Roel F. Maas-Bakker

PXR-mediated induction of P-glycoprotein by anticancer drugs in a human colon adenocarcinoma-derived cell line

PurposeThe development of multidrug resistance (MDR) is one of the major limitations in the treatment of cancer. Induction of P-glycoprotein (Pgp) has been regarded as one of the main mechanisms underlying anticancer drug-induced MDR. Since the induction of Pgp is (in part) regulated by the pregnane X receptor (PXR), the ability of several widely used anticancer drugs to activate PXR-mediated Pgp induction was investigated.MethodsA Pgp-reporter gene assay was employed to determine the ability of a panel of widely used anticancer drugs to induce Pgp. To further assess whether PXR could be involved in the induction of Pgp by anticancer drugs, Pgp protein expression after treatment with the anticancer drugs was determined in both wild-type and PXR-knocked down LS180 cells. Furthermore, the effect of the anticancer drugs on the intracellular accumulation of the Pgp-probes rhodamine 123 and doxorubicin was determined.ResultsOur study showed that vincristine, tamoxifen, vinblastine, docetaxel, cyclophosphamide, flutamide, ifosfamide and paclitaxel activate PXR-mediated Pgp induction, and were additionally shown to affect the intracellular accumulation of the Pgp probe rhodamine 123. Moreover, PXR activation was also shown to reduce the cytotoxic activity of the Pgp substrate doxorubicin in colon cancer cells.ConclusionOur results indicate that several anticancer drugs can activate PXR-mediated induction of Pgp and affect the accumulation of Pgp substrates.

Effect of Chinese herbs on CYP3A4 activity and expression in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE Traditional Chinese Medicine (TCM) has become more popular among cancer patients in the Western world, who often use Chinese herbs as adjuvant therapy to reduce the adverse effects of conventional chemotherapy. However, pharmacokinetic (PK) interactions between Chinese herbs and anticancer drugs can occur and have dramatic consequences for these patients. Currently, only a few possible PK interactions between Chinese herbs and conventional Western drugs have been documented. AIM OF THE STUDY Since the drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) contributes to most of the PK interactions with (anticancer) drugs, the effect of four Chinese herbs (Oldenlandia diffusa, Codonopsis tangshen, Rehmannia glutinosa and Astragalus propinquus) on the activity and expression of CYP3A4 was investigated in vitro. MATERIALS AND METHODS Ethanol and water-ethanol extracts of the four Chinese herbs were prepared from raw material. CYP3A4 inhibition was assessed by the use of Supersomes™ in a fluorescence assay. Furthermore, CYP3A4 induction was evaluated in a human pregnane X receptor (hPXR)-mediated CYP3A4 reporter gene assay and a quantitative real time PCR assay, both in human colon adenocarcinoma-derived LS180 cells (LS180). RESULTS Extracts of Oldenlandia diffusa, Codonopsis tangshen, Rehmannia glutinosa and Astragalus propinquus inhibited CYP3A4 in human CYP3A4 Supersomes™ (IC50 values: 17-83 µg/mL). Oldenlandia diffusa and Rehmannia glutinosa significantly induced PXR-mediated CYP3A4 (p<0.001). Oldenlandia diffusa also significantly induced CYP3A4 mRNA levels (p<0.001 at 250 µg/mL). CONCLUSIONS Concomitant use of Oldenlandia diffusa and Rehmannia glutinosa could result in induction of CYP3A4, leading to a reduced efficacy of drugs that are CYP3A4 substrates and have a narrow therapeutic window. Because of the possible enhanced toxicity caused by CYP3A4 inhibition, clinical effects of CYP3A4 inhibition by Astragalus propinquus and Codonopsis tangshen must also be taken into account. In conclusion, herb-drug interactions between Chinese herbs and various CYP3A4 substrates can occur. Further research to investigate the clinical relevance of the interactions caused by Oldenlandia diffusa, Codonopsis tangshen, Rehmannia glutinosa and Astragalus propinquus is required.

PXR-mediated P-glycoprotein induction by small molecule tyrosine kinase inhibitors

The rapid development of drug resistance as a result of exposure to small molecule tyrosine kinase inhibitors (TKIs) is an important drawback to the successful use of these agents in the clinic. Although one of the most established mechanisms by which cells acquire drug resistance to anticancer drugs is the up regulation of drug efflux transporters such as P-glycoprotein (PGP), it is currently still unknown whether TKIs have the propensity to induce PGP. The effect of TKIs on the protein expression and activity of PGP was assessed after treatment of LS180 cells with clinically relevant concentrations of the TKIs. In addition, the involvement of the nuclear pregnane X receptor (PXR), a known regulator of PGP expression, was determined. At least five out of the nine tested TKIs (erlotinib, gefitinib, nilotinib, sorafenib, vandetanib) were able to induce the expression of PGP within 48 h in LS180 cells. Accordingly, these TKIs were also shown to affect the accumulation of a P-glycoprotein specific probe substrate. Furthermore, we showed that the pregnane X receptor (PXR), which is an important regulator of PGP induction, is involved in the upregulation of PGP protein expression following exposure to these TKIs. Our data show that PXR-mediated upregulation of PGP expression by TKIs might be a possible mechanism underlying acquired drug resistance in cancer cells.

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 (β-carotene, Echinacea purpurea, garlic, Ginkgo biloba, ginseng, grape seed, green tea, milk thistle, saw palmetto, valerian, St. Johns 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.

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.

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

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 (β‐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.

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

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.

Development and validation of a LC‐MS/MS method for the in vitro analysis of 1‐hydroxymidazolam in human liver microsomes: application for determining CYP3A4 inhibition in complex matrix mixtures

Complementary and alternative medicines (CAM) can affect the pharmacokinetics of anticancer drugs by interacting with the metabolizing enzyme cytochrome P450 (CYP) 3A4. To evaluate changes in the activity of CYP3A4 in patients, levels of 1-hydroxymidazolam in plasma are often determined with liquid chromatography-quadrupole mass spectrometry (LC-MS/MS). However, validated LC-MS/MS methods to determine in vitro CYP3A4 inhibition in human liver microsomes are scarce and not optimized for evaluating CYP3A4 inhibition by CAM. The latter is necessary because CAM are often complex mixtures of numerous compounds that can interfere with the selective measurement of 1-hydroxymidazolam. Therefore, the aim was to validate and optimize an LC-MS/MS method for the adequate determination of CYP3A4 inhibition by CAM in human liver microsomes. After incubation of human liver microsomes with midazolam, liquid-liquid extraction with tert-butyl methyl ether was applied and dried samples were reconstituted in 50% methanol. These samples were injected onto a reversed-phase chromatography consisting of a Zorbax Extend-C18 column (2.1 × 150 mm, 5.0 µm particle size), connected to a triple quadrupole mass spectrometer with electrospray ionization. The described LC-MS/MS method was validated over linear range of 1.0-500 nm for 1-hydroxymidazolam. The results revealed good inter-assay accuracy (≥85% and ≤115%) and within-day and between-day precisions (coefficient of variation ≤ 4.43%). Furthermore, the applicability of this assay for the determination of CYP3A4 inhibition in complex matrix mixtures was successfully demonstrated in an in vitro experiment in which CYP3A4 inhibition by known CAM (β-carotene, green tea, milk thistle and St. Johns wort) was determined.

Abstract 3791: Milk thistle as an inhibitor of PXR-mediated CYP3A4 induction

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction Many anticancer drugs, such as paclitaxel and erlotinib, are able to induce the expression of cytochrome P450 (CYP450) 3A4.1 Induction of intestinal and hepatic CYP3A4 can result in a decreased uptake and an increased metabolism of anticancer drugs. Consequently, systemic levels decrease, possibly resulting in a lower efficacy. The major mechanism behind this CYP3A4 induction is the activation of the pregnane X receptor (PXR)1 and therefore inhibition of PXR might prevent CYP3A4 induction. As 54-77% of the cancer patients use complementary and alternative medicine (CAM) including herbs and dietary supplements, these CAM are interesting to screen for their ability to inhibit PXR-mediated CYP3A4. For the current study, the following CAM were selected: α-carotene, Echinacea, garlic, Ginkgo biloba, P. ginseng, grape seed, green tea, milk thistle, saw palmetto, St. Johns Wort, valerian, vitamin B6+12 and vitamin C.2,3 Aim The aim was to screen CAM for their ability to inhibit PXR-mediated CYP3A4 induction in LS180 cells, a human colon adenocarcinoma-derived cell line. Method The selected CAM were screened for possible inhibition of PXR-mediated CYP3A4 induction, using a CYP3A4 reporter gene assay and a real-time polymerase chain reaction (RT-PCR) assay. In both assays, LS180 cells were exposed to fresh medium containing 0.1% DMSO or model inducers of CYP3A4 (10 μM rifampicin, 20 μM erlotinib or 20 μM paclitaxel) with or without addition of known PXR inhibitors (10 μM ketoconazole and 10 μM A792611) or 50-100 μg/ml standardized CAM (table 1) for 24 hours. Data were analyzed by using the Students unpaired t-test and were considered statistically significant when p< 0.05. Results Milk thistle was the most potent inhibitor of PXR-mediated CYP3A4 induction compared to the other CAM. The reporter gene assay demonstrated that 100 and 75 μg/ml milk thistle significantly inhibited PXR-mediated CYP3A4 induction by rifampicin by respectively 88% and 75%; by paclitaxel by respectively 90% and 81%; and by erlotinib by 82% and 49%. According to the RT-PCR assay 100 and 75 μg/ml milk thistle significantly inhibited PXR-mediated CYP3A4 induction by rifampicin by respectively 91% and 90%. Conclusion Milk thistle significantly inhibited PXR-mediated CYP3A4 induction at the transcriptional level, demonstrated by a reporter gene and RT-PCR assay. Thus, milk thistle is a suitable candidate for the inhibition of PXR-mediated CYP3A4 induction. Consequently, the concomitant use of milk thistle with anticancer drugs can possibly prevent the decrease in systemic levels of anticancer drugs resulting in a higher efficacy, but also a higher risk for toxicity. References 1. Harmsen S, et al., Cancer Chemother Pharmacol 2009 Jun; 64(1): 35-43. 2. Gupta D, et al., Support Care Cancer 2005 Nov; 13(11): 912-9. 3. McCune J, et al., Support Care Cancer 2004 Jun;12(6):454-62. Acknowledgements The present study was supported by the Dutch Cancer Society grant UU2007-3795. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3791. doi:1538-7445.AM2012-3791

Quality Comparison of Biosimilar and Copy Filgrastim Products with the Innovator Product

PurposeFilgrastim, a recombinant human granulocyte-colony stimulating factor, is widely used to treat congenital and acquired neutropenia. Following patent expiration of the innovator filgrastim product, biosimilar filgrastim products have been approved in the EU and shown to be comparable with the innovator with respect to quality, safety and efficacy. In less regulated markets, copy filgrastim products are available but data about their quality are scarce. In the present study, we provide a head-to-head comparative study on the quality of biosimilar and copy filgrastim products.MethodsInnovator filgrastim product, Neupogen®, two EU-licensed biosimilars, Zarzio® and Tevagrastim®, and two copy filgrastim products, Biocilin® and PDgrastim®, were subjected to peptide mapping, circular dichroism spectroscopy, fluorescence spectroscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis, high performance size-exclusion chromatography, reversed-phase ultra-performance liquid chromatography, endotoxin test, flow imaging microscopy and in vitro potency assay.ResultsZarzio® and Tevagrastim® have comparable quality to Neupogen®, while Biocilin® showed a significantly lower and PDgrastim® a higher specific activity. Moreover, PDgrastim® showed a higher level of impurities and a lower thermo stability than the other products.ConclusionsExcept for the deviating specific activities of the two copy filgrastim products, we found no substantial differences in product quality between the filgrastim products studied.