Comparison of the Inhibitory Effect of Ketoconazole, Voriconazole, Fluconazole, and Itraconazole on the Pharmacokinetics of Bosentan and Its Corresponding Active Metabolite Hydroxy Bosentan in Rats
Mengchun Chen1#, Xufei Zhang2#, Yijie Chen3, Wei Sun1, Zhe Wang1, Chengke Huang1, Guoxin Hu4, and Ruijie Chen1*
Abstract
1. This study aimed to investigate the inhibitory effect of azole antifungal agents, including ketoconazole, voriconazole, fluconazole, and itraconazole, on the pharmacokinetics of bosentan (BOS) and its active metabolite hydroxy bosentan (OHBOS) in Sprague–Dawley (SD) rats.
2. A total of 25 healthy male SD rats were divided into five groups and treated with various azole antifungal agents by gavage, followed by a single dose of BOS after 30 min.
3. The study found that ketoconazole led to a significant increase (5.1-fold) in the AUC(0-t) of BOS, associated with a 5.8-fold elevation in the Cmax, which was greater than that for fluconazole (2.6- and 2.9-fold) and voriconazole (1.1- and 1.7-fold). Accordingly, the Vz/F and CLz/F of BOS reduced by 89.2% and 83.7%, respectively, on administering ketoconazole concomitantly. However, fluconazole caused a decrease in Vz/F and CLz/F by 77.4% and 72.2%, respectively, compared with voriconazole that exhibited a decrease in CLz/F by 51.7% with a negligible change in Vz/F. Also, obvious differences were observed in the pharmacokinetic parameters of OHBOS between the control and treated groups.
4. Collectively, treatment with ketoconazole resulted in a prominent inhibitory effect on the metabolism of BOS, followed by treatment with fluconazole, voriconazole, and itraconazole. Therefore, these details of animal studies may help draw more attention to the safety of BOS while combining it with ketoconazole, voriconazole, fluconazole, or itraconazole clinically.
Keywords: Bosentan; Hydroxy bosentan; Ketoconazole; Voriconazole; Fluconazole; Itraconazole
1. Introduction
Drug interaction is one of the commonest factors that may induce adverse reactions in patients in clinic(Canu et al., 2011; Siniscalchi et al., 2011). Therefore, optimizing therapeutic strategies using mixed drugs to mitigate the adverse effect is urgently needed. Bosentan (BOS) (Tracleer), a dual-receptor antagonist involved in the blockade of endothelin-A and endothelin-B, has been widely applied in treating pulmonary artery hypertension (PAH) (Funke et al., 2010) and effectively inhibiting the growth of digital ulcers around the fingertips or knuckles in patients with systemic sclerosis (Abraham and Steen, 2015). However, the adverse effects of BOS have led to additional observations or examinations to monitor its systemic toxicity. In the clinic, patients are always asked to undergo a blood test monthly for predicting elevated liver aminotransferase levels while they are taking BOS (Barst et al., 2003; Channick et al., 2001; Fattinger et al., 2001; Humbert et al., 2007; Rubin et al., 2002; Sitbon et al., 2004). The BOS metabolic pathway in humans involves hepatic catalysis, followed by biliary excretion (Weber et al., 1999). As shown in Figure 1, BOS is metabolized by the cytochrome P450 system in the liver and subsequently converted into three metabolites, namely, hydroxy bosentan (OHBOS) (Ro 48-5033), desmethyl BOS (Ro 47-8634), and hydroxy desmethyl BOS (Ro 64-1056) (Markova et al., 2013; van Giersbergen et al., 2002; Weber et al., 1999), of which OHBOS is a primary metabolite of BOS that assists BOS pharmacologically, retaining 10%–20% activities (Weber et al., 1999). A previous study also showed that two subtypes of cytochrome P450 system, cytochrome P450 3A4 (CYP3A4) and 2C9 (CYP2C9), were responsible for approximately 60% and 40% of BOS metabolism, respectively (Dingemanse and van Giersbergen, 2004). Therefore, changes to, or competition with, CYP2C9- and CYP3A4-mediated metabolism of bosentan would affect the pharmacokinetic profiles of BOS and metabolites.
Systemic antifungal drugs are essential to endow prophylaxis or treat invasive fungal infections. In most cases, these antifungal drugs, given either singly or in multiple combinations, exhibited an inhibitory effect on the CYP450 system (Hamberg et al., 2011; Kunze et al., 1996; Spriet et al., 2013; Venkatakrishnan et al., 2000; von Moltke et al., 1996). In these scenarios, antifungal drugs have been reported to not only inhibit CYP2C9 and CYP3A4 enzymatic activities but also indirectly exert an influence on the downstream substrates (Isoherranen et al., 2004; Niwa et al., 2005; Pea and Furlanut, 2001; Sakaeda et al., 2005). BOS is determined to be the substrate of both CYP2C9 and CYP3A4 enzymes (Dingemanse and van Giersbergen, 2004). Thus, any changes for activities of these two enzymes would affect the metabolism of BOS. Based on this fact, clinicians who prescribe both BOS and azole antifungal agents should deeply understand their pharmacokinetic profiles to manipulate a better outcome in the clinic. Considering that ketoconazole damages hepatic functions dramatically, other azole antifungal agents, including fluconazole, voriconazole, and itraconazole, which share large similarities with ketoconazole in structure, should be examined so that the metabolism of BOS can be optimized using the expected azole antifungal agent for the future standard of care. Indeed, if patients with fungal infections are suffering from pulmonary hypertension, the combination of BOS with azole antifungal agents will be highly recommended to achieve expected efficacy. Therefore, parent drug BOS and its offspring active metabolite OHBOS were employed in this study to provide insight into their pharmacokinetic changes after treating rats with ketoconazole, voriconazole, fluconazole, and itraconazole.
2. Methods and Materials
2.1 Chemicals and Reagents
Ketoconazole, voriconazole, fluconazole, and itraconazole were purchased from Melone Biotechnology Co., Ltd(Beijing, China). Bosentan (purity >98.0 %), Ro 48-5033 (OHBOS, purity >98.0 %), and losartan (internal standard, IS, purity >98.0 %) were procured from Sigma-Aldrich (St. Louis, MO). LC-grade organic solvents and LC/MS-grade acetonitrile were obtained from Merck (Darmstadt, Germany), and LC/MS-grade formic acid (FA, 98 % purity) was purchased from Sigma-Aldrich (Munich, Germany). All chemical agents used in this study were analytical grade. Ultra-pure water was freshly purified by Milli-Q A10 System (Millipore, Billerica, MA) that also was applied to the mobile phase and all other water relative solutions.
2.2 Instrumentation
Plasma levels of BOS and OHBOS were quantitated by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) using a Waters ACQUITY I-Class and a Waters XEVO TQD triple-quadrupole mass spectrometer (Waters Corp., Milford, MA) with an electrospray ionization source. Instrument control and data acquisition were performed using Masslynx 4.1 software (Waters Corp., Milford, MA).
2.3 Chromatographic Conditions
The chromatographic separation was carried out through employing an ACQUITY UPLC-MS/MS and combined a Waters ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7-μm particle size, Waters Corp.) with inline 0.2-mm stainless steel frit filter (Waters Corp.). The initial mobile phase consisted of solvent A (water containing 0.1 % formic acid) and solvent B (acetonitrile) with gradient elution at a flow rate of 0.45 mL/min, and the injection volume was 5 μL. Elution presented in a linear gradient as follows: 20 % B (0-0.5 min), 20- 80 % B (0.5-2.5 min), maintained at 80 % B (2.5-3.5 min), 20-80 % B (3.5-4 min), and maintained at 20 % B (4-5 min). The entire run for each sample cost about 5 min. Afterward, the sample manager executed a rigorous needle wash procedure, including a strong wash (methanol-water, 50/50, v/v) and a weak wash (methanol- water, 10/90, v/v) before the upcoming sample loading. Under these experimental settings, the determined retention times of BOS, OHBOS, and IS were 2.72, 2.16, and 2.23 min, respectively. In order to optimize the experimental process, the column and sample temperature were kept at 40 and 4 °C.
2.4 Mass Spectrometric Settings
The sample ionization was achieved by Waters XEVO TQD triple-quadrupole mass spectrometer with electrospray ionization source interface in a positive mode, and the subsequent quantitative analysis on BOS, OHBOS and IS was performed using the multiple reaction monitoring modes. The optimized ionization conditions were set as follows: 3.5 KV of capillary voltage, source temperature at 150 °C, and nitrogen was used as the desolvation gas (1000 liter/h) at 400 °C temperature and as well as cone gas (50 liter/h). The collision energy voltage was configured separately: 35 V for both BOS and OHBOS, and 25 V for losartan alone. Meanwhile, the cone energy voltage was fixed at 60 V for BOS and OHBOS and 35 V for losartan. Quantification was analyzed by multiple reaction monitoring with the transitions of m/z 552.2→202.1, m/z 568.2→202.1, and m/z 423.2→207.2 for BOS, OHBOS, and IS respectively.
2.5 Animals
Male Sprague–Dawley (SD) rats (220-250 g, n=25) were procured from Laboratory Animal Center of Wenzhou Medical University (Wenzhou, China, License No. SCXK [ZJ] 2005-0019) and were fed in ideal laboratory conditions which had free access to food and fresh drinking water with a 12 h light/dark cycle at constant temperatures (24-26 °C). Animal experimental protocols were approved by Institutional Animal Experimentation Committee of Wenzhou Medical University.
2.6 Experimental designs
25 healthy male SD rats were randomly divided into five groups, including four experimental groups (A, B, C, D, n=5 per group, A=ketoconazole, B=voriconazole, C=fluconazole, and D=itraconazole) and control group E (n=5). All rats were kept on fasting for 12 h before azole antifungal agents administration without the restriction of drinking water. BOS, ketoconazole, voriconazole, fluconazole, and itraconazole were dissolved in 0.5 % carboxymethylcellulose. The above five groups were treated with various azole antifungal agents at the identical dosage of 30 mg/kg by gavage, and 0.5 % carboxymethyl cellulose as a negative control. 30 mg/kg of azole antifungal agents on rats (Chen et al., 2016; Lin et al., 2014; Wang et al., 2015) are approximately equivalent to 336 mg on a person with 70 kg body weight. Also, 20 mg/kg BOS (i.g.) was used in our early study to establish the methods for BOS and glimepiride detection (Chen et al., 2016). According to the drug instruction, people with PAH is recommended oral maintenance dosage at 125 mg each, twice time per day. In our study, rats were orally administered 20 mg/kg BOS, which was equivalent to 224 mg on a person with 70 kg body weight. Thereafter, blood samples (~0.3 mL) were collected from each group at 0 (before drug administration), 0.17, 0.33, 0.67, 1, 1.5, 2, 3, 4, 6, 8, 10, 12 and 24 h and carefully harvested plasma by 13,000 rpm centrifugation. All samples were stored in -80 °C before use.
2.7 Plasma Sample Pretreatment
The stock solutions of BOS, OHBOS, and internal standard (IS) were prepared at 1 mg/mL in methanol respectively, all of which were stored at 4 °C and recovered to room temperature before use. Calibration standards and quality control (QC) samples were obtained by diluting the stock solution using blank rat plasma. The calibration samples were prepared at series of concentrations of 150, 300, 750, 1,500, 3,000, 7,500 and 15,000 ng/mL for BOS, and 0.5, 1, 2.5, 5, 10, 25 and 50 ng/mL for OHBOS in rat plasma. Meanwhile, QC samples were prepared at concentrations of 300, 1500, and 7,500 ng/mL for BOS, and 1, 5 and 25 ng/mL for OHBOS in rat plasma. Frozen samples were tabled at room temperature until completely thawed. Losartan (20 μL of 1 μg/mL in methanol solution) was added to 70 μL of collected plasma samples as an IS, following by addition of 140 μL of acetonitrile for protein precipitation. The mixed samples were vortex well for 2 min and centrifuged at 13,000 rpm at 4 °C for 10 min. 100 μL supernatant was collected and diluted with ultra-purified water with a ratio of 1: 1. Finally, 5 μL aliquot of the obtained solution was injected into the UPLC-MS/MS system for further analysis.
2.8 Pharmacokinetic and Statistical Evaluations
The results were expressed as mean ± standard deviation. The noncompartmental analysis was used to calculate the pharmacokinetic parameters by DAS version 3.0 (Bontz Inc., Beijing, China). One-way ANOVA and a Dunnet’s multiple comparisons test were used for the unpaired data analysis (GraphPad Prime 7.0a). Ap value of < 0.01 was considered statistically significant. 3. Results The UPLC-MS/MS chromatogram results of BOS and OHBOS quantifications in rat plasma standards, rat plasma samples, and blank rat plasma showed that blank plasma samples exhibited a negligible impact on peak shift during the elution time window, indicating no endogenous perturbation in experimental samples (Fig. 2). Following the blank plasma test, a variety of azole antifungal agents affecting the metabolism of BOS and OHBOS were analyzed. First, the changes in the pharmacokinetic parameters of BOS co-administered with ketoconazole were deciphered. The area under the plasma concentration-time curve (AUC(0–t)) and peak plasma concentration (Cmax) of BOS dramatically increased 5.1 and 5.8 times due to the combination of BOS and ketoconazole, compared with fluconazole (2.6- and 2.9-fold) and voriconazole (1.1- and 1.7-fold). Accordingly, the Vz/F and CLz/F of BOS significantly decreased by 89.2% and 83.7%, respectively, by introducing a certain dose of ketoconazole compared with a decrease in Vz/F and CLz/F after treatment with fluconazole (77.4% and 72.2%, respectively) and voriconazole (51.7% for CLz/F and no significant change in Vz/F). Meanwhile, the time to Cmax (Tmax) of BOS dropped to 4 and 3 h with the use of ketoconazole and voriconazole, respectively, whereas the Tmax of the other three groups was approximately 6 h. In addition, no statistically significant changes in the mean residence time (MRT(0-t)) were observed after ketoconazole treatment. Also, OHBOS, as the metabolite of BOS, indirectly changed various pharmacokinetic parameters in the presence of ketoconazole. Compared with BOS, AUC(0–t), and Cmax of OHBOS decreased by 20.9% and 27.9%, respectively, after co-administration of ketoconazole (Fig. 3 and Table 1). Other parameters, including Tmax and MRT(0-t) of OHBOS, maintained a consistent level as that in the control group. Collectively, the results showed that ketoconazole had a significant impact on the disposition of BOS and OHBOS in vivo. Next, voriconazole received attention when co-administered with BOS. Briefly, the AUC(0–t) and Cmax of BOS presented 1.1- and 1.7-fold elevation, and CLz/F reduced by 51.7% with a negligible change in Vz/F. It was worth noting that the Tmax of BOS had a noticeable decrease (3-h decrease) accompanied by shorter MRT(0-t) in the presence of voriconazole. On the contrary, the AUC(0–t) and Cmax of OHBOS decreased by 64.6% and 70.0%, respectively. In parallel, the Tmax of OHBOS showed a 2-h decrease compared with the BOS-alone group. Impressively, the MRT(0-t) of BOS dropped by 16.3%, whereas the MRT(0-t) of OHBOS showed a 16.0% increase after voriconazole treatment (Fig. 3 and Table 1). Consequently, voriconazole could influence the bioavailability of both BOS and OHBOS in vivo. As an important azole antifungal agent, fluconazole could significantly regulate the metabolism of both BOS and OHBOS. The AUC(0–t) and Cmax of BOS exhibited a relatively moderate increment (2.6- and 2.9-fold, respectively) after fluconazole treatment compared with the results obtained after ketoconazole treatment (5.1- and 5.8-fold). Conversely, the Vz/F and CLz/F of BOS decreased by 77.4 and 72.2%, respectively, after fluconazole treatment, which was close to the results obtained after ketoconazole treatment. Fluconazole did not disturb the Tmax of BOS but prolonged the MRT(0-t) (1 h) to some extent compared with the BOS-alone setting. Also, significant differences were detected in major pharmacokinetic parameters of OHBOS after administering fluconazole. Compared to the control group, the AUC(0–t) of OHBOS was decreased by 55.1 % and Cmax was decreased by 63.5 % in rats co-treated with fluconazole. Other pharmacokinetic profiles were all elevated compared with fluconazole treatment; Tmax (1.2-h increase) and MRT(0-t) increased by approximately 30.0%. Overall, fluconazole inhibited to the metabolism of BOS and OHBOS in vivo. Finally, itraconazole exhibited encouraging results clinically when co-administered with BOS owing to its effect on the pharmacokinetic values of BOS and OHBOS, except for only a 28.8% decrease in the CLz/F of BOS, a 27.5% increase in the AUC (0–t), and a 22.6% increase in the MRT(0-t) of OHBOS compared with those in the control group. The results indicated that itraconazole was more compatible with BOS metabolism. Hence, itraconazole was probably safer to use in combination with BOS, at least in the present rat model. 4. Discussion This study demonstrated that four azole antifungal agents (ketoconazole, voriconazole, fluconazole, and itraconazole) exhibited significantly varying effects on the pharmacokinetic parameters of BOS and its active metabolite OHBOS in rats. It further discussed the mechanism of action of these azole antifungal agents in interfering with the pharmacokinetics of BOS and OHBOS. CYP2C11 in rats is equivalent to CYP2C9 in humans, and CYP3A2 in rats is equivalent to CYP3A4 in humans. CYP2C9 and CYP3A4 were therefore used in this study to examine the changes in enzymatic activity in rats. The instructions regarding the use of BOS in clinic show that it reaches a maximum plasma concentration within 3–5 h after oral administration and has an elimination half-life of around 5 h in healthy adult participants (Venitz et al., 2012; Weber et al., 1996). BOS metabolism mostly occurred by hepatic, and to a lesser extent intestinal, CYP3A4 and CYP2C9-mediated hydroxylation and/or demethylation, followed by biliary excretion (Venitz et al., 2012; Weber et al., 1999). Regarding this metabolic feature of BOS, the CYP2C9 and CYP3A4 inhibitors, such as azole antifungal agents, can partially or wholly influence BOS metabolism theoretically. In the present study, the changes in the pharmacokinetics of BOS and its metabolite OHBOS in rats after administering ketoconazole revealed dramatic interactions between these compounds compared with the control group. The conspicuous increase in AUC(0-t) and Cmax associated with a sharp reduction in Vz/F, CLz/F, and Tmax of BOS, along with the decrease in AUC(0-t) and Cmax of OHBOS, was observed in the presence of ketoconazole that predominantly interfered with BOS elimination and metabolism. Intriguingly, AUC(0-t) and Cmax of BOS significantly enhanced in the present study (5.1- and 5.8-fold, respectively) compared with those in a similar study by van Giersbergen (van Giersbergen et al., 2002), in which AUC(0-t) and Cmax of BOS increased 2.3- and 2.1-fold, respectively. The differences in the findings of these two studies might be due to different experimental settings, including subjects selection, dosing regimens, and administration period. In van Giersbergen’s work (van Giersbergen et al., 2002), authors performed the experiments on healthy male subjects that were divided into two groups: Group A regimen consisted of a single dose of 62.5 mg BOS on day one and followed by 62.5 mg twice daily for 5.5 days; Group B regimen was comprising of BOS of 62.5 mg twice daily for 5.5 days plus concomitant ketoconazole (200 mg per day) for 6 days. In our study, BOS dose at 20 mg/kg on rats was equal to 224 mg on a person of 70 kg body weight, which was close to the clinical use that people with PAH is recommended oral maintenance of BOS at a dosage of 125 mg, twice per day. Also, the dose of azole antifungal agents at 30 mg/kg on rats was theoretically equivalent to 336 mg in the person of 70 kg body weight. Moreover, the drug interactions of BOS and azole antifungal agents in our study were determined by one single administration, whereas there were multiple administrations of either BOS or ketoconazole in van Giersbergen’s work. Further, the varied metabolism of BOS and OHBOS after ketoconazole treatment was determined mainly based on three aspects in the present study. First of all, the CYP3A4 enzyme was inhibited by ketoconazole, which caused a higher BOS concentration in circulation. Midazolam, a drug has a similar absolute bioavailability to that of BOS (Weber et al., 1999; Garzone, PD et al., 1989), is exclusively metabolized by CYP3A4, and consequently, the AUC of midazolam was dramatically increased by 16-fold in the presence of ketoconazole (Tsunoda, SM et al., 1999). However, in our work, the AUC of BOS was increased by 5.1-fold when treated with ketoconazole. This modest increase indicated that BOS metabolism is not solely dependent on CYP3A4. Second, ketoconazole itself may bind to plasma protein directly to divert BOS to that protein by the competitive way, which leads to enhanced BOS concentration in the bloodstream (George, L Trainor, 2007). Third, ketoconazole served as an inhibitor of the bile salt export pump (Zhang et al., 2016) that reduced the biliary excretion of BOS to some extent. Taken together, these mechanisms allowed the ketoconazole to disturb the metabolism and excretion of BOS. Voriconazole was thought to be a substitute for ketoconazole because of its weaker hepatic toxicity (Mikus et al., 2011). As reported, voriconazole had a selective influence on CYP2C9 and CYP3A4 as a result of their sensitivities to different hepatic enzymes; it inhibited CYP3A4 more extensively than CYP2C9 (Hyland et al., 2003). In the present study, the AUC(0–t) and Cmax of BOS after treatment with voriconazole showed a relative decrease accompanied by increased CLz/F profile compared with the ketoconazole and fluconazole groups (Fig. 3). In contrast, significant differences in OHBOS pharmacokinetics were observed in the voriconazole group compared with the ketoconazole and fluconazole groups. The AUC(0–t) and Cmax of OHBOS were significantly lower on co-administering BOS and voriconazole compared with those in the ketoconazole and fluconazole groups, leading to an augmentation in MRT(0-t). The reduced AUC(0–t) and Cmax and elevated MRT(0- t) in OHBOS metabolism were probably a result of an impaired hepatic injury induced by voriconazole. Also, a previous study demonstrated that voriconazole led to liver toxicity (Hulin et al., 2011). Thus, despite moderate hepatic damage caused by voriconazole compared with ketoconazole, the hepatic function needs long-term examinations when BOS and voriconazole are taken simultaneously. Fluconazole had a relevantly higher inhibitory effect on hepatic enzymes, especially on CYP2C9 (Niwa et al., 2005). In the present study, the AUC(0–t) and Cmax of BOS were higher after fluconazole treatment than after voriconazole treatment but lower than after ketoconazole treatment. The varied response to these azole antifungal agents was due to their different inhibitory effects on CYP2C9 and CYP3A4 enzymes. Additionally, a clinically relevant report showed that fluconazole was primarily eliminated by renal excretion; approximately 80% of administered fluconazole was still intact in urine (Brammer et al., 1990). The remaining 20% of fluconazole solely engaged in CYP-mediated metabolism, which was corroborated by its compromised inhibition of CYP2C9 and CYP3A4 in the present study (Fig. 3). Itraconazole has a considerable inhibitory effect on the CYP3A4 enzyme (Niwa et al., 2005). Further, several itraconazole metabolites were also CYP3A4 inhibitors. Although itraconazole possessed a higher plasma protein–binding affinity (99.8%), it induced negligible changes in BOS pharmacokinetic parameters except for the CLz/F value, which decreased by 28.8%. Moreover, itraconazole was metabolized predominantly by CYP3A4 in the liver and competed for the CYP3A4 enzyme that always regulated BOS disposition. Intriguingly, in the present study, itraconazole barely interfered with BOS metabolism. Therefore, the readily accessible biosafety of itraconazole in combination with BOS may account for its widespread use. The results in this study favored the extensive application of BOS combined with azole antifungal agents. In summary, itraconazole exhibited the lowest inhibitory effects on BOS metabolism, followed by voriconazole, fluconazole, and ketoconazole. Moreover, the liver aminotransferase levels were monitored more frequently when BOS was initiated, adjusted, or discontinued in patients taking voriconazole and fluconazole. However, further studies are needed because the present study was conducted on rats, which are slightly different from humans. 5. Conclusions In this study, ketoconazole, voriconazole, fluconazole, and itraconazole exhibited varied inhibitory effects on BOS metabolism after the co-administration of BOS. Their inhibitory effect followed the order: ketoconazole > fluconazole > voriconazole > itraconazole. Considering the existing toxicity of BOS, clinicians should pay more attention to BOS prescription, irrespective of reduction, adjustment, or discontinuation of its use, during its combined treatment with ketoconazole, fluconazole, and voriconazole.
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