Effect of Naltrexone Hydrochloride on Cytochrome P450 1A2, 2C9, 2D6, and 3A4 Activity in Human Liver Microsomes

Eur J Drug Metab Pharmacokinet https://doi.org/10.1007/s13318-018-0482-x

ORIGINAL RESEARCH ARTICLE

1 2 1 2 Haitham AlRabiah • Abdul Ahad • Gamal A. E. Mostafa • Fahad I. Al-Jenoobi

Ó Springer International Publishing AG, part of Springer Nature 2018

Abstract inhibition concentration (IC50) values for CYP2C9 and Background and Objective Cytochrome P450 (CYP) 1A2, CYP2D6 inhibition were 3.40 ± 1.78 and 2C9, 2D6, and 3A4 are the most important phase I drug- 5.92 ± 1.58 lM, respectively. metabolizing enzymes in the liver, but there is a dearth of Conclusion These outcomes advocate that there is a great literature available on the effects of naltrexone possibility of drug interactions resulting from the concur- hydrochloride on these major enzymes present in the rent administration of naltrexone hydrochloride with human liver. Thus, in the present study, the effect of nal- actives that are metabolized by these CYP enzymes, par- trexone hydrochloride on the activity of CYP1A2, 2C9, ticularly CYP2C9 and CYP2D6. Nevertheless, further 2D6, and 3A4 using human liver microsomes (HLM) was clarification is needed through detailed in vivo pharma- investigated. cokinetic studies. Methods A selective probe for CYP1A2, 2C9, 2D6, and 3A4 was incubated with HLM with or without naltrexone hydrochloride. Phenacetin O-deethylation, tolbutamide Key Points 4-hydroxylation, dextromethorphan O-demethylation, and testosterone 6b-hydroxylation reactions were monitored for Naltrexone hydrochloride inhibited the activity of enzyme activity. CYP1A2, 2C9, 2D6, and 3A4 in human liver Results The activity of all the studied CYP enzymes microsomes. except 1A2 was significantly inhibited by naltrexone hydrochloride 1 lM. Furthermore, 1 lM naltrexone Naltrexone hydrochloride has strongly inhibited hydrochloride inhibited CYP3A4 enzyme activity, the most activity with IC50 values of 3.40 and 5.92 lM for by 37.9% followed by CYP2C9 (36.5%) and CYP2D6 CYP2C9 and 2D6, respectively. (31.8%). The CYP2C9 and CYP2D6 metabolic activities were greatly affected by naltrexone hydrochloride, which even at the lowest concentration of naltrexone hydrochloride (0.01 lM) significantly decreased the metabolic activity by 34.9 and 16.0%, respectively. The half maximal 1 Introduction

Naltrexone hydrochloride is an opioid antagonist that was & Abdul Ahad [email protected]; [email protected] approved by the US Food and Drug Administration (FDA) for the treatment of heroin addiction in 1984 and alcohol 1 Department of Pharmaceutical Chemistry, College of dependence in 1995 [1]. For the past 2 decades, medical Pharmacy, King Saud University, P.O. Box 2457, Riyadh practitioners have believed that low-dose naltrexone 11451, Saudi Arabia hydrochloride (B 5 mg/day) could have valuable thera- 2 Department of Pharmaceutics, College of Pharmacy, King peutic actions on autoimmune diseases such as multiple Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia H. AlRabiah et al. sclerosis and inflammatory bowel disease [2], chronic pain 2.2 Phenacetin O-Deethylation Activity [3], and fibromyalgia [4, 5]. Following oral administration, naltrexone hydrochloride HLM were incubated in triplicate at 37 °C as reported is rapidly and almost completely absorbed, and approxi- previously [10, 11]. The drug phenacetin was used as a mately, 96% of the dose is absorbed from the gastroin- CYP1A2 marker substrate at 100 lM. The CYP marker testinal tract [6]. Although naltrexone hydrochloride is well substrate was incubated for 30 min with the HLM in 0.1 M absorbed orally, it is subject to extensive hepatic first-pass potassium phosphate buffer (pH 7.4) in the presence or metabolism and, therefore, has an oral bioavailability absence of naltrexone hydrochloride [final concentration ranging from 5 to 40% [7]. The effectiveness of naltrexone (FC) = 1, 10, and 100 lM]. The negative control samples hydrochloride is believed to be mediated by both naltrex- contained 10% methanol, and all samples were keep aside one hydrochloride, and its 6-b-naltrexol metabolite. The for approximately 10 min to allow organic solvent to elimination half-life of naltrexone hydrochloride and its evaporate. The reactions were initiated by the addition of metabolite is 4 and 13 h, respectively, and they achieved 1 mM NADPH (FC) to a total volume of 500 ll. The their peak plasma levels within 1 h of dosing [8]. microsomal protein concentration in the reaction mixture Naltrexone hydrochloride has been used for decades, was 0.5 mg/ml. The reactions were stopped by the addition and its safety profile, when used for approved indications of 10 ll perchloric acid (70%). Caffeine (25 llofa (alcohol and opioid dependence), is well known. According 100 lM stock solution) was added to each tube as the to the Norwegian Prescription Database, the number of internal standard, the samples were centrifuged at naltrexone hydrochloride users increased from \ 20 in 12,000 rpm for 10 min, and the resulting supernatants were 2012 to [ 15,000 in 2013 and 2014 [1, 9]. Considering that transferred to HPLC vials for the HPLC analysis of acet- naltrexone hydrochloride could be co-prescribed with aminophen (phenacetin metabolite). For metabolite analy- drugs that are metabolized by the liver, there appears to be sis, the mobile phase (acetonitrile: 50 mM phosphate scope for investigating the effect of naltrexone buffer, 15:85, v/v) was pumped at flow rate of 1.3 ml/min hydrochloride on the metabolic activity of major cyto- through a Nucleodur C18, (4.6 9 250 mm, 5.0 lm) column chrome P450 (CYP) enzymes in pooled human liver and the metabolite was detected at 245 nm [10]. microsomes (HLM). 2.3 Tolbutamide 4-Hydroxylation Activity

2 Materials and Methods The microsomal tolbutamide 4-hydroxylation activity was determined by measuring the formation of 4-hydroxy- 2.1 Materials and Reagents tolbutamide at 37 °C using an HPLC method [12, 13]. Briefly, a Shimadzu HPLC system with an ultraviolet (UV) Ò All chemicals and reagents used were of high-performance detector and C18 Symmetry (5.0 lm, 4.6 9 150 mm) liquid chromatography (HPLC) analytical grade. Ultra- HPLC column was used for the metabolite analysis. Ace- PoolTM HLM from 150 donors (20 mg/ml) was purchased tonitrile and potassium dihydrogen phosphate buffer from Corning (Woburn, MA, USA). Naltrexone (0.02 M, pH 3.4, 25:75, v/v) were used as mobile phase hydrochloride, tolbutamide, testosterone, and 6b-hydrox- and were delivered at a flow rate of 1.5 ml/min. 4-hy- ytestosterone were obtained from Sigma-Aldrich (St. droxytolbutamide was monitored at a wavelength of Louis, MO, USA). Reduced b-nicotinamide adenine dinu- 230 nm. The incubation mixture comprised 0.25 mg HLM cleotide 20-phosphate (NADPH) tetrasodium salt hydrate protein, 0.1 M phosphate buffer (pH 7.4), 1 mM NADPH, was procured from Chem-Implex Int’l Inc. (Wood Dale, and tolbutamide (FC = 0.15 mM) in the presence or IL, USA). 4-hydroxytolbutamide was procured from Cay- absence of naltrexone hydrochloride (FC = 0.01, 0.5, 0.1, man Chemical Company (MI, USA). Dextromethorphan 1, 10, and 100 lM) in a final volume of 500 ll. The neg- hydrobromide, dextrorphan-D-tartrate, and phenacetin ative control samples contained 10% methanol, and all were purchased from ICN Biomedicals, Inc. (Eschwege, samples were keep aside for approximately 10 min to Germany). Caffeine was purchased from Alfa Aesar (Ward allow organic solvent to evaporate. The final reaction Hill, MA, USA). HPLC-grade acetonitrile was obtained mixture was incubated at 37 °C for 30 min. After the from Winlab (Leicestershire, UK) and the potassium reaction was stopped by adding 250 ll of cold methanol, dihydrogen phosphate was from Fisher Scientific (Leices- 25 ll nitrazepam (4.0 lg/ml in methanol) was added to the tershire, UK). samples as an internal standard. The samples were centrifuged at 12,000 rpm for 10 min, and the resulting supernatants were pipetted and analyzed for the tolbutamide metabolite using HPLC as described above [12, 13]. Effect of Naltrexone on Cytochrome P450s

2.4 Dextromethorphan O-Demethylation Activity using GraphPad Prism 6 (GraphPad, Software Inc., San Diego, CA, USA). The statistical analysis was performed Dextromethorphan O-demethylation activity was assessed using the GraphPad Instat (v3.06, San Diego, CA, USA). with dextromethorphan as the specific substrate probe for An analysis of variance (ANOVA) followed by Dunnett’s CYP2D6 using a previously described method [14, 15]. test was performed and P value \ 0.05 was considered as Briefly, the incubation mixture containing dextromethor- significant. phan (FC = 25 lM), HLM (FC = 0.5 mg/ml), NADPH

(FC = 1 mM), and magnesium chloride (MgCl2,FC=6 mM) in a total volume of 500 ll phosphate buffer (0.1 M, 3 Results pH 7.4) was incubated at 37 °C for 30 min in the presence or absence of naltrexone hydrochloride (FC = 0.01, 0.05, 3.1 Phenacetin O-Deethylation 0.1, 1, 10, and 100 lM). The negative control samples contained 10% methanol, and all samples were keep aside In this study, we monitored the formation of acet- for approximately 10 min to allow organic solvent to aminophen by phenacetin O-deethylation to represent evaporate. The addition of 70% perchloric acid (10 ll) CYP1A2 activity in HLM. At 1 lM naltrexone stopped the reaction [14, 15]. The final mixture was cen- hydrochloride concentration, the CYP1A2 activity was not trifuged for 10 min at 12,000 rpm, and the supernatant was inhibited. Naltrexone at 10 and 100 lM inhibited the pipetted and the metabolite formed (dextrorphan-D-tartrate) phenacetin metabolite formation by 21.8 ± 0.6 and was analyzed using an HPLC system consisting of a col- 27.7 ± 7.0% (P \ 0.01), respectively (Fig. 1a). umn (C18 Nucleodur, 5.0 lm, 4.6 9 250 mm), a mobile phase of acetonitrile: HPLC-grade water containing 1.5% 3.2 Tolbutamide 4-Hydroxylation glacial acetic acid and 0.1% trimethylamine (25:75, v/v, pH 3, flow rate 1 ml/min) and a fluorescence detector In the present study, tolbutamide 4-hydroxylation was used (kEX = 280 nm and kEM = 330 nm) [16]. as the preferred probe reaction to evaluate CYP2C9 activity. It was observed that, at 1 lM naltrexone 2.5 Testosterone 6b-Hydroxylation Activity hydrochloride, tolbutamide metabolite formulation was decreased by 36.5 ± 1.5% (P \ 0.01). At 10 and 100 lM Testosterone 6b-hydroxylation activity was evaluated naltrexone hydrochloride concentration, there was using testosterone (FC = 50 lM) as CYP3A4-specific 52.5 ± 1.4% (P \ 0.01) and 66.7 ± 2.0% (P \ 0.01) marker substrate. Testosterone was incubated for 30 min decreased in tolbutamide metabolite formulation was with HLM (FC of protein = 0.25 mg/ml) in 0.1 M potas- observed, respectively (Fig. 1b). sium phosphate buffer (pH 7.4) in the presence or absence A further investigation was done using an extensive of naltrexone hydrochloride (FC = 1, 10, and 100 lM). range of six naltrexone hydrochloride concentrations

The reaction was initiated by the addition of 1 mM (0.01–100 lM) to determine the IC50 value. It was NADPH to obtain a total volume of 500 ll. The negative observed that, at the lowest naltrexone hydrochloride control samples contained 10% methanol and all samples concentration of 0.01 lM, CYP2C9 activity was inhibited were keep aside for approximately 10 min to allow organic by 34.9 ± 5.7% (P \ 0.01), while 0.05 and 0.1 lM nal- solvent to evaporate. The reactions were terminated by the trexone hydrochloride showed 37.2 ± 0.9 and addition of 250 ll cold methanol, and then, 8 ll phenacetin 39.3 ± 4.7% (both P \ 0.01) inhibition of tolbutamide

(50 lg/ml in methanol) was added to each sample as the metabolite formation, respectively, and the IC50 value was internal standard [17–19]. The samples were centrifuged at 3.40 ± 1.78 lM (Fig. 2a). 12,000 rpm for 10 min, and the resulting supernatants were transferred to HPLC vials for the analysis of 6-b-hydrox- 3.3 Dextromethorphan O-Demethylation ytestosterone. For the analysis, a mixture of methanol and HPLC-grade water (70/30, v/v) was delivered at a ﬂow rate In the CYP2D6 study, dextromethorphan O-demethylation of 0.5 ml/min through the C18 Nucleodur (5.0 lm, was the preferred reaction used to examine CYP2D6-based 4.6 9 250 mm) HPLC column. The testosterone metabo- drug interaction potential. In this study, dextromethorphan lite was detected at a wavelength of 242 nm [20]. was incubated with HLM in the absence and presence of different naltrexone hydrochloride concentrations, and the 2.6 Data Analysis inhibitory effect of naltrexone hydrochloride on the formation of the dextromethorphan metabolite (dextrorphan- The results were expressed as a percentage of the control D-tartrate) was measured. activity. The determinations of the IC50 values were done H. AlRabiah et al.

Fig. 1 Inhibitory effect of naltrexone hydrochloride on a phenacetin Values are presented as mean ± SEM, n =3.*P \ 0.01, compared O-deethylation, b tolbutamide 4-hydroxylation, c dextromethorphan to control. HLM human liver microsomes O-demethylation d testosterone 6b-hydroxylation activity in HLM.

The results showed a potent and concentration-depen- 3.4 Testosterone 6b-Hydroxylation dent inhibition of CYP2D activity by naltrexone hydrochloride in HLM (Fig. 1c). The naltrexone In the present study, testosterone 6b-hydroxylation was hydrochloride at 1 and 10 lM significantly inhibited dex- chosen as the probe reaction for measuring the CYP3A4- tromethorphan metabolite formation by 31.8 ± 1.1 and mediated drug interaction potential. The naltrexone 47.3 ± 0.4% (P \ 0.01). The maximum inhibition of the hydrochloride concentration (1 lM) significantly inhibited formation of dextrorphan-D-tartrate of 82.4 ± 0.4% was the testosterone metabolite formation by 37.9 ± 7.6% observed following treatment with 100 lM naltrexone (P \ 0.01, Fig. 1d). A similar inhibition (37.2 ± 3.6%, hydrochloride (Fig. 1c). P \ 0.01) of the testosterone metabolite was observed at In the CYP2D6 study, a further investigation was done 10 lM naltrexone hydrochloride. Furthermore, the maxi- using an extensive range of six naltrexone hydrochloride mum naltrexone hydrochloride concentration (100 lM) concentrations (0.01–100 lM) to determine the IC50 value. significantly decreased the testosterone metabolite forma- The lowest concentration of naltrexone hydrochloride tion by 38.5 ± 5.7% (P \ 0.01, Fig. 1d). (0.01 lM) significantly (P \ 0.01) inhibited the formation of the dextromethorphan metabolite by 16.0 ± 5.5%. Furthermore, 0.05 and 0.1 lM naltrexone hydrochloride 4 Discussion inhibited the dextromethorphan metabolite formation by

16.3 ± 4.0 and 18.2 ± 4.4%, respectively, and the IC50 CYP inhibition and induction are the major mechanisms value of naltrexone hydrochloride was 5.92 ± 1.58 lM arbitrating drug–drug interaction in clinical pharma- (Fig. 2b). cotherapy. Induction of CYPs by drugs results in enhanced Effect of Naltrexone on Cytochrome P450s

suggested by preclinical data. It well absorbed orally and is subject to significant first-pass metabolism with oral bioavailability estimates ranging from 5 to 40%. The activity of naltrexone is believed to be due to both parent and the 6-b-naltrexol metabolite. It is indicated in the treatment of alcohol dependence and for the blockade of the effects of exogenously administered opioids. It is contraindicated in acute hepatitis or liver failure [23, 24]. CYP performs significant functions in the biotransfor- mation of various endogenous substances including different categories of drugs. The main human hepatic CYP subfamilies are CYP2C and CYP3A, which account for 20% and 30% of total CYP activity, respectively, in addition to CYP1A2 (13%), CYP2E1 (7%), CYP2A6 (4%), CYP2D6 (2%), and CYP2B6 (\ 1%) [11]. CYP1A2 metabolizes numerous important drugs such as phenacetin, amitriptyline, imipramine, theophylline, clozapine, tacrine, and zileuton. Several previous investi- gations have shown that the high-affinity component of phenacetin O-deethylation is CYP1A2 [25–27]. Another important CYP is CYP2C; numerous important drugs including tolbutamide, carbamazepine, phenytoin, and warfarin are metabolized by this enzyme. Several studies demonstrated the main role of CYP2C9 in tolbutamide 4-hydroxylation reaction [28]. CYP2D is also an important CYP investigated in this study, CYP2D6 constitutes approximately 2% of the total CYP enzymes in the liver [29]. Approximately 25% of the currently marketed drugs have been reported to be substrates for CYP2D6. Several classes of drugs such as antitussives, antiemetics, antidepressants, antiarrhythmics, antipsychotics, b-blockers, and analgesics are metabolized by the CYP2D6 enzyme [30]. Fig. 2 Inhibition of a CYP2C9 and b CYP2D6 by naltrexone Furthermore, among the major CYP enzymes, CYP3A hydrochloride in HLM. Representing half maximal inhibition con- constitutes approximately 30–40% of total CYP protein in centration (IC50) curve and values of naltrexone hydrochloride against the liver. The previous data indicate that approximately CYP2C9 and CYP2D6 (n = 3, mean ± SEM). CYP cytochrome 50% of marketed drugs are metabolized by CYP3A4 [31]. P450, HLM human liver microsomes Mason et al. performed human pharmacokinetic and clearance of co-administered drugs, which can lead to pharmacodynamic drug interaction study of acamprosate decreased drug action if the parent compound is pharma- and naltrexone. In the study, 24 healthy adult volunteers cologically active, while inhibition of CYPs by drug con- participated. The therapeutic dose of acamprosate (2 g/day) sequences in a raised concentration of drug in plasma leads and naltrexone (50 mg/day) was given alone and in com- to the toxicity of drugs [21]. It is estimated that about 70% bination. The authors observed that the co-administration of drug–drug interactions associated with CYP-mediated of acamprosate with naltrexone substantially augmented metabolism are the result of enzyme inhibition [22]. the rate and extent of absorption of acamprosate. The Tmax Therefore, the present study investigated the inhibitory of acamprosate reduced by 33%. While the Cmax and AUC potential of naltrexone on CYP1A2, CYP2C9, CYP2D6, of acamprosate were increased by 33 and 25%, respec- and CYP3A4 in HLM. tively, when concurrently administered with naltrexone. In brief, naltrexone hydrochloride is a pure opioid On the other hand, acamprosate did not affect the phar- receptor antagonist; it blocks the effects of opioids by macokinetic parameters of naltrexone or its metabolite competitive binding at opioid receptors. The mechanism of [32]. action of naltrexone in alcoholism is not understood; In another study, naltrexone increased the sedative however, involvement of the endogenous opioid system is effects of diazepam (CYP2C19 is involved in the metabolism of diazepam) and delayed the Tmax of diazepam in H. AlRabiah et al. blood. Likewise, naltrexone enhanced the half-life of dia- major enzymes, particularly CYP2C9 and CYP2D6. The zepam from 4.0 to 4.3 h. The AUC of diazepam remains modulation of the metabolic activity of these CYP enzymes unaltered. The specific mechanism by which naltrexone by naltrexone hydrochloride could alter the pharmacoki- modifies the Tmax is not known, but it could be due to a netics profile of co-administered drugs, especially those delay in diazepam absorption [33]. There are, however, with narrow therapeutic range and, thereby, enhance the case reports of interactions between thioridazine and nal- prevalence of drug-induced toxicity. trexone [34]. In the present study, it was observed that, at 1 lM, naltrexone hydrochloride significantly inhibited all the 5 Conclusion investigated CYP activities except for that of CYP1A2. At 1 lM, naltrexone hydrochloride inhibited CYP3A4 In conclusion, the outcomes of the present study demon- enzyme activity the most by 37.9%, followed by CYP2C9 strate that naltrexone hydrochloride substantially inhibited (36.5%), and CYP2D6 (31.8%). the metabolic activity of CYP1A2, 2C9, 2D6, and 3A4 On increasing the concentration of naltrexone to 10 lM, enzymes in HLM. These findings demonstrate the drug– the maximum activity of CYP2C9 was inhibited by 52.5%, drug interaction potential of naltrexone hydrochloride and followed by CYP2D6 (47.3%) [ CYP3A4 indicate that the inhibition of these CYP enzymes could (37.2%) [ CYP1A2 (21.8%). It was observed that all cause potential problems of drug toxicity in patients. investigated CYP activities were significantly (P \ 0.01) inhibited at 10 lM concentration of naltrexone. While on Acknowledgement The authors thank the Deanship of Scientific increasing the concentration of naltrexone from 10 to Research and RSSU at King Saud University for their technical support. 100 lM, CYP2D6 activity was maximum inhibited by 82.4%, followed by CYP2C9 (66.7%) [ CYP3A4 Compliance with Ethical Standards (38.5%) [ CYP1A2 (27.7%). At 100 lM naltrexone concentration, all CYP activities were significantly (P \ 0.01) Funding The authors extend their appreciation to the deanship of scientific research and the research center, college of Pharmacy, King inhibited. Saud University for funding this research. We have observed that, at 1 lM naltrexone concentration, the CYP3A4 activity was maximum inhibited by Conflict of interest H. AlRabiah, A. Ahad, G. A. E. 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