German, Polina PharmD*; Parikh, Sunil MD, MPH†; Lawrence, Jody MD†; Dorsey, Grant MD, PhD†; Rosenthal, Philip J MD†; Havlir, Diane MD†; Charlebois, Edwin PhD†; Hanpithakpong, Warunee PhD‡; Lindegardh, Niklas PhD‡§; Aweeka, Francesca T PharmD*
Malaria and HIV are 2 of the most important global health problems. Malaria causes close to 500 million episodes of illness resulting in more than a million deaths annually.1,2 Ninety percent of these deaths occur in Africa, with pregnant women and children younger than 5 years most affected.3 Together, HIV and malaria account for over 4 million deaths per year.
Treatment of malaria- and HIV-coinfected patients remains a challenge, and the evidence of interactions between these infections is accumulating. Clinical studies indicate an increase in plasma HIV viral load in the setting of acute malaria and a greater incidence of malaria in HIV-infected patients with low CD4− cell counts. In addition, the incidence of clinical malaria increases with advanced HIV immunosuppression.4-6
Historically, treatment of uncomplicated Plasmodium falciparum malaria relied on chloroquine or sulfadoxine-pyrimethamine. However, these treatments are now discouraged due to an increase in parasite resistance to the older drugs. Currently, nearly all countries in Africa advocate use of artemisinin-based combination therapies (ACTs) that consist of a short-acting artemisinin derivative that rapidly reduces parasite burden and fever and a longer acting partner drug that removes residual parasites. ACTs are administered as 3-day treatment courses and offer improved efficacy over older regimens.
Currently, the 2 leading ACTs are artesunate plus amodiaquine and artemether/lumefantrine (AL), each of which has been adopted as first-line therapy for uncomplicated malaria by many malaria-endemic countries in Africa, Asia, and South America.7 Recent studies have shown artesunate plus amodiaquine to have somewhat worse antimalarial efficacy compared with AL,8 hepatotoxicity after coadministration with efavirenz in normal volunteers,9 and neutropenia in the setting of concurrent antiretroviral (ARV) use in African children.10 A recent in vitro study has demonstrated that efavirenz is a potent inhibitor of cytochrome (CYP) 2C8, the primary enzyme involved in amodiaquine metabolism, and this finding may partially explain the toxicities described above.11 In contrast, AL remains safe and highly efficacious. However, because both artemether (AR) and lumefantrine (LR) also undergo metabolism via CYP450 isozymes making them prone to drug-drug interactions, their coadministration with ARV drugs, specifically protease inhibitors (PIs), has not been recommended.12 The objective of this study was to investigate the pharmacokinetics (PK) of AL when administered with the PI combination lopinavir/ritonavir (LPV/r) in HIV-uninfected healthy volunteers.
MATERIALS AND METHODS
The study was conducted at the General Clinical Research Center, San Francisco General Hospital. All subjects read and signed the informed consent before participation. Healthy HIV-uninfected volunteers between ages 18 and 60 years who had no evidence of systemic illness and required no medications that had known potential for drug interactions were consented and screened for the study. Enrolled subjects received 6 doses of coformulated AL (Coartem), 80/480 mg twice daily (study days 1-4). This regimen was followed by a washout period of 2 weeks and then a 26-day course of LPV/r (Kaletra) 400/100 mg twice daily (study days 16-41). The LPV/r was initially given alone and then given concomitantly with a second 6-day course of AL 80/480 mg twice daily (study days 28-31) (Fig. 1). Subjects were instructed to take all drugs with a meal to optimize absorption and to record the time of dose and food intake in a diary. Subjects were not allowed to take any medications known to modulate CYP450 isozymes or interfere with study completion. Study personnel contacted subjects by phone or e-mail before each study visit to evaluate medication/dietary compliance and to address questions. Subjects were reminded of the upcoming study visits by phone and during this time had the opportunity to ask any questions. Study participants were admitted into the inpatient unit of the General Clinical Research Center the night before the intensive PK collections. The subjects remained inpatient until discharge after the 24-hour blood collection.
Sample Collection and Processing
Intensive serial PK sampling for AR, dihydroartemisinin (DHA-active AR metabolite), and LR was conducted on study days 4 (alone) and 31 (in the context of LPV/r). Blood samples were collected before the sixth AL dose (days 4 and 31) and at 0.5, 1, 2, 4, 6, 8, 12, 24, 48, and 72 hours post dose for the analyses of plasma levels of AR, DHA, and LR (Fig. 2). Blood sampling continued at 96, 120, 168, 216, and 264 hours post dosing for the analysis of LR. Steady state PK sampling for the analyses of plasma levels of LPV/r alone (study day 25) and in the context of AL (study day 31) was conducted before and at 1, 2, 4, 6, 8, and 12 hours post dosing.
Whole blood samples were drawn into sodium heparin and EDTA-containing tubes for the analyses of AR/DHA/LR and LPV/r, respectively. AR/DHA/LR tubes were immediately placed on ice and then centrifuged at 2000g for 10 minutes at 4°C. The resulting plasma was split into aliquots and kept at −70°C until analysis. LPV/r samples were centrifuged at 3000 rpm for 10 minutes at room temperature. The plasma was aliquoted and stored at −70°C until analysis.
Concentrations of AR/DHA and LR were measured by the Clinical Pharmacology Laboratory in the Faculty of Tropical Medicine Bangkok. AR/DHA were analyzed using solid phase extraction and liquid chromatography-mass spectrometry/mass spectrometry. Stable isotope-labeled AR and DHA were used as internal standards. AR and DHA were quantified using an API 5000 triple quadrupole mass spectrometer (Applied Biosystems/MDS SCIEX, Foster City, CA) with a TurboV ionization source interface operated in the positive ion mode. Quantification was performed using selected reaction monitoring for the transitions m/z 302-163 and 307-166 for DHA and stable isotope-labeled DHA, respectively, and 316-163 and 320-163 for AR and stable isotope-labeled AR, respectively. Total-assay coefficients of variation (CV) for DHA and AR during analysis were less than 5% at all QC levels. Lower limit of quantification for both drugs was 1.43 ng/mL.13 LR concentrations were determined using a solid phase extraction liquid chromatographic assay with UV detection.14 A hexyl analogue of desbutyl-lumefantrine (Novartis No. TA 213/435/16) was used as internal standard. The CV% during the analysis was less than 6 at all QC levels. The lower limit of quantification for the assay was 25 ng/mL. For quantification of LPV/r, liquid chromatography/tandem mass spectrometry methods were employed within the Drug Research Unit of the University of California, San Francisco. Methods were approved by the Quality Assurance/Quality Control Committee of the National Institutes of Health-sponsored AIDS Clinical Trials Group. LPV/r interassay CV was less than 11%. Lower limits of quantification for lopinavir and ritonavir (RTV) were 0.04 and 0.025 μg/mL, respectively.
Noncompartmental analysis was performed via the linear up-log down trapezoidal rule in conjunction with an oral input model using WinNonlin 5.0.1 (Pharsight Corporation, Mountain View, CA). Samples below the limit of quantification of the bioanalytical assays were treated as missing data. PK parameters, such as elimination rate constant (λz) and half-life (t1/2) were evaluated upon inspection of PK data on a profile-by-profile basis. The t1/2 was calculated as ln2/λz. LR area under the curve (AUC)0-264 (end of the sampling period) and AUC0-inf, AR and DHA AUClast and AUC0-inf, and LPV/r steady state AUC0-12 were estimated. Cmax, median Tmax, and t1/2 (if applicable) were calculated. Geometric means and their associated 90% confidence intervals were calculated for all parameters of interest. Ten subjects provided >80% power to reject the null hypothesis that LR AUC0-inf estimated during AL administration alone is equivalent to that of AUC0-inf during concomitant administration with LPV/r (1-sided alpha = 0.05). Statistical analysis was conducted using STATA version 9.2 (StataCorp, College Station, TX).
A physical examination, complete blood count with differential, comprehensive metabolic chemistry panel (including serum electrolytes, glucose, blood urea nitrogen, creatinine, and liver function tests), fasting lipid panel, and electrocardiogram were performed before enrollment and on the last day of the study. A serum HIV antibody test and urine tests for drugs of abuse were carried out at the screening visit. Female subjects were asked to take a pregnancy test within 48 hours before receiving any study drugs. Laboratory tests (complete blood count with differential, metabolic chemistry panel, and lipid panel) and electrocardiogram were repeated after completion of the 2 courses of AL, during the washout period, after 10 days of LPV/r regimen, and at the end of the study. Standard audiometry tests were conducted at baseline, during the washout period, and after completion of the second course of AL. All subjects returned for repeat laboratory tests 2 weeks and 4 weeks after completing the study.
Compliance was monitored by examining the medication diary and by counting the number of remaining pills. Subjects were interviewed during their inpatient stays and asked to report any adverse events to a nurse or study investigator. Adverse events were recorded throughout the study, and the onset, duration, severity, and relationship to the study drug was assessed after each event. All events were classified according to the Division of AIDS (DAIDS) Table for Grading the Severity of Adult Adverse Experiences.15 Follow-up of serious adverse events was at the discretion of the study physicians and included additional laboratory tests, physical examinations, and workup for alternative etiologies.
Demographics and Safety
A total of 13 subjects were enrolled in the study with 10 subjects (6 males and 4 females) completing the study. The mean age of subjects who completed the study was 31.4 years (range, 21-45 years). All were within 30% of their ideal body weight (mean, 82.9 kg; range, 55.8-104.7 kg). Of the 10 subjects completing the study, ethnicity was as follows: Hispanic 1, Asian 1, African American 1, and white 7. All subjects tolerated study medications well, with all reported adverse events consistent with what has previously been reported for AL and LPV/r treatment. The most commonly reported adverse events for LPV/r and AL/LPV/r were gastrointestinal disturbances reported in 3 and 2 subjects, respectively, whereas for AL, fatigue was reported in 2 subjects. None of the clinical laboratory abnormalities measured in any of the subjects were considered to be clinically significant by the study physician.
Effects of LPV/r on AR/DHA
PK results for AR/DHA/LR are summarized in Table 1 (Fig. 1). Trends toward decreased AR Cmax, AUClast, and AUC0-inf were noted in the context of LPV/r coadministration compared with AL administration alone, although none of these differences reached statistical significance. Specifically, with coadministration, Cmax decreased by 22%, whereas AUClast and AUC0-inf decreased by 39% and 34%, respectively (P > 0.05). The median t1/2 remained similar between AL alone and AL with LPV/r (3.5 versus 3.4 hours). In contrast, significant differences were observed in the PK parameters for active DHA during LPV/r coadministration, with decreases in DHA Cmax, AUClast, and AUC0-inf of 36%, 45%, and 45%, respectively (P < 0.02) The ratio of active DHA to AR AUC remained unchanged.
Effects of LPV/r on LR
Coadministration of AL with LPV/r led to 2.3-fold increases in the LR AUC0-264 and AUC0-inf when compared with AUC estimates for AL alone (P < 0.01). LR Cmax, Tmax, and t1/2 were not significantly altered with LPV/r, although a trend toward increased Cmax was observed (1.4-fold increase).
Effects of AL on LPV/r
LPV and RTV AUC0-12, Cmax, Tmax, and t1/2 were not significantly altered in the presence of AL relative to LPV/r administration by itself, with all PK parameters being similar between study phases (Table 2 and Fig. 3).
We investigated potential PK interactions between ACTs used to treat malaria and PIs used to treat HIV. Of note, therapies for both infections are susceptible to drug-drug interactions due to their involvement in CYP450 metabolism, and the high prevalence of both malaria and HIV infection in Africa suggests that antimalarial and ARV drugs will be coadministered in very large numbers of patients. This study revealed a 2.3-fold increase in the PK exposure, as assessed by AUC, of the longer acting antimalarial, LR, and small decreases in the exposure (AUC and Cmax) of AR/DHA after coadministration with LPV/r. The change in AR AUC in the setting of ARVs did not reach statistical significance. This finding may be explained by the large AR variability observed in this study and the small sample size. A 1-sided alpha in the sample size calculation was selected because an increase in LR exposure upon coadministration with the strong CYP3A4 inhibitor ketoconazole was noted previously.16 As ritonavir is another potent CYP3A inhibitor, an increase in LR PK in the setting of LPV/r was expected.
The 6-dose regimen of AL utilized in this study is widely prescribed for malaria due to the rapid spread of P. falciparum resistance to other drugs and its excellent efficacy and safety profile. AL is currently adopted by more than 60 countries in Africa, Asia, and South America as either the first- or the second-line ACT, and it is used to treat millions of cases of malaria annually.7 For patients coinfected with HIV, ARV treatment options are expanding for individuals residing in the developing world. In Africa, PI-based ARV regimens including LPV/r will be increasingly used as second-line therapy and are already used as first-line therapy among children who are at high risk for nevirapine resistance in some countries.
PK data evaluating interactions between ACTs and potentially interacting drugs are scarce. A clinical study evaluating the PK of a single AL dose in the setting of multiple doses of ketoconazole revealed modest increases in the exposure (AUC) of AR (131.6% ↑), DHA (51.3% ↑), and LR (60.9% ↑), changes not considered to be clinically significant.16 In their report, the authors recommended that dose adjustment of AL in malaria-infected patients is unnecessary when AL is administered together with ketoconazole or other potent CYP3A4 inhibitor.16 Despite the recommendations from this earlier study, treatment guidelines have discouraged use of AL in HIV-coinfected patients requiring ARV treatment due to the concern that a clinically significant interaction between AL and ARVs may occur.12 Therefore, a definitive PK study, best achieved through enrollment of healthy volunteers, was urgently needed.
Both AR and LR are metabolized via CYP3A4 pathways. AR is rapidly demethylated into its active metabolite DHA by CYP3A4/CYP3A5, which in turn is converted to inactive metabolites primarily by glucuronidation via uridine diphosphate-glucuronosyltransferases 1A1, 1A8-9, and 2B7.16-18 In vitro, AR may also induce CYP3A4.17,18 LR is desbutylated into desbutyl-lumefantrine by CYP3A4 isozymes and inhibits CYP2D6 in vitro.19 RTV is a potent inhibitor of gastrointestinal and hepatic CYP3A enzymes, resulting in improved oral bioavailability and slowing of the systemic hepatic clearance of drugs that are metabolized via CYP3A pathways. In addition, RTV is an inhibitor of p-glycoprotein, an efflux transporter that pumps drugs back into the gastrointestinal tract. The use of RTV as a PK booster has become important in the treatment of HIV infection, as it is used to increase systemic concentrations of several protease inhibitors. However, coadministration of RTV-boosted PIs with drugs that are predominantly metabolized by CYP3A4 isozymes may lead to clinically important drug-drug interactions. Because single-dose studies may underestimate the impact of multiple doses on P450 isozymes, this study explored an interaction between AL administered as a standard 6-dose regimen and multiple doses of LPV/r.
Coadministration of AL and LPV/r resulted in more than doubling of the LR AUC in healthy volunteers. The magnitude of LPV/r effect on LR in malaria-infected patients may be different as LR bioavailability is largely dependent on food and is thus poor in acute malaria but improves with recovery.20 The observed increase in this study is not surprising because RTV is a potent mechanism-based inhibitor of CYP3A4 and is more likely to participate in PK drug interactions relative to competitive inhibitors.21 However, in this study, we formally evaluate the interactions using multiple dosing at standard doses of both therapies to more precisely quantify the impact. Data, suggesting that LR AUC is a key parameter with respect to malarial cure20 combined with safety profile of AL, suggest that the observed increase in LR AUC in the presence of LPV/r may be beneficial in the treatment of malaria. Careful monitoring of patients with respect to tolerability and incidence of adverse events upon AL coadministration with strong CYP3A4 inhibitors is, nevertheless, advised. Because the elimination half-life of LR in the setting of LPV/r was not altered, inhibition of CYP3A4 enzymes by RTV may have occurred at the level of the gut rather than the liver. These data are consistent with the results from the ketoconazole study.16
In contrast to LR exposure, coadministration of AL with LPV/r resulted in a small but insignificant decrease in AR exposure. These results are not completely unexpected because AR may autoinduce its own metabolism over time. In addition, in vitro studies suggest involvement of CYP isoforms, other than CYP3A4 in AR metabolism.22,23 Slight decreases in DHA AUC and Cmax were noted in the setting of LPV/r. The change in DHA PK upon coadministration with LPV/r could be attributed to the effect of RTV on CYP3A4 and/or UGT1A1 and UGT2B7 that are involved in the metabolism of AR to DHA and conversion of DHA to inactive metabolites, respectively.16-18 Clinical impact of the observed changes in AR/DHA PK is unclear as the treatment cure (assessed on days 28 and 42 after treatment) is largely attributed to higher LR AUC and the duration for which LR plasma concentration exceeds in vivo minimum inhibitory concentration.20,24 Higher AUCs of AR and DHA were found to decrease parasite clearance time using population PK data.24 However, because parasite clearance in all patients in that study was achieved in 48 hours,24 the relevance of exposure estimates around the last dose is unknown.
LPV and RTV PK were not altered in the presence of AL. However, the absence of observed changes in LPV/r PK during concomitant therapy may also be influenced by study design because the antimalarial combination was administered for only a 3-day period.
In summary, this is the first known study to examine the drug-drug interactions between LPV/r and AL with standard multidose administration of both therapies The results from this study suggest that coadministration of AL with LPV/r can be carried out safely for patients coinfected with malaria parasites and HIV. Although these combinations were well tolerated by the study participants, the formal safety analysis of concomitant therapy should be addressed by future studies among individuals living in malaria-endemic regions.
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