- Email: [email protected]
Effects of concurrent administration of efavirenz on the disposition kinetics of amodiaquine in healthy volunteers
j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 2 7 5 e2 7 9
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jopr
Original Article
Effects of concurrent administration of efavirenz on the disposition kinetics of amodiaquine in healthy volunteers Julius O. Soyinka a,*, Cyprian O. Onyeji a, Thomas I. Nathaniel b, Olugbenga O. Odunfa c, Benjamin U. Ebeshi d a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria Department of Biomedical Sciences, University of South Carolina School of Medicine e Greenville, 701 Grove Road, Greenville, SC 29605, United States c Federal Medical Centre, Idi-Aba, Abeokuta, Nigeria d Department of Pharmaceutical & Medicinal Chemistry, Faculty of Pharmacy, Niger Delta University, Wilberforce Island, Bayelsa State, Nigeria b
article info
abstract
Article history:
Background/Objectives: Efavirenz and amodiaquine are likely to be administered concur-
Received 19 December 2012
rently for the treatment of patients with HIV and malaria. The metabolism of amodiaquine
Accepted 6 February 2013
is mediated principally by CYP2C8 while efavirenz is known to inhibit this enzyme. This
Available online 28 February 2013
study therefore investigated the effect of efavirenz on amodiaquine disposition. Methods: Fourteen healthy volunteers were each given 600 mg single oral doses of amodia-
Keywords:
quine alone or with the 9th dose of efavirenz (400 mg daily for 12 days) in a crossover fashion.
Amodiaquine
Blood samples were collected at pre-determined time intervals and analyzed for amodia-
Desethylamodiaquine
quine and its major metabolite, desethylamodiaquine, using a validated HPLC method.
Efavirenz
Results: Co-administration of amodiaquine and efavirenz resulted in significant increases
Interactions
(p < 0.05) in Cmax, Tmax, AUCT and elimination half-life (T1/2) of amodiaquine compared with values for amodiaquine alone. Also, efavirenz caused a pronounced decrease in the AUC (metabolite)/AUC (unchanged drug) ratio of amodiaquine along with a significant decrease (p < 0.05) in Cmax and AUC of the metabolite. Conclusion: Efavirenz significantly alters the pharmacokinetics of amodiaquine, exposure to amodiaquine is increased leading to toxic effect, and reduction in the antimalarial activity since amodiaquine is a prodrug that relies on its active metabolite against malaria parasites. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
1.
Introduction
Amodiaquine is a 4-aminoquinoline derivative that has been widely used for treatment of malaria over the past 50 years.1 It is
intrinsically more active than the other 4-aminoquinoline, chloroquine, against Plasmodium falciparum parasites, which are moderately chloroquine resistant. The drug is therefore increasingly being considered as a replacement for chloroquine
* Corresponding author. 1, Akodu Cresent, Felele Ibadan, Oyo State, Nigeria. Tel.: þ234 8035822785. E-mail addresses: [email protected], [email protected] (J.O. Soyinka). 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.02.008
276
j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 2 7 5 e2 7 9
as a first line drug in Africa because of widespread chloroquine resistance.1 Since amodiaquine is rapidly cleared and the formed desethylamodiaquine attains high plasma concentrations for a long time, it is considered a prodrug, which is bioactivated to desethylamodiaquine. This metabolic transformation has been shown to be mediated by CYP2C8.2 In countries with high prevalence of malaria and HIV infections, co-infection is common. Thus, in these regions, there is a very high possibility of a patient taking an antimalarial and an antiretroviral drug concurrently.3 Efavirenz, a nonnucleoside reverse transcriptase inhibitor (NNRTI), is metabolized principally by CYP2B6 and to a lesser degree by CYP3A4.4 Although most drug interaction studies done with efavirenz have demonstrated the effects of the drug on CYP3A4 and CYP2B6 substrates, there are studies indicating that the NNRTI can also inhibit CYP2C8, CYP2C9 and CYP2C19.5e7 For example, concurrent administration of proguanil with efavirenz resulted in elevated plasma proguanil levels and was attributed to inhibition of CYP2C9 and CYP2C19 that mediate proguanil metabolism.8 Since amodiaquine is mainly metabolized by CYP2C8 and activity of this isozyme has been demonstrated to be modulated by efavirenz,9 there is a potential for pharmacokinetic interaction between both drugs when taken concurrently. Therefore, this study determined whether, and to what magnitude, efavirenz influences the disposition kinetics of amodiaquine in man.
2.
Materials and methods
2.1.
Subjects
Fourteen healthy volunteers (8 males and 6 females) between the ages of 26 and 38 years weighing 60e78 kg were enrolled into the study after giving written informed consent. The volunteers had a Body Mass Index of 19.46 1.68 (range 16e22) kg/m2 and were certified healthy by a physician on the basis of medical history, clinical examination, laboratory baseline investigations and serum chemistry tests, prior to enrollment into the study. Subjects were excluded from participating in the study if they met any of the following additional criteria: pregnancy, breast feeding, serum creatinine greater than 1.5 times the upper limit of normal, any liver function test more than 3 times the upper limit of normal. None of the subjects was receiving any drugs for at least one month before the study and none was a smoker. Approval for the study was obtained from the Obafemi Awolowo University Teaching Hospitals Research Ethics Board and Safety committee.
2.2.
Drug administration and blood sample collection
The study was an open-label, randomized, multiple antiretroviral dosing, two-period crossover pharmacokinetic study. After an overnight fast, each of the 14 volunteers received a single oral dose of 600 mg amodiaquine (Amodiaquine dihydrochloride tablets, Parke-Davis, USA) either alone or with the 9th dose of efavirenz. Efavirenz (Aviranz Capsules, Ranbaxy Laboratory Ltd, India) was given as 400 mg oral dose daily for 12 days. A washout period of 3 weeks was allowed between the two arms of the study. Blood samples (5 ml) were withdrawn
by venipuncture from the forearm of each subject prior to and at 0.08, 0.25, 0.5, 1.5, 3, 5, 24, 48 and 192 h after drug administration into heparinised tubes. They were immediately centrifuged (3000 g at 20 C for 10 min) to separate plasma. The plasma aliquots were stored at 20 C until analyzed. The plasma samples were analyzed for amodiaquine and monodesethylamodiaquine to obtain their baseline pharmacokinetics and then to evaluate the effect of concurrent administration of efavirenz on the pharmacokinetics of amodiaquine. No other drugs or alcohol was allowed to be taken throughout the duration of the study.
2.3.
Chemicals and reagents
Amodiaquine dihydrochloride and desethylamodiaquine dihydrochloride were obtained from Parke-Davis, USA and quinidine from BDH Laboratory Supplies, Poole, England. Amodiaquine dihydrochloride tablets (Parke-Davis, USA) were purchased from a retail pharmacy in Nigeria. HPLC grade acetonitrile and methanol, and analytical grade diethyl ether, perchloric acid, sodium hydroxide and hydrochloric acid were purchased from Sigma (SigmaeAldrich chemical company, Germany).
2.4.
Instruments and chromatographic conditions
A Mersham Pharmacia Biotech IP-900 liquid chromatography (USA) (AKTA) fitted with a variable UV detector (model P-900) was used for the analysis. The stationary phase was a reversed-phase C18 column {Eclipse-XDBC18e3.5 mm (200 4.6 mm I.D.)}. The solvent system for HPLC consisted of acetonitrile: 0.02 M potassium dihydrogen phosphate (10:90). The pH of the mobile phase was adjusted to 4.0 with orthophosphoric acid. The mobile phase was pumped through the column at a flow rate of 1.0 ml/min. The experiments were performed at ambient temperature. The method was a slight modification of Gitau et al (2004).10 Whirl mixer (Fissions), precisions pipettes (MLA), table centrifuge (Gallenkamp) and digital sonicator (Gallenkamp) were used for the extraction procedure.
2.5.
Analytical procedure
To 1 ml of plasma placed in a 15-ml screw capped extraction tube were added 20 mL of 500 mg/ml quinidine solution (internal standard) and 2 ml of acetonitrile before mixing for about 15 s, followed by mechanical tumbling for 15 min. After centrifuging for 10 min at 3000 g, the liquid phase was transferred to a clean tube, to which was added 2 ml of ammonia. The mixture was then extracted by mechanical tumbling for 15 min, with 2 5 ml of diethyl ether. After centrifugation and separation, the combined organic phases were evaporated to dryness and the residue was reconstituted in 100 mL of methanol while a 50 mL aliquot was injected onto the HPLC column. Calibration curve based on peak area ratio was prepared by spiking drug-free plasma with standard solutions of amodiaquine and monodesethylamodiaquine to give concentration ranges of 2e30 ng/ml and 20e300 ng/ml respectively. The samples were taken through the extraction procedure described above.
j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 2 7 5 e2 7 9
2.6.
Pharmacokinetic evaluation
277
A
The pharmacokinetic (PK) parameters for amodiaquine and monodesethylamodiaquine were calculated with the computer program WinNonLin (version 1.5). The data were analyzed using noncompartmental analysis. The parameters that could be established were as follows: time point of maximum observed concentration in plasma (Tmax); concentration in plasma corresponding to Tmax (Cmax); terminal half-life (T1/2); area under the plasma concentration versus time (Cet) curve (AUCT).
3.
Result
The mean (SD) plasma concentration versus time profiles of amodiaquine and desethylamodiaquine following oral administration of single doses of 600 mg of amodiaquine hydrochloride alone, and with multiple doses of efavirenz, to each of 14 volunteers are shown in Fig. 1A and B. The derived pharmacokinetic parameters for amodiaquine following administration of the drug with and without efavirenz are presented in Table 1. Concurrent administration of efavirenz was associated with a significant (p < 0.05) prolongation of the Tmax and marked increase in Cmax, AUCT, and elimination T1/2 of amodiaquine compared with values obtained following administration of the antimalarial alone (Table 1). These show a 125%, 78%, 80%, and 42.15% increase in the Tmax, Cmax AUCT and T1/2 of amodiaquine respectively. Also, the apparent oral clearance (Cl/F) of amodiaquine decreased about 72% in the presence of efavirenz. Pharmacokinetic parameters of desethylamodiaquine following administration of amodiaquine with and without efavirenz are also shown in Table 1. There was a significant decrease in the mean Cmax (40% decrease) and mean AUC0e192 h (25.92% decrease) in the presence of efavirenz (p < 0.05). Concurrent efavirenz administration also resulted in a marked reduction in the metabolic ratio by about 74%.
4.
B
Fig. 1 e Plasma concenentrationetime profile of (A) Amodiaquine and (B) Desethylamodiaquine following administration of 600 mg amodiaquine to 14 healthy volunteers with or without concurrent administration of efavirenz.
Discussion
In addition to antiretroviral regimens, HIV patients are treated with a variety of other drugs for concurrent diseases. The resulting combinations may include antimalarials, antibiotics, analgesics, etc.11 and this can render HIV patients prone to drug interactions. All NNRTIs are extensively metabolized by specific cytochrome P450 enzymes and have been reported to inhibit or induce these enzymes resulting in alterations of the pharmacokinetics of other concurrently administered drugs.12 This study was designed to evaluate the in vivo interaction between amodiaquine and efavirenz. The results from the present study indicate that amodiaquine is rapidly absorbed after oral administration in all subjects with a Tmax in the range of 0.5e1.2 h. The pharmacokinetic parameters obtained for the drug when administered alone such as Tmax, elimination T1/2, Cl/F, and AUCT are generally in agreement with the values obtained in other single dose pharmacokinetic studies.9,13,14 With concurrent efavirenz administration, the observed marked increase in the Tmax of amodiaquine
(Table 1) which is indicative of a slower rate or prolongation of absorption of the antimalarial may be attributable to the modulation of intestinal P-glycoprotein by efavirenz. It has been demonstrated that efavirenz is not a P-glycoprotein substrate but can slightly induce P-glycoprotein functionality and expression probably through induced cell stress.15 Since amodiaquine is a substrate for P-glycoprotein,16 it is possible for its absorption to be prolonged by P-glycoprotein upregulation caused by efavirenz. This speculation is based on reports indicating that drug-induced increase in P-glycoprotein expression can result in prolongation of Tmax of a coadministered drug. For example, the Tmax of levofloxacin was prolonged by 50% following efavirenz concurrent administration and this was ascribed to up-regulation of Pglycoprotein induced by efavirenz.17 Moreover, in our previous study, the Tmax of proguanil was prolonged significantly following efavirenz concurrent administration and this was ascribed to up-regulation of P-glycoprotein induced by efavirenz.8 The total systemic exposure (AUCT) of amodiaquine
278
j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 2 7 5 e2 7 9
Table 1 e Effect of co-administration of efavirenz on the pharmacokinetic parameters of amodiaquine and desethylamodiaquine. Parameters Amodiaquine Cmax (ng/ml) Tmax (h) AUCT (ng.h/ml) T1/2 (h) Desethylamodiaquine Cmax (ng/ml) Tmax (h) AUCT (ng.h/ml)
Amodiaquine alone 50.0 0.8 160.80 6.21
1.2 0.08 15.22 0.82
192 17.24 4.20 0.82 2054 86.21
was substantially increased (mean of about 80%) in the presence of efavirenz (Table 1) and, this is quite evident in the significant difference in the plasma concentration profiles of amodiaquine with or without efavirenz (Fig. 1A). The increased systemic drug exposure coupled with the markedly diminished oral drug clearance (Cl/F) and significantly prolonged elimination T1/2 of amodiaquine suggests a systemic inhibition of metabolism of the drug by efavirenz. This assertion is buttressed by the observation of an evident marked reduction in plasma levels of the major metabolite (desethylamodiaquine) (Fig. 1B), which is reflected in significant decreases in the Cmax and AUC of the metabolite. Previous studies have shown that both CYP2C8 and CYP3A4 contribute to the metabolism of amodiaquine but the former is the major contributor in the biotransformation.2,16 Since efavirenz has been demonstrated as an inhibitor of CYP2C8 as well as a mixed inducer/inhibitor of CYP3A4,9 the increase in plasma levels of amodiaquine following coadministration with efavirenz is most likely due to the inhibition of CYP2C8 and probably a contribution from CYP3A4 inhibition. In a study,18 looking at amodiaquine pharmacokinetics of following co-administration of efavirenz (600 mg once daily) and amodiaquine/artesunate (600/250 mg once daily) in HIV-subjects had to be terminated after the first two subjects developed asymptomatic but significant elevations of liver transaminases. Addition of efavirenz increased amodiaquine AUC by 114% and 302% in the 1st and 2nd subjects respectively. Table 1 shows a pronounced decrease (68%) in the ratio of AUC of metabolite to that of unchanged drug, the metabolic ratio (MR). This further strengthens the point that a metabolic interaction occurs between amodiaquine and efavirenz, and that efavirenz inhibits the metabolism of amodiaquine. The increased plasma levels of amodiaquine with efavirenz coadministration may increase the toxicity of amodiaquine. After oral administration, amodiaquine is rapidly absorbed from the gastrointestinal tract. In the liver it undergoes rapid and extensive metabolism to N-desethyl-amodiaquine (DEAQ) which concentrates in blood cells.2 Amodiaquine is threetimes more potent than DEAQ but the concentration of amodiaquine in blood is quite low.2 Therefore, DEAQ is responsible for most of the observed antimalarial activity, thus inhibition of amodiaquine metabolism by concurrent administration of efavirenz may also lead to decreased antimalarial activity. In conclusion, this study has demonstrated that there is a significant pharmacokinetic interaction between amodiaquine and efavirenz. Co-administration of efavirenz, a mixed
Amodiaquine þ Efavirenz 68.2 1.08 254.68 5.92
5.6 0.06 22.14 0.74
226 24.28 5.53 0.96 2671 84.34
Significance P< P< P< P>
0.05 0.05 0.05 0.05
P < 0.05 P < 0.05 P < 0.05
inducer/inhibitor of CYP3A4 and inhibitor of CYP2C8, with amodiaquine that is a substrate of the same isoenzymes results in significant elevation in plasma levels of the antimalarial. The plasma concentrations of DEAQ, the major metabolite of amodiaquine, are markedly diminished in the presence of efavirenz. Thus, the protection against malaria may be decreased, and toxic effects of amodiaquine may be increased when efavirenz and amodiaquine are concurrently administered.
Conflicts of interest All authors have none to declare.
Acknowledgment This work was supported by Obafemi Awolowo University, Ile-Ife, Nigeria, Research Grant No. 11813 AEC.
references
1. Juliana MS, Olivia T. Protecting the malarial drug arsenal: halting the rise and spread of amodiaquine resistance by monitoring the PFCRT, SVMNT type. Malar J. 2010;9:374. 2. Li XQ, Bjorkman A, Anderrson TB, Ridderstrom M, Masimirembwa CM. Amodiaquine clearance and its metabolism to n-desethylamodiaquine is mediated by CYP2C8: a new high affinity and turnover enzyme-specific probe substrate. J Pharmacol Exp Ther. 2002;300:399e407. 3. World Health Organization. Malaria and HIV/AIDS Interactions and Implications: Conclusions of a Technical Consultation Convened by WHO, June 23e24, 2004. 4. Ward BA, Gorski JC, Jones DR, Hall SD, Flockhat DA, Desta Z. The cytochrome P 450 2B6 is the main catalyst of efavirenz primary and secondary metabolism: implication for HIV/AIDS therapy and utility of efavirenz as a substrate marker of CYP 2B6 catalytic activity. J Pharmacol Exp Ther. 2003;306:287e300. 5. Smith PF, Dicenzo R, Morse GD. Clinical pharmacokinetics of non-nucleoside reverse transcriptase inhibitors. Clin Pharmacokinet. 2001;40:893e905. 6. Robertson SM, Penzack SR, Lane J, Pau AK, Mican JM. A potentially significant interaction between efavirenz and phenytoin: a case report and review of the literature. Clin Infect Dis. 2005;41:15.
j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 2 7 5 e2 7 9
7. Liu P, Foster G, LaBadie RR, Gutierrez MJ, Sharma A. Pharmacokinetic interaction between voriconazole and efavirenz at steady state in healthy male subjects. J Clin Pharmacol. 2008;48(1):73e84. 8. Soyinka JO, Onyeji CO. Alteration of pharmacokinetics of proguanil in healthy volunteers following concurrent administration of efavirenz. Eur J Pharm Sci. 2010;39:213e218. 9. Parikh S, Onedraogo JB, Rosenthal PJ, Kroctz DL. Amodiaquine metabolism is impaired by common polymorphisms in CYP2C8: implications for malaria treatment in Africa. Clin Pharmacol Ther. 2007;88(2):218e224. 10. Gitau EN, Muchohi SN, Ogutu BR, Githiga LM, Kokwaro GO. Selective and sensitive liquid chromatographic assay of amodiaquine and desethylamodiaquine in whole blood spotted on filter paper. J Chromatogr B. 2004;799:173e177. 11. Fogelman SX, Daily JP, Shader RI. Protease inhibitors as inhibitors of human cytochromes P450: high risk associated with ritonavir. J. Clin Pharmacol. 1998;38(2):106e111. 12. Barry M, Gibbons S, Mulcahy F, Merry C, Back D. Pharmacokinetics and potential interactions amongst antiretroviral agents used to treat patients with HIV infections. Clin Pharm. 1999;36:289e304.
279
13. Orrell C, Little F, Smith P, et al. Pharmacokinetics and tolerability of artesunate and amodiaquine alone and in combination in healthy volunteers. Eur J Clin Pharmacol. 2008;64(7):683e690. 14. Soyinka JO, Odunfa O, Ademisoye AA. Effects of food on the pharmacokinetics of amodiaquine in healthy volunteers. Asian J Pharm Res Healthc. 2011;3(4):109e113. 15. Chandler B, Almond L, Ford J, et al. The effects of protease inhibitors and nonnucleoside reverse transcriptase inhibitors on p-glycoprotein expression in peripheral blood mononuclear cells in vitro. J Acquir Immune Defic Syndr. 2003;5:551e556. 16. Zuluaga L, Pabon A, Lopez C, Ochua A, Blair S. Amodiaquine failure associated with erythrocytic gluthathione in Plasmodium falciparum malaria. Malar J. 2007;6:47. 17. Villani P, Viale P, Signorini L, et al. Pharmacokinetic evaluation of oral levofloxacin in human immunodeficiency virus-infected subjects receiving concomitant antiretroviral therapy. Antimicrob Agents Chemother. 2001;45(7):2160e2162. 18. Martin-Carbonero L, Nunez M, Gonzalez-Lahoz J, Soriano V. Incidence of liver injury after beginning antiretroviral therapy with efavirenz or nevirapine. HIV Clin Trials. 2003;4:115e120.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jopr
Original Article
Effects of concurrent administration of efavirenz on the disposition kinetics of amodiaquine in healthy volunteers Julius O. Soyinka a,*, Cyprian O. Onyeji a, Thomas I. Nathaniel b, Olugbenga O. Odunfa c, Benjamin U. Ebeshi d a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria Department of Biomedical Sciences, University of South Carolina School of Medicine e Greenville, 701 Grove Road, Greenville, SC 29605, United States c Federal Medical Centre, Idi-Aba, Abeokuta, Nigeria d Department of Pharmaceutical & Medicinal Chemistry, Faculty of Pharmacy, Niger Delta University, Wilberforce Island, Bayelsa State, Nigeria b
article info
abstract
Article history:
Background/Objectives: Efavirenz and amodiaquine are likely to be administered concur-
Received 19 December 2012
rently for the treatment of patients with HIV and malaria. The metabolism of amodiaquine
Accepted 6 February 2013
is mediated principally by CYP2C8 while efavirenz is known to inhibit this enzyme. This
Available online 28 February 2013
study therefore investigated the effect of efavirenz on amodiaquine disposition. Methods: Fourteen healthy volunteers were each given 600 mg single oral doses of amodia-
Keywords:
quine alone or with the 9th dose of efavirenz (400 mg daily for 12 days) in a crossover fashion.
Amodiaquine
Blood samples were collected at pre-determined time intervals and analyzed for amodia-
Desethylamodiaquine
quine and its major metabolite, desethylamodiaquine, using a validated HPLC method.
Efavirenz
Results: Co-administration of amodiaquine and efavirenz resulted in significant increases
Interactions
(p < 0.05) in Cmax, Tmax, AUCT and elimination half-life (T1/2) of amodiaquine compared with values for amodiaquine alone. Also, efavirenz caused a pronounced decrease in the AUC (metabolite)/AUC (unchanged drug) ratio of amodiaquine along with a significant decrease (p < 0.05) in Cmax and AUC of the metabolite. Conclusion: Efavirenz significantly alters the pharmacokinetics of amodiaquine, exposure to amodiaquine is increased leading to toxic effect, and reduction in the antimalarial activity since amodiaquine is a prodrug that relies on its active metabolite against malaria parasites. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
1.
Introduction
Amodiaquine is a 4-aminoquinoline derivative that has been widely used for treatment of malaria over the past 50 years.1 It is
intrinsically more active than the other 4-aminoquinoline, chloroquine, against Plasmodium falciparum parasites, which are moderately chloroquine resistant. The drug is therefore increasingly being considered as a replacement for chloroquine
* Corresponding author. 1, Akodu Cresent, Felele Ibadan, Oyo State, Nigeria. Tel.: þ234 8035822785. E-mail addresses: [email protected], [email protected] (J.O. Soyinka). 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.02.008
276
j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 2 7 5 e2 7 9
as a first line drug in Africa because of widespread chloroquine resistance.1 Since amodiaquine is rapidly cleared and the formed desethylamodiaquine attains high plasma concentrations for a long time, it is considered a prodrug, which is bioactivated to desethylamodiaquine. This metabolic transformation has been shown to be mediated by CYP2C8.2 In countries with high prevalence of malaria and HIV infections, co-infection is common. Thus, in these regions, there is a very high possibility of a patient taking an antimalarial and an antiretroviral drug concurrently.3 Efavirenz, a nonnucleoside reverse transcriptase inhibitor (NNRTI), is metabolized principally by CYP2B6 and to a lesser degree by CYP3A4.4 Although most drug interaction studies done with efavirenz have demonstrated the effects of the drug on CYP3A4 and CYP2B6 substrates, there are studies indicating that the NNRTI can also inhibit CYP2C8, CYP2C9 and CYP2C19.5e7 For example, concurrent administration of proguanil with efavirenz resulted in elevated plasma proguanil levels and was attributed to inhibition of CYP2C9 and CYP2C19 that mediate proguanil metabolism.8 Since amodiaquine is mainly metabolized by CYP2C8 and activity of this isozyme has been demonstrated to be modulated by efavirenz,9 there is a potential for pharmacokinetic interaction between both drugs when taken concurrently. Therefore, this study determined whether, and to what magnitude, efavirenz influences the disposition kinetics of amodiaquine in man.
2.
Materials and methods
2.1.
Subjects
Fourteen healthy volunteers (8 males and 6 females) between the ages of 26 and 38 years weighing 60e78 kg were enrolled into the study after giving written informed consent. The volunteers had a Body Mass Index of 19.46 1.68 (range 16e22) kg/m2 and were certified healthy by a physician on the basis of medical history, clinical examination, laboratory baseline investigations and serum chemistry tests, prior to enrollment into the study. Subjects were excluded from participating in the study if they met any of the following additional criteria: pregnancy, breast feeding, serum creatinine greater than 1.5 times the upper limit of normal, any liver function test more than 3 times the upper limit of normal. None of the subjects was receiving any drugs for at least one month before the study and none was a smoker. Approval for the study was obtained from the Obafemi Awolowo University Teaching Hospitals Research Ethics Board and Safety committee.
2.2.
Drug administration and blood sample collection
The study was an open-label, randomized, multiple antiretroviral dosing, two-period crossover pharmacokinetic study. After an overnight fast, each of the 14 volunteers received a single oral dose of 600 mg amodiaquine (Amodiaquine dihydrochloride tablets, Parke-Davis, USA) either alone or with the 9th dose of efavirenz. Efavirenz (Aviranz Capsules, Ranbaxy Laboratory Ltd, India) was given as 400 mg oral dose daily for 12 days. A washout period of 3 weeks was allowed between the two arms of the study. Blood samples (5 ml) were withdrawn
by venipuncture from the forearm of each subject prior to and at 0.08, 0.25, 0.5, 1.5, 3, 5, 24, 48 and 192 h after drug administration into heparinised tubes. They were immediately centrifuged (3000 g at 20 C for 10 min) to separate plasma. The plasma aliquots were stored at 20 C until analyzed. The plasma samples were analyzed for amodiaquine and monodesethylamodiaquine to obtain their baseline pharmacokinetics and then to evaluate the effect of concurrent administration of efavirenz on the pharmacokinetics of amodiaquine. No other drugs or alcohol was allowed to be taken throughout the duration of the study.
2.3.
Chemicals and reagents
Amodiaquine dihydrochloride and desethylamodiaquine dihydrochloride were obtained from Parke-Davis, USA and quinidine from BDH Laboratory Supplies, Poole, England. Amodiaquine dihydrochloride tablets (Parke-Davis, USA) were purchased from a retail pharmacy in Nigeria. HPLC grade acetonitrile and methanol, and analytical grade diethyl ether, perchloric acid, sodium hydroxide and hydrochloric acid were purchased from Sigma (SigmaeAldrich chemical company, Germany).
2.4.
Instruments and chromatographic conditions
A Mersham Pharmacia Biotech IP-900 liquid chromatography (USA) (AKTA) fitted with a variable UV detector (model P-900) was used for the analysis. The stationary phase was a reversed-phase C18 column {Eclipse-XDBC18e3.5 mm (200 4.6 mm I.D.)}. The solvent system for HPLC consisted of acetonitrile: 0.02 M potassium dihydrogen phosphate (10:90). The pH of the mobile phase was adjusted to 4.0 with orthophosphoric acid. The mobile phase was pumped through the column at a flow rate of 1.0 ml/min. The experiments were performed at ambient temperature. The method was a slight modification of Gitau et al (2004).10 Whirl mixer (Fissions), precisions pipettes (MLA), table centrifuge (Gallenkamp) and digital sonicator (Gallenkamp) were used for the extraction procedure.
2.5.
Analytical procedure
To 1 ml of plasma placed in a 15-ml screw capped extraction tube were added 20 mL of 500 mg/ml quinidine solution (internal standard) and 2 ml of acetonitrile before mixing for about 15 s, followed by mechanical tumbling for 15 min. After centrifuging for 10 min at 3000 g, the liquid phase was transferred to a clean tube, to which was added 2 ml of ammonia. The mixture was then extracted by mechanical tumbling for 15 min, with 2 5 ml of diethyl ether. After centrifugation and separation, the combined organic phases were evaporated to dryness and the residue was reconstituted in 100 mL of methanol while a 50 mL aliquot was injected onto the HPLC column. Calibration curve based on peak area ratio was prepared by spiking drug-free plasma with standard solutions of amodiaquine and monodesethylamodiaquine to give concentration ranges of 2e30 ng/ml and 20e300 ng/ml respectively. The samples were taken through the extraction procedure described above.
j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 2 7 5 e2 7 9
2.6.
Pharmacokinetic evaluation
277
A
The pharmacokinetic (PK) parameters for amodiaquine and monodesethylamodiaquine were calculated with the computer program WinNonLin (version 1.5). The data were analyzed using noncompartmental analysis. The parameters that could be established were as follows: time point of maximum observed concentration in plasma (Tmax); concentration in plasma corresponding to Tmax (Cmax); terminal half-life (T1/2); area under the plasma concentration versus time (Cet) curve (AUCT).
3.
Result
The mean (SD) plasma concentration versus time profiles of amodiaquine and desethylamodiaquine following oral administration of single doses of 600 mg of amodiaquine hydrochloride alone, and with multiple doses of efavirenz, to each of 14 volunteers are shown in Fig. 1A and B. The derived pharmacokinetic parameters for amodiaquine following administration of the drug with and without efavirenz are presented in Table 1. Concurrent administration of efavirenz was associated with a significant (p < 0.05) prolongation of the Tmax and marked increase in Cmax, AUCT, and elimination T1/2 of amodiaquine compared with values obtained following administration of the antimalarial alone (Table 1). These show a 125%, 78%, 80%, and 42.15% increase in the Tmax, Cmax AUCT and T1/2 of amodiaquine respectively. Also, the apparent oral clearance (Cl/F) of amodiaquine decreased about 72% in the presence of efavirenz. Pharmacokinetic parameters of desethylamodiaquine following administration of amodiaquine with and without efavirenz are also shown in Table 1. There was a significant decrease in the mean Cmax (40% decrease) and mean AUC0e192 h (25.92% decrease) in the presence of efavirenz (p < 0.05). Concurrent efavirenz administration also resulted in a marked reduction in the metabolic ratio by about 74%.
4.
B
Fig. 1 e Plasma concenentrationetime profile of (A) Amodiaquine and (B) Desethylamodiaquine following administration of 600 mg amodiaquine to 14 healthy volunteers with or without concurrent administration of efavirenz.
Discussion
In addition to antiretroviral regimens, HIV patients are treated with a variety of other drugs for concurrent diseases. The resulting combinations may include antimalarials, antibiotics, analgesics, etc.11 and this can render HIV patients prone to drug interactions. All NNRTIs are extensively metabolized by specific cytochrome P450 enzymes and have been reported to inhibit or induce these enzymes resulting in alterations of the pharmacokinetics of other concurrently administered drugs.12 This study was designed to evaluate the in vivo interaction between amodiaquine and efavirenz. The results from the present study indicate that amodiaquine is rapidly absorbed after oral administration in all subjects with a Tmax in the range of 0.5e1.2 h. The pharmacokinetic parameters obtained for the drug when administered alone such as Tmax, elimination T1/2, Cl/F, and AUCT are generally in agreement with the values obtained in other single dose pharmacokinetic studies.9,13,14 With concurrent efavirenz administration, the observed marked increase in the Tmax of amodiaquine
(Table 1) which is indicative of a slower rate or prolongation of absorption of the antimalarial may be attributable to the modulation of intestinal P-glycoprotein by efavirenz. It has been demonstrated that efavirenz is not a P-glycoprotein substrate but can slightly induce P-glycoprotein functionality and expression probably through induced cell stress.15 Since amodiaquine is a substrate for P-glycoprotein,16 it is possible for its absorption to be prolonged by P-glycoprotein upregulation caused by efavirenz. This speculation is based on reports indicating that drug-induced increase in P-glycoprotein expression can result in prolongation of Tmax of a coadministered drug. For example, the Tmax of levofloxacin was prolonged by 50% following efavirenz concurrent administration and this was ascribed to up-regulation of Pglycoprotein induced by efavirenz.17 Moreover, in our previous study, the Tmax of proguanil was prolonged significantly following efavirenz concurrent administration and this was ascribed to up-regulation of P-glycoprotein induced by efavirenz.8 The total systemic exposure (AUCT) of amodiaquine
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Table 1 e Effect of co-administration of efavirenz on the pharmacokinetic parameters of amodiaquine and desethylamodiaquine. Parameters Amodiaquine Cmax (ng/ml) Tmax (h) AUCT (ng.h/ml) T1/2 (h) Desethylamodiaquine Cmax (ng/ml) Tmax (h) AUCT (ng.h/ml)
Amodiaquine alone 50.0 0.8 160.80 6.21
1.2 0.08 15.22 0.82
192 17.24 4.20 0.82 2054 86.21
was substantially increased (mean of about 80%) in the presence of efavirenz (Table 1) and, this is quite evident in the significant difference in the plasma concentration profiles of amodiaquine with or without efavirenz (Fig. 1A). The increased systemic drug exposure coupled with the markedly diminished oral drug clearance (Cl/F) and significantly prolonged elimination T1/2 of amodiaquine suggests a systemic inhibition of metabolism of the drug by efavirenz. This assertion is buttressed by the observation of an evident marked reduction in plasma levels of the major metabolite (desethylamodiaquine) (Fig. 1B), which is reflected in significant decreases in the Cmax and AUC of the metabolite. Previous studies have shown that both CYP2C8 and CYP3A4 contribute to the metabolism of amodiaquine but the former is the major contributor in the biotransformation.2,16 Since efavirenz has been demonstrated as an inhibitor of CYP2C8 as well as a mixed inducer/inhibitor of CYP3A4,9 the increase in plasma levels of amodiaquine following coadministration with efavirenz is most likely due to the inhibition of CYP2C8 and probably a contribution from CYP3A4 inhibition. In a study,18 looking at amodiaquine pharmacokinetics of following co-administration of efavirenz (600 mg once daily) and amodiaquine/artesunate (600/250 mg once daily) in HIV-subjects had to be terminated after the first two subjects developed asymptomatic but significant elevations of liver transaminases. Addition of efavirenz increased amodiaquine AUC by 114% and 302% in the 1st and 2nd subjects respectively. Table 1 shows a pronounced decrease (68%) in the ratio of AUC of metabolite to that of unchanged drug, the metabolic ratio (MR). This further strengthens the point that a metabolic interaction occurs between amodiaquine and efavirenz, and that efavirenz inhibits the metabolism of amodiaquine. The increased plasma levels of amodiaquine with efavirenz coadministration may increase the toxicity of amodiaquine. After oral administration, amodiaquine is rapidly absorbed from the gastrointestinal tract. In the liver it undergoes rapid and extensive metabolism to N-desethyl-amodiaquine (DEAQ) which concentrates in blood cells.2 Amodiaquine is threetimes more potent than DEAQ but the concentration of amodiaquine in blood is quite low.2 Therefore, DEAQ is responsible for most of the observed antimalarial activity, thus inhibition of amodiaquine metabolism by concurrent administration of efavirenz may also lead to decreased antimalarial activity. In conclusion, this study has demonstrated that there is a significant pharmacokinetic interaction between amodiaquine and efavirenz. Co-administration of efavirenz, a mixed
Amodiaquine þ Efavirenz 68.2 1.08 254.68 5.92
5.6 0.06 22.14 0.74
226 24.28 5.53 0.96 2671 84.34
Significance P< P< P< P>
0.05 0.05 0.05 0.05
P < 0.05 P < 0.05 P < 0.05
inducer/inhibitor of CYP3A4 and inhibitor of CYP2C8, with amodiaquine that is a substrate of the same isoenzymes results in significant elevation in plasma levels of the antimalarial. The plasma concentrations of DEAQ, the major metabolite of amodiaquine, are markedly diminished in the presence of efavirenz. Thus, the protection against malaria may be decreased, and toxic effects of amodiaquine may be increased when efavirenz and amodiaquine are concurrently administered.
Conflicts of interest All authors have none to declare.
Acknowledgment This work was supported by Obafemi Awolowo University, Ile-Ife, Nigeria, Research Grant No. 11813 AEC.
references
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