Stefan Harmsen

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.

Drug targeting to the kidney: Advances in the active targeting of therapeutics to proximal tubular cells

Activated signaling cascades in the proximal tubular cells of the kidneys play a crucial role in the development of tubulointerstitial fibrosis. Inhibition of these signaling cascades with locally delivered therapeutics is an attractive approach to minimize the risk of unwanted side effects and to enhance their efficacy within the renal tissue. This review describes the potential avenues to actively target drugs to proximal tubular cells by recognition of internalizing receptors and how drug carriers can reach this cell type from either the apical or basolateral side. Important characteristics of drug carrier systems such as size and charge are discussed, as well as linking technologies that have been used for the coupling of drugs to the presented carrier systems. Lastly, we discuss the cellular handling of drugs by proximal tubular cells after their delivery to the kidneys.

Nuclear receptor mediated induction of cytochrome P450 3A4 by anticancer drugs: a key role for the pregnane X receptor

PurposeInduction of cytochrome P450 (CYP) 3A4, an enzyme that is involved in the biotransformation of more than 50% of all drugs, by xenobiotics is an important cause of pharmacokinetic drug–drug interactions in oncology. In addition to rifampicin and hyperforin, the anticancer drug paclitaxel has also been shown to be an inducer of CYP3A4 via activation of the pregnane X receptor (PXR). We therefore screened 18 widely used anticancer drugs for their ability to activate PXR-mediated CYP3A4 induction.MethodsA CYP3A4 reporter gene assay was employed to identify PXR agonists among the eighteen anticancer drugs. Subsequently CYP3A4 mRNA and protein expression following treatment with these PXR agonists was assessed. Finally, the effect of pre-treatment with these agents on the 1’-hydroxylation of midazolam (a specific CYP3A4 probe) was determined.ResultsPaclitaxel, erlotinib, tamoxifen, ifosfamide, flutamide and docetaxel are able to activate PXR, while only strong PXR activation leads to significant induction of CYP3A4 activity.ConclusionsThe identified PXR agonists may have the propensity to cause clinically relevant drug–drug interactions as a result of CYP3A4 induction.

Comparison of two immortalized human cell lines to study nuclear receptor-mediated CYP3A4 induction.

Since CYP3A4 is responsible for the biotransformation of over 50% of all clinically used drugs, induction results in an increased clearance of many concomitantly administered drugs, thereby decreasing treatment efficacy or, in the case of prodrugs, lead to severe intoxications. CYP3A4 induction is regulated by the pregnane X receptor, constitutive androstane receptor, and vitamin D receptor. Since these nuclear receptors show large interspecies differences, accurate prediction of nuclear receptor-mediated CYP3A4 induction in humans requires the use of human systems. Because primary cultures of human hepatocytes or enterocytes have major drawbacks like poor availability and poor reproducibility, human cell lines are a good alternative. In this study, the widely used HepG2 cell line was compared with the LS180 cell line to serve as a model to study CYP3A4 induction. There was a clear difference between the cell lines with respect to CYP3A enzyme expression and induction. In LS180, CYP3A4 was expressed and was found to be induced by prototypical nuclear receptor agonists, whereas in HepG2, CYP3A4 was nonresponsive to treatment with rifampicin, CITCO [6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-3,4-dichlorobenzyl) oxime], or calcitriol. We subsequently evaluated whether these host-cell differences also have an effect on CYP3A4 reporter gene activity. We clearly show that there are differences in CYP3A4 reporter activity between the cell lines, and based on these results and those found on mRNA and protein level, we conclude that LS180 is a more suitable cell line to study CYP3A4 induction than the widely used HepG2.

Characterization of drug-lysozyme conjugates by sheathless capillary electrophoresis–time-of-flight mass spectrometry

Drug-protein conjugates have been widely used for the cell-specific targeting of drugs to cells that can bind and internalize the proteinaceous carrier. For renal drug targeting, lysozyme (LZM) can be used as an effective carrier that accumulates in proximal tubular cells. We used capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF-MS) for the characterization of different drug-LZM conjugates. A recently developed prototype porous tip sprayer was employed for sheathless electrospray ionization (ESI) CE-MS interfacing. In order to prevent adsorption of LZM conjugates to the capillary wall, a positively charged polyethylenimine capillary coating was used in combination with a low-pH background electrolyte. Drug-LZM products had been prepared by first coupling BOC-l-methionine hydroxysuccinimide ester (BOCmet) to lysine residues of LZM followed by conjugation with the kinase inhibitors LY364947, erlotinib, or Y27632 via a platinum(II)-based linker. CE-TOF-MS of each preparation showed narrow symmetrical peaks for the various reaction products demonstrating that drug-LZM conjugates remained stable during the CE analysis and subsequent ESI. Components observed in the drug-LZM products were assigned based on their relative migration times and on molecular mass as obtained by TOF-MS. The TOF-MS data obtained for the individual components revealed that the preparations contained LZM carrying one or two drug molecules, next to unmodified and BOCmet-modified LZM. Based on relative peak areas (assuming an equimolar response for each component) a quantitative conjugate profile could be derived for every preparation leading to drug loading values of 0.4-0.6 mol drug per mole protein.

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.

PXR-mediated induction of human CYP3A4 and mouse Cyp3a11 by the glucocorticoid budesonide

Budesonide, a glucocorticoid with a high first-pass metabolism, is used for the oral treatment of inflammatory bowel disease. Cytochrome P450 3A4 (CYP3A4) is an enzyme involved in the metabolism of numerous drugs, including budesonide. Since inhibition or induction of CYP3A4 is often the cause of drug-drug interactions we analyzed how budesonide affects the activity and expression of this enzyme. CYP3A4 activity was assessed by the metabolism of a luminogenic substrate (luciferin-benzylether) using recombinant human CYP3A4 protein. We observed no inhibition of the metabolism in the presence of budesonide at concentrations up to 25 microM. Induction experiments in human LS180 colon carcinoma cells showed an increased expression of CYP3A4 mRNA after budesonide treatment. Transactivation assays revealed that budesonide activates the CYP3A4 promoter via the pregnane X receptor (PXR). In mice, oral budesonide administration (25mg/kg) for 4 days induced the murine homolog Cyp3a11 in the intestine 3-fold, whereas liver expression was notably less influenced. In knockout mice devoid of PXR, budesonide-mediated inductions were reduced compared to wild-type mice. In conclusion, we could demonstrate that budesonide is not an efficient inhibitor but rather an inducer of CYP3A via a PXR-mediated mechanism. In vivo, however, oral budesonide administration to mice showed only modest gene induction, which occurred mainly in the intestine. Therefore, the risk for budesonide-mediated drug interactions seems to be low but cannot be ruled out entirely.

Targeting of a platinum-bound sunitinib analog to renal proximal tubular cells

Background Activated proximal tubular cells play an important role in renal fibrosis. We investigated whether sunitinib and a kidney-targeted conjugate of sunitinib were capable of attenuating fibrogenic events in tubulointerstitial fibrosis. Methods A kidney-targeted conjugate was prepared by linkage of a sunitinib analog (named 17864) via a platinum-based linker to the kidney-specific carrier lysozyme. Pharmacological activity of 17864-lysozyme was evaluated in human kidney proximal tubular cells (HK-2); the capability of the kidney-directed conjugate to accumulate in the kidneys was studied in mice. Potential antifibrotic effects of a single-dose treatment were evaluated in the unilateral ureteral obstruction (UUO) model in mice. Results The 17864-lysozyme conjugate and its metabolites strongly inhibited tyrosine kinase activity. Upon intravenous injection, 17864-lysozyme rapidly accumulated in the kidneys and provided sustained renal drug levels for up to 3 days after a single dose. Renal drug level area under the curve was increased 28-fold versus an equimolar dose of sunitinib malate. Daily treatment of UUO mice with a high dose of sunitinib malate (50 mg/kg) resulted in antifibrotic responses, but also induced drug-related toxicity. A single dose of 17864-lysozyme (equivalent to 1.8 mg/kg sunitinib) was safe but showed no antifibrotic effects. Conclusion Multikinase inhibitors like sunitinib can be of benefit in the treatment of fibrotic diseases, provided that their safety can be improved by strategies as presented in this paper, and sustained renal levels can be achieved.

Development of a cell-selective and intrinsically active multikinase inhibitor bioconjugate.

Multikinase inhibitors are potent anticancer drugs that simultaneously intervene in multiple related signaling cascades, thus being capable of blocking salvage pathways that may play a role in the development of drug resistance. Multikinase inhibitors are increasingly evaluated for indications other than cancer, but long-term safety risks dictated by off-organ toxicities of these agents may prevent their safe and effective use. Here, we describe a new approach in which platinum coordination chemistry is applied for the development of a cell-selective multikinase inhibitor bioconjugate. The platinum(II) kinase inhibitor bioconjugate was designed to be active with the linker attached to the inhibitor and displayed improved activity by enhanced cell specificity as well as enhanced intracellular retention, thereby prolonging its pharmacological activity. In addition, the utilized platinum-based linkage technology potentiated the inhibitory activity of the multikinase inhibitor. These features in combination with carrier-mediated uptake in the target cells may revolutionize dosing regimens and safety profiles of (multi)kinase inhibitors.

Kinase Inhibitor Conjugates

In the last decade kinase inhibitors have emerged as a new class of very potent and relatively safe anticancer drugs. However, despite their success, off-target toxicities and induction of rapid resistance are major drawbacks that highly compromise successful longterm administration. In the current review, we provide an overview of the approaches that have been undertaken to overcome these issues by conjugation of these agents to carriers that enable their cellular and/or intracellular targeted delivery. Eventually, targeting kinase inhibitors will provide powerful treatment options not only for cancer, but also for other less-life threatening diseases that involve deregulated kinase signaling pathways.