Abstract
CYP2A6, CYP2B6, and UGT1A9 genetic polymorphisms and treatment response after a three-day course of artesunate-mefloquine was investigated in 71 Burmese patients with uncomplicated Plasmodium falciparum malaria. Results provide evidence for the possible link between CYP2A6 and CYP2B6 polymorphisms and plasma concentrations of artesunate/dihydroartemisinin and treatment response. In one patient who had the CYP2A6*1A/*4C genotype (decreased enzyme activity), plasma concentration of artesunate at one hour appeared to be higher, and the concentration of dihydroartemisinin was lower than for those carrying other genotypes (415 versus 320 ng/mL). The proportion of patients with adequate clinical and parasitologic response who had the CYP2B6*9/*9 genotype (mutant genotype) was significantly lower compared with those with late parasitologic failure (14.0% versus 19.0%). Confirmation through a larger study in various malaria-endemic areas is required before a definite conclusion on the role of genetic polymorphisms of these drug-metabolizing enzymes on treatment response after artesunate-based combination therapy can be made.
Introduction
Emergence and spread of artemisinin-resistant Plasmodium falciparum is a major concern for malaria control in malaria-endemic areas, particularly in areas bordering Myanmar and Cambodia.1–5 In Thailand and most countries in Southeast Asia, a three-day artesunate-mefloquine combination is currently used as first-line treatment for acute uncomplicated P. falciparum malaria. However, because of the limitation in the study design of various studies, it was not possible to attribute treatment failures after this regimen in these studies to true parasite resistance.
Several factors contribute to antimalarial treatment response, including the pharmacokinetics of the drugs (plasma/blood drug concentrations) in malaria patients. In our previous study,6 identification of treatment failure cases caused by an intrinsic parasite factor to each component of a three-day artesunate-mefloquine was reported based on integrated information on clinico-pathologic assessment, together with in vitro sensitivity (intrinsic parasite resistance) and systemic drug exposure (pharmacokinetic factor) in 17 patients with recrudescence response (late parasitologic failure [LPF]). Excluding the pharmacokinetic factor, results demonstrated only one confirmed case with reduced sensitivity to artesunate alone, and three cases were identified that had reduced sensitivity to artesunate and resistance to mefloquine. A pharmacokinetic factor was shown to account for approximately 59% of the total recrudescence cases, of which approximately half had inadequate dihydroartemisinin systemic drug exposure. Information on the impact of pharmacogenetics on antimalarial drug concentrations and treatment outcome after treatment with artemisinin-based combination therapy (ACT) in populations from various malaria-endemic regions, particularly the Thailand–Myanmar and Thailand–Cambodia borders, have been limited.
Cytochrome P450 (CYP) and uridine diphosphate glucuronosyltransferase (UGT) are the major enzyme superfamilies involved in biotransformation of xenobiotics in humans, including antimalarial drugs. Both enzymes are highly polymorphic and several of them, either singularly or in combination, are functionally of therapeutic significance with regards to clinical efficacy and safety.7 After oral or parenteral route administration, artesunate is rapidly and extensively converted by the liver enzyme CYP2A6, and to a lesser extent CYP2B6, to form the major active plasma metabolite dihydroartemisinin. Elimination of dihydroartemisinin is largely through the conversion to inactive glucuronide conjugates by UGT1A9 and UGT2B7 enzymes.8 Therefore, the polymorphisms in CYP2A6 and UGT1A9 genes may be of clinical relevance to treatment response after artesunate-based combination therapy as a result of inadequate plasma concentrations of dihydroartemisinin in some patients. Patients with poor metabolic activity of CYP2A6 and/or CYP2B6 may have unusually low concentrations of the active metabolite dihydroartemisinin. In addition, persons with high metabolic activity of UGT1A9 and/or UGT2B7 may also have unusually low concentrations of dihydroartemisinin.
There is no direct evidence for the association between this poor metabolic genotype and the decrease in antimalarial activity. Although, the in vitro antimalarial potency of both the parent drug artesunate and dihydroartemisinin have been shown to be similar, the residence time (reflected by half-life) of the later is relatively longer, and thus more sustainable antimalarial activity.9,10 In either case, the situation may lead to the decrease in antimalarial activity and as a consequence, an increase in the potential of resistance development of P. falciparum parasite to artesunate.
In the present study, the genetic polymorphisms of CYP2A6, CYP2B6, and UGT1A9, and their relationship with plasma concentrations of artesunate/dihydroartemisinin and treatment response were investigated in Burmese patients with acute uncomplicated P. falciparum malaria after a three-day course of artesunate-mefloquine therapy.
Materials and Methods
Chemicals and reagents.
Phenol, chloroform, isoamyl alcohol, and magnesium chloride were obtained from Merk (Hesse, Darmstadt, Germany). Trizma base, sodium dodecyl sulfate, sodium chloride, and 7.5 M ammonium acetate were obtained from Sigma (Missouri, St. Louis, MO). EDTA and proteinase K were obtained from Bio-Rad (Hercules, CA). Taq DNA polymerase and deoxyribonucletides were obtained from Fermentas (Vilnius, Lithuania). All restriction enzymes were obtained from New England BioLabs (Ipswich, MA). Agarose was obtained from Axygen Scientific Inc. (Union City, CA).
Patients, treatment, and sample collection.
The study was conducted at the Mae Tao clinic for migrant workers, Tak Province, Thailand during 2008–2009. This area is along the Thailand–Myanmar border and had highly multidrug-resistant P. falciparum.6 The study was approved by the Ethics Committee of the Ministry of Public Health of Thailand. Written informed consents for study participation were obtained from all patients before study.
A total of 71 (34 men and 37 women, age range = 16–50 years) Burmese patients with acute uncomplicated P. falciparum malaria were included in the study. Inclusion criteria for patient enrollment were according to the World Health Organization protocol for areas with low-to-moderate malaria transmission11: axillary temperature ≥ 37.5°C or recent history of fever; and a slide-confirmed P. falciparum mono-infection with a parasite density of 1,000–100,000 asexual parasites/μL. All were treated with a three-day course of artesunate-mefloquine combination therapy (4 mg/kg body weight of artesunate daily for three days; 750 and 500 mg mefloquine on the first and second day, respectively). Patients were admitted to the clinic during the three-day course of treatment or until signs and symptoms of malaria disappeared. Blood samples (3 mL each) were collected into heparinized tubes before treatment (H0) and at 1, 6, and 12 hours after the first dose for determination of artesunate/dihydroartemisinin concentrations. Plasma samples were separated within 30 minutes after collection by centrifugation at 1,200 × g for 15 min and stored at −180°C in a liquid nitrogen tank until analysis.
Patients were requested to return for follow-up on days 7, 14, 21, 28, and 42 or at any time if fever or symptoms suggestive of malaria developed. At each visit, a parasite count was performed (blood smear stained with Giemsa), and a detailed questionnaire for general symptoms was completed. Malaria blood smears were prepared at enrollment and thereafter, twice daily until two consecutive slides were confirmed to be negative, as well as at every follow-up visit. Thick blood films were screened for 200 oil-immersion fields before declaring a slide negative. Asexual parasites and gametocytes were separately counted against 200 leukocytes. If the parasite density was too numerous to count on the thick blood film, the number of parasites per 2,000 erythrocytes on the thin blood film was counted. The clinical outcome of a three day course of artesunate-mefloquine was evaluated in all patients who completed the 42-day follow-up period.
The classification of the therapeutic outcome was according to the World Health Organization protocol9 as adequate clinical and parasitologic response (ACPR), early treatment failure, and late treatment failure, which was further classified as late parasitologic failure (LPF) or late clinical failure. Plasmodium falciparum genotyping of the three polymorphic genes for merozoite surface antigen 1, merozoite surface antigen 2, and glutamate-rich protein was performed in paired samples obtained before treatment and at the time of parasite re-appearance to distinguish between re-infection and recrudescence.12–14
Determination of artesunate and dihydroartemisinin concentrations in plasma.
Plasma concentrations of artesunate and its active metabolite dihydroartemisinin were measured by using liquid chromatography mass-spectrometry.15 In brief, the liquid-liquid extraction of plasma samples was carried out by using dichloromethane and tert.-methyl butyl ether (at a ratio of 8:2 v/v). Chromatographic separation and mass analysis were performed on the 1100 Series Liquid Chromatography/Mass Spectrometer Detector Trap System (Agilent Technologies, Santa Clara, CA) by using electrospray ionization as an interface. The stationary phase was an Elipse XDB-C18 column. The mobile phase consisted of acetonitrile and 0.003 M glacial acetic acid at a ratio of 62:38 (v/v) delivered at a flow rate of 0.5 mL per minute. Positive ion mode was selected to detect extracted ions at m/z 407 and 261 for artesunate, at m/z 307 and 261 for dihydroartemisinin, and at m/z 305 for artemisinin (internal standard). The limit of detection was 2 ng/mL for artesunate and dihydroartemisinin.
Detection of polymorphisms in CYP2A6, CYP2B6, and UGT1A9 alleles.
Genomic DNA was extracted from all samples using phenol/chloroform method.16 DNA samples were analyzed for CYP2A6 alleles CYP2A6*1A, *1B, *4C,17 *2 and *3,18 and *619; CYP2B6 alleles CYP2B6*1,*2, *3, *4, *5, *6, *7, and *920; and UGT1A9 alleles UGT1A9*1, *4, and *521 using a polymerase chain reaction–restriction fragment length polymorphism method.
Data analysis.
Statistical analysis was performed by using the SPSS version 15 (SPSS, Chicago, IL). Median (95% confidence interval [CI]) values were used to summarize quantitative data that were not normally distributed, and statistical analysis was performed by using Kruskal Wallis and/or Mann-Whitney U tests. The frequencies of CYP2A6, CYP2B6, and UGT1A9 alleles and genotypes are summarized as the number of cases and percentage values. Association between the prevalence of CYP2A6, CYP2B6, and UGT1A9 alleles and treatment response was evaluated by using chi square and Fisher exact tests. The statistical significance level was set at α = 0.05 for all tests.
Results
Clinical response.
Median (95% CI) admission parasitemia and body weights of the patients were 4,970 (4,268–5,672) parasites/μL and 53.5 (49.0–58.0) kg, respectively. Fifty (70.42%) patients had ACPR, and 21 (29.58%) patients had LPF (confirmed by polymerase chain reaction as recrudescence) during days 20–35 of treatment. Patients with LPF and ACPR were well matched with respect to sex, age, and body weight. Significantly higher admission parasitemia, longer parasite clearance time (time until the first series of negative blood smears occurred), and fever clearance time (time from start of treatment until the axillary temperature decreased to below 37.5°C and remained below this temperature for the next 48 hours) were observed in patients with LPF compared with those with ACPR (median [95% CI] admission parasitemia = 6,615 [2,407–10,823] versus 4,690 [4,160–5,220] parasites/μL; P = 0.02; parasite clearance time = 32 [29–35] versus 26 [24–28] hours; P = 0.02; and fever clearance time = 34 [31–37] versus 26 [25–27] hours; P = 0.01).
Plasma concentrations of artesunate and dihydroartemisinin.
Plasma artesunate and dihydroartemisinin concentrations at one hour after the first dose of artesunate were comparable between patients with LPF (n = 14) and ACPR (n = 27) (Table 1). In addition, the proportions of patients with LPF and ACPR who had inadequate concentrations of artesunate (< 300 ng/mL, the lower limit of 95% CI for median concentration in the ACPR group) and dihydroartemisinin (< 479 ng/mL, the lower limit of 95% CI for median concentration in the ACPR group) concentrations at one hour after the first dose were comparable.
Table 1.
Plasma concentrations of artesunate and dihydroartemisinin at one hour after a three-day course of artesunate-mefloquine and proportions of patients (ACPR and LPF) with inadequate concentrations*
| Drug | Variable | Treatment response | |
|---|---|---|---|
| ACPR (n = 27) | LPF (n = 14) | ||
| Artesunate | Plasma concentration at 1 hour after dosing (ng/mL) | 350 (300–400) | 337 (237–438) |
| Proportion of patients with inadequate plasma concentrations at 1 hour† after dosing (%) | 9/27 (33.3) | 5/14 (35.7) | |
| Dihydroartemisinin | Plasma concentration at 1 hour after dosing (ng/mL) | 536 (479–593) | 555 (461–649) |
| Proportion of patients with inadequate plasma concentrations at 1 hour‡ after dosing (%) | 6/27 (22.2) | 5/15 (33.3) | |
Values are median (95% confidence interval) or no. positive/no. tested (%). ACPR = adequate clinical and parasitologic response; LPF = late parasitologic failure.
< 300 ng/mL (lower limit of 95% confidence interval for median concentration in the ACPR group).
< 479 ng/mL (lower limit of 95% confidence interval for median concentration in the ACPR group).
CYP2A6, CYP2B6, and UGT1A9 allele frequencies and genotypes and relationship with treatment response.
The allele frequencies of CYP2A6, CYP2B6, and UGT1A9 were determined in 50 and 21 patients with ACPR and LPF, respectively (Table 2). The allele frequencies of CYP2A6*1A (80.0 versus 90.5%) and CYP2A6*1B (12.0 versus 9.5%) in patients with ACPR and LPF were similar. Heterozygous mutant of CYP2A6*4C was observed in one patient (1.4%). CYP2A6*2, CYP2A6*3, and CYP2A6*6 alleles were not found in any patient. For CYP2B6, most (84.5%) had the wild-type allele CYP2B6*1, and 15.5% had the mutant CYP2B6*9 allele. CYP2B6*2, CYP2B6*3, CYP2B6*4, CYP2B6*5, CYP2B6*6, and CYP2B6*7 alleles were not found in any patient. For UGT1A9, all patients had the homozygous wild-type allele.
Table 2.
CYP2A6, CYP2B6, and UGT1A9 allele frequencies in 50 patients with ACPR and 21 patients with LPF after treatment with a three-day course of artesunate-mefloquine*
| Allele | Artesunate-mefloquine treatment response | Total (n = 71) | |
|---|---|---|---|
| ACPR (n = 50) | LPF (n = 21) | ||
| CYP2A6 | |||
| CYP2A6*1A | 86.0 (23, 20) | 90.5 (14, 5) | 87.3 (37, 25) |
| CYP2A6*1B | 12.0 (3, 3) | 9.5 (2, 0) | 11.3 (5, 3) |
| CYP2A6*2 | 0 (0, 0) | 0 (0, 0) | 0 (0, 0) |
| CYP2A6*3 | 0 (0, 0) | 0 (0, 0) | 0 (0, 0) |
| CYP2A6*4C | 2.0 (1, 0) | 0 (0, 0) | 1.4 (1, 0) |
| CYP2A6*6 | 0 (0, 0) | 0 (0, 0) | 0 (0, 0) |
| CYP2B | |||
| CYP2B6*1 | 86.0 (22, 21) | 81.0 (8, 9) | 84.5 (30, 30) |
| CYP2B6*2 | 0 (0, 0) | 0 (0, 0) | 0 (0, 0) |
| CYP2B6*3 | 0 (0, 0) | 0 (0, 0) | 0 (0, 0) |
| CYP2B6*4 | 0 (0, 0) | 0 (0, 0) | 0 (0, 0) |
| CYP2B6*5 | 0 (0, 0) | 0 (0, 0) | 0 (0, 0) |
| CYP2B6*6 | 0 (0, 0) | 0 (0, 0) | 0 (0, 0) |
| CYP2B6*7 | 0 (0, 0) | 0 (0, 0) | 0 (0, 0) |
| CYP2B6*9 | 14.0 (3, 4) | 19.0 (4, 0) | 15.5 (7,4) |
| UGT1A9 | |||
| UGT1A9*1 | 100 (25, 25) | 100 (12, 9) | 100 (37, 34) |
| UGT1A9*4 | 0 (0, 0) | 0 (0, 0) | 0 (0, 0) |
| UGT1A9*5 | 0 (0, 0) | 0 (0, 0) | 0 (0, 0) |
Values are percentages (no. females, no. males). CYP = cytochrome P450; UGT = uridine diphosphate glucuronosyltransferase; ACPR = adequate clinical and parasitologic response; LPF = late parasitologic failure.
The genotype frequencies of CYP2A6 and CYP2B6 are summarized in Table 3. Four CYP2A6 genotypes were observed with frequencies in descending order as follows: CYP2A6*1A/*1B (46.2%) > CYP2A6*1B/*1B (27.7%) > CYP2A6*1A/*1A (24.6%) > CYP2A6*1A/*4C (1.5%). The frequencies of these genotypes in patients with ACPR and LPF were not significantly different. Two CYP2B6 genotypes were observed, i.e., CYP2B6*1/*1 (84.6%) and CYP2B6*9/*9 (15.4%). The proportion of patients with ACPR who had the CYP2B6*9/*9 genotype was significantly lower than those with LPF (14.0% versus 19.0%; P = 0.015).
Table 3.
CYP2A6 and CYP2B6 genotypes in 46 patients with ACPR and 19 patients with LPF after treatment with a three-day course of artesunate-mefloquine*
| Genotype | Artesunate-mefloquine treatment response | Total (n = 65) | |
|---|---|---|---|
| ACPR (n = 46) | LPF (n = 19) | ||
| CYP2A6 | |||
| CYP2A6†1A/†1A | 21.7 (7, 3) | 31.6 (5, 1) | 24.6 (12, 4) |
| CYP2A6†1A/†1B | 47.8 (7, 15) | 42.1 (3, 5) | 46.2 (10, 20) |
| CYP2A6†1B/†1B | 30.4 (10, 4) | 21.1 (2, 2) | 27.7 (12, 6) |
| CYP2A6†1A/†4C | 0.0 (0, 0) | 5.2 (0, 1) | 1.5 (0, 1) |
| CYP2B6 | |||
| CYP2B6†1/†1 | 86 (21,19) | 81.0 (8, 7) | 84.6 (29, 26) |
| CYP2B6†9/†9 | 14.0 (3, 3) | 19.0 (2, 2)* | 15.4 (5, 5) |
Values are percentage (no. females, no. males). CYP = cytochrome P450; ACPR = adequate clinical and parasitologic response; LPF = late parasitologic failure.
Statistically significant difference from ACPR (P = 0.015, by Fisher exact test).
CYP2A6 and CYP2B6 genotypes and relationship with plasma concentrations of artesunate and dihydroartemisinin.
The CYP2A6 and CYP2B6 genotypes in relationship with plasma concentrations of artesunate and dihydrortemisinin at 1, 6, and 12 hours after the first dose of artesunate are summarized in Tables 4 and 5. For one patient who had the CYP2A6*1A/*4C genotype, plasma concentration of artesunate at one hour (415 ng/mL) appeared to be higher, and the concentration of dihydroartemisinin (320 ng/mL) was lower than in persons who had other three genotypes (CYP2A6*1A/*1A, CYP2A6*1A/*1B and CYP2A6*1B/*1B) (Table 4). Plasma concentrations of artesunate and dihydroartemisinin in patients who had CYP2A6*1A/*1A, CYP2A6*1A/*1B, CYP2A6*1B/*1B (Table 4), CYP2B6*1/*1 and CYP2B6*9/*9 (Table 5) genotypes were similar.
Table 4.
CYP2A6 genotypes and relationship with concentrations of artesunate and dihydroartemisinin*
| Drug | Time | Drug concentration (ng/mL) | |||
|---|---|---|---|---|---|
| CYP2A6*1A/*1A | CYP2A6*1A/*1B | CYP2A6*1B/*1B | CYP2A6*1A/*4C | ||
| Artesunate | H0 | 0 (0–0) [10] | 0 (0–0) [15] | 0 (0–0) [10] | 0 [1] |
| H1 | 364 (285–443) [10] | 311 (219–403) [15] | 339 (241–437) [10] | 415 [1] | |
| H6 | 1.5 (0–7) [6] | 1 (0–5) [11] | 7 (1–13) [5] | ND | |
| H12 | 0 (0–0) [10] | 0 (0–0) [15] | 0 (0–0) [10] | 0 [1] | |
| Dihydro-artemisinin | H0 | 0 (0–0) [11] | 0 (0–0) [16] | 0 (0–0) [10] | 0 [1] |
| H1 | 555 (473–637) [11] | 519 (457–581) [16] | 606 (439–774) [10] | 320 [1] | |
| H6 | 123 (74–172) [10] | 84 (69–99) [16] | 99 (73–125) [9] | 40 [1] | |
| H12 | 11 (0–62) [6] | 10 (6–14) [13] | 14 (5–22) [8] | ND | |
Values are median (95% confidence interval) [no. of cases]. CYP = cytochrome P450; ND = not determined. Data are presented as median (95% CI) [number of cases].
Table 5.
| Drug | Time | Drug concentration (ng/mL) | |
|---|---|---|---|
| CYP2B6*1 | CYP2B6*9 | ||
| Artesunate | H0 | 0.0 (0.0–0.0) [33] | 0.0 (0.0–0.0) [8] |
| H1 | 315.0 (259.0–371.0) [33] | 402.0 (276.0–528.0) [8] | |
| H6 | 1.0 (0.0–2.0) [26] | 10.0 (5.0–15.0) [7] | |
| H12 | 0.0 (0.0–0.0) [33] | 0.0 (0.0–0.0) [8] | |
| Dihydro-artemisinin | H0 | 0.0 (0.0–0.0) [33] | 0.0 (0.0–0.0) [8] |
| H1 | 555.0 (490.0–620.0) [34] | 493.5 (395.0–592.0) [8] | |
| H6 | 98.5 (77.5–119.5) [32] | 70.0 (27.0–113.0) [8] | |
| H12 | 10.0 (8.0–12.0) [26] | 12.5 (0.0–28.0) [8] | |
Values are median (95% confidence interval [no. cases]. CYP = cytochrome P450.
Discussion
The artemisinin group of antimalarial drugs markedly exhibit variable pharmacokinetic profiles with rapid absorption and elimination phases. Their bioavailability is highly variable depending on drug formulations, route of administration, health status, and severity of malaria disease.22 Biotransformation into its active metabolite dihydroartemisinin occurs almost immediately after artesunate administration and the drug is considered as a prodrug. The elimination half-lives of artesunate and dihydroartemisinin range from 0.5 to 3 hours.9,10 CYP2A6, CYP2B6, and UGT1A9 in human livers are drug-metabolizing enzymes involved in the biotransformation of artesunate. Results from the relatively limited sample size in our present study provide evidence for the possible contribution of CYP2A6 and CYP2B6 polymorphisms on artesunate/dihydroartemisinin concentrations and treatment response in patients with uncomplicated P. falciparum malaria after a three-day course of artesunate-mefloquine therapy.
The influence of UGT1A9 polymorphism on treatment response could not be assessed because all patients had the wild-type genotype of this gene. Most of the patients had the two CYP2A6 variants with normal (*1A) or increased (*1B) activity. The frequency distribution of these two allelic variants in Burmese population appears to be similar to those of other Asian ethnicities, particularly the Thai ethnic groups, where the frequencies of CYP2A6*1A and CYP2A6*1B alleles were 34.0 and 35.5%, respectively.23 The gene allele frequencies in Japanese and Koreans were relatively lower, i.e., 20.1 versus 27.7% and 24.4 versus and 36.8%, respectively.19 It was noted that in one patient who had the CYP2A6*1A/*4C genotype (decreased enzyme activity), plasma concentration of artesunate at one hour after the first dose (415 ng/mL) appeared to be higher, and the concentration of dihydroartemisinin (320 ng/mL) was lower than the median concentrations for those who had the other three genotypes. However, with adequate whole blood concentration of mefloquine and adequate parasite sensitivity to mefloquine and artesunate,6 the patient still responded well to treatment (ACPR).
Polymorphism of CYP2A6 has been reported at high frequency in Asian populations than in Caucasian and African populations.24 Several polymorphisms with functional significance have been identified, and there are at least 13 CYP2A6 variants with decreased metabolizing function and 5 variants with absent activity in vivo. CYP2A6*1B allele occurs when the 3′ flanking region of CYP2A6 gene is converted with CYP2A7 gene. CYP2A6*1B is believed to increase the stability of the mRNA transcript, resulting in a greater amount of the CYP2A6 enzyme.25 Previous studies showed that persons who had the CYP2A6*1B allele tended to have higher metabolic ratios than those who did not have this allele.26–28 Therefore, persons who had the CYP2A6*1/*1 genotype would have 100% activity of CYP2A6 enzyme. CYP2A6*4C is a null gene with deleted CYP2A6.29,30 Persons who have one or two copies of the CYP2A6*2 or CYP2A6*4 alleles, together with one of CYP2A6*9 or CYP2A6*12 alleles were shown to have reduced enzyme activity in nicotine metabolism by 50%.31 For CYP2B6, which plays a secondary role in artesunate metabolism, the defective mutant CYP2B6*9 was the only mutant variant detected in the samples at a frequency of approximately 15.5%.
Although no direct association between CYP2B6*9/*9 genotypes and artesunate and dihydroartemisinin plasma concentrations was found, there was a significantly lower proportion of patients with ACPR (cure) who had the CYP2B6*9/*9 genotype compared with LPF (14% versus 19%). CYP2B6 functional polymorphisms are highly prevalent in several African32–35 and Asian countries.35,36 In most of these countries, artemisinin-based combination therapy has been used as first-line treatment for acute uncomplicated P. falciparum malaria. The CYP2B6*9 allele is commonly found in all ethnic groups, but more frequently in Hispanics compared with African-Americans, Caucasians, and Asians.37 Previous studies have shown an effect of this variant on CYP2B6-mediated metabolism of non-nucleoside reverse transcriptase inhibitors.38 The 516T allele has been associated with higher plasma exposure to efavirenz in patients infected with human immunodeficiency virus.39
Results from the present study provide evidence for the possible contribution of CYP2A6 and CYP2B6 genetic polymorphisms on clinico-parasitologic treatment response after a three-day course of artesunate-mefloquine therapy. Apart from the contribution of genetic polymorphism in drug metabolism, the possible link between treatment failure and the newly discovered K13 protein gene is also worth exploring. Whole-genome sequencing of an artemisinin-resistant parasite line from Africa and clinical parasite isolates from Cambodia has associated the mutation in this parasite gene with artemisinin resistance in vitro and in vivo.40 Confirmation through a larger study in various malaria-endemic areas is required before a definite conclusion on the role of genetic polymorphisms in drug metabolizing enzymes on treatment response after artesunate-based combination therapy can be made and a translational use of pharmacogenetic for supporting surveillance and monitoring of their clinical efficacy can be implemented.
ACKNOWLEDGMENTS
We thank all patients for participating in the study and Kalaya Ruengweerayut and the staff of Mae Tao Clinic for their support.
Footnotes
Financial support: The study was supported by the Commission on Higher Education, Ministry of Education of Thailand; the National Research University Project of Thailand, Office of Higher Education Commission; the Royal Golden Jubilee Ph.D. Program; the Thailand Research Fund–Thammasat University Joint Fund; and a Graduate Student Grant to Papichaya Phompradit from Thammasat University.
Authors' addresses: Papichaya Phompradit, Anurak Cheoymang, and Kesara Na-Bangchang, Chulabhorn International College of Medicine, Thammasat University (Rangsit Campus), Patumthani 12121, Thailand, E-mails: bphompradit@yahoo.com, anurak_ch9@yahoo.com, and kesaratmu@yahoo.com. Poonuch Muhamad, Thailand Center of Excellence on Drug Discovery and Development, Thammasat University (Rangsit Campus), Patumthani 12121, Thailand, E-mail: nurah_ab@yahoo.com.
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