Modeled Hepatic/Plasma Exposures of Fluvastatin Prescribed Alone in Subjects with Impaired Cytochrome P450 2C9*3 as One of Possible Determinant Factors Likely Associated with Hepatic Toxicity Reported in a Japanese Adverse Event Database

Abstract

Fluvastatin is a 3-hydroxy-3-methylglutaryl CoA reductase inhibitor that competitively inhibits human cytochrome P450 (P450) 2C9 in vitro. Drug interactions between a variety of P450 2C9 substrates/inhibitors and fluvastatin can increase the incidence of fluvastatin-related hepatic or skeletal muscle toxicity in vivo. In this survey, the prescribed dosage of fluvastatin was reduced or discontinued in 133 of 164 patients receiving fluvastatin alone, as recorded in the Japanese Adverse Drug Event Report database of spontaneously reported events. The median days to onset of fluvastatin-related disorders were in the range 30–35 d in the 87 patients. Therefore, we aimed to focus on fluvastatin and, using the pharmacokinetic modeling technique, estimated the virtual plasma and hepatic exposures in subjects harboring the impaired CYP2C9*3 allele. The plasma concentrations of fluvastatin modeled after a virtual oral 20-mg dose increased in homozygotes with CYP2C9*3; the area under the plasma concentration curve was 4.9-fold higher than that in Japanese homozygotes for wild-type CYP2C9*1. The modeled hepatic concentrations of fluvastatin in patients with CYP2C9*3/*3 after virtual daily 20-mg doses for 7 d were 31-fold higher than those in subjects with CYP2C9*1/*1. However, heterozygous Chinese patients with CYP2C9*1/*3 reportedly have a limited elevation (1.2-fold) in plasma maximum concentrations. Virtual hepatic/plasma exposures in subjects harboring the impaired CYP2C9*3 allele estimated using pharmacokinetic modeling indicate that such exposure could be a causal factor for hepatic disorders induced by fluvastatin prescribed alone in a manner similar to that for interactions with a variety of co-administered drugs.

INTRODUCTION

Individual differences in drug-metabolizing enzymes or transporters may affect the pharmacokinetics of a drug and result in impaired therapeutic responses. Polymorphic cytochrome P450 (P450 or CYP) 2C9 is a major enzyme involved in the metabolism of a variety of marketed medicines such as phenytoin, celecoxib, diclofenac, and some statins.^1–3) Guidelines for statin use for the primary prevention of cardiovascular disease have recently been updated in the United States.⁴⁾ Pharmacokinetic interactions between typical P450 2C/3A substrate/inhibitor medicines and some statins,⁵⁾ specifically fluvastatin, may lead to statin-induced hepatic and skeletal muscle disorders,⁵⁾ so-called statin intolerance⁶⁾; in Japan, these facts were summarized in a clinical guide in 2018.⁷⁾ The Japanese Adverse Drug Event Report (JADER) database,⁸⁾ a spontaneous reporting system, is generally used to evaluate the time to onset of drug-associated adverse events^9–11) such as statin intolerance.

Patients receiving P450 2C9-dependent drugs, such as phenytoin, are carefully controlled by therapeutic drug monitoring based on individual plasma concentrations.^12,13) Siponimod, a treatment for secondary progressive multiple sclerosis,^14,15) was first approved in the U.S.A. and then in the EU. The importance of CYP2C9 genotypes has recently been added to the package insert of siponimod in Japan, despite the limited number of cases with information on CYP2C9 polymorphisms. Individual variations in the in vivo P450-dependent intrinsic clearance of drugs may be a causal factor for adverse events associated with the prescription of a single drug. Against this background, events reported in the JADER database associated with the prescription of a single drug only^16–18) were the focus of the current study. We recently reported that high virtual exposure to atorvastatin, another statin, in subjects with an amino acid substitution resulting in impaired CYP3A4*16 may indicate a causal factor for statin intolerance.¹⁶⁾

Fluvastatin is a 3-hydroxy-3-methylglutaryl CoA reductase inhibitor that competitively inhibits human cytochrome P450 (P450) 2C9 in vitro.^19,20) The aim of the current study was to estimate virtual hepatic/plasma exposures to fluvastatin in subjects with impaired CYP2C9 using simplified physiologically based pharmacokinetic (PBPK) modeling, as was done in recent related reports.^16–18) A P450 2C9 Ile359Leu missense variant, CYP2C9*3 (rs1057910), has been found in East Asians with allele frequencies of 2.5% (Tohoku Medical Megabank database 54 K Japanese population). Possible roles of organic anion transporting polypeptides 1B1 also have been reported in fluvastatin pharmacokinetics.^21,22) CYP2C9 rs77760615 (c.-5813A > G) and SLCO1B1 rs58310495 (intronic) variants were reportedly related to high fluvastatin exposures in a genome-wide association study.²³⁾ Therefore, we focused on fluvastatin and, using the PBPK modeling technique, estimated the virtual plasma and hepatic exposures in subjects harboring the impaired CYP2C9*3 allele; we also analyzed the statin intolerance data recorded in the JADER database.

MATERIALS AND METHODS

JADER database items recorded between April 2004 and December 2022 were surveyed in a manner similar to that described previously^16–18) for adverse events in patients treated with fluvastatin alone, and a relational database was constructed. The number of days to onset was calculated from the date of treatment initiation and the date of occurrence of an adverse event (recorded as drug regimen “discontinued” or “reduced”) for 20 or 30 mg fluvastatin. Adverse events were summarized in this study either as all adverse events or as selected events related to hepatic or skeletal muscle disorders (Table 1). The cumulative incidence was calculated according to the Kaplan–Meier method using JMP Pro 13 (SAS Institute, Cary, NC, U.S.A.) and Prism 10 (GraphPad Software, La Jolla, CA, U.S.A.).

Table 1. Adverse Events (n = 172) Associated with Fluvastatin Therapy in 133 Subjects Receiving No Other Prescription Drugs Who Had to Discontinue Therapy or Receive a Reduced Dose

Adverse event	Number	Percentage, %
Total	172	100
Hepatic function abnormal	52	30
Liver disorder	33	19
Rhabdomyolysis	14	8
Drug-induced liver injury	9	5
Blood creatine phosphokinase increased	6	4
Liver function test abnormal	4	2
Gamma-glutamyl transferase increased	3	2
Others	51	30

The data were obtained from the JADER database (Fig. 1). Some subjects experienced multiple adverse events.

Reported plasma concentration-versus-time data for fluvastatin after oral administration in Japanese subjects²⁴⁾ were used to establish the pharmacokinetics of wild-type CYP2C9*1 homozygotes. The procedure for calculating the input parameters for a simplified human PBPK model, i.e., the absorption rate constant (k_a), the volume of the systemic circulation (V₁), and the in vivo hepatic intrinsic clearance (CL_h,int), to provide the best fit to the published data was described previously^16–18,25) and is briefly outlined in the footnote to Table 2. A simple hepatic clearance model was adopted in this study based on the intrinsic clearance achieved by polymorphic CYP2C9; however, it has been suggested that liver uptake transport may be a rate-limiting step.^23,26) To estimate the CL_h,int values in subjects harboring CYP2C9*1 or CYP2C9*3 alleles, the reported in vitro hepatic intrinsic clearance values for fluvastatin by recombinant variant P450 2C9.3 relative to wild type P450 2C9.1 were adopted, as summarized in Table 3.

Table 2. Chemical Properties and Calculated Parameters for PBPK Modeling of Fluvastatin Based on Reported Pharmacokinetic Data

Parameter	Abbreviation (unit)	Value
Acid dissociation constant	pKa	4.34
Octanol–water partition coefficient	log P	4.05
Plasma unbound fraction	fu,p	0.0126
Blood–plasma concentration ratio	Rb	0.685
Liver (kidney)–plasma concentration ratio	Kp,h (Kp,r)	6.44
Fraction absorbed × intestinal availability	Fa·Fg	0.448
Absorption rate constant	ka (1/h)	3.21 ± 0.31a)
Volume of systemic circulation	V1 (L)	11.6 ± 1.1a)
Hepatic intrinsic clearance	CLh,int (L/h)	3080 ± 10a)
Hepatic clearance	CLh (L/h)	27.7
Renal clearance	CLr (L/h)	0.3
Maximum concentration in plasma	Cmax (ng/mL)	190 (0.98)b)
Area under the concentration curve from 0 to 6 h in plasma	AUC6 (ng h/mL)	231 (1.10)b)

The acid dissociation constant (pK_a), plasma unbound fraction (f_u,p,), and octanol–water partition coefficient (log P) values were obtained by in silico estimation using ACD/Percepta, Simcyp, and ChemDraw software, respectively. The liver (kidney)-to-plasma concentration ratios (K_p,h (K_p,r)) and the blood-to-plasma concentration ratio (R_b) were calculated from the f_u,p, and log P values as follows³⁴⁾:

where

a) Data of fitting estimations (mean ± standard deviation) were obtained from reported pharmacokinetic data in 11 Japanese subjects²⁴⁾ with 1% renal elimination (U.S. FDA drug label). The following set of differential equations was solved for the amounts and concentrations:　

when at

where X_g, V_h, V_r, C_h, C_r, and C_b are the amount of drug in the gut compartment; liver and kidney volumes; and hepatic, renal, and blood substrate concentrations, respectively.²⁵⁾ V_h, V_r, and Q_h (Q_r) are the liver (1.5 L) and kidney (0.28 L) volumes and the blood flow rates of the systemic circulation to the hepatic (renal) compartments (96.6 L/h) in humans (70 kg body weights).²⁵⁾ b) Estimated PBPK model outputs after virtual oral administration of 20 mg fluvastatin. The values in parentheses are the ratios to the observed values. C_max, maximum concentration; AUC₆, area under the concentration curve from 0 to 6 h.

Table 3. In Vitro Hepatic Intrinsic Clearance Values of Fluvastatin by P450 2C9 for Estimating in Vivo Hepatic Intrinsic Clearance Values as Input Parameters for PBPK Modeling in Subjects Harboring CYP2C9*1 or CYP2C9*3

Parameter	Value
Fitted in vivo hepatic intrinsic clearance for CYP2C9.1, L/h, taken from the literature	3080 ± 10 from Table 2
Reported in vitro fraction metabolized by CYP2C9	0.835)
Vmax and Km for 2C9.1	0.27 min−1 and 1.1 µM30)
Vmax and Km for 2C9.3	0.19 min−1 and 4.1 µM30)
2C9.3/2C9.1 ratio of Vmax/Km	0.19
Estimated in vivo hepatic intrinsic clearance for CYP2C9.3, L/h	1080

RESULTS

Adverse Events with Fluvastatin Prescribed Alone

As shown in Fig. 1A, of the 3345 patients in the JADER database treated with fluvastatin, 164 patients treated with fluvastatin alone were surveyed. Discontinued or reduced dosages were recorded in 133 of these patients, who recorded a total of 172 adverse events (Table 1); the apparent incidence rate was high at 81%. Among the 93 of these patients with data on the timing of adverse events, the median (interquartile range) number of days to onset of adverse events was 30 (19–53) and 35 (28–56) d for 20 and 30-mg daily doses of fluvastatin, respectively (Fig. 1B). Regarding selected events related to hepatic and skeletal muscle disorders (Table 1), 87 of the 93 patient records indicated a similar number of days to onset. There were no significant differences between the two fluvastatin dose groups on the days to onset (Fig. 1B).

Fig. 1. Case Selection Process (A) and Time to Onset of Selected Events in Patients Prescribed Fluvastatin Alone (B) Based on Data Taken from the Japanese Adverse Drug Event Report Database

(A) Patient number (n) is shown. (B) Number of events (n) is indicated.

Model for Decreased P450 2C9 Activity

The reported mean plasma concentration plots of fluvastatin after a single oral dose of 20 mg²⁴⁾ or 40 mg²⁷⁾ and the estimated time-dependent plasma concentration curves generated using the current PBPK model are shown in Figs. 2A and B. The input parameters (Table 2) modeled on pharmacokinetic data from 11 Japanese subjects (plots in Fig. 2A)²⁴⁾ were applied to estimate the reported plasma concentrations of fluvastatin in nine Chinese subjects genotyped as CYP2C9*1/*1 after a single oral dose of 40 mg.²⁷⁾ To the best of our knowledge, we were unable to search the genotyped Japanese data in the literature. We then moved to the reported data of the same East Asian Chinese population. Three Chinese subjects heterozygous for CYP2C9*1/*3 experienced negligible effects on the reported plasma concentrations of fluvastatin (Fig. 2B). To fit additional reported pharmacokinetic data for fluvastatin in nine Chinese subjects with CYP2C9*1/*1, the calculated input parameters were similar to those for the 11 Japanese subjects. To fit the pharmacokinetic data for fluvastatin to concentration data from an additional nine reported Chinese subjects with CYP2C9*1/*1 and three subjects with CYP2C9*1/*3,²⁷⁾ the k_a, V₁, and CL_h,int values for these two groups were calculated as 2.87 ± 0.16 and 2.09 ± 0.40 (1/h), 9.5 ± 0.2 and 11.0 ± 3.1 (L), and 3420 ± 10 and 2250 ± 10 (L/h), respectively. These estimated values for the wild-type subjects were considered reasonable when compared with the data shown in Table 2. The in vivo hepatic intrinsic clearance values for fluvastatin subjects harboring the impaired allele CYP2C9*3 relative to wild-type CYP2C9*1 were estimated, as shown in Table 3, as input parameters for PBPK modeling. Time-dependent plasma concentration curves after a virtual oral dose of 40 mg fluvastatin were generated using the PBPK model for CYP2C9*3/*3 (Fig. 2C). After virtual repeated doses of 20 mg fluvastatin, the maximum plasma concentration (C_max) and area under the concentration curve from 0 to 168 h (AUC₁₆₈) in subjects homozygous for CYP2C9*3 were 2.6-fold and 4.9-fold higher, respectively, compared to those in subjects homozygous for CYP2C9*1 (Figs. 2D, E, Table 4). CYP2C9*3 might result in modification of the catalytic function similar to that resulting from the co-administration of fluvastatin and some other medicines like celecoxib.

Fig. 2. Reported and Estimated Human Plasma and Liver Concentrations of Fluvastatin

(A, B) Mean reported plasma concentrations after oral administration of 20 or 40 mg fluvastatin for (A) eleven subjects homozygous for CYP2C9*1 and (B) nine subjects homozygous for CYP2C9*1 (circles) and three subjects heterozygous for CYP2C9*3 (triangles). The line shows the PBPK model results for subjects homozygous for CYP2C9*1. (C) Estimated virtual plasma concentrations of fluvastatin after a virtual dose of 40 mg in CYP2C9*3 homozygotes based on the in vitro fraction metabolized by polymorphic P450 2C9 and the reported impairment ratio of in vitro CL_h,int values. (D, E) PBPK model results for plasma (solid lines) and liver (dotted lines) concentrations of fluvastatin in subjects harboring CYP2C9*1/*1 (black) or CYP2C9*3/*3 (red) after 20 mg daily for 7 d.

Table 4. Estimated Plasma and Liver C_max and AUC₁₆₈ Values Obtained Using Human PBPK Models after Virtual Oral Doses of 20 mg Fluvastatin Daily for 7 d in Virtual Subjects Harboring CYP2C9*1/*1 or CYP2C9*3/3

Plasma/liver	Genotype	Cmax, µg/mL or µg/g	AUC168, µg h/mL or µg h/g
Plasma	CYP2C9*1/*1	0.19 (1.0)	1.6 (1.0)
	CYP2C9*3/*3	0.49 (2.6)	7.8 (4.9)
Liver	CYP2C9*1/*1	1.4 (7.4)	10 (6.3)
	CYP2C9*3/*3	3.3 (17)	50 (31)

Modified in vivo hepatic intrinsic clearance values were used for the two genotypes (Table 3). C_max, maximum concentration; AUC₁₆₈, area under the concentration curve from 0 to 168 h. The numbers in parentheses indicate the percentages of plasma C_max or AUC₁₆₈ values with respect to subjects with CYP2C9*1/*1.

The virtual hepatic concentrations of fluvastatin after daily doses of 20 mg (the standard recommended dose in Japan) for 7 d were estimated (Table 4). The plasma and hepatic C_max values generated by the PBPK models were 0.19 µg/mL and 1.4 µg/g, respectively, in homozygous subjects harboring CYP2C9*1/*1. The plasma and hepatic C_max concentrations increased to 0.49 µg/mL and 3.3 µg/g, respectively, in subjects homozygous for CYP2C9*3/*3. Under the present conditions, the virtual hepatic concentrations of fluvastatin after daily doses of 20 mg for 7 d were higher than the plasma concentrations in subjects harboring CYP2C9*3/*3, as illustrated in Fig. 2E. The high hepatic/plasma exposures to fluvastatin in this distinct population (Table 4) indicated that impaired CYP2C9 activity could be a causal factor associated with statin intolerance (Fig. 1).

DISCUSSION

A clear dose-dependent transient increases in liver transaminases in patients taking statins, such as atorvastatin, lovastatin, and simvastatin, has been proven, although idiosyncratic liver injury due to statins is very rare and causality difficult to prove.²⁸⁾ The factors causing adverse events in patients administered fluvastatin alone could be related to liver malfunction, specifically, the reduced function of organic anion transporter OATP1B1 as a result of genetic variants or the reduced metabolic activity of CYP2C9 variants.^21,22) Although various factors contribute to drug-dependent adverse events, we speculate that, when a drug is prescribed alone, increased hepatic exposure resulting from polymorphism in the responsible enzyme gene may also induce hepatic adverse events, resulting in dose reduction or discontinuation of the drug, as also happens when concomitant therapy causes enzyme inhibition.

Based on in vitro systems, estimates of the reduced activity possessed by the variant CYP2C9*3 allele relative to wild-type CYP2C9*1 have been proposed; the fraction of activities of CYP2C9*1/*3 and CYP2C9*3/*3 compared with CYP2C9*1/*1 are reportedly 0.6 and 0.1–0.2, respectively.^29,30) The observed plasma AUC values of fluvastatin in Caucasian subjects heterozygous for CYP2C9*1/*3 were limited to a 1.3-fold elevation in vivo,¹⁾ in contrast to a 3-fold C_max elevation in virtual subjects homozygous for CYP2C9*3/*3. As illustrated in Fig. 2B, a similar 1.2-fold increase in the plasma C_max of fluvastatin in Chinese subjects heterozygous for CYP2C9*1/*3 compared with wild-type CYP2C9*1/*1 has been reported.²⁷⁾ Although homozygotes with CYP2C9*3 (rs1057910, with allele frequencies of 2.5%) would be relatively rare, these limited effects of heterozygous for CYP2C9*1/*3 could be attributed to the abundance of CYP2C9.1 in human livers³¹⁾ because of the reduced effects of heterozygous CYP2C9*1/*3 on the expression or catalytic activity in vivo, which differs from the clinical usefulness of a typical proton pump inhibitor dosage³²⁾ for low hepatic contents of CYP2C19.1³³⁾ that were modified in CYP2C19 heterozygous and homozygous poor metabolizers.

In our previous JADER studies, 258, 150, 56, and 14 patients in the JADER registry who were prescribed each of atorvastatin,¹⁶⁾ celecoxib,¹⁷⁾ diclofenac,¹⁷⁾ and atomoxetine¹⁸⁾ alone and who had their dosages reduced or their medication discontinued were included, in approximately 53, 19, 13, and 24% of single-prescription patients, respectively.^16–18) In virtual patients homozygous for responsive P450 3A4.16, 2C9.3, 2C9.3, and 2D6.10, the estimated plasma and hepatic concentrations of atorvastatin and celecoxib both increased several-fold compared with wild-type homozygotes, although the enzyme contributions to the metabolism of diclofenac and atomoxetine diverged, showing slightly lower values in vivo than in vitro.^16–18) In the current study, hepatic intrinsic clearance values for fluvastatin were used to estimate hepatic and plasma drug concentrations after virtual oral administrations, taking into account the in vitro or in vivo contributions of the enzyme responsible for the drug oxidation reaction and the in vivo loss of enzyme function.

In conclusion, the administration of fluvastatin alone caused statin intolerance in some patients recorded in the JADER database. The high virtual hepatic exposures in subjects harboring the CYP2C9*3 allele may indicate impaired CYP2C9 activity as a possible causal factor for statin intolerance, in a manner similar to well-known drug interactions. To the best of our knowledge, no cases of fluvastatin intolerance have been monitored in actual subjects or in patients with CYP2C9*3. In future studies, establishing the relationship between adverse events and drug exposure in actual patients will be most informative. Ongoing accumulation of pharmacogenomic information in clinical practice in Japan is needed to confirm the present pharmacogenetic hypothesis for adverse events related to these drugs.

Acknowledgments

The authors thank Manato Hosoi, Hina Nakano, and Norie Murayama for their assistance. This work was supported in part by the Japan Agency for Medical Research and Development under Grant No. 23mk0101253h0102, and by the Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (23K14393 and 23K06217). We are also grateful to David Smallbones for copyediting a draft of this article.

Conflict of Interest

The authors declare no conflict of interest.

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