Effects of Concomitant Administration of PXR Ligand Drugs on the Anticoagulant Effects of Warfarin

Abstract

We encountered cases in which the anticoagulant effects of warfarin (CYP2C9 substrate) were reversibly attenuated by the concomitant administration of rifampicin or bosentan, which are potent pregnane X receptor (PXR) ligands. The purpose of the present study is to report the previous case with rifampicin, and to evaluate the changes in the warfarin anticoagulant effects when withdrawing or switching bosentan treatment. The former is a case study of a 4-year-old girl undergoing warfarin treatment. The latter is a longitudinal study of 20 pediatric patients receiving stable warfarin treatment. The prothrombin time and international normalized ratio (PT-INR) values were extracted from the medical records and normalized by the daily-dose per body size as an index for the warfarin anticoagulant effects. Rifampicin treatment resulted in a 52.0% decrease in the anticoagulant index. On the other hand, 10 of 20 patients started bosentan and their anticoagulant index was reduced by a median of 2.00. Bosentan was withdrawn in 4 of 20 patients and their anticoagulant index increased by a median of 3.67. Six of 20 patients switched from bosentan to macitentan, which is considered not to activate PXR in clinical settings. However, switching from bosentan to macitentan resulted in a median of 2.25 reduction of the anticoagulant index rather than recovery of the response to warfarin. This study suggests not only the possibility of heterogeneity in the response to PXR activation and deactivation, but also the importance of long-term monitoring of drug–drug interactions when switching from bosentan to macitentan.

INTRODUCTION

The pregnane X receptor (PXR) is an orphan nuclear receptor involved in mediating the effects of compounds that induce both CYP3A and CYP2C gene expression.^1,2) Several drugs are known to be PXR ligands,²⁾ and drug–drug interactions via PXR activation or deactivation are of concern. Rifampicin is a potent PXR ligand and affects the clinical pharmacokinetics of drugs used concomitantly to different extents.³⁾ For example, rifampicin decreased the total area under the plasma concentration–time curve (AUC) of simvastatin and simvastatin acid by 87 and 93%, respectively, which are CYP3A substrates.⁴⁾ As for CYP2C substrates, rifampicin decreased the mean AUC of pioglitazone by 54% and shortened its dominant elimination half-life from 4.9 to 2.3 h.⁵⁾ Thus, assessment of changes in drug-metabolizing activity via PXR is necessary to provide useful information on effective and safe drug treatment.

Warfarin is an oral anticoagulant drug and a substrate of CYP2C9. Careful adjustment of the dose based on the prothrombin time international normalized ratio (PT-INR) is essential because of the narrow therapeutic index and large individual variability in the relationship between the warfarin dose and its anticoagulant effect. We previously demonstrated that the rational pediatric dosage of warfarin can be well-described by a body size (SIZE) parameter that includes an allometry exponent of weight.^6,7) The SIZE parameter is useful to describe the response to warfarin in pediatric patients. We encountered the interaction between warfarin and rifampicin via PXR in a pediatric patient with congenital heart disease in whom rifampicin was used for infective endocarditis (Fig. 1). When the patient was treated with rifampicin from day −63 to day 0, PT-INR values normalized by the daily-dose per SIZE (PT-INR/(Dose/SIZE)) decreased compared with before rifampicin treatment. Of note, after withdrawal of rifampicin on day 0, normalized PT-INR values returned to pre-rifampicin levels (Fig. 1). Martins et al.⁸⁾ reported that the dosage of warfarin needed to be doubled at the start of rifampicin therapy and a markedly high PT-INR was observed (7.22) 4 weeks after the discontinuation of rifampicin in a 59-year-old Brazilian woman who was chronically treated with warfarin for atrial fibrillation. Our observation (Fig. 1) is consistent with that reported by Martins et al.,⁸⁾ suggesting that the PXR-mediated interactions occurred in pediatric patient. Therefore, caution should be exercised in the concomitant use of PXR ligand drugs and warfarin in the process of anticoagulant index returning to the normal range.

Fig. 1. The Effects of Rifampicin on the Response to Warfarin

Double arrow indicates period of rifampicin treatment (from day −63 to day 0). The dose of rifampicin was between 19.9 and 21.6 mg/kg/d.

Pediatric patients with congenital heart disease undergoing warfarin treatment sometimes require an additional endothelin receptor antagonist (ERA) for the treatment of pulmonary arterial hypertension. Bosentan and macitentan are ERAs approved for use in children and both drugs have PXR-stimulating activity in nature.⁹⁾ We previously reported that the combined administration of bosentan reversibly reduced the warfarin anticoagulant effects to 87.5% in pediatric patients.^6,7) Thus, we proposed paying attention to the changes in response to warfarin after the withdrawal of bosentan or switching to other drugs. On the other hand, the PXR activation effects of macitentan are thought to be negligible in clinical settings because the EC₅₀ value for PXR activation is higher than the therapeutic plasma concentrations of macitentan.⁹⁾ In recent years, macitentan has been reported to be preferable to bosentan due to its greater improvements in hemodynamic parameters, heart rate, arterial oxygen saturation and brain natriuretic peptide.¹⁰⁾ To our knowledge, however, the changes in warfarin response have not been documented when switching from bosentan to macitentan.

The purpose of the present study is to report the previous clinical case of drug–drug interactions with warfarin via PXR activation by rifampicin, and to evaluate the changes in the anticoagulant effects of warfarin when switching from bosentan to macitentan in clinical settings.

PATIENTS AND METHODS

Subjects and Data Collection

Case Report

The patient was a 4-year-old girl with congenital heart disease. The patient received rifampicin from September 7, 2006 to November 9, 2006 for the treatment of infective endocarditis. Thirty-six data points were collected retrospectively. During the study period, the patient age changed from 3.77 to 5.61 years old and body weight ranged from 8.81 to 11.8 kg. The dosage of warfarin ranged from 0.070 to 0.278 mg/kg/d. The patient took other drugs concomitantly with warfarin: in particular, aspirin, beraprost and sildenafil. Some data where sulfamethoxazole/trimethoprim was used in combination were excluded in the present study. Of note is that there was no concomitant use that affected the effects of warfarin.

Longitudinal Study

A retrospective follow-up study was conducted on 20 Japanese pediatric patients (13 boys and 7 girls). The study group consisted of 10 patients who started initial treatment of bosentan, 4 patients who withdrew bosentan treatment, and 6 patients who switched from bosentan to macitentan. No patients initiated ERA treatment with macitentan in the study group. Duration of bosentan treatment in individuals ranged from 22 to 463 d in initial treatment. Duration of bosentan treatment in individuals ranged from 197 to 2301 d until withdrawal. Duration of bosentan treatment in individuals ranged from 1001 to 2941 d before switching to macitentan. The mean dose of bosentan was 1.80 ± 0.983 mg/kg/d.

We extracted information relating anticoagulant therapy from the medical records, including demographics, the corresponding date for each PT-INR measurement, indication for warfarin usage, concomitant drugs and other medical assessments. When patients received a stable warfarin dosage for at least 1 week, their data were eligible for analysis unless the PT-INR value was unsettled and/or unpredictably exceeded the therapeutic range. A total of 138 data points was collected during the follow-up period of 1.16 years in average. All patients received oral administration of warfarin at Toyama University Hospital. They routinely received oral administration of warfarin (Warfarin Tablet; Eisai Co., Ltd., Tokyo, Japan) at doses between 0.0342 and 0.171 mg/kg/d. The dose of warfarin was adjusted based on the anticoagulant response measured by the PT-INR in each patient. The patients and/or parents provided written informed consent to participate in the present study, which was approved by the ethics committee of the University of Toyama (#EG21-6, #EG22-1).

Index for Response to Warfarin

In this study, the PT-INR values normalized by daily-dose per SIZE⁶⁾ was used as an anticoagulant index for the warfarin effects.

  

(1)

  

(2)

Dose is the daily dose of warfarin (mg/d), SIZE is the hypothetical body size, WT is the individual body weight in kg and 0.559 is the allometric index value reported in our previous study.⁷⁾

Calculation of Estimated Glomerular Filtration Rate (eGFR)

The eGFR value was calculated by using the Schwartz formula.¹¹⁾

  

(3)

L is body length in cm, P_cr is plasma creatinine concentration (mg/dL) and k is a proportionality constant that reflects the relationship between urinary creatinine excretion and body size units.

Genotyping of CYP2C9 and Vitamin K Epoxide Reductase Complex 1 (VKORC1)

Genotypes of CYP2C9 and VKORC1 were determined in this study and our previous study.^6,12) We determined CYP2C9 genotypes by using the PCR-restriction fragment length polymorphism (PCR-RFLP) method and confirmed the VKORC1 1173 C > T variant by direct sequencing.¹²⁾

Data Analysis

Values were shown as the mean ± standard deviation (S.D.). Since the number of data sampling points was varied in each patient, the mean value was determined based on individual averages of observations. The significance of the differences between two groups was assessed using the Student’s t-test when the variances of the groups were similar. p < 0.05 was considered to be significant.

RESULTS

The effects of rifampicin on the anticoagulant activity of warfarin where PT-INR values normalized by daily-dose per SIZE are shown in Fig. 1. During the rifampicin treatment period, the anticoagulant index decreased by 52.0%. When rifampicin was discontinued, the anticoagulant index recovered to the level before rifampicin treatment (Fig. 1). This outcome suggested the activation of PXR by rifampicin and its reversibility.

Next, we retrospectively collected data from 20 patients to clarify the changes in the warfarin anticoagulant effects associated with the switch from bosentan to macitentan. The demographics of the 20 patients are summarized in Table 1. Fourteen patients had the VKORC1 1173TT genotype, 5 had the 1173CT genotype and 1 had the 1173CC genotype. One patient was heterozygous for CYP2C9*1/*3. Six of 20 patients switched from bosentan to macitentan. The ages ranged from 0.853 to 18.9 years old, and was 6.78 ± 5.18 years on average. Body weight ranged from 5.75 to 45.4 kg, and was 19.1 ± 11.0 kg on average. The daily dose of warfarin normalized by body weight for patients with the VKORC1 1173TT genotype was significantly lower than that for those with the 1173CT or 1173CC genotype, although there was no significant difference in the PT-INR values. This suggested the effects of the VKORC1 genotype on the optimal dose of warfarin (Table 1).

Table 1. Demographics and Data of Japanese Pediatric Patients Who Underwent Bosentan Therapy

	Total	VKORC1 genotypes
		1173CT or 1173CC	1173TT
Number of patients	20	6	14
Male	13	5	8
CYP2C9*3	1	0	1
Bosentan therapy
Initial treatment	10	2	8
Withdrawal	4	2	2
Switch to macitentan	6	2	4
Data sampling points	138	46	92
Age (years)	6.78 ± 5.18 (0.853–18.9)	5.42 ± 2.37 (2.28–9.30)	7.36 ± 5.99 (0.853–18.9)
Body weight (kg)	19.1 ± 11.0 (5.75–45.4)	15.1 ± 4.41 (9.63–20.7)	20.9 ± 12.5 (5.75–45.4)
Warfarin dose (mg/kg/d)	0.0919 ± 0.0313 (0.0494–0.162)	0.123 ± 0.0228 (0.0978–0.163)	0.0784 ± 0.0239* (0.0494–0.134)
PT-INR	1.54 ± 0.171 (1.24–2.03)	1.49 ± 0.0856 (1.38–1.63)	1.56 ± 0.196 (1.24–2.03)
Serum creatinine (mg/dL)	0.342 ± 0.102 (0.217–0.625)	0.343 ± 0.0683 (0.275–0.443)	0.341 ± 0.117 (0.217–0.625)
eGFR (mL/min/1.73 m2)	110 ± 27.0 (53.1–183)	104 ± 13.3 (56.5–139)	112 ± 31.6 (53.1–183)

Values were expressed as the mean ± S.D. with range in parentheses. * p < 0.05 compared with the 1173CT or 1173CC genotype.

The effects of ERAs and VKORC1 genotype on the response to warfarin in 20 patients are shown in Fig. 2, where the arrows indicate the time course. The anticoagulant index of patients receiving bosentan slightly decreased. However, switching from bosentan to macitentan slightly reduced the anticoagulant index rather than restoring the response to warfarin. These decreases in the anticoagulant index did not depend on the VKORC1 genotype (Fig. 2). Therefore, the anticoagulant index in the two VKORC1 genotype groups combined to perform subset analysis on patients who received ERA treatment. Differences in the anticoagulant index among patients undergoing several patterns of ERA treatment are shown in Fig. 3. Ten patients (7.48 ± 6.23 years) started bosentan and their anticoagulant index decreased by a median of 2.00. Bosentan was withdrawn in 4 patients (3.89 ± 2.42 years) and their anticoagulant index increased by a median of 3.67. Six patients (7.45 ± 4.50 years) switched from bosentan to macitentan and their anticoagulant index decreased by a median of 2.25 (Fig. 3).

Fig. 2. The Effects of ERAs and VKORC1 Genotype on the Anticoagulant Effects of Warfarin

The direction of arrows corresponds to the time course. Open symbols represent the data from patient with the CYP2C9*3 allele. The dotted lines represent the mean value. The mean dose of bosentan was 1.80 ± 0.983 mg/kg/d. The mean dose of macitentan was 0.196 ± 0.0370 mg/kg/d.

Fig. 3. Difference in the Response to Warfarin in Patients Receiving ERAs

Boxes indicate the median and interquartile ranges. Vertical lines above and below the boxes indicate the minimum and maximum values. Outliers are denoted with small circles.

DISCUSSION

The purpose of the present study is to report the previous case with rifampicin, and to evaluate the changes in the anticoagulant effects of warfarin when withdrawing or switching bosentan treatment. The former is a case study of a 4-year-old girl with congenital heart disease undergoing warfarin treatment. The latter is a longitudinal study of 20 Japanese pediatric patients receiving stable warfarin treatment. Rifampicin resulted in a 52.0% decrease in the anticoagulant index and the anticoagulant index returned to original levels after discontinuation of rifampicin (Fig. 1). For reference, the PT-INR values increased to 2.17 and 7.75 on day 11 and day 16 after the end of rifampicin administration, respectively. After that, anticoagulant indexes during the period from day 16 to day 38 and from day 96 to day 168 were unstable (data not shown). Therefore, unfortunately, we could not estimate the time that the anticoagulant index returns to the pre-dose levels after the end of rifampicin treatment. To our knowledge, however, this is the first pediatric case report highlighting the reversibility of PXR activation by rifampicin. On the other hand, 10 of 20 patients (7.48 ± 6.23 years) started bosentan and their anticoagulant index was reduced by a median of 2.00 (Fig. 3). Bosentan was withdrawn in 4 of 20 patients (3.89 ± 2.42 years) and their anticoagulant index increased by a median of 3.67. Six of 20 patients (7.45 ± 4.50 years) switched from bosentan to macitentan, which is considered not to activate PXR in clinical settings. Unexpectedly, switching from bosentan to macitentan resulted in a median of 2.25 reduction of the anticoagulant index rather than restoring the response to warfarin (Fig. 3).

PXR contains a DNA-binding domain linked to a ligand-binding domain by a probably flexible hinge region and the ligand-binding domain contains the ligand-dependent activation function region.¹³⁾ PXR is heterodimerized with retinoic X receptor-α (RXRα) and binds to various response elements (direct repeats DR-3, DR-4 and DR-5, and everted repeats ER-6 and ER-8) in the promoter regions of target genes.^13,14) The promoter region of CYP3A4 contains DR-3 and ER-6, and that of CYP2C9 contains DR-4.¹⁴⁾ PXR controls transcription events and regulates CYP3A4, CYP2C8, CYP2C9 and CYP2C19.^13,14) PXR also controls the expression of genes encoding CYP2B6, carboxylesterases, dehydrogenases, enzymes implicated in heme production, uridine 5′-diphosphate (UDP)-glucuronosyltransferases, glutathione-S-transferases, multidrug resistance protein 1 and multidrug resistance protein 2.^13,14) Chen et al.¹⁵⁾ reported that PXR activated by rifampicin induced CYP3A4 more strongly than CYP2C9; rifampicin induced approximately 30-fold higher CYP3A4 activation and 2.3-fold higher CYP2C9 activation in HepG2 cells.¹⁵⁾ As shown in Table 2, the mean EC₅₀ values for PXR activation by rifampicin, bosentan and macitentan were reported to be 0.7, 4.8 and 1.0 µM, respectively.^9,16) Of note, macitentan is an even more potent PXR ligand than bosentan (Table 2).

Table 2. Comparison of ERA Characteristics

	Rifampicin16,21,22)	Bosentan9,23,24)	Macitentan9,25)
EC50 for PXR activation (µM)	0.7	4.8 ± 1.0	1.0 ± 0.02
Maximum plasma concentration (µM)	9.71	5.67	0.63
Protein binding (%)	87–91	98	>99

In contrast to our results, Sidharta et al.¹⁷⁾ reported that macitentan has no effect on the pharmacokinetics or pharmacodynamics of a single dose of warfarin in healthy subjects. However, we should consider important differences of the study design between the two studies. First, the study by Sidharta et al. was aimed at healthy adults, whereas our study was aimed at pediatric patients with heart disease. Second, the subjects received a single dose of warfarin in the study by Sidharta et al., but our subjects underwent routine and stable treatment with warfarin. Third, our subjects had been pretreated with bosentan prior to macitentan treatment. Fourth, the treatment period of macitentan was only 4 d in the Sidharta’s study, while our subjects received macitentan for at least 65 d. Lepist et al.¹⁸⁾ reported that hepatocyte accumulation and cellular uptake of macitentan were higher than those of bosentan. When compared with the extracellular test concentration, the accumulated values were approximately 20-times and >100-times for bosentan and macitentan in sandwich-cultured human hepatocytes, respectively. The cellular uptake of macitentan was about 7-times higher than that of bosentan (31.9 pmol/million cells vs. 4.6 pmol/million cells) in human hepatocytes.¹⁸⁾ Macitentan being a potent PXR ligand and accumulating in hepatocytes more than bosentan may explain the decrease in anticoagulant index by switching from bosentan to macitentan (Fig. 3). In addition, Hedrich et al.¹⁹⁾ reviewed polymorphisms within the non-coding region that may affect the overall expression of the CYP2B6 gene, revealing that some of the SNPs identified in the promoter region of CYP2B6 are within the binding sites of several transcription factors.¹⁹⁾ Indeed, Li et al.²⁰⁾ demonstrated that individuals with the −82T > C mutation may become hypersensitive to drugs which are CYP2B6 substrates when co-applied with PXR-type inducers. Heterogeneity in PXR activation by genetic polymorphisms may explain the discordance in anticoagulant index observed in this study (Fig. 3).

There are several study limitations that must be acknowledged. First, the number of patients was limited. Second, although long-term data analysis was performed, age-related changes in organ function and body composition are dynamic; therefore, the effects of growth may not be negligible. Third, the plasma concentration of warfarin and ERAs were not directly evaluated in this study. Fourth, PXR polymorphisms were not determined in this study. Additional studies may be required to clarify PXR activation mechanisms.

In conclusion, starting bosentan resulted in a reduction of the anticoagulant index, bosentan withdrawal resulted in an increase, and switching from bosentan to macitentan resulted in a reduction. This suggests not only the possibility of heterogeneity in the response to PXR activation and deactivation, but also the importance of long-term monitoring of drug–drug interactions when switching from bosentan to macitentan.

Acknowledgments

This study was supported in part by JSPS KAKENHI Grant No. 18K06780.

Conflict of Interest

The authors declare no conflict of interest.

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