No detrimental association between antibiotic use and immune checkpoint inhibitor therapy: an observational cohort study comparing patients with ICI-treated and TKI-treated melanoma and NSCLC.

BACKGROUND: The role of antibiotics in malignancies treated with immune checkpoint inhibitors (ICI) remains unclear. Several studies suggested a detrimental impact of antibiotic use on the response to ICI, but were susceptible to confounding by indication. Our objective was therefore to assess whether the relationship between antibiotic use and ICI response is causative or merely associative. METHODS: A large, single-center observational cohort study was performed with individuals treated for either non-small cell lung carcinoma (NSCLC) or metastatic melanoma. An effect modification approach was used, aiming to estimate the association between antibiotic use and overall survival (OS) and compare these estimates between individuals receiving first-line ICI treatment versus those receiving first-line tyrosine kinase inhibitors (TKIs). Exposure of interest was antibiotic use within 30 days before the start of anticancer treatment. HRs for OS were estimated for antibiotics versus no antibiotics in each cohort using multivariable propensity adjusted analysis. The "true antibiotic effect" within the ICI versus TKI cohort was modeled using an interaction term. RESULTS: A total of 4534 patients were included, of which 1908 in the ICI cohort and 817 in the TKI cohort. Approximately 10% of patients in each cohort used antibiotics within 30 days before the start of anticancer treatment. Our results demonstrate a lack of synergistic interaction between current antibiotic use and ICI therapy in relation to OS: although antibiotic use was significantly associated with OS decline in the ICI cohort (HR=1.26 (95% CI 1.04 to 1.51)), a similar magnitude in OS decline was found within the TKI cohort (HR=1.24 (95% CI 0.95 to 1.62)). This was reflected by the synergy index (HR=0.96 (95% CI 0.70 to 1.31)), which implied no synergistic interaction between current antibiotic use and ICI. CONCLUSION: This study strongly suggests that there is no causal detrimental association between antibiotic use and ICI therapy outcome when looking at OS in individuals with malignant melanoma or NSCLC. The frequently observed inverse association between antibiotics and ICI response in previous studies is most likely driven by confounding by indication, which was confirmed by the findings in our reference TKI cohort.

A robust mRNA signature obtained via recursive ensemble feature selection predicts the responsiveness of omalizumab in moderate-to-severe asthma.

BACKGROUND: Not being well controlled by therapy with inhaled corticosteroids and long-acting ß2 agonist bronchodilators is a major concern for severe-asthma patients. The current treatment option for these patients is the use of biologicals such as anti-IgE treatment, omalizumab, as an add-on therapy. Despite the accepted use of omalizumab, patients do not always benefit from it. Therefore, there is a need to identify reliable biomarkers as predictors of omalizumab response. METHODS: Two novel computational algorithms, machine-learning based Recursive Ensemble Feature Selection (REFS) and rule-based algorithm Logic Explainable Networks (LEN), were used on open accessible mRNA expression data from moderate-to-severe asthma patients to identify genes as predictors of omalizumab response. RESULTS: With REFS, the number of features was reduced from 28,402 genes to 5 genes while obtaining a cross-validated accuracy of 0.975. The 5 responsiveness predictive genes encode the following proteins: Coiled-coil domain- containing protein 113 (CCDC113), Solute Carrier Family 26 Member 8 (SLC26A), Protein Phosphatase 1 Regulatory Subunit 3D (PPP1R3D), C-Type lectin Domain Family 4 member C (CLEC4C) and LOC100131780 (not annotated). The LEN algorithm found 4 identical genes with REFS: CCDC113, SLC26A8 PPP1R3D and LOC100131780. Literature research showed that the 4 identified responsiveness predicting genes are associated with mucosal immunity, cell metabolism, and airway remodeling. CONCLUSION AND CLINICAL RELEVANCE: Both computational methods show 4 identical genes as predictors of omalizumab response in moderate-to-severe asthma patients. The obtained high accuracy indicates that our approach has potential in clinical settings. Future studies in relevant cohort data should validate our computational approach.

A novel undergraduate biomedical laboratory course concept in synergy with ongoing faculty research.

Optimal integration of education and ongoing faculty research in many undergraduate science programs is limited to the capstone project. Here, we aimed to develop a novel course-based undergraduate research experience (CURE) in synergy with ongoing faculty research. This 10-week course called Biomedical Research Lab is embedded in the curriculum of the undergraduate program Biomedical Sciences and grounded in the theoretical framework of research-based learning. Four groups of four students work together in a dedicated laboratory on an actual ongoing research problem of faculty. All groups work on the same research problem, albeit from different (methodological) perspectives, thereby stimulating interdependence between all participants. Students propose new research, execute the experiments, and collectively report in a single research article. According to students, the course enhanced scientific, laboratory, and academic skills. Students appreciated ownership and responsibilities of the research, laboratory teachers as role models, and they were inspired and motivated by doing authentic actual research. The course resulted in a better understanding of what doing research entails. Faculty valued the didactical experience, research output and scouting opportunities. Since topics can change per course edition, we have showcased a widely applicable pedagogy creating synergy between ongoing research and undergraduate education.

Implementation of Competency-Based Pharmacy Education (CBPE).

Implementation of competency-based pharmacy education (CBPE) is a time-consuming, complicated process, which requires agreement on the tasks of a pharmacist, commitment, institutional stability, and a goal-directed developmental perspective of all stakeholders involved. In this article the main steps in the development of a fully-developed competency-based pharmacy curriculum (bachelor, master) are described and tips are given for a successful implementation. After the choice for entering into CBPE is made and a competency framework is adopted (step 1), intended learning outcomes are defined (step 2), followed by analyzing the required developmental trajectory (step 3) and the selection of appropriate assessment methods (step 4). Designing the teaching-learning environment involves the selection of learning activities, student experiences, and instructional methods (step 5). Finally, an iterative process of evaluation and adjustment of individual courses, and the curriculum as a whole, is entered (step 6). Successful implementation of CBPE requires a system of effective quality management and continuous professional development as a teacher. In this article suggestions for the organization of CBPE and references to more detailed literature are given, hoping to facilitate the implementation of CBPE.

The in-vitro effect of complementary and alternative medicines on cytochrome P450 2C9 activity.

OBJECTIVES: The aim of this study is to establish the inhibitory effects of 14 commonly used complementary and alternative medicines (CAM) on the metabolism of cytochrome P450 2C9 (CYP2C9) substrates 7-methoxy-4-trifluoromethyl coumarine (MFC) and tolbutamide. CYP2C9 is important for the metabolism of numerous drugs and inhibition of this enzyme by CAM could result in elevated plasma levels of drugs that are CYP2C9 substrates. Especially for anticancer drugs, which have a narrow therapeutic window, small changes in their plasma levels could easily result in clinically relevant toxicities. METHODS: The effects of CAM on CYP2C9-mediated metabolism of MFC were assessed in Supersomes, using the fluorometric CYP2C9 inhibition assay. In human liver microsomes (HLM) the inhibition of CYP2C9-mediated metabolism of tolbutamide was determined, using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). KEY FINDINGS: The results indicated milk thistle as the most potent CYP2C9 inhibitor. For milk thistle, silybin (main constituent of milk thistle) was mainly responsible for the inhibition of CY2C9. CONCLUSIONS: Milk thistle and green tea were confirmed as potent inhibitors of CYP2C9-mediated metabolism of multiple substrates in vitro. Clinical studies with milk thistle are recommended to establish the clinical relevance of the demonstrated CYP2C9 inhibition.

The effect of complementary and alternative medicines on CYP3A4-mediated metabolism of three different substrates: 7-benzyloxy-4-trifluoromethyl-coumarin, midazolam and docetaxel.

OBJECTIVE: Concomitant use of complementary and alternative medicine (CAM) and anticancer drugs can affect the pharmacokinetics of anticancer drugs by inhibiting the metabolizing enzyme cytochrome P450 3A4 (CYP3A4) (EC 1.14.13.157). Several in vitro studies determined whether CAM can inhibit CYP3A4, but these studies revealed contradictory results. A plausible explanation for these conflicting results is the use only of a single model CYP3A4 substrate in each study. Therefore, the objective was to determine the potential of selected CAM (ß-carotene, Echinacea, garlic, Ginkgo biloba, ginseng, grape seed extract, green tea extract, milk thistle, saw palmetto, valerian, vitamin B6, B12 and C) to inhibit CYP3A4-mediated metabolism of different substrates: 7-benzyloxy-4-trifluoromethyl-coumarin (BFC), midazolam and docetaxel. The effect of CAM on CYP3A4-mediated metabolism of an anticancer drug has never been determined before in vitro, which makes this study unique. The oncolytic CYP3A4 substrate docetaxel was used to establish the predictive value of the model substrates for pharmacokinetic interactions between CAM and anticancer drugs in vitro, and to more closely predict these interactions in vivo. METHODS: The inhibition of CYP3A4-mediated metabolism of 7-benzyloxy-4-trifluoromethyl-coumarin (BFC) by CAM was assessed in Supersomes, using the fluorometric CYP3A4 inhibition assay. In human liver microsomes (HLM) the inhibition of CYP3A4-mediated metabolism of midazolam and docetaxel was determined, using liquid-chromatography coupled to tandem mass spectrometry (LC-MS/MS). KEY FINDINGS: The results confirmed grape seed and green tea as potent inhibitors and milk thistle as moderate inhibitor of CYP3A4-mediated metabolism of BFC, midazolam and docetaxel. CONCLUSION: Clinical studies are required to determine the clinical relevance of the determined CYP3A4 inhibition by grape seed, green tea and milk thistle.

The effect of St John's wort on the pharmacokinetics of docetaxel.

BACKGROUND AND OBJECTIVE: St John's wort (SJW), a herbal antidepressant, is commonly used by cancer patients, and its component hyperforin is a known inducer of the cytochrome P450 (CYP) isoenzyme 3A4. Here, the potential pharmacokinetic interaction between SJW and the sensitive CYP3A4 substrate docetaxel was investigated. METHODS: In ten evaluable cancer patients, the pharmacokinetics of docetaxel (135 mg administered intravenously over 60 min) were compared before and after 14 days of supplementation with SJW (300 mg extract [Hyperiplant(®)] three times daily). RESULTS: SJW supplementation resulted in a statistically significant decrease in the mean area under the docetaxel plasma concentration-time curve extrapolated to infinity (AUC∞) from 3,035 ± 756 to 2,682 ± 717 ng · h/mL (P = 0.045). Furthermore, docetaxel clearance significantly increased from 47.2 to 53.7 L/h (P = 0.045) after SJW intake. The maximum plasma concentration and elimination half-life of docetaxel were (non-significantly) decreased after SJW supplementation. In addition, the incidence of docetaxel-related toxicities was lower after SJW supplementation. CONCLUSION: These results suggest that concomitant use of docetaxel and the applied SJW product should be avoided to prevent potential undertreatment of cancer patients.

The effect of grape seed extract on the pharmacokinetics of dextromethorphan in healthy volunteers.

PURPOSE: Grape seed extract (GSE) has been shown to inhibit the cytochrome P450 (CYP) 2D6 isoenzyme in vitro. To determine the clinical effect of GSE on CYP2D6, the pharmacokinetic interaction between GSE and the sensitive CYP2D6 probe dextromethorphan in healthy adult volunteers was examined. METHODS: In this open label, randomized, cross-over study, 30 subjects were assigned to cohort A or B. Both cohorts ingested 30 mg dextromethorphan hydrobromide on day 1 and day 10. Cohort A received 100 mg GSE capsules three times daily on days 8, 9 and 10, while cohort B started with GSE on day -1 until day 1. After urine collection (0-8 h) on day 1 and day 10, the urinary dextromethorphan to dextrorphan metabolic ratio was determined. RESULTS: Among 28 evaluable subjects, an increase of the urinary metabolic ratio was observed in 16 subjects (57 %). The mean metabolic ratio (± standard deviation) before and after GSE supplementation was 0.41 (± 0.56) and 0.48 (± 0.59), respectively. This result was neither statistically (P = 0.342) nor clinically [geometric mean ratio 1.10, 90 % CI (0.93-1.30)] significant. Further, the majority (73 %) of the included subjects did not experience any adverse events after intake of dextromethorphan or GSE. CONCLUSIONS: Supplementation of GSE did not significantly affect the urinary dextromethorphan to dextrorphan metabolic ratio in healthy volunteers. The results of this clinical study indicate that GSE appears to be safe to combine with drugs extensively metabolized by CYP2D6, such as dextromethorphan and tamoxifen.

The effect of Echinacea purpurea on the pharmacokinetics of docetaxel.

AIMS: The herbal medicine Echinacea purpurea (E. purpurea) has been shown to induce cytochrome P450 3A4 (CYP3A4) both in vitro and in humans. This study explored whether E. purpurea affects the pharmacokinetics of the CYP3A4 substrate docetaxel in cancer patients. METHODS: Ten evaluable cancer patients received docetaxel (135 mg, 60 min IV infusion) before intake of a commercially available E. purpurea extract (20 oral drops three times daily) and 3 weeks later after a 14 day supplementation period with E. purpurea. In both cycles, pharmacokinetic parameters of docetaxel were determined. RESULTS: Before and after supplementation with E. purpurea, the mean area under the plasma concentration-time curve of docetaxel was 3278 ± 1086 and 3480 ± 1285 ng ml(-1) h, respectively. This result was statistically not significant. Nonsignificant alterations were also observed for the elimination half-life (from 30.8 ± 19.7 to 25.6 ± 5.9 h, P = 0.56) and maximum plasma concentration of docetaxel (from 2224 ± 609 to 2097 ± 925 ng ml(-1) , P = 0.30). CONCLUSIONS: The multiple treatment of E. purpurea did not significantly alter the pharmacokinetics of docetaxel in this study. The applied E. purpurea product at the recommended dose may be combined safely with docetaxel in cancer patients.

Milk thistle's active components silybin and isosilybin: novel inhibitors of PXR-mediated CYP3A4 induction.

Because cancer is often treated with combination therapy, unexpected pharmacological effects can occur because of drug-drug interactions. Several drugs are able to cause upregulation or downregulation of drug transporters or cytochrome P450 enzymes, particularly CYP3A4. Induction of CYP3A4 may result in decreased plasma levels and therapeutic efficacy of anticancer drugs. Since the pregnane X receptor (PXR) is one of the major transcriptional regulators of CYP3A4, PXR antagonists can possibly prevent CYP3A4 induction. Currently, a limited number of PXR antagonists are available. Some of these antagonists, such as sulphoraphane and coumestrol, belong to the so-called complementary and alternative medicines (CAM). Therefore, the aim was to determine the potential of selected CAM (ß-carotene, Echinacea purpurea, garlic, Ginkgo biloba, ginseng, grape seed, green tea, milk thistle, saw palmetto, valerian, St. John's Wort, and vitamins B6, B12, and C) to inhibit PXR-mediated CYP3A4 induction at the transcriptional level, using a reporter gene assay and a real-time polymerase chain reaction assay in LS180 colon adenocarcinoma cells. Furthermore, computational molecular docking and a LanthaScreen time-resolved fluorescence resonance energy transfer (TR-FRET) PXR competitive binding assay were performed to explore whether the inhibiting CAM components interact with PXR. The results demonstrated that milk thistle is a strong inhibitor of PXR-mediated CYP3A4 induction. The components of milk thistle responsible for this effect were identified as silybin and isosilybin. Furthermore, computational molecular docking revealed a strong interaction between both silybin and isosilybin and PXR, which was confirmed in the TR-FRET PXR assay. In conclusion, silybin and isosilybin might be suitable candidates to design potent PXR antagonists to prevent drug-drug interactions via CYP3A4 in cancer patients.

Relevance of in vitro and clinical data for predicting CYP3A4-mediated herb-drug interactions in cancer patients.

The use of complementary and alternative medicines (CAM) by cancer patients is increasing. Concomitant use of CAM and anticancer drugs could lead to serious safety issues in patients. CAM have the potential to cause pharmacokinetic interactions with anticancer drugs, leading to either increased or decreased plasma levels of anticancer drugs. This could result in unexpected toxicities or a reduced efficacy. Significant pharmacokinetic interactions have already been shown between St. John's Wort (SJW) and the anticancer drugs imatinib and irinotecan. Most pharmacokinetic CAM-drug interactions, involve drug metabolizing cytochrome P450 (CYP) enzymes, in particular CYP3A4. The effect of CAM on CYP3A4 activity and expression can be assessed in vitro. However, no data have been reported yet regarding the relevance of these in vitro data for the prediction of CAM-anticancer drug interactions in clinical practice. To address this issue, a literature research was performed to evaluate the relevance of in vitro data to predict clinical effects of CAM frequently used by cancer patients: SJW, milk thistle, garlic and Panax ginseng (P. ginseng). Furthermore, in clinical studies the sensitive CYP3A4 substrate probe midazolam is often used to determine pharmacokinetic interactions. Results of these clinical studies with midazolam are used to predict pharmacokinetic interactions with other drugs metabolized by CYP3A4. Therefore, this review also explored whether clinical trials with midazolam are useful to predict clinical pharmacokinetic CAM-anticancer drug interactions. In vitro data of SJW have shown CYP3A4 inhibition after short-term exposure and induction after long-term exposure. In clinical studies using midazolam or anticancer drugs (irinotecan and imatinib) as known CYP3A4 substrates in combination with SJW, decreased plasma levels of these drugs were observed, which was expected as a consequence of CYP3A4 induction. For garlic, no effect on CYP3A4 has been shown in vitro and also in clinical studies garlic did not affect the pharmacokinetics of both midazolam and docetaxel. Milk thistle and P. ginseng predominantly showed CYP3A4 inhibition in vitro. However, in clinical studies these CAM did not cause significant pharmacokinetic interactions with midazolam, irinotecan, docetaxel and imatinib. Most likely, factors as poor pharmaceutical availability, solubility and bioavailability contribute to the lack of significant clinical interactions. In conclusion, in vitro data are useful as a first indication for potential pharmacokinetic drug interactions with CAM. However, the discrepancies between in vitro and clinical results for milk thistle and P. ginseng show that clinical studies are required for confirmation of potential interactions. At last, midazolam as a model substrate for CYP3A4, has convincingly shown to correctly predict clinical interactions between CAM and anticancer drugs.

The bioanalysis of the major Echinacea purpurea constituents dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides in human plasma using LC-MS/MS.

Alkylamides are a group of active components of the widely used herb Echinacea purpurea (E. purpurea), which have immunostimulatory and anti-inflammatory effects. For the most abundant alkylamides, dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides (DTAI), an LC-MS/MS assay has been developed and validated for quantification in human plasma. This assay will be used to support a clinical interaction study with E. purpurea. A 300 µL plasma aliquot underwent liquid-liquid extraction with diethylether-n-hexane (50:50, v/v). After evaporization and reconstitution in 100 µL of acetonitrile-water (50:50, v/v) 20 µL of sample were injected into the HPLC system. Chromatographic separation was achieved with a Polaris 3 C18-A column (50 mm × 2 mm ID, particle size 3 µm), a flow rate of 0.3 mL/min and isocratic elution with acetonitrile-water (50:50, v/v) containing 0.1% formic acid during the first 5 min. Hereafter, gradient elution was applied for 0.5 min, followed by restoration of the initial isocratic conditions. The total run time was 7.5 min. The assay was validated over a concentration range from 0.01 to 50 ng/mL for DTAI, with a lower limit of quantification of 0.01 ng/mL. Validation results show that DTAI can be accurately and precisely quantified in human plasma. DTAI also demonstrated to be chemically stable under relevant conditions. Finally, the applicability of this assay has been successfully demonstrated by measuring the plasma concentration of DTAI in patients after ingestion of a commercial extract of E. purpurea.

3,4-methylenedioxymethamphetamine (MDMA) interacts with therapeutic drugs on CYP3A by inhibition of pregnane X receptor (PXR) activation and catalytic enzyme inhibition.

Metabolism of MDMA (3,4-methylenedioxymethamphetamine, Ecstasy) by the major hepatic drug-metabolizing enzyme cytochrome P450 3A (CYP3A), plays an important role in MDMA-induced liver toxicity. In the present study, we investigated interactions between MDMA and several therapeutic and recreational drugs on CYP3A and its regulator pregnane X receptor (PXR), using a human PXR-mediated CYP3A4-reporter gene assay, rat primary hepatocytes and microsomes. MDMA significantly inhibited hPXR-mediated CYP3A4-reporter gene expression induced by the human PXR activator rifampicin (IC50 1.26 ± 0.36 mM) or the therapeutic drugs paroxetine, fluoxetine, clozapine, diazepam and risperidone. All these drugs concentration-dependently inhibited CYP3A activity in rat liver microsomes, but in combination with MDMA this inhibition became more efficient for clozapine and risperidone. In rat primary hepatocytes that were pretreated with or without the rodent PXR activator pregnenolone 16alpha-carbonitrile (PCN), MDMA inhibited CYP3A catalytic activity with IC50 values of 0.06 ± 0.12 and 0.09 ± 0.13 mM MDMA, respectively. This decrease appeared to be due to decreased activation of PXR and subsequent decreased CYP3A gene expression, and catalytic inhibition of CYP3A activity. These data suggest that in situations of repeated MDMA use in combination with other (therapeutic) drugs, adverse drug-drug interactions through interactions with PXR and/or CYP3A cannot be excluded.

A sensitive LC-MS/MS method for the quantitative analysis of the Echinacea purpurea constituent undeca-2-ene-8,10-diynoic acid isobutylamide in human plasma.

Echinacea purpurea is one of the most popular herbal medicines and is known for its immunostimulatory effects. Alkylamides are the main lipophilic components of E. purpurea that contribute to its pharmacological actions. For quantification in human plasma of one of these alkylamides, undeca-2-ene-8,10-diynoic acid isobutylamide, a sensitive LC-MS/MS assay has been developed and validated. Plasma samples were pretreated using liquid-liquid extraction with a mixture of diethyl ether and n-hexane (50:50, v/v). Dried extracts were reconstituted in 50 µL of acetonitrile-water (50:50, v/v) after which 15 µL of sample was injected into the HPLC system. HPLC was performed using a Polaris 3 C18-A column (50 mm×2 mm ID) and isocratic elution with acetonitrile-water (50:50, v/v) containing 0.1% formic acid at a flow rate of 0.3 mL/min. Subsequently, electrospray ionization in the positive ion mode followed by tandem mass spectrometry was performed for detection. The total run time was 3 min. The assay was validated over a concentration range from 0.05 to 50 ng/mL for undeca-2-ene-8,10-diynoic acid isobutylamide, with 0.05 ng/mL being the lower limit of quantification using 1.0 mL plasma samples. Inter-assay inaccuracy (±12.7%), within-day and between-day precisions (CV≤8.23%) were acceptable. Further, undeca-2-ene-8,10-diynoic acid isobutylamide was found to be chemically stable under relevant conditions. Finally, the applicability of this assay has been successfully demonstrated in a pharmacokinetic experiment in which a human volunteer ingested a commercial extract of E. purpurea.

Combined action and regulation of phase II enzymes and multidrug resistance proteins in multidrug resistance in cancer.

A major limitation in the treatment of cancer patients is the ability of cancer cells to become resistant to chemotherapeutic drugs, a phenomenon known as multidrug resistance (MDR). Two important mechanisms involved in multidrug resistance are the increased activity of efflux pumps, such as those of the multidrug resistance proteins (MRPs) and the detoxification by phase II conjugating enzymes, like glutathione S-transferases and UDP-glucuronosyltransferases. A synergistic interaction between these two mechanisms, MRPs and phase II enzymes, in conferring MDR has been shown for multiple anticancer drugs. In addition, there is substantial evidence of a coordinate regulation of the expression of phase II enzymes and MRPs, most likely mediated by the nuclear factor-erythroid 2 p45-related factor (Nrf2) and antioxidant response elements. Further elucidation of the combined action and regulation of phase II enzymes and MRPs in MDR will be an aid in the improvement of the chemotherapeutic treatment of cancer patients.

Pharmacogenetic screening of the gene deletion and duplications of CYP2D6.

Cytochrome P450 (CYP) 2D6 is one of the most important enzymes involved in the metabolism of drugs. Multiple, clinically relevant, genetic variants of this gene have been identified and, among them, a gene deletion as well as multiplications of the gene. These large structural mutations in CYP2D6 occur at a relatively high frequency in several populations. Genotyping of CYP2D6 could therefore be applied to individualize drug therapy to improve therapeutic efficacy and decrease adverse effects in patients. However, a prerequisite for the pharmacogenetic screening of CYP2D6 in a clinical setting is the development of fast, reliable and cost-effective techniques for the routine genotyping of patients. In the case of CYP2D6, besides the general problems that arise in the detection of large gene deletions and multiplications, the presence of two highly homologous pseudogenes, CYP2D7 and CYP2D8, forms an extra challenge. This review provides an overview of the techniques that have been described to detect the CYP2D6 gene deletion and multiplication: Southern-blotting RFLP, long-template PCR, and real-time PCR. Of these techniques, real-time PCR is the only technique giving quantitative information about the exact copy number of the gene. Considering all of the other advantages of this method over other methods, such as cost-effectiveness and suitability for high throughput screening, real-time PCR is the most promising method for the genotyping of large structural alterations in the CYP2D6 gene in a routine clinical setting.

In vitro characterization of the human biotransformation pathways of aplidine, a novel marine anti-cancer drug.

Aplidine is a potent marine anti-cancer drug and is currently being investigated in phase II clinical trials. However, the enzymes involved in the biotransformation of aplidine and thus its pharmacokinetics are not known yet. To assess the biotransformation pathways of aplidine and their potential implications for human pharmacology and toxicology, the in vitro metabolism of aplidine was characterized using incubations with human plasma, liver preparations, cytochrome P450 (CYP) and uridine diphosphoglucuronosyl transferase (UGT) supersomes in combination with HPLC analysis and cytotoxicity assays with cell lines. Aplidine was metabolised by carboxyl esterases in human plasma. Using CYP supersomes and liver microsomes, it was shown that aplidine was metabolised mainly by CYP3A4 and also by CYP2A6, 2E1 and 4A11. Four metabolites were observed after incubation with human liver microsomes, one formed by CYP2A6 (C-demethylation) and three by CYP3A4 (hydroxylation and/or C-dealkylation). No conjugation was observed in human liver S9 fraction. However, the aplidine metabolites formed by CYP were further conjugated by the phase II enzymes UGT, GST and SULT. In accordance with the findings in microsomes and CYP supersomes, a significant effect of specific CYP2A6, 2E1, 3A4 and 4A11 inhibitors on the cytotoxicity of aplidine in Hep G2 and IGROV-1 cells could be observed. These results provide evidence that CYP3A4 has a major role in metabolising aplidine in vitro with additional involvement of CYP2A6, 2E1, and 4A11. Further, the metabolites formed by CYPs can be conjugated by UGT, SULT and GST. These findings could help interpret the in vivo pharmacokinetics of aplidine.

Herb-drug interactions in oncology: focus on mechanisms of induction.

An increasing number of cancer patients are using complementary and alternative medicines (CAM) in combination with their conventional chemotherapeutic treatment. Considering the narrow therapeutic window of oncolytic drugs, this CAM use increases the risk of clinically relevant herb-anticancer drug interactions. Such a relevant interaction is that of St. John's wort with the anticancer drugs irinotecan and imatinib. It is, however, estimated that CAM-anticancer drug interactions are responsible for substantially more unexpected toxicities of chemotherapeutic drugs and possible undertreatment seen in cancer patients. Induction of drug-metabolizing enzymes and ATP-binding cassette drug transporters can be one of the mechanisms behind CAM-anticancer drug interactions. Induction will often lead to therapeutic failure because of lower plasma levels of the anticancer drugs, and will easily go unrecognized in cancer treatment, where therapeutic failure is common. Recently identified nuclear receptors, such as the pregnane X receptor, the constitutive androstane receptor, and the vitamin D-binding receptor, play an important role in the induction of metabolizing enzymes and drug transporters. This knowledge has already been an aid in the identification of some CAM probably capable of causing interactions with anticancer drugs: kava-kava, vitamin E, quercetin, ginseng, garlic, beta-carotene, and echinacea. Evidently, more research is necessary to prevent therapeutic failure and toxicity in cancer patients and to establish guidelines for CAM use.

Genetic polymorphisms of drug-metabolising enzymes and drug transporters in the chemotherapeutic treatment of cancer.

There is wide variability in the response of individuals to standard doses of drug therapy. This is an important problem in clinical practice, where it can lead to therapeutic failures or adverse drug reactions. Polymorphisms in genes coding for metabolising enzymes and drug transporters can affect drug efficacy and toxicity. Pharmacogenetics aims to identify individuals predisposed to a high risk of toxicity and low response from standard doses of anti-cancer drugs. This review focuses on the clinical significance of polymorphisms in drug-metabolising enzymes (cytochrome P450 [CYP] 2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, dihydropyrimidine dehydrogenase, uridine diphosphate glucuronosyltransferase [UGT] 1A1, glutathione S-transferase, sulfotransferase [SULT] 1A1, N-acetyltransferase [NAT], thiopurine methyltransferase [TPMT]) and drug transporters (P-glycoprotein [multidrug resistance 1], multidrug resistance protein 2 [MRP2], breast cancer resistance protein [BCRP]) in influencing efficacy and toxicity of chemotherapy. The most important example to demonstrate the influence of pharmacogenetics on anti-cancer therapy is TPMT. A decreased activity of TPMT, caused by genetic polymorphisms in the TPMT gene, causes severe toxicity with mercaptopurine. Dosage reduction is necessary for patients with heterozygous or homozygous mutation in this gene. Other polymorphisms showing the influence of pharmacogenetics in the chemotherapeutic treatment of cancer are discussed, such as UGT1A1*28. This polymorphism is associated with an increase in toxicity with irinotecan. Also, polymorphisms in the DPYD gene show a relation with fluorouracil-related toxicity; however, in most cases no clear association has been found for polymorphisms in drug-metabolising enzymes and drug transporters, and pharmacokinetics or pharmacodynamics of anti-cancer drugs. The studies discussed evaluate different regimens and tumour types and show that polymorphisms can have different, sometimes even contradictory, pharmacokinetic and pharmacodynamic effects in different tumours in response to different drugs. The clinical application of pharmacogenetics in cancer treatment will therefore require more detailed information of the different polymorphisms in drug-metabolising enzymes and drug transporters. Larger studies, in different ethnic populations, and extended with haplotype and linkage disequilibrium analysis, will be necessary for each anti-cancer drug separately.