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Five 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins), (e.g. atorvastatin, fluvastatin, lovastatin, pravastatin and simvastatin), were investigated for their ability to reverse P-glycoprotein (P-gp) mediated rhodamine 123 (R123) transport in a murine monocytic leukaemia cell line that over-expresses the multi-drug resistance protein 1a/b (mdr1a/1b).
P-gp modulation was studied by a fluorimetric assay and confocal microscopy by means of R123 efflux and uptake experiments, respectively.
Atorvastatin acid, methyl ester and lactone, lovastatin lactone and simvastatin lactone inhibited R123 transport in a concentration-dependent manner. Lovastatin acid, simvastatin acid, fluvastatin and pravastatin did not show a significant inhibition of the R123 transport in our cell system. Atorvastatin methyl ester and lactone showed the highest affinities for P-gp and results were comparable for both methods.
In conclusion, monitoring of R123 transport in living cells by confocal microscopy in addition to fluorimetric assay is a sensitive tool to study P-gp affinity in drug screening that is especially useful for early phases of drug development.
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P-glycoprotein (P-gp, mdr1-gene product), a member of the superfamily of ATP-binding cassette (ABC) transporters, has been intensively examined because of its multi-drug resistance (MDR) phenotype in oncology. This multi-drug transporter prevents the intracellular accumulation of anti-cancer drugs by actively removing them from the cell membrane before they reach their intracellular targets.
In the body, P-gp is widely expressed on the luminal surface of capillary endothelial cells in brain and testis, adrenal cortex, brush border membrane of the proximal renal tubule epithelium, canalicular membranes of hepatocytes, mucosa of the small and large intestine, pancreatic ductules, endometrium of gravid uterus and placenta (Ambudkar et al., 1999). Strong morphological and genetic evidence exist demonstrating a role of P-gp in absorption, distribution and excretion of certain hydrophobic, amphiphatic drugs and xenobiotics in mice and probably in humans (Schinkel, 1997). This may also lead to pharmacokinetic drug – drug interactions in human as described by different authors (Boyd et al., 2000; Hunter & Hirst, 1997; Yu, 1999). Thus two drugs that are transported by P-gp could compete for the transporter and as a result increase oral absorption, decrease hepatic/renal excretion and/or alter distribution into tissues where there is expression of this protein.
It is of interest to note that the cytochrome P450 system, especially the isoform P450 3A, has an overlapping substrate specificity to that of P-gp (Kim et al., 1999; Wacher et al., 1995).
The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) represent an established class of drugs for the treatment of hypercholesterolaemia, with potentially fatal adverse events (such as rhabdomyolysis). The lipophilic drugs lovastatin, simvastatin, atorvastatin and fluvastatin are metabolized via the cytochrome P450 system in the liver and the gut, making them subject to potential interactions with concomitantly administered drugs that are competing for metabolism via this system (Christians et al., 1998). Pravastatin is water-soluble and does not undergo metabolism via CYP450 to any significant extent. Both lovastatin and simvastatin are inactive lactone pro-drugs and are converted by hydrolytic enzymes in plasma and liver to the active acid form (Tang & Kalow, 1995) whereas atorvastatin, fluvastatin and pravastatin are administrated in their active acid form (Transon et al., 1996). All statins included in this study, except pravastatin, show a high hepatic extraction ratio. Furthermore it is known that fluvastatin (Lennernäs & Fager, 1997; Lindahl et al., 1996), lovastatin (Halpin et al., 1993) and simvastatin metabolites (Cheng et al., 1994) are excreted into bile, and that pravastatin has an important glomerular filtration and tubular secretion.
Numerous factors contributing to the risk for adverse drug interactions with statins have been reported recently (Bottorff, 1999; Christians et al., 1998; Corsini et al., 1999), and should be considered when patients are receiving additional drugs. Knowledge about the differences in the adverse drug interaction profile of statins is an important determinant of safety in long-term therapy of hypercholesterolaemia.
In the present study, we developed an in vitro fluorimetric assay and a confocal microscopy method to study the ability of five statins and their derivates for modulation of transport of a fluorescent P-gp substrate rhodamine 123 (R 123). As cell system, a murine mdr1a/1b over-expressing monocytic leukaemia cell line and its parental analogue (Boesch et al., 1991) was used.
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Because of its hydrophilicity, pravastatin's passive diffusion into hepatocytes is limited and transport across the sinusoidal membrane is facilitated by the sodium dependent organic anion transporter, hOATP2; this process can be inhibited by atorvastatin, simvastatin and lovastatin, suggesting that these statins possibly compete for this transport (Hsiang et al., 1999). Bile excretion of pravastatin is facilitated by MRP2 (Yamazaki et al., 1997).
When drugs compete for a common transporter, drug interactions are likely to occur. Until now, effects on metabolism explained most clinical interactions with HMG-CoA reductase inhibitors. Pravastatin is not metabolized by the CYP450 3A4 system, therefore it is likely that the increased pravastatin plasma concentration after co-administration with cyclosporin A is caused by interaction on one or more transporter proteins.
We established two in vitro assay systems that can be used complementary to assess drug mediated P-gp modulation. The confocal microscopy based assay can be used as a first-line highly sensitive method to analyse new substances for their effect on R123 accumulation properties. The need for only small amounts of compounds and cells and the real-time process monitoring makes it an appropriate method for early preclinical drug screening. Potential cellular toxicity and changes in the dye accumulation pattern is readily detectable. The microtiter plate based fluorimetric assay performs a fast further screening of extended concentrations and saturation kinetics. The latter assay allows a higher throughput of samples.
The differences in IC50 values between the two assays (Table 1) were most likely under-estimations of the inhibitory effect measured by the fluorimetric based assay. They could be explained by the different experimental designs, efflux versus uptake assays. During the efflux phase after loading of the cells, the direction of R123 passive diffusion and P-gp mediated extrusion was the same and sink conditions were maintained. Whereas in the uptake assay the two fluxes were in opposite directions. The same observations were reported in flow cytometry assays (Wang et al., 2000). Also an intracellular trapping of dye had to be considered, a fraction that would not be available anymore for active extrusion. Furthermore, efflux was stopped after 5 min, where uptake was analysed at least for 10 – 30 min after addition of modulator. In addition, a 6.7-times lower R123 concentration was used in confocal microscopy.
In confocal microscopy, calculated IC50 values for the statins may be over-estimated, due to the small number of samples in the low concentration range of the dose-response curve. The latter method showed an unexpectedly high IPrel−50 value for simvastatin (268%) and lovastatin (215%) in their lactone forms that can not be explained yet. Nevertheless, the overall ranking from strong over weak to none P-gp inhibition and affinity was: SDZ-PSC833 > atorvastatin methyl ester atorvastatin lactone > verapamil > simvastatin lactone ∼percnt; lovastatin lactone ∼percnt; atorvastatin acid > pravastatin ∼percnt; fluvastatin. This ranking was comparable for both methods and consistent with results of other groups for known inhibitors (Pourtier-Manzanedo et al., 1992).
In previous structure – activity – relationship studies on modulators of P-glycoprotein (Ecker & Chiba, 1995; Khan et al., 1998; Zamora et al., 1988), it has been shown that a correlation exists between the log P values and the chemosensitizing activity of a compound. In the present study, high affinity (low IC50) correlates with rather high log P values (Table 1) with the exception of fluvastatin, which has log P higher than 3. Our findings suggest that fluvastatin does not interact with P-gp, but the exact mechanism of interaction with cell membranes has to be elucidated.
Its ability to decrease the surface tension at the membrane surface and postulated interaction with lipids that could result in an altered membrane fluidity has been reported (Lindahl et al., 1999). Involvement of other transporters such as cMOAT and OATP, both anion transporters, cannot be excluded and will be investigated in future experiments.
Looking at the R123 accumulation rate initiated by a modulator, fast and slow processes can be distinguished. In addition, solubility of the modulator, transport rate through membrane bilayer and affinity for the transporter can influence P-gp interaction. The reported lower trans-membrane movement rate for SDZ-PSC833 compared to the one for verapamil (Ambudkar et al., 1999; Eytan & Kuchel, 1999; Smith et al., 1998), is well reflected in its high t50 value of 29.3 min in comparison to that of verapamil (t50=9.6 min). Furthermore, the fast onset of P-gp inhibition initiated by verapamil was proven in intestinal perfusion studies with talinolol in healthy volunteers (Grammaté & Oertel, 1999).
Our data suggested that the more lipophilic HMG-CoA reductase inhibitors had a higher R123 uptake rate (lower t50) (Table 1).
Drug – drug interactions
What is the value of this information to predict potential pharmacokinetic drug interactions? Recently, it was demonstrated that mibefradil is an inhibitor of CYP450 3A4 (Prueksaritanont et al., 1999). Mibefradil is also shown to be a P-gp inhibitor (Wandel et al., 2000).
Itraconazole, a P-gp inhibitor and CYP450 3A4 substrate, has a clinical interaction pattern with statins, that could be explained by both P-gp and CYP450 3A4 effects. Among the reported drug interactions (Table 2), the increase in digoxin (a P-gp substrate which is not metabolized by CYP450 3A4) plasma levels under concomitant administration of atorvastatin and simvastatin can be explained by P-gp inhibition alone.
Table 2. Reported drug interactions with HMG-CoA reductase inhibitors
Increased statin levels after co-administration with cyclosporin A can be explained both by P-gp and CYP450 3A4 inhibition. However the 5 – 23-fold increased pravastatin bioavailability is more likely caused by cMOAT inhibition rather than an effect on P-gp and CYP450 3A4. In addition, statins that showed P-gp modulating properties in our assays, are presumably also transported by the latter transporter.
Our in vitro cell system can be used to screen potential drug interactions, but well-designed clinical studies will still be needed to prove the hypothesis. A variety of other factors such as genetic polymorphism and interindividual variation (Hoffmeyer et al., 2000; Lown et al., 1997) or susceptibility to adverse effects are further determinants of clinical outcome.
In conclusion, the present study demonstrated that the described methods are able to analyse P-gp modulating properties of test compounds in vitro. We have shown that atorvastatin acid, atorvastatin methyl ester and lactone, simvastatin lactone and lovastatin lactone are P-gp modulators. No effect was seen with simvastatin acid, lovastatin acid, fluvastatin and pravastatin. Clinical and in vitro data suggest that P-gp modulating statins have a general trend to high probability for drug interactions.