39. Both were found to inhibit the P‐gp and are also subject to efflux by the P‐gp. Assessment of the extent of BBB transport in vivo in rats from unbound brain to plasma concentration ratios (Kp,uu,brain) revealed extensive efflux of both compounds, with less than 10 percent of unbound mitragynine and 7‐hydroxymitragynine in plasma crossing the BBB. By contrast, the extent of intra‐brain distribution was significantly different, with mitragynine having 18‐fold higher brain tissue uptake in brain slice assay compared with 7‐hydroxymitragynine. Mitragynine showed a moderate capacity to accumulate inside brain parenchymal cells, while 7‐hydroxymitragynine showed restricted cellular barrier transport. The presented findings from this systematic investigation of brain pharmacokinetics of mitragynine and 7‐hydroxymitragynine are essential for design and interpretation of in vivo experiments aiming to establish exposure–response relationship.

Quantitative Assessment of Drug Delivery to Tissues and Association with Phospholipidosis: A Case Study with Two Structurally Related Diamines in Development

Drug induced phospholipidosis (PLD) may be observed in the preclinical phase of drug development and pose strategic questions. As lysosomes have a central role in pathogenesis of PLD, assessment of lysosomal concentrations is important for understanding the pharmacokinetic basis of PLD manifestation and forecast of potential clinical appearance. Herein we present a systematic approach to provide insight into tissue-specific PLD by evaluation of unbound intracellular and lysosomal (reflecting acidic organelles) concentrations of two structurally related diprotic amines, GRT1 and GRT2. Their intratissue distribution was assessed using brain and lung slice assays. GRT1 induced PLD both in vitro and in vivo. GRT1 showed a high intracellular accumulation that was more pronounced in the lung, but did not cause cerebral PLD due to its effective efflux at the blood-brain barrier. Compared to GRT1, GRT2 revealed higher interstitial fluid concentrations in lung and brain, but more than 30-fold lower lysosomal trapping capacity. No signs of PLD were seen with GRT2. The different profile of GRT2 relative to GRT1 is due to a structural change resulting in a reduced basicity of one amino group. Hence, by distinct chemical modifications, undesired lysosomal trapping can be separated from desired drug delivery into different organs. In summary, assessment of intracellular unbound concentrations was instrumental in delineating the intercompound and intertissue differences in PLD induction in vivo and could be applied for identification of potential lysosomotropic compounds in drug development.

P.1.g.052 Understanding blood-brain barrier penetration and link to target engagement

V. Sinha, I. Loryan, P. De Boer, X. Langlois, C. Mackie, A. Van Peer, W. Drinkenburg, A. Vermeulen, D. Heald, M. Hammarlund-Udenaes Janssen Research and Development, Clinical Pharmacology, Beerse, Belgium Uppsala University, Translational PKPD Group Department of Pharmaceutical Biosciences, Uppsala, Sweden Janssen Research and Development, Experimental Medicine, Beerse, Belgium Janssen Research and Development, Neurosciences, Beerse, Belgium Janssen Research and Development, Pharmaceutical Development and Manufacturing Sciences, Beerse, Belgium Janssen Research and Development, Model Based Drug Development, Beerse, Belgium Janssen Research and Development, Clinical Pharmacology, Titusville, USA