Altered drug disposition of the platelet activating factor antagonist apafant in mdr1a knockout mice
Introduction
It is now well established that the expression of the multidrug resistance MDR1 gene product P-glycoprotein not only confers drug resistance to cancer cells but also exerts significant influence on oral absorption, bioavailability, tissue distribution and pharmacodynamic effects of drugs (Schinkel, 1997). P-gp is a transmembrane, ATP-dependent drug efflux transporter which accepts a wide range of structurally unrelated compounds including anticancer drugs such as taxol, doxorubicin and vinblastine but also other clinically relevant drugs like the immunosuppressive agent cyclosporine A and the HIV-1 protease inhibitor amprenavir (Yu, 1999, Seelig and Landwojtowicz, 2000). P-gp is located in the apical membrane of endothelial and epithelial cells of different tissues and functions as a drug efflux pump by transporting its substrates through the apical membrane out of the cell. In the enterocytes of the intestinal mucosa and capillary endothelial cells forming the blood–brain barrier P-gp limits the access of drugs to the systemic circulation and the brain, respectively (Cordon-Cardo et al., 1990, Schinkel, 1999). In the human, a single gene, MDR1, encodes P-gp. In mice, there are two genes, mdr1a and mdr1b, encoding P-gp together probably fulfilling the same function(s) (Schinkel et al., 1997). The expression pattern of mdr1a is different from mdr1b. The mouse mdr1a gene is predominantly expressed in intestine, liver and blood capillaries of brain and testis, the mdr1b gene in adrenal glands, placenta and ovaries (Gottesmann and Pastan, 1993). Similar levels of the mdr1a and mdr1b expression are observed in the kidney (Schinkel et al., 1994). Transgenic mice lacking the mdr1a gene (Schinkel et al., 1994) have been generated in order to get a better understanding of the physiological function of P-gp and its influence on the pharmacokinetics of transported drugs. The human colon carcinoma-derived cell line Caco-2 has been shown to be a suitable in vitro model for the investigation of passive transcellular permeability and active transport of drugs. When grown on semiporous filters, Caco-2 cells develop into polarized, tight monolayers which express P-gp on their apical cell surface (Hunter et al., 1993, Artursson and Karlsson, 1991). This allows to study the absorptive apical-to-basal (A–B) and secretory basal-to-apical (B–A) transcellular permeability of drugs. Vectorial, B–A directed and saturable transport which can be inhibited by P-gp substrates like verapamil and cyclosporin A (Seelig, 1998) is indicative of P-gp mediated drug efflux.
Apafant (WEB 2086, CAS 105219-56-5) is a platelet activating factor (PAF) receptor antagonist which has been developed for the treatment of asthma or acute pancreatitis. It has been shown to displace PAF from binding sites in human platelet membranes and is therefore a potent inhibitor of PAF-induced human platelet aggregation in vitro (Tahraoui et al., 1990). Clinical studies showed apafant to be well tolerated at low doses (Adamus et al., 1988) with a bioavailability of at least 44% (Brecht et al., 1991). But, early autoradiography studies showed a limited distribution of apafant into the brain of rats.
This raised the question whether apafant is subject to active drug efflux. The aim of the present study was to investigate whether apafant is a P-gp substrate and whether the tissue distribution and pharmacokinetics of apafant are influenced by P-gp. Such information could significantly impact the clinical development of apafant especially the need for clinical drug interaction studies.
To address this question, in vitro transport experiments in Caco-2 monolayers and in vivo studies in mdr1a(−/−) mice and wildtype mice were performed. The dose of 2 mg/kg of apafant administered to the mice in this study corresponds to the relevant dose employed in the clinical studies.
Digoxin is a widely used medication for the treatment of congestive heart failure and is a well characterised substrate of P-gp (Mayer et al., 1996, Kawahara et al., 1999). Therefore, we have chosen digoxin to evaluate both our transgenic animal model and the Caco-2 cell model and to compare our results with published data. For reasons of historical data comparison, we have also tested digoxin in NMRI mice.
Section snippets
Animals
All experiments were performed with male mdr1a(−/−) mice (P-gp deficient) or wildtype mice (breeder: Taconic Farm Inc., Germantown, NY) or with NMRI mice (breeder: Boehringer Ingelheim Pharma KG, Biberach, Germany). The animals were between 5 and 10 weeks of age (body weight 17 to 36 g). The animals were housed and handled according to institutional guidelines issued by the government of the Federal Republic of Germany (novel of June 1, 1998, BGB I). Food (supplier: Eberle Nafag AG, Gossau,
Transport experiments in Caco-2 monolayers
PEG4000, mannitol, atenolol and theophylline were selected as reference compounds to check the integrity and permeability characteristics of the Caco-2 monolayer for passively permeating compounds. These compounds are recommended as model drugs for the Caco-2 system in an FDA Guidance (FDA Guidance for Industry, 1999) and often described in the literature (Artursson and Karlsson, 1991). For these reference compounds extents of oral absorption in humans of 0, 16, 50 and 100%, respectively, have
Discussion
Digoxin has been established as a substrate for P-glycoprotein by transport experiments in Caco-2 monolayers and tissue distribution studies in mdr1a(−/−) and wildtype mice. We observed a brain radioactivity ratio for digoxin of more than 70 between mdr1a(−/−) and wildtype mice 4 h after intravenous administration. This is comparable to the recently published ratio of 66 (Mayer et al., 1996). Similar results were reported by other groups (Kawahara et al., 1999, Fromm et al., 1999). Furthermore,
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