Introduction

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Lovastatin and pravastatin are structurally related (Fig. 1) inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting step in de novo cholesterol synthesis. In the long term, HMG-CoA reductase inhibitors slow the progression or even cause regression of coronary atherosclerosis, resulting in fewer new lesions and total occlusions compared with untreated hypercholesterolemic patients (MAAS Investigators, 1994; Jukema et al., 1995). The oral bioavailability of lovastatin is approximately 5% and highly variable (Henwood and Heel, 1988). In the liver, lovastatin is metabolized by cytochrome P-450 (CYP) 3A enzymes (Wang et al., 1991), and more than 90% is eliminated as metabolites in the bile (Henwood and Heel, 1988). One of the major side effects of HMG-CoA reductase inhibitors is skeletal muscle toxicity (Hsu et al., 1995); the incidence of lovastatin skeletal muscle toxicity in patients with HMG-CoA reductase inhibitor monotherapy is 0.1% (Bradford et al., 1991) but increases up to 30% when lovastatin is combined with a drug that is a CYP3A substrate/inhibitor, such as cyclosporine (Tobert, 1988). In clinical studies, itraconazole and cyclosporine, both of which are known to interfere with the CYP3A-dependent metabolism of lovastatin, increased lovastatin blood concentrations by 20-fold (Neuvonen and Jalava, 1996; Olbricht et al., 1997). In skeletal muscle cells, high concentrations of HMG-CoA reductase inhibitors cause damage as a result of a reduced formation of HMG-CoA metabolites such as geranylgeraniol (Flint et al., 1997).

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Structures of pravastatin (A), its major metabolites, and degradation products (Everett et al., 1991) and lovastatinlovastatin and its major metabolites formed by CYP3A (Wang et al., 1991). Alternative names used for the pravastatin metabolites in the literature (Kitazawa et al., 1993; Quion and Jones, 1994) are shown.

Until recently, poor oral bioavailability was mainly attributed to poor solubility in gastrointestinal fluids, poor permeability through the mucosal membrane, and/or extensive hepatic first-pass metabolism (Benet et al., 1996). CYP-dependent drug interactions were generally assumed to take place mainly in the liver. It has recently been recognized that CYP3A enzymes in the small intestinal mucosa are a significant drug metabolism and drug interaction site and, in combination with intestinal countertransporters, may play a major role in the low and variable oral bioavailability of several drugs that are CYP3A substrates (Lampen et al., 1995, 1996; Wu et al., 1995; Benet et al., 1996; Paine et al., 1997).

To date, only the liver metabolism of lovastatin has been evaluated (Greenspan et al., 1988; Vyas et al., 1988, 1990a,b; Wang et al., 1991). Because lovastatin is a known CYP3A substrate in the liver (Wang et al., 1991), we hypothesize that the small intestine is also a lovastatin drug metabolism site and that intestinal lovastatin metabolism is involved in drug interactions. Thus, it was our primary goal to study the intestinal metabolism of lovastatin. As a secondary goal, we compared the small intestinal metabolism of lovastatin with that of the structurally related HMG-CoA reductase inhibitor pravastatin.

	Experimental Procedures

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HPLC Equipment. For HPLC-UV analysis of lovastatin and its metabolites, a model 1090 M liquid chromatograph equipped with a diode array detector and an autosampler (Hewlett-Packard, Waldbronn, Germany) was used. The HPLC system was controlled and data were processed using Hewlett-Packard ChemStation Version C.02.02.

HPLC-Mass Spectrometry (MS) Equipment. Lovastatin and pravastatin metabolites were identified and pravastatin and its metabolites were quantified by HPLC-electrospray/MS. A model 1090 M HPLC (Hewlett-Packard) was connected to a model 5989B mass spectrometer equipped with an Iris Hexapole Ion Guide (Analytica of Branford, Branford, CT) by a model 59987A electrospray interface. The mass spectrometer and interface were controlled, and data were processed using ChemStation Revision A04.02 (Hewlett-Packard).

Materials. Lovastatin was the kind gift of Merck, Sharp & Dohme (Rahway, NJ). Pravastatin and its metabolites 3'-iso-hydroxy- and 3',5',6'-trihydroxypravastatin were the kind gifts of Bristol-Myers Squibb (Princeton, NJ). Cyclosporine was the kind gift of of Novartis (Basel, Switzerland). Ketoconazole was purchased from Research Biochemicals Inc. (Natick, MA). Estrone, dehydroepiandrosterone, mevastatin, adenosine-3'-phosphate 5'-phosphosulfate (PAPS), and troleandomycin were obtained from Sigma Chemical Co. (Deisenhofen, Germany).

Tissue Samples. To study human small intestinal metabolism, samples were collected from two male and two female patients undergoing surgery for tumor resection at the Klinik für Abdominal- und Transplantationschirurgie (Medizinische Hochschule Hannover, Hannover, Germany). For patient safety, it was necessary to remove healthy tissue beyond the tumor border. Portions of this healthy tissues were used for our study. All samples were collected from anatomically equivalent positions of the duodenum. Only patients who did not take drugs known to interfere with CYP3A enzymes for at least 2 weeks before surgery were included. Human liver samples were collected after liver transplantation in children who received part of an adult liver (Klinik für Abdominal- und Transplantationschirurgie).

Isolation of Microsomes. Human and pig intestinal epithelial cells were isolated according to the methods of Porteous et al. (1979) and Pinkus (1981). Enterocytes and liver samples were frozen at 80°C until the preparation of microsomes. Each preparation step was carried out at 4°C. Enterocytes and the liver samples were taken up in 4 times their volume of a buffer containing 0.1 M Na⁺/K⁺-phosphate, 0.1 mM KCl, 1 mM EDTA, and 0.1 mM dithiothreitol. Samples were homogenized using a glass-Teflon manual homogenizer and were treated with an ultrasound disintegrator (Branson, Danbury, CT; four 1-min pulses, 80 W). Microsomes were isolated by differential centrifugation as described by Guengerich (1982) with the following modification: Instead of Tris buffer, 0.1 M phosphate buffer (pH 7.4) was used. After ultracentrifugation, the supernatant (cytosol) was collected to study the hepatic and intestinal pravastatin phase II metabolism. The residue (microsomes) was reconstituted in four times its volume of a buffer solution containing 0.1 mM Na⁺/K⁺ phosphate buffer, 0.1 mM pyrophosphate, 1 mM EDTA, and 0.1 mM dithiotreitol and stored at 80°C. No protease inhibitors were used during the isolation of small intestinal microsomes. The protease inhibitor phenylmethylsulfonyl fluoride was found to inhibit lovastatin metabolism. The use of a protease inhibitor mixture of 1 µM leupeptin and 0.3 µM aprotinin had no beneficial effect on CYP3A activity; however, it was essential to maintain the microsomes at 4°C during isolation. Protein concentrations were determined using the bicinchoninic acid method described by Smith et al. (1985) with BSA as standard. Protein concentrations of the microsomal suspensions were adjusted with 0.1 M phosphate buffer, pH 7.4. The CYP concentrations were determined using the method described by Omura and Sato (1964) following the protocol of Estabrook and Werringloer (1978).

Microsomal Metabolism of Lovastatin and Pravastatin. Microsomal protein was adjusted to the following concentrations: human liver, 0.25 g/l; human small intestine, 1.5 g/l; pig liver, 1.5 g/l; and pig small intestine, 1.5 g/l. Then 1-ml microsomal preparation and lovastatin [in acetonitrile/water (2:1, v/v); final concentrations, see below] were preincubated for 5 min. Of an NADPH-producing system containing 2 mM EDTA, 10 mM MgCl₂, 0.84 mM NADP, 18 mM isocitric acid, and 700 U/l isocitrate dehydrogenase in 0.1 M phosphate buffer (pH 7.4), 0.5 ml was added. The assays were incubated for 20 min, and the reaction was stopped by protein precipitation after the addition of 0.5 ml of acetonitrile (liver microsomes) or methanol/0.2 M ZnSO₄ (7:3, v/v; small intestinal microsomes).

Pravastatin Phase II Metabolism in Liver and Small Intestine. To study cytosolic sulfation of pravastatin in the cytosol of small intestinal mucosa cells and hepatocytes, the assay described for hepatic cytosol by Kitazawa et al. (1993) was used. Small intestinal cytosol preparations were based on isolated mucosa cells as described for the isolation of intestinal microsomes. Pravastatin (50 µM) was incubated with either human liver cytosol (20 mg protein) or human small intestinal mucosa cytosol (3 mg protein) and 0.5 M PAPS in 0.1 M Na⁺/Na⁺ phosphate buffer at 37°C for 90 min. Reactions were stopped by protein precipitation after the addition of 500 µl of methanol/0.2 M ZnSO₄ (7:3, v/v). Negative controls contained heat-inactivated (10 min, 95°C) cytosol preparations or cytosol preparations were incubated with 0.1 M Na⁺/Na⁺ phosphate buffer without PAPS.

Determination of Unbound Fraction. To compensate for nonspecific binding during the microsomal incubation (Obach, 1997), the unbound fraction was determined using the Microcon YM-3 (Millipore, Bedford, MA) filtration system. Lovastatin and pravastatin were incubated with liver and intestinal microsomes and pravastatin in addition to cytosol from intestinal mucosa cells as described above but without NADP or PAPS, respectively. A 500-µl sample was transferred into the reservoir of the Microcon filter devices. After centrifugation at 10,900g for 30 min, the concentrations of pravastatin and lovastatin (after the addition of an equal volume of acetonitrile because lovastatin is water insoluble) were measured in the filtrate as described below.

Quantification of Lovastatin and Its Metabolites. After protein precipitation (see above), the internal standard mevastatin (600 ng) was added. The samples were centrifuged at 4°C (3400g, 5 min), and the supernatant was extracted with 3 ml of ethyl acetate/acetone (2:1, v/v). The samples were vortexed for 15 s, and 2.5 ml of organic phase was transferred into a glass centrifuge tube. The solvent was evaporated under a stream of nitrogen at 40°C. The residues were reconstituted in 100 µl of acetonitrile; 50 µl of water was added, and the samples were transferred into glass HPLC vials.

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Quantification of Pravastatin and Its Metabolites. After protein precipitation to stop the metabolism reaction, samples were centrifuged at 4°C (3400g, 5 min). The supernatant was drawn through extraction columns filled with C₁₈ material of 25- to 40-µm particles (Merck/Recipe) that had been primed by subsequently washing the columns with 2 ml of methanol and 2 ml of water. The vacuum was adjusted to result in a flow rate of 1 ml/min. After the supernatants were loaded onto the extraction columns, the samples were washed with 2 ml of water. Pravastatin and its metabolites were eluted using 400 µl of acetonitrile/formic acid, pH 4 (2:1 v/v), and the eluates were transferred into glass HPLC microvials.

Identification of Lovastatin and Pravastatin Metabolites. Lovastatin metabolites were identified by their characteristic UV absorption spectra (Vyas et al., 1990a) and HPLC-MS. The mass spectrometer was run in the scan mode (m/z 50-600). Isolated metabolite fractions were introduced into the mass spectrometer by flow injection using a manual injection valve connected between the analytical column and the electrospray interface. The electrospray interface and the mass spectrometer were adjusted as described for quantification of pravastatin and its metabolites above. For pravastatin, authentic standards of 3',5',6'-trihydroxypravastatin and 3'-iso-pravastatin were available. Pravastatin metabolite peaks in the ion-chromatograms were identified by comparison of mass spectra and HPLC retention times with the authentic standards.

Determination of Apparent K_m and Apparent V_max Values. To determine the apparent K_m and apparent V_max values of lovastatin metabolite formation, microsomes were incubated with the following lovastatin concentrations (n = 4 for each concentration): human liver microsomes, 5, 10, 15, 20, 30, 40, and 50 µM; and human small intestinal, pig liver, and pig small intestinal microsomes, 10, 20, 30, 40, 50, 60, 70, and 100 µM. Human liver (n = 4) and small intestinal (n = 3) microsomes were incubated with pravastatin concentrations of 250, 500, 750, 1000, 1250, 1500, and 2000 µM. Heat-inactivated microsomes (95°C, 10 min) were used as negative controls. Apparent K_m and V_max values were determined after data fitting (SigmaPlot Version 4.0; Jandel Scientific, San Rafael, CA) using the Hanes-Woolf linearization method. Intrinsic clearances (V_max/K_m) were calculated after correction of the apparent K_m values for the unbound fraction.

Inhibition of Liver and Small Intestinal Lovastatin Metabolite Formation by CYP3A Antibodies and Inhibitors. Microsomal protein (100 µg) isolated from human intestinal mucosa was incubated with 0, 0.5, 1, 2.5, or 10 µl CYP3A antibody solution (1 µl  10 µg protein; Gentest, Woburn, MA) on ice for 15 min. Then, 10 µM lovastatin or 1000 µM pravastatin and the NADPH-producing system were added. Samples were incubated and extracted as described above.

	Results

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Lovastatin Metabolism by Liver and Small Intestinal Microsomes. The two major lovastatin metabolites 6'-hydroxy- and 6'-exomethylene lovastatin were formed during incubation with human liver and intestinal microsomes. Incubation with pig small intestinal microsomes also generated 3'-hydroxylovastatin.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Representative Hanes-Woolf plots of lovastatin metabolite formation by human liver (A) and small intestinal microsomes (B). The results of all experiments are summarized in Table 1.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   CYP3A4/5 antibody concentration-dependent immunoinhibition of the formation of lovastatin (A) and pravastatin (B) metabolites by human small intestinal microsomes. Each data point represents the mean ± S.D. (n = 4).

Pravastatin Metabolism by Liver and Small Intestinal Microsomes. Incubation of pravastatin with human small intestinal microsomes resulted in the formation of two metabolites. One metabolite could be identified as 3',5',6'-trihydroxypravastatin. The other metabolite was a hydroxylated pravastatin metabolite. Because no standard material for hydroxylated pravastatin metabolites was available, its exact structure was not identified. The metabolite pattern generated by small intestinal microsomes equaled that after incubation with human liver microsomes. The apparent K_m and apparent V_max values are shown in Table 2. Apparent K_m values of 3',5',6'-trihydroxypravastatin in the human small intestine and in the liver were not significantly different. Compared with the liver, the mean K_m value of hydroxypravastatin in the small intestine was 2.4-fold lower. The mean V_max value of 3',5',6'-trihydroxypravastatin was 1.5-fold and that of hydroxypravastatin was 2.5-fold lower in the human small intestinal microsomes than in the human liver microsomal preparations. This was at least in part due to the lower CYP concentrations/g microsomal protein. Although the mean CYP concentration in the liver microsomes was 0.11 ± 0.09 µmol/g protein (mean ± S.D., n = 4), the CYP concentrations in the small intestinal microsomes were below the detection limit. Compared with lovastatin metabolites, the mean K_m values of the pravastatin metabolites were approximately 400-fold higher and the V_max values were 2.5-fold lower. Formations of 3',5',6'-trihydroxypravastatin and hydroxypravastatin were significantly inhibited by CYP3A4/5 antibodies. However, at the highest antibody concentration (100 µg/100 µg microsomal protein), compared with uninhibited controls, 3',5',6'-trihydroxypravastatin formation was inhibited by only 57.6% (Fig. 3B). Hydroxypravastatin formation was inhibited by 71.2%.

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View larger version (20K): [in this window] [in a new window]   Fig. 4.   Formation rates of 3'-iso-pravastatin in human liver (A) and small intestinal mucosa (B) cytosol. Pravastatin (50 µM) was incubated with 0.5 M PAPS at 37°C for 90 min. 3'-Iso-pravastatin was quantified by HPLC-MS. Each bar represents the mean ± S.D. (n = 3).

Comparison of CYP-Dependent Metabolism of Lovastatin and Pravastatin. To compare lovastatin and pravastatin metabolism, intrinsic metabolic clearances were calculated after correction of the K_m values for the unbound protein fraction. The unbound protein fractions for lovastatin were 59.7 ± 5.0% (n = 3) in liver microsomal preparations and 13.4 ± 0.8% in small intestinal microsomal preparations. The unbound fractions for pravastatin were 98.7 ± 5.0 and 80.1 ± 4.4%, respectively. Protein binding was linear over the protein and study drug concentration ranges used in this study.

Inhibition of Small Intestinal Metabolism of Lovastatin by Specific CYP3A Inhibitors Cyclosporin, Ketoconazole, and Troleandomycin. Cyclosporin, ketoconazole, and troleandomycin were effective inhibitors of 6'-exomethylene lovastatin and 6'-hydroxylovastatin formation in human liver and pig small intestinal microsomes (Table 4). Only ketoconazole also inhibited 3'-hydroxylovastatin formation in pig small intestinal microsomes. Ketoconazole was the most potent inhibitor of lovastatin metabolism with apparent K_i values in the liver and small intestine 4- to 30-fold lower than cyclosporine. Both cyclosporine and troleandomycin were better inhibitors of lovastatin metabolism in pig small intestinal than in human liver microsomes. In human liver microsomes, troleandomycin was a 3-fold weaker inhibitor than cyclosporine. In the pig small intestinal microsomes, however, troleandomycin K_i values were up to 50-fold lower than those in human liver microsomes.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

We demonstrated that lovastatin is metabolized in the small intestine to its major metabolites and that the generated metabolism pattern is similar to that in the liver. As in the liver, lovastatin metabolism was inhibited by CYP3A inhibitors and antibodies, indicating that in the small intestine, CYP3A enzymes are involved in lovastatin metabolism and that the small intestine is a potential site of lovastatin drug interactions.

It has to be taken into account that only apparent enzyme kinetic parameters were determined for the following reasons: microsomes do not represent an isolated enzyme, but a mixture of different enzymes and more than one enzyme of the CYP3A subfamily may be involved in the metabolism of lovastatin or pravastatin. In addition, the inhibitors used are also CYP3A substrates and are metabolized during incubation. Nonspecific binding to microsomal proteins has been recognized as an important factor when comparing the metabolism of different substrates. Evidence has been reported that the results of in vitro upscaling based on the intrinsic metabolic clearance correlated better with the in vivo situation when K_m values were corrected for the unbound fraction (Obach, 1997). Accordingly, we compared intrinsic clearances of lovastatin and pravastatin after correction of the K_m values for the unbound fractions.

The highest CYP3A concentration in the gastrointestinal tract is present in the duodenum and decreases in the following sequence: duodenum > jejunum > ileum > colon (Kolars et al., 1994; Paine et al., 1997). To avoid interference with variability resulting from the different locations of small intestinal samples, it was essential to collect samples from equivalent anatomical positions of the duodenum. Furthermore, patients with a history of ingesting drugs known to influence the activity of CYP3A enzymes were excluded. Duodenal samples were chosen because they have the highest CYP3A concentration and the proximal small intestine is the primary location at which lovastatin is predominantly absorbed.

For lovastatin drug interaction studies in the small intestine, pig small intestinal microsomes were used because the quantities of human small intestinal samples available were insufficient. Because some differences in midazolam metabolism in pigs and humans have been reported (Ochs et al., 1987; Gorski et al., 1994), the metabolite patterns and enzymatic parameters of human and pig liver and small intestinal microsomes were cross-validated in our study. The results proved that pig small intestinal microsomes were a valid model for the study of lovastatin drug metabolism and drug interactions. This is supported by other studies evaluating drug metabolism and interactions of CYP3A substrates in the small intestine. Thus, Lampen et al. (1995, 1996) found similar K_m and V_max values for the formation of tacrolimus and cyclosporine metabolites in pig and human liver and small intestinal microsomes and almost identical K_i values for drug interactions in human liver and pig small intestinal microsomes. Lampen et al. (1995) showed that pig CYP3A enzymes in the liver and small intestine cross-react with a human CYP3A antibody, indicating structural homologies of the respective human and pig enzymes. Although the inhibition constants probably cannot be directly extrapolated from pigs to humans, effective and potent inhibitors of lovastatin metabolism in the pig small intestine can be expected to also be effective and potent inhibitors of lovastatin metabolism in the human gut mucosa.

Although the intrinsic metabolic clearance of lovastatin metabolites in human intestinal microsomes was 4.2- to 8.3-fold lower than that in liver microsomes, it is impossible to estimate the contribution of intestinal metabolism to lovastatin first pass metabolism and drug interactions. The lower intrinsic clearance by intestinal than by liver microsomes in our study was mainly due to the lower concentration of microsomal protein in the intestinal microsomal preparations and the resulting lower V_max value. Despite a well established lower metabolic capacity of the small intestine in comparison with the liver, recent results demonstrated that the small intestine may play a significant role in first-pass metabolism (Benet et al., 1996). The most important unresolved issue is the role of intestinal countertransport and its functional interaction with intestinal CYP enzymes. Lovastatin interacts with P-glycoprotein (Dimitroulakos and Yeger, 1996), an ATP-binding cassette transporter, that is involved in intestinal countertransport (Benet et al., 1996; Hunter and Hirst, 1997). It can be expected that countertransport limits the access of drugs to the CYP enzymes and may prevent CYP enzymes from being overwhelmed by the high drug concentrations present in the small intestine. On the other hand, with a drug being repeatedly transported out of the mucosa cells and being reabsorbed, repeated exposure to CYP3A enzymes may lead to more efficient metabolism (Benet et al., 1996; Gan et al., 1996). In addition, the intestinal transporter/CYP barrier may control substrate availability to liver CYP enzymes. Another factor that complicates an estimate of the relative role of intestinal in comparison with liver first-pass metabolism is the high interindividual variability of intestinal CYP3A enzymes and transporters as well as liver CYP3A enzymes (Kolars et al., 1994; Lown et al., 1997). Because in vivo the functional interaction between intestinal transporters and CYP3A enzymes seems important (Benet et al., 1996) and intestinal transporters control substrate availability to the CYP3A enzymes, it is unclear whether the intrinsic metabolic clearance, which was calculated based on isolated intestinal microsomal preparations, alone is a valid parameter to decide whether intestinal metabolism of lovastatin is clinically relevant and significantly involved in the low oral bioavailability of lovastatin and lovastatin drug interactions.

Pravastatin is the only HMG-CoA reductase inhibitor that is mainly eliminated unchanged (Everett et al., 1991; Quion and Jones, 1994). Its main metabolite, 3'-iso-pravastatin, is inactive and has a terminal plasma half-life slightly shorter than that of pravastatin (Quion and Jones, 1994). Our study showed that small intestinal microsomes generated the same metabolite pattern as liver microsomes. As shown before (Jacobsen et al., 1999), CYP3A4 and CYP3A5 are involved in the formation of these metabolites in the liver and, as shown in our study using CYP3A antibodies and specific chemical inhibitors, also in the small intestine. However, as in the case of formation of 3',5',6'-trihydroxypravastatin, inhibition at the highest CYP3A antibody doses was incomplete, and the involvement of other microsomal enzymes in the formation of this metabolite may be possible. A similar result was described for 3',5',6'-trihydroxypravastatin in the liver (Jacobsen et al., 1999). When compared with lovastatin, however, the small intestinal intrinsic clearance (V_max/K_m) of pravastatin metabolites was approximately 5000-fold lower (Table 3). The difference in the intrinsic clearances indicated that in contrast to lovastatin and although both drugs are structurally related, CYP-dependent small intestinal metabolism of pravastatin cannot be expected to play a significant role in pravastatin pharmacokinetics. The results of our study also suggest that mechanisms other than CYP-dependent metabolism must be responsible for the low oral bioavailability of pravastatin of an average 15% (Quion and Jones, 1994). The major pravastatin metabolite 3'-iso-pravastatin is generated by both acidic degradation in the stomach (Triscari et al., 1995) and, in the liver, by sulfation at the 6'-hydoxy group by sulfotransferases, followed by a nucleophilic attack of hydroxy anions at the 3' position (Kitazawa et al., 1993). Our study showed that the gut wall is an additional site of 3'-iso-pravastatin formation.

It is concluded from our study that lovastatin is metabolized in the small intestine by CYP3A enzymes, resulting in a similar metabolite pattern as in the liver. Thus, as described for other CYP3A substrates (Benet et al., 1996), gut wall metabolism is a potential factor in the low and variable oral bioavailability of lovastatin and its pharmacokinetic interactions with other drugs that are CYP3A substrates and/or inhibitors. In comparison, the gut intrinsic clearance of pravastatin is 5000-fold lower, and only minor pravastatin metabolites are generated. Based on our results, it is unlikely that intestinal CYP3A-mediated metabolism plays a clinically relevant role in pravastatin pharmacokinetics. However, as in the liver, pravastatin undergoes phase II metabolism in the small intestine, which results in formation of its major metabolite 3'-iso-pravastatin.