Microsomal protein per gram of liver (MPPGL) in paediatric biliary atresia patients.

The microsomal protein per gram of liver (MPPGL) is an important scaling factor in the in vitro-in vivo extrapolation of metabolic data obtained in liver microsomes. This study aimed to determine the MPPGL in four biliary atresia patients (0.6-1.6 years old) undergoing liver transplantation, as it is known that the MPPGL is affected by age and possibly by liver disease. Due to the presence of bilirubin in the homogenates and microsomes, the NADPH-cytochrome reductase activity was used to determine the recovery factor, rather than methods using the dithionite difference spectrum. A mean value of 18.73 (± 2.82) mg/g (geometric mean ± SD, n = 4) was observed, which is lower than the expected MPPGL based on the age of the patients (26.60 ± 0.40 mg/g). This suggests a decreased amount of microsomal protein in the livers of biliary atresia patients. Moreover, no differences in MPPGL between different zones of the liver could be detected.

In vitro cytochrome p450 activity decreases in children with high pediatric end-stage liver disease scores.

To improve the modeling and simulation of pharmacokinetics in pediatric patients, research into developmental and disease-specific determinants is needed. This article describes the evaluation of the activity of in vitro cytochrome P450 (P450), an important enzyme family in drug metabolism, in children with hepatic dysfunction. The activity of six P450 isoforms (CYP1A2, 2C9, 2C19, 2D6, 2E1, and 3A4) was evaluated in 31 patients with different pathologies, predominantly biliary atresia (n = 23). Hypervariable activity was observed for all the isoforms. Compared with average adult activity, low activity levels were seen for CYP1A2, 2C19, 2E1, and 3A4. For CYP2E1 and 3A4, a positive correlation between activity and abundance was observed. Age, comedication, and genotype could not be used as predictors for P450 activity in this patient population. In contrast, the pediatric end-stage liver disease score was negatively correlated with the ln(activity). This finding suggests a decrease in P450 activity with deteriorating hepatic function. Moreover, the activity of all isoforms was correlated, demonstrating a concomitant decrease of all isoforms in young patients with liver disease. To our knowledge, this is the first study to evaluate P450 activity in children with hepatic impairment. The presented data may provide support in the further optimization of a disease-specific model in this patient population.

The Introduction of a Full Medication Review Process in a Local Hospital: Successes and Barriers of a Pilot Project in the Geriatric Ward.

For the majority of Belgian hospitals, a pharmacist-led full medication review process is not standard care and, therefore, challenging to introduce. With this study, we aimed to evaluate the successes and barriers of the implementation of a pharmacist-led full medication review process in the geriatric ward at a local Belgian hospital. To this end, we carried out an interventional study, performing a full medication review on older patients (≥70 years) with polypharmacy (≥5 drugs) who had an unplanned admission to the geriatric ward. The process consisted of 3 steps: (1) medication reconciliation upon admission; (2) medication review using an explicit reviewing tool (STOPP/START criteria or GheOP³S tool), followed by a discussion between the pharmacist and the geriatrician; and (3) medication reconciliation upon discharge. Ethical approval was obtained from the Ethical Commission of the Ghent University Hospital. Outcomes included objective data on the interventions (e.g., number of drug discrepancies; number of potentially inappropriate prescriptions (PIP)); as well as subjective experiences (e.g., satisfaction with service; opinion on inter-professional communication). There was a special focus on communication aspects within the introduction of this process. In total, 52 patients were included in the study, taking a median of 10 drugs (IQR 8-12). Upon admission, 122 drug discrepancies were detected. During medication review, 254 PIPs were detected and discussed, leading to an improvement in the appropriateness of medication use. The satisfaction of community pharmacists concerning additional communication and the satisfaction of the patients after counselling at discharge were positive. However, several barriers were encountered, such as the time-consuming process to gather necessary information from different sources, the non-continuity of the service due to the lack of trained personnel or the lack of safe, electronic platforms to share information. The communicative and non-communicative successes and hurdles encountered during this project need to be addressed in order to improve the full medication review process and to strengthen the role of the clinical pharmacist.

The Ontogeny of Cytochrome P450 Enzyme Activity and Protein Abundance in Conventional Pigs in Support of Preclinical Pediatric Drug Research.

Since the implementation of several legislations to improve pediatric drug research, more pediatric clinical trials are being performed. In order to optimize these pediatric trials, adequate preclinical data are necessary, which are usually obtained by juvenile animal models. The growing piglet has been increasingly suggested as a potential animal model due to a high degree of anatomical and physiological similarities with humans. However, physiological data in pigs on the ontogeny of major organs involved in absorption, distribution, metabolism, and excretion of drugs are largely lacking. The aim of this study was to unravel the ontogeny of porcine hepatic drug metabolizing cytochrome P450 enzyme (CYP450) activities as well as protein abundances. Liver microsomes from 16 conventional pigs (8 males and 8 females) per age group: 2 days, 4 weeks, 8 weeks, and 6-7 months were prepared. Activity measurements were performed with substrates of major human CYP450 enzymes: midazolam (CYP3A), tolbutamide (CYP2C), and chlorzoxazone (CYP2E). Next, the hepatic scaling factor, microsomal protein per gram liver (MPPGL), was determined to correct for enzyme losses during the fractionation process. Finally, protein abundance was determined using proteomics and correlated with enzyme activity. No significant sex differences within each age category were observed in enzyme activity or MPPGL. The biotransformation rate of all three substrates increased with age, comparable with human maturation of CYP450 enzymes. The MPPGL decreased from birth till 8 weeks of age followed by an increase till 6-7 months of age. Significant sex differences in protein abundance were observed for CYP1A2, CYP2A19, CYP3A22, CYP4V2, CYP2C36, CYP2E_1, and CYP2E_2. Midazolam and tolbutamide are considered good substrates to evaluate porcine CYP3A/2C enzymes, respectively. However, chlorzoxazone is not advised to evaluate porcine CYP2E enzyme activity. The increase in biotransformation rate with age can be attributed to an increase in absolute amount of CYP450 proteins. Finally, developmental changes were observed regarding the involvement of specific CYP450 enzymes in the biotransformation of the different substrates.

Strategies for Determining Correct Cytochrome P450 Contributions in Hepatic Clearance Predictions: In Vitro-In Vivo Extrapolation as Modelling Approach and Tramadol as Proof-of Concept Compound.

BACKGROUND AND OBJECTIVE: Although the measurement of cytochrome P450 (CYP) contributions in metabolism assays is straightforward, determination of actual in vivo contributions might be challenging. How representative are in vitro for in vivo CYP contributions? This article proposes an improved strategy for the determination of in vivo CYP enzyme-specific metabolic contributions, based on in vitro data, using an in vitro-in vivo extrapolation (IVIVE) approach. Approaches are exemplified using tramadol as model compound, and CYP2D6 and CYP3A4 as involved enzymes. METHODS: Metabolism data for tramadol and for the probe substrates midazolam (CYP3A4) and dextromethorphan (CYP2D6) were gathered in human liver microsomes (HLM) and recombinant human enzyme systems (rhCYP). From these probe substrates, an activity-adjustment factor (AAF) was calculated per CYP enzyme, for the determination of correct hepatic clearance contributions. As a reference, tramadol CYP contributions were scaled-back from in vivo data (retrograde approach) and were compared with the ones derived in vitro. In this view, the AAF is an enzyme-specific factor, calculated from reference probe activity measurements in vitro and in vivo, that allows appropriate scaling of a test drug's in vitro activity to the 'healthy volunteer' population level. Calculation of an AAF, thus accounts for any 'experimental' or 'batch-specific' activity difference between in vitro HLM and in vivo derived activity. RESULTS: In this specific HLM batch, for CYP3A4 and CYP2D6, an AAF of 0.91 and 1.97 was calculated, respectively. This implies that, in this batch, the in vitro CYP3A4 activity is 1.10-fold higher and the CYP2D6 activity 1.97-fold lower, compared to in vivo derived CYP activities. CONCLUSION: This study shows that, in cases where the HLM pool does not represent the typical mean population CYP activities, AAF correction of in vitro metabolism data, optimizes CYP contributions in the prediction of hepatic clearance. Therefore, in vitro parameters for any test compound, obtained in a particular batch, should be corrected with the AAF for the respective enzymes. In the current study, especially the CYP2D6 contribution was found, to better reflect the average in vivo situation. It is recommended that this novel approach is further evaluated using a broader range of compounds.

Age-related Differences in CYP3A Abundance and Activity in the Liver of the Göttingen Minipig.

In view of paediatric drug development, regulatory authorities often request safety studies in juvenile animals, including minipigs. Unfortunately, knowledge on the ontogeny of the biotransformation processes in animal models remains scarce and impedes a correct interpretation of the toxicity findings. CYP3A4 is one of the most important drug-metabolizing enzymes in human beings and shows important similarities with CYP3A in the minipig. Therefore, the aim of this study was to assess the abundance and activity of CYP3A in liver microsomes from foetal, juvenile (days 1, 3, 7 and 28) and adult male and female Göttingen minipigs. CYP3A abundance was studied by an indirect enzyme-linked immunosorbent assay (ELISA), whereas CYP3A activity was assessed by a biotransformation assay with Luciferin-IPA. CYP3A abundance could not be detected until day 3. From day 7 onwards, a gradual increase in expression was noted, leading to the highest abundance in adult animals. CYP3A activity was not detectable in foetuses and 1-day-old animals. The CYP3A activity was detectable, but below the LLOQ in day 3 animals and increased gradually with age to reach the highest level in adults. The CYP3cide and ketoconazole inhibition, and testosterone and midazolam reduction of Luciferin-IPA metabolism in minipig liver microsomes substantiate that Luciferin-IPA is metabolized by CYP3A in minipigs. A positive correlation was found between CYP3A abundance and biotransformation of Luciferin-IPA (Pearson r = 0.863; p < 0.0001). In conclusion, both abundance and activity of CYP3A increased gradually in juvenile minipigs, but remained below the levels observed in adult animals.

Quantification of cytochrome 2E1 in human liver microsomes using a validated indirect ELISA.

CYP2E1 is an important cytochrome P450 isoform in many endogenous processes and in the metabolism of organic solvents, a number of drugs and pre-carcinogens. Information on the abundance of the enzyme may be valuable in various types of research in the field of toxicology and pharmacology. An indirect ELISA for the quantification of CYP2E1 in human liver microsomes was developed and successfully validated. All samples, including validation samples and calibrators, were diluted to a final concentration of microsomal protein of 10µg/ml. Detection of the antigen was obtained through binding of a polyclonal antibody raised against the full length protein, followed by the addition of horseradish peroxidase conjugated secondary antibodies and enzymatic detection. A five-parameter logistics function with 1/x weighting was used for quantification within the concentration range of 4-256pmol CYP2E1/mg microsomal protein. The method showed acceptable intra- and inter-assay precision, with calculated coefficients of variation of 6.3-15.2% and 11.3-21.0%, respectively. The relative error varied between -2.3 and 8.9%, and the total error between 16.0 and 27.2%. No significant cross reactivity with other abundant CYP isoforms was observed. The method was evaluated through the analysis of samples from a pharmacokinetic study, and the comparison with the CYP2E1 activity in those samples.

Enzymatic tumour tissue digestion coupled to SPE-UPLC-Tandem Mass Spectrometry as a tool to explore paclitaxel tumour penetration.

Paclitaxel is a good compound for regional (intraperitoneal) chemotherapy of peritoneal carcinomatosis. During IPEC, a cytotoxic solution is circulated in the peritoneal cavity, thereby promoting close contact between the cytotoxic agent and the exposed (residual) tumour tissue. To further explore the role of PTX in this type of treatment and study the impact of treatment modalities on tumour tissue penetration, in-vivo animal experiments were set-up. In literature, PTX tumour uptake is frequently studied using autoradiography and/or fluorescence microscopy techniques. Owing to their semi-quantitative nature on one hand and the difficulty of incorporating imaging data within a pharmacokinetic-pharmacodynamic modelling framework on the other hand, we set out to develop a validated assay for the quantification of PTX in tumour tissue samples. Furthermore, in order to maximise spatial resolution, care was taken to minimise the sample weight necessary for the analysis. Based on an enzymatic tumour tissue digestion protocol, an easy, less labour-intensive, when compared to mechanical tissue disruption techniques, method was developed. Through validation experiments we showed that our method reliably quantifies PTX in a working range of 30-8000 ng/g tumour tissue. Finally, using samples from the in-vivo experiments we demonstrated the suitability of the developed method.

Development and validation of a fast and uniform approach to quantify ß-lactam antibiotics in human plasma by solid phase extraction-liquid chromatography-electrospray-tandem mass spectrometry.

Monitoring of plasma antibiotic concentrations is necessary for individualization of antimicrobial chemotherapy dosing in special patient populations. One of these special populations of interest are the post-bariatric surgery patients. Until today, little is known on the effect of this procedure on drug disposition and efficacy. Therefore, close monitoring of antimicrobial plasma concentrations in these patients is warranted. A fast and uniform ultra-high-performance liquid chromatography (UPLC) method with tandem mass spectrometric detection (MS/MS) has been developed and qualified for the simultaneous quantification of ß-lactam antibiotics in human plasma. Compounds included in this multi-component analysis are: amoxicillin, ampicillin, phenoxymethylpenicillin, piperacillin, cefuroxime, cefadroxil, flucloxacillin, meropenem, cefepime, ceftazidime, tazobactam, linezolid and cefazolin. After spiking of five different stable isotope labelled internal standards, plasma samples were prepared for UPLC-MS/MS analysis by mixed-mode solid phase extraction. The developed method was proven to be free of (relative) matrix effects and proved to be reliable for the quantification of 12 out of 13 ß-lactam antibiotics. As a proof of concept the method has been applied to plasma samples obtained from a healthy volunteer treated with amoxicillin. The analytical method is suitable for use in a therapeutic drug monitoring setting, providing the clinician with reliable measurements on ß-lactam antibiotic plasma concentrations in a timely manner.

Metabolism of hop-derived bitter acids.

In this study, in vitro metabolism of hop-derived bitter acids was investigated. Besides their well-known use as bitter compounds in beer, in several studies, bioactive properties have been related to these types of molecules. However, scientific data on the absorption, distribution, metabolism, and excretion aspects of these compounds are limited. More specific, in this study, α-acids, ß-acids, and iso-α-acids were incubated with rabbit microsomes, and fractions were subjected to LC-MS/MS analysis for identification of oxidative biotransformation products. Metabolism of ß-acids was mainly characterized by conversion into hulupones and the formation of a series of tricyclic oxygenated products. The most important metabolites of α-acids were identified as humulinones and hulupones. Iso-α-acids were found to be primarly metabolized into cis- and trans-humulinic acids, next to oxidized alloiso-α-acids. Interestingly, the phase I metabolites were highly similar to the oxidative degradation products in beer. These findings show a first insight into the metabolites of hop-derived bitter acids and could have important practical implications in the bioavailability aspects of these compounds, following ingestion of hop-based food products and nutraceuticals.

Development and validation of an enzyme-linked immunosorbent assay for the quantification of cytochrome 3A4 in human liver microsomes.

Little is known about the influence of hepatic pathologies on cytochrome P450 (CYP) mediated drug metabolism in children. The determination of the abundance of the different isoforms in pediatric microsomes may provide valuable information on the mechanisms of possible changes in activity. Until now, western blotting was mostly used for abundance measurements, but this technique only provides semi-quantitative data. Therefore, this study aimed to develop and validate an indirect ELISA for the quantification of the most important CYP isoform, CYP3A4, in human liver microsomes, using commercially available reagents. Samples, calibrators and validation samples were diluted to a final concentration of 10 µg microsomal protein/ml. A polyclonal antibody raised against the full length human protein was used as primary antibody; horseradish peroxidase conjugated secondary antibodies for detection. The assay was validated for sensitivity, working range and calibration, accuracy and precision. Amounts of CYP3A4 between 2 and 300 pmol/mg microsomal protein could be quantified with a 5-parameter logistics function with 1/x weighting factor. Coefficients of variation of intra and inter assay variability were between 9.54 and 13.98% (16.34% at LLOQ), and between 10.51 and 14.55% (19.44% at LLOQ), respectively. The relative error (%RE) varied between -5.96 and 6.68% (11.53% at LLOQ), and the total error between 11.93 and 21.23% (30.97% at LLOQ). The cross-reactivity of the method with human CYP2E1 showed to have no significant effect on the accuracy of the results. Successful analysis of five samples from an ongoing study demonstrated the usefulness of the method.

Development and validation of a fast and sensitive UPLC-MS/MS method for the quantification of six probe metabolites for the in vitro determination of cytochrome P450 activity.

A fast and sensitive UPLC-MS/MS method was developed and validated for the simultaneous quantification of six probe metabolites for the in vitro cytochrome P450 activity determination in hepatic microsomes from patients with hepatic impairment. The metabolites acetaminophen (CYP1A2), 4'-hydroxy-mephenytoin (CYP2C19), 4-hydroxy-tolbutamide (CYP2C9), dextrorphan (CYP2D6), 6-hydroxy-chlorzoxazone (CYP2E1) and 1-hydroxy-midazolam (CYP3A4), together with the internal standard chlorpropamide, were separated on a Waters Acquity UPLC BEH C18 column (50 mm × 2.1mm, 1.7 µm particle size) with VanGuard pre-column (5 mm × 2.1mm, 1.7 µm particle size). A short gradient elution (total run time of 5.25 min), using water with 0.1% formic acid (eluent A) and acetonitrile with 0.1% formic acid (eluent B) at a flow rate of 400 µl/min, was used. The metabolites were detected with a triple quadrupole mass spectrometer in the multiple reaction monitoring mode. Two runs, one in the positive ionization mode and one in the negative mode, were necessary for the detection of all metabolites. The method was selective and showed good accuracy (84.59-109.83%) and between-day (RSD%<5.13%) and within-day (RSD%<9.60%) precision. The LOQ was in full accordance with the intended application, and no relative matrix effects were observed. Also, the sample incubation extracts were stable after three freeze-thaw cycles. The usability of the method was demonstrated by the incubation of pediatric microsomes with subsequent quantification of the formed metabolites and CYP activity calculation.

In vitro cytochrome P450 activity: development and validation of a sensitive high performance liquid chromatography-tandem mass spectrometry method for the quantification of six probe metabolites after derivatization with pyridine-3-sulfonyl chloride in an aqueous environment.

For the determination of the in vitro cytochrome P450 activity in microsomes, a quantification method for the probe metabolites, formed during incubation, is required. Due to insufficient sensitivity of a previously developed high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for some of the metabolites, a fast and easy derivatization method with pyridine-3-sulfonyl chloride (PS) is described. Acetaminophen (CYP1A2), dextrorphan (CYP2D6), hydroxy-chlorzoxazone (CYP2E1) and hydroxy-mephenytoin (CYP2C19) can be derivatized because of the presence of a phenolic OH, whereas hydroxy-midazolam (CYP3A4) and hydroxy-tolbutamide (CYP2C9) remain unchanged. As PS improves the ionization efficiency in the positive electrospray ionization (ESI) mode, the sensitivity of the detection is improved significantly and meets requirements for the activity determination. Native negative electrospray type molecules, moreover, become positive ESI candidates. The direct derivatization in the aqueous incubation medium, without any other sample pre-treatment steps, such as evaporation or extraction, makes this procedure easy to perform. The method using 20s microwave irradiation was shown to equal a 10min reaction in a 60°C heating block, consequently simplifying and shortening the process. Collision induced fragmentation of the derivatives resulted in at least one native compound, rather than derivative, specific product ion, thereby improving the selectivity of the method in the multiple reaction monitoring mode. The HPLC-MS/MS method was validated, and was demonstrated to be sensitive, selective, precise and accurate. The absence of a relative matrix effect was established, notwithstanding that an absolute matrix effect was observed. The analysis of a sample after microsomal incubation, from which some of the metabolites could not be quantified using the method without derivatization, proved the usefulness of the method.