Drug Interactions between Dolutegravir and Artemether-Lumefantrine or Artesunate-Amodiaquine

Across sub-Saharan Africa, patients with HIV on antiretrovirals often get malaria and need cotreatment with artemisinin-containing therapies. We undertook two pharmacokinetic studies in healthy volunteers, using standard adult doses of artemether-lumefantrine or artesunate-amodiaquine given with 50 mg once daily dolutegravir (DTG) to investigate the drug-drug interaction between artemether-lumefantrine or artesunate-amodiaquine and dolutegravir.


RESULTS
Forty-eight participants were enrolled into both studies: 18 in study A and 30 in study B. In study A, two participants withdrew consent, and another two were terminated from the study due to nonadherence to study procedures. In study B, one participant was withdrawn due to safety concerns, three participants withdrew consent, and one was terminated from the study due to nonadherence to study procedures. Statistical analysis was performed on 39 subjects who completed study procedures and their demographic variables are presented in Table 1.
Similarly lumefantrine showed peak concentrations approximately 4 h after drug administration, with a 12% increase in C max (GMR, 1.12; 90% CI, 0.97 to 1.29) and 10% increase in AUC 0 -t (GMR, 1.10; 90% CI, 0.96 to 1.27). The lumefantrine metabolite   Table 2. Coadministration of artemether-lumefantrine with dolutegravir did not significantly alter the C max , the AUC 0 -t , or the clearance of artemether, lumefantrine, or their metabolites. Furthermore, the time of the last measurable concentration (t) for all artemetherlumefantrine components did not significantly differ when administered alone or in combination with DTG. Interestingly, the terminal elimination half-life of artemether was increased in combination with DTG (GMR, 1.57; 90% CI, 1.09 to 2.27), but this appeared to be largely driven by two subjects who had detectable artemether levels beyond 24 h in combined phase. The analysis of variance (ANOVA) showed no evidence of a significant sequence or period effect upon artemether-lumefantrine PK.

Dolutegravir Interactions with Antimalarials
Antimicrobial Agents and Chemotherapy sequence effect upon dolutegravir PK. Furthermore, there was no significant period effect (DTG alone versus DTGϩAL) for the dolutegravir C 24 in both arms.
Safety. No deaths or serious adverse events were reported during the study. In all, 111 adverse events were observed and reported, with 80 adverse events judged as having a temporal relationship with study medication. All adverse events were grade 1 or 2 in severity. Gastrointestinal adverse events were more common among participants receiving artesunate-amodiaquine. No clinically significant changes were observed during safety assessments (laboratory blood assessments, ECG, or vital signs).

DISCUSSION
This is the first study to examine for drug interactions between dolutegravir with artemether-lumefantrine and artesunate-amodiaquine. We found no significant impact of dolutegravir upon drug exposure of either antimalarial regimen, suggesting that standard doses of artemether-lumefantrine and artesunate-amodiaquine should be used when coadministered with dolutegravir. These findings are important given the increasing use of dolutegravir in first-line antiretroviral therapy regimens. The results confirm the low propensity for dolutegravir to perpetrate clinically significant DDIs, as judged by in vitro observations of minimal effects on drug transporters and cytochrome P450 enzymes (5,13).
We observed that artemether-lumefantrine was not associated with any significant change in dolutegravir exposure parameters (C max and AUC 0 -24 ). However, dolutegravir C 24 was 37% lower with artemether-lumefantrine than when given alone. The reasons for this are unclear but may have been driven in part by an unexplained rise in dolutegravir C 24 in some participants. Additional intake of dolutegravir prior to the last sampling point at 24 h was unlikely, since subjects were instructed not to take the next dose before this time point and were issued with an exact number of pills, which precluded such additional intake.
With artesunate-amodiaquine, we observed an unexplained statistically significant reduction of 42 and 24% for dolutegravir C 24 and AUC 0 -24 , respectively. However, in all subjects who received DTG with artemether-lumefantrine or artesunate-amodiaquine, the dolutegravir C trough was comparable to or above 1,100 ng/ml (10), the mean C trough observed in prior dolutegravir phase 3 adult trials. The target minimum effective concentrations for dolutegravir are unknown, although a DTG protein-adjusted effective concentration (EC 90 ) above 300 ng/ml has been proposed (11,12). In a phase II study, C trough concentrations over 324 ng/ml after 10 days of DTG monotherapy were associated with virological efficacy (13). All subjects in our study had C trough concentrations exceeding these targets, suggesting that the modest pharmacokinetic changes observed have unlikely clinical significance, especially given the short duration of antimalarial therapy.
Notably, dolutegravir concentrations in this study of black African healthy volunteers were somewhat higher than previously reported in Caucasians where exposures of 4,560 ng/ml and 7,080 ng · h/ml for C max and AUC 0 -t , respectively, were observed (11). However, it should be noted that in our study, dolutegravir was dosed with a moderate fat meal, and concentrations observed are consistent with reports on the food effect upon DTG bioavailability (14).
We observed a 65% increase in T max for lumefantrine (GMR, 1.65; 90% CI, 1.02 to 2.69) and a 3-fold increase in T max for desbutyl-lumefantrine (GMR, 3.00; 90% CI, 2.06 to 4.36) during dolutegravir/artemether-lumefantrine coadministration. The reasons for the increase in T max for lumefantrine and desbutyl-lumefantrine are unclear, but the observed differences are very unlikely to be clinically significant.
This intensive pharmacokinetic study evaluated the drug-drug interactions using a two-way crossover study design with a 21-day washout period and a parallel study design for dolutegravir/artemether-lumefantrine and dolutegravir/artesunateamodiaquine, respectively. While a crossover study design would be theoretically ideal for investigating both artemisinin containing therapies in combination with dolutegravir, the amodiaquine active metabolite, N-desethylamodiaquine, has an extensive terminal half-life of approximately 9 to 18 days; therefore, it was not feasible to undertake a crossover design for this arm of the study since the washout period between the two phases would exceed two months, risking subject attrition. Furthermore, the crossover study design was chosen to allow for a two-way analysis for potential drug interactions between dolutegravir and artemether-lumefantrine. However, the elimination half-life for lumefantrine active metabolite desbutyl-lumefantrine was unexpectedly increased to ϳ142 h, suggesting that the washout period for desbutyl-lumefantrine may not have been sufficiently long. Although this finding highlights a limitation in our study, the analysis of variance found no period or sequence effects upon artemether-lumefantrine pharmacokinetics.
Dolutegravir is a weak inhibitor of organic cation transporter 2 (OCT2) and multidrug and toxin extrusion 1 (MATE1) transporter with reported interactions with substrates of both transporters. However, neither lumefantrine nor desbutyl-lumefantrine is a substrate of OCT2 and MATE1. It remains unclear by which mechanism dolutegravir might have influenced the observed lumefantrine/desbutyl-lumefantrine pharmacokinetics.
The combination of dolutegravir with artemether-lumefantrine and artesunateamodiaquine was well tolerated. Nausea, the most common study drug-related adverse event, was reported predominantly in the artesunate-amodiaquine arm. This safety profile was consistent with the approved drug labeling (14)(15)(16).
In conclusion, standard treatment regimens of artesunate-amodiaquine and of artemether-lumefantrine should be prescribed when treating malaria in HIV-infected patients receiving a dolutegravir-based antiretroviral regimen.

MATERIALS AND METHODS
Ethics. The study was approved by the Joint Clinical Research Centre Institutional Review Board, Kampala, Uganda, and the University of Liverpool Research Ethics Committee, Liverpool, United Kingdom, and registered at ClinicalTrials.gov (NCT02242799). The study was conducted in compliance with International Council for Harmonization Good Clinical Practice guidelines, the current ethical principles in the Declaration of Helsinki, and applicable local regulatory requirements.
Study design. Two open-label, fixed sequence studies between dolutegravir (Tivicay; ViiV Healthcare, Research Triangle Park, NC) given at 50 mg once daily and artemether-lumefantrine (Coartem; Novartis Pharma AG, Basel, Switzerland) (study A) or artesunate-amodiaquine (Winthrop; Sanofi-Aventis, Casablanca, Morocco) (study B) were conducted at the Infectious Diseases Institute, Kampala, Uganda (Fig. 3). Inclusion of 16 subjects in study A was calculated to have a Ͼ80% power to detect a change in AUC outside U.S. Food and Drug Administration (FDA) limits for bioequivalence (with the 90% CI for AUC falling within 80 to 125%) for dolutegravir and lumefantrine (assuming a coefficient of variation of Յ30%) and to detect a Ն32% change in dihydroartemisinin levels. Including 30 subjects in study B would yield an 80% power to detect an AUC difference of Ͼ25% to 30% (DTG and DEAQ), and a Ն42% change for dihydroartemisinin.
Eligibility criteria. Consenting healthy adults (Ն18 years) weighing Ͼ40 kg, without malaria and HIV, were eligible to participate if they were willing to use mosquito bed nets and able to comply with study procedures. Subjects were excluded if they had evidence of significant systemic disease, serum liver transaminases greater than three times the upper limits of normal, serum creatinine grater than two times the upper limits of normal, positive hepatitis B surface antigen, and evidence of QT prolongation on ECG exceeding 450 ms (men) or 470 ms (women) and were taking medications, which are well-known inhibitors or inducers of CYPs or glucuronyltransferase UGT1A1. Pregnant or breastfeeding women and female volunteers unwilling to use reliable contraception during the study were also excluded. A (artemether-lumefantrine). Study A used a random sequence two-way crossover study design with participants randomized to sequence 1 or sequence 2. In sequence 1, participants received six doses of oral artemether-lumefantrine tablets (80/480 mg twice daily taken with food) for 3 days (regimen used for treatment of uncomplicated malaria) with PK sampling predose (0 h) and 1,2,3,4,8,12,24,48,72,96,168, and 264 h after the final dose. After a 21-day washout, selected from the expected terminal half-lives of lumefantrine and desbutyl-lumefantrine, they received dolutegravir 50 mg once daily alone for 6 days with PK sampling on day 6 predose (0 h) and 1, 2, 3, 4, 8, 12, and 24 h postdosing. Subsequently, they received 3 days of twice daily AL plus dolutegravir, with PK sampling predose (0 h) and 1,2,3,4,8,12,24,48,72,96,168, and 264 h after the final doses of both drugs using the sampling time points described. In sequence 2, participants received the dolutegravir and DTG-AL combination with PK sampling as detailed for sequence 1, followed by artemetherlumefantrine alone after the 21-day washout period.

Dolutegravir Interactions with Antimalarials
Antimicrobial Agents and Chemotherapy (ii) Study B (artesunate-amodiaquine). Study B used a parallel group design due to the long terminal half-life of approximately 9 to 18 days for the amodiaquine active metabolite N-desethylamodiaquine. Participants were randomized to receive AS-AQ (4 mg/kg AS, 10 mg/kg AQ) once daily for 3 days with PK sampling predose (0 h) and 1,2,3,4,8,12,24,48,72,96,120,228, and 624 h after the last dose (arm 1) or dolutegravir for 7 days with PK sampling at predose (0 h) and at 1, 2, 3, 4, 8, 12, and 24 h after the last dose, followed by AS-AQ once daily, together with dolutegravir 50 mg once daily for 3 days, with PK sampling after the last dose of both drugs using the sampling time points listed (arm 2).
All study drugs prior to intensive PK sampling were administered after an overnight fast. A standard moderate fat breakfast was provided after the predose (0 h) sampling.
Safety assessments. At screening, a medical history, physical examination, urine pregnancy test, rapid malaria and HIV tests, and safety bloods (hemoglobin, white cell count, platelets, urea, creatinine, electrolytes, and ALT [alanine transaminase]) were performed. A 12-lead ECG was performed at screening, intensive PK and at the end of the study. Safety bloods were repeated at every intensive PK visit and prior to discharge from the study. Laboratory and clinical abnormalities were graded for severity according to the U.S National Institutes for Health Division of AIDS table for grading severity of adult and pediatric adverse events.
Pharmacokinetic analysis. Dolutegravir blood samples were collected in ethylenediaminetetraacetic acid (K 2 EDTA)-coated tubes (17). Samples for artemether/dihydroartemisinin, lumefantrine/ desbutyl-lumefantrine, and amodiaquine/desethylamodiaquine were collected in lithium heparin tubes, whereas artesunate/dihydroartemisinin was collected in fluoride-oxalate tubes to minimize ex vivo degradation of artemisinins to dihydroartemisinin by plasma esterases (18,19). Blood samples were delivered within 15 min of collection to the laboratory for separation and storage at Ϫ80°C until shipment to the Liverpool Bioanalytical Facility and Mahidol University for quantification of dolutegravir and ACTs, respectively. Both laboratories participate in external quality assurance programs for antiretrovirals (Association for Quality Assessment in Therapeutic Drug Monitoring and Clinical Toxicology [KKGT], The Netherlands) and antimalarials (Quality Assurance/Quality Control proficiency testing program supported by the Worldwide Antimalarial Resistance Network) and operate to Good Clinical Practice with assays validated according to published FDA guidelines.
Dolutegravir was extracted using liquid-liquid extraction and analyzed using validated reversed phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a lower limit of quantification (LLOQ) set at 10 ng/ml and precision of 5% at low quality control (30 ng/ml) (17).
Statistical analysis. Pharmacokinetic parameters, including the area under the concentration-time curve to the last measurable time point (AUC 0 -t ), terminal elimination half-life (t 1/2 ), maximum concentration (C max ), and time to C max (T max ), were estimated using noncompartmental analysis (WinNonlin, Phoenix, version 6.1; Pharsight, Mountain View, CA). The elimination clearance (CL/F) of the parent drug was calculated using the equation dose/AUC 0 -t . PK data were log transformed to calculate the GMR, with 90% CI values evaluated using paired (study A) or unpaired (study B) t tests and backtransformed to FIG 3 Study design. Two intensive PK studies using standard treatment doses of artemether-lumefantrine and artesunateamodiaquine with 50 mg of dolutegravir given once daily.
absolute ng/ml concentrations. The changes in PK parameters were considered statistically significant for a drug-drug interaction when the CI did not cross the value of one. An ANOVA was performed by SPSS (Windows standard version 22; SPSS, Inc., Chicago, IL) on PK parameters (AUC 0 -t , C max , and C 24 ) using generalized linear models procedures to assess potential sequence and period-related effects.

SUPPLEMENTAL MATERIAL
Supplemental material for this article may be found at https://doi.org/10.1128/AAC .01310-18.