In Vivo And In Vitro Metabolism And Drug Interactions Of Fluvastatin

Fluvastatin, the first fully synthetic HMG-CoA reductase inhibitor, is structural analogues of the intermediate formed by HMG-CoA reductase in the synthesis of mevalonate, and block cholesterol synthesis by competitively and reversibly inhibiting the activity of HMG-CoA redutase. Enzyme inhibition results in decreased in the expression of high-affinity receptors of LDL on hepatocyte membrances. This, in turn, results in the increased clearance of LDL-C and LDL precursors leading to decrease serum concentrations of total cholesterol and LDL-C. Fluvastatin has been shown to reduce cholesterol in patients with essential hypercholesterolemia, to prevent subsequent coronary heart disease. Since lipid-lowering therapy for the middle and aged patients often is life-long, the use of fluvastain will be freguently accompanied by the use of multiple drugs, which may course adverse effect by drug-drug interactions. Hence considering of the in vivo and in vitro metabolism and the risk for drug interaction is very important to instruct clinical medication. Our study focused on the in vivo and in vitro metabolism and drug-drug interactions of fluvastatin to explain the in vivo and in vitro correlation and species difference of fluvastatin metabolism, and evaluate the security and rationality of co-administration of fluvastatin with western medicine and traditional Chinese medicine. 1. In vivo pharmacokinetics study of fluvastatin in rats and Beagle dogs AIM: To establish an HPLC method for determination of fluvastatin in plasma andto investigate its pharmacokinetic characteristics in SD rats and Beagle dogs. METHODS: Six rats or dogs received a single oral dose of fluvadtatin, respectively, and blood samples were collected at different time. One week later, each subject received a single intravenous dose of fluvastatin and the blood samples were collected. The plasma was mixed with the same volume of KH2PO4 buffer (pH2), and then the fluvastatin in the mixture was extracted with ether. The organic layer was dried. The residue was dissolved in mobile phase and determined by RP-HPLC method. The data obtained was processed with 3P87 program on computer for pharmacokinetic parameters. RESULTS: The assay was linear from 20.16~5040.0 ng-mL"1 for fluvastatin. The LOQ and LOD were 20.2 ng-mL"1 (S/N=10, recovery 117.6±1.9%, RSD 1.6%, n=5) and 5.04 ng-mL"1 (S/N=3) , respectively. The mean method recovery was 93.30%~ 110.6%, and the extracted recovery was over 90% for the low, middle, high control sample. The relative standard deviations obtained for inter- and intra-day precisions were less than 5%. The concentration-time curves of fluvastatin after oral or intravenous administration in rats and dogs fitted two-compartment model. The absolute bioavailability of rats and dogs was 45.29±22.48%, 65.13±25.20%, respectively. CONCLUTION: The bioavailability of fluvastatin was different in different species.2. In vitro metabolism of fluvastatin in liver microsomes of rat, dog and human AIM: Study the in vitro metabolism of fluvastatin in different source of liver microsomes to verify the metabolism difference of fluvastatin in different species of rat, Beagle dog and human. METHODS: The metabolic reaction of fluvastatin was performed in the native liver microsomes of rat, dog, huaman and the rat liver microsomes pretreated with P-naphthoflavone (BNF), phenobarbital (PB), dexamethasone (Dex), respectively. After incubation for a given time, the reactions were stopped with the same volume of cold methanol, as well as to denature the protein. The remained substrate and produced metabolites in incubates were detected by HPLC. RESULTS: The assay was linear from 10.0 ~ 2500.0 ng-mL'1 forfluvastatin in incubates. The LOQ and LOD were 10.0 ng-mL"1 (S/N=10, recovery 110.0±2.5%, RSD2.3%, n=5) and 3.0 ng-mL"1 (S/N=3) , respectively. The mean method recovery was 99.3%~105.9%, and the extracted recovery was over 95% for the low, middle, high control sample. The relative standard deviations obtained for inter- and intra-day precisions were less than 5%. After 60min incubation, the metabolic rate of fluvastatin was 14%, 21% ,32%, by dog, rat and human liver microsomes, respectively. The Km, Vmax and Clmt of fluvastatin in rat and human microsomes were 3.79±1.00umol- L"1, 97.5±20.1 nmol- L"1- min"1- mg protein'1, 0.026±0.003 ul -mm4- mg protein"1 and 1.47±0.10umol- L"1, 106.4±13.6 nmol- L"1- min"1- mg protein"1 > 0.073±0.008ul -min"1- mg protein'1, respectively. No significant difference was found for the metabolism of fluvastatin in different pretreated rat microsomes, comparing with the native (control) group (p>0.05). The three primary metabolites of fluvastatin, 5-hydroxy-fluvastin, 6-hydroxy-fluvastatin and N-desisopropyl-fluvastatin were observed and verified by LC-MS. CONCLUTION: There were significant difference for the metabolism of fluvastatin in different species, the fastest in human liver microsome, second in rat liver and the slowest in dog liver. The result was coincidence with that of in vivo pharmacokinetics study from part 1 and literature.3. The metabolic interaction between fluvastatin and some commonly used drugs or compoundsAIM: Study the metabolic drug interaction of fluvastatin with some commonly used western medicine and traditional Chinese medicine and the inhibition of fluvastatin to CYP enzymes, to provide some useful information for clinical medicate. METHODS: Fluvastatin was co-incubated with several kinds of commonly used drugs or compounds, respectively. The selected medicine and compounds were 5 antipyretic/ analgesic drugs such as phenacetin etc, 15 cardiovascular drugs such as felodipine etc, 8 antibacterial drugs such as ofloxacin etc, 4 antidepressant drugs such as fluvoxamine etc, as well as 26 other drugs or compounds, and the selectedtraditional Chinese medicines were 14 flavonoids components such as quercetin etc, 11 alkaloids such as ephedrine hydrochloride etc, 3 phenylpropanoids such as o-coumaric acid etc, 10 terpenes such as ginsenoside Rgl etc. The interactions were evaluated by IC50 or K\ of the co-incubated drugs or compounds on the metabolism of fluvastatin, and that of fluvastatin on the metabolism of the classic substrates of CYP enzymes. RESULTS: Flavonoids components showed fairly strong inhibition on the metabolism of fluvastatin, with K\ value between 0.15-85.18 umol- L"1. Among these flavonoids, isoflavone (daidzein and glycitein) had the most strong inhibition, with K\ value about 0.15 umol- L"1. The inhibition of glycoside to fluvastatin was significantly weakened compared with the corresponding aglycone. Among the cardiovascular drugs, both felodipine and nimodipine were almost equally inhibitory of all three metabolites (IC50 5~9umol-L'1). Among the antidepressant drugs, fluvoxamine and fluoxetine hydrochloride, had small effect on fluvastatin metabolism, with IC50 value 27-43 umol- L"1 and 65-78 umol- L"1, respectively. The substrates or inhititors of CYP2C9, diclofenac potassium, flurbiprofen and sulphaphenazole inhibited all pathways of fluvastatin metabolism, and the specific CYP2C9 inhititor sulphaphenazole had a very strong inhibition on the 6-hydroxy-and N-desisopropyl- fluvasatin formation, with K\ value O^OiO.OSumol-L"1 and 0.44±0.04umol-L'1, respectively. Similarly, fluvastatin had a strong inhibition on the metabolism of CYP2C9 substrates, diclofenac potassium and flurbiprofen, with K\ value between 0.32~0.48 umol-L"1. Typical CYP3A specific inhibitors such as ketoconazol and cyclosporine A only inhibited the formation of 5-hydro-fluvastatin, with IC50 value 27.63 umol- L"1 and 44.20 umol-L"1, respectively. The specific substrates or inhititors of CYP1A2, CYP2C19, CYP2D6 and CYP2E1 had no relevant effect on the metabolism of fluvastatin, and the same for fluvastatin on the metabolism of those substrates above (including CYP3A4). CONCLUTION: There were strong interactions between flavonoids, substrates or inhibitors of CYP2C9 and fluvastatin, special attention must be paid when co-administrated. It was of interest tonote the strongly inhibition of flavonoids on the metabolism of fluvastatin, and worth investigation further.