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CYP3A4-mediated metabolism of artemisinin to 10β-hydroxyartemisinin with comparable anti-malarial potency

Malaria Journal volume 23, Article number: 328 (2024) Cite this article

Abstract

Background

The most widely used anti-malarial drug artemisinin (ART) is metabolized extensively, but the therapeutic capacity of its major metabolite remains unknown. Whether the major metabolite of ART (ART-M) contributes to its antiplasmodial potency was investigated in this study.

Methods

The metabolite identification and enzyme phenotyping of ART were performed using human liver microsomes (HLMs). The stereostructure of the major metabolite ART-M was elucidated by spectroscopic and X-ray crystallographic analysis. The anti-malarial activity of ART-M against two reference Plasmodium strains (Pf3D7 and PfDd2) was evaluated. The pharmacokinetic profiles of ART and its metabolite ART-M were investigated in healthy Chinese subjects after a recommended two-day oral dose of ART plus piperaquine. Pharmacodynamic parameters based on minimum inhibitory concentration (MIC50) and free plasma concentration were employed to evaluate the therapeutic potency of ART-M, including fAUC0-t/MIC50, fCmax/MIC50 and T > MIC50.

Results

A major metabolite 10β-hydroxyartemisinin (ART-M) was found for ART in human, and CYP3A4/3A5 was the major enzymes responsible for ART 10β-hydroxylation. Compared with ART (MIC50, 10.1 nM against Pf3D7), weaker antiplasmodial activity was found for ART-M (MIC50, 61.4 nM against Pf3D7). However, a 3.5-fold higher maximal free plasma concentration was achieved for ART-M (fCmax, 180.0 nM vs. 51.8 nM for ART). ART-M displayed comparable antiplasmodial potency to ART, in terms of fAUC0-t/MIC50 (12.5 h), fCmax/MIC50 (2.8) and T > MIC50 (5 h).

Conclusions

The major metabolite 10β-hydroxyartemisinin contributes to the antiplasmodial efficacy of ART, which should be considered when evaluation of ART dosing regimens and/or clinical outcomes.

Background

Artemisinin (ART; Qing-Hao-Su; Fig. S1) and its derivatives (e.g., dihydroartemisinin) are the most important anti-malarial drugs used in artemisinin-based combination therapy (ACT), i.e., an artemisinin drug is responsible for the rapid clearance of Plasmodium falciparum and another long-acting partner anti-malarial drug is involved for eliminating residual parasites [1]. However, parasite resistance to ACT drugs (e.g., dihydroartemisinin plus piperaquine) has emerged in Southeast Asia [2, 3], and suboptimal drug concentrations can also lead to parasite recurrence. As relatively inexpensive artemisinin-based combinations containing ART instead of its synthetic derivatives, a two-day fixed-dose of ART plus piperaquine (Artequick) and a single fixed-dose of ART plus naphthoquine are commercially available [4, 5]. To avoid the rapid development of ART resistance, the dosing regimens of ART should be optimized based on the understanding of its pharmacokinetic-pharmacodynamic characteristics.

ART has not been used to a great extent in artemisinin-based combinations because of its low bioavailability (~ 30%), a short elimination half-life (t1/2 ~ 2 h), and time-dependent pharmacokinetics, i.e., lower plasma concentration of ART was observed after repeated oral administration of ART [6, 7]. ART underwent extensive metabolism, with < 1% unchanged ART in human urine after an oral administration [8]. A total of thirteen phase I (e.g., five monohydroxylated metabolites) and twelve phase II metabolites were detected for ART in liver microsomal incubates and plasma samples based on mass spectrometry [9, 10]. The major metabolic pathways were proposed to be hydroxylation, loss of oxygen, and subsequent glucuronidation (Fig. S1). Several metabolites were identified for ART in human urine [8], including 9,10-dihydroxyartemisinin, deoxyartemisinin and a “crystal 7” (Fig. S1). The structure–activity relationship showed that the endoperoxide group is essential for the anti-malarial activity of ART [11], and the absolute configuration of ART can influence the anti-malarial activity [12]. However, the exact structures of monohydroxylated metabolites of ART and whether the hydroxylated metabolites contribute to ART efficacy remain unclear.

The underlying mechanism for the time-dependent pharmacokinetics of ART was suggested to be the autoinduction metabolism mediated by CYP2B6/CYP3A4 enzymes [13, 14]. The ART depletion in human liver microsomes (HLMs) was primarily mediated by CYP2B6, with a minor role of CYP3A4 and CYP2A6 [15]. On the other hand, ART could induce CYP2B6 (bupropion hydroxylation and mephenytoin N-demethylation) [14, 16] and CYP3A4 (midazolam hydroxylation) [17, 18], via activation of constitutive androstane receptor (CAR) and pregnane X receptor (PXR) [19].

In this study, whether the hydroxylated metabolite of ART displays any therapeutic potency was investigated. The metabolite identification and enzyme phenotyping of ART were first performed. The structure and stereo-configuration of the major hydroxylated metabolite (ART-M) were elucidated by spectroscopic techniques and X-ray single crystal diffraction. The anti-malarial activity of ART-M against two reference Plasmodium strains (chloroquine-sensitive strain Pf3D7 and chloroquine-resistant strain PfDd2) was evaluated. The pharmacokinetic profiles of ART and its metabolite ART-M were investigated in healthy Chinese subjects after a recommended two-day oral dose of Artequick (ART plus piperaquine). Pharmacokinetic/pharmacodynamic (PK/PD) indices based on minimal inhibitory concentration (MIC50) and free plasma concentration (e.g., fAUC0-t/MIC50, fCmax/MIC50) were calculated to evaluate the therapeutic potency of ART-M.

Methods

Chemicals and reagents

Artemisinin (ART) was purchased from Kunming Pharmaceutical Co. (Yunnan, China; purity > 99.0%). Artemisinin-d3 (purity > 98.0%) was purchased from TRC Canada (Ontario, Canada). Pooled liver microsomes derived from human (HLMs) were purchased from RILD Research Institute for Liver Diseases (Shanghai, China). Recombinant P450 isoforms were purchased from Cypex Ltd. (Dundee, UK). Chloroquine diphosphate (CQ), NADPH, probe substrates and (non)selective inhibitors of CYP enzymes were purchased from Sigma-Aldrich (St. Louis, MO). Artequick tablets (artemisinin plus piperaquine) were provided by Artepharm Co., Ltd (Guangzhou, China). Chloroquine-sensitive strain Pf3D7 and chloroquine-resistant strain PfDd2 were obtained from the Malaria Research and Reference Reagent Resource Center (MR4) as a part of the BEI Resources Repository, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Preparation of 10β-hydroxyartemisinin

10β-Hydroxyartemisinin (ART-M; Fig. S1) was prepared by microbial transformation, based on a previous report [20]. Briefly, ART (250 mg/mL) was incubated with fungal mycelia of Cunninghamella elegans ATCC 9245. The broth filtrate was extracted with ethyl acetate. 10β-Hydroxyartemisinin was then purified by an Agilent 1260 HPLC system equipped with a DAD detector.

The structure of 10β-hydroxyartemisinin (purity > 98.0%) was confirmed by HRMS, NMR (Table S1) and X-ray single crystal diffraction (Fig. S2). The HRMS was performed using a ThermoScientific LTQ-Orbitrap Velos mass spectrometer. 1H-NMR and 13C-NMR spectra were recorded on a Bruker Avance 600 MHz spectrometer. The X-ray diffraction experiments were performed on a Bruker D8 X-ray diffractometer.

In vitro metabolism of ART and its enzyme kinetics in HLMs

For metabolite profiling, ART (1 μM) was incubated with HLMs (1 mg/mL) in potassium phosphate buffer (0.1 M, pH 7.4) and NADPH (1 mM) at 37 °C for 30 min. The incubation was initiated by adding NADPH after a preincubation of 3 min, and stopped by adding two volumes of cold acetonitrile.

For the kinetic analysis, ART was incubated with HLMs (0.5 mg/mL; pre-optimized) at six concentrations (1–32 μM) concentrations. Reactions were initiated by adding NADPH (1 mM) and terminated after 15 min (pre-optimized). All incubations were carried out in triplicate. The formation rate of ART-M was calculated and plotted against ART concentration. The depletion rate of ART was also determined. Nonlinear regression analysis (WinNonlin 7.0, Pharsight, NC) was performed using the Michaels-Menten kinetic equation for the calculation of Km and Vmax. The intrinsic hepatic clearance of ART (CLint,H) was calculated by Vmax/Km, derived from both metabolite formation and substrate depletion.

Enzyme phenotyping

To assess the contribution of CYP enzymes to ART metabolism, incubations of ART (1 μM) with HLMs (1 mg/mL) were carried out as described above in the presence of (non)selective chemical inhibitors of total P450 enzymes (1 mM 1-ABT; preincubated for 15 min), CYP1A2 (5 μM α-naphthoflavone), CYP2B6 (10 μM ticlopidine), CYP2C8 (25 μM quercetin), CYP2C9 (20 μM sulfaphenazole), CYP2C19 (10 μM ticlopidine), CYP2D6 (10 μM quinidine) or CYP3A4 (1 μM ketoconazole).

ART (1 μM) was incubated with recombinant CYPs (100 pmol/mL) in potassium phosphate buffer (0.1 M, pH 7.4) and NADPH (1 mM) at 37 °C for 30 min. The incubation was initiated by adding NADPH and stopped by adding two volumes of cold acetonitrile.

In vitro drug susceptibility

The determination of 50% or 90% growth inhibitory concentration (MIC50 or MIC90) values of the metabolite ART-M against Pf3D7 or PfDd2 was performed using a previously reported protocol [21]. Briefly, synchronous ring-stage parasite culture was prepared with a final 0.5% parasitaemia and 2% haematocrit. CQ and 0.1% DMSO were used as the positive and negative control, respectively. The antiplasmodial activity was determined by a fluorometric method using SYBR Green I. MIC50 and MIC90 values obtained from OriginPro 9.0 were the average of two independent measurements each performed in duplicate. Drug susceptibility was analyzed by a nonlinear regression of logarithmically transformed concentrations.

The pharmacokinetic study in humans

The clinical experiment was carried out in accordance with the Declaration of Helsinki, and the experimental protocol was approved (KYLL-202307–025) by the Ethics Committee of Qilu Hospital (Shandong University, China). Six healthy and non-smoking male adults (20–26 years; body mass index of 20–25 kg/m2) received a recommended two-day oral dose of Artequick (125 mg of ART plus 750 mg of piperaquine). Venous blood (2 mL) was taken from an indwelling intravenous catheter at 0, 0.25, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 8.0, 12.0, 24.0, 36.0 (second dose), and 48.0 h (second dose) after each dose, and collected in anticoagulant tubes drawn from forearm venous catheters. Plasma was separated by centrifugation at 4000 g for 15 min at 4 °C. The plasma samples were stored at − 80 °C until analysis.

Sample processing and data analysis

After termination of microsomal or rCYP incubation by adding two volumes of cold acetonitrile (containing the internal standard artemisinin-d3), 50 μL of incubates were centrifuged at 7200 g for 15 min. The supernatant was dried under N2 at room temperature, and then reconstituted with initial mobile phase before analysis. For the pharmacokinetic study, plasma samples were pretreated by protein precipitation with acetonitrile based on a previous study [9]. Briefly, a 25 μL aliquot of human plasma was mixed with 105 μL of acetonitrile (containing the internal standard artemisinin-d3). The mixture was mixed and centrifuged at 7200 g for 15 min. Aliquots (25 μL) of the supernatant were injected into the LC-HRMS system.

All samples were analysed by liquid chromatography tandem high-resolution mass spectrometric (LC-HRMS) methods, which were performed on an LTQ-Orbitrap Velos hybrid mass spectrometer (ThermoScientific, Bremen, Germany) equipped with an electrospray ionization interface. The mass spectrometer was calibrated allowing for mass accuracy < 5 ppm in external calibration mode. The ionization voltage was + 4.5 kV and the capillary temperature was set at 300 °C. Nitrogen was used as both the sheath gas (40 units) and auxiliary gas (10 units). The resolving power was 15, 000 for the MS full scan. The analytical methods for quantification were fully validated, which included specificity, linearity, intra and inter-day accuracy and precision, and stability. The representative LC-HRMS chromatograms for quantification of ART and its metabolite in human plasma are shown in Fig. S3.

The pharmacokinetic parameters were analysed by a non-compartmental model using the software TOPFIT 2.0 (Thomae GmbH, Germany). The maximum plasma concentration (Cmax) and time-to-peak concentration (Tmax) were obtained from experimental observations. The area under the plasma concentration time curve (AUC0-t) was calculated using the linear trapezoidal rule to approximately the last point. Total oral body clearance (CL/F) was calculated as the dose/AUC0-t. The terminal elimination half-life (t1/2) was estimated by log-linear regression in the terminal phase. The metabolic capability for formation of the metabolite ART-M was calculated by the ratio of AUCART-M/AUCART. The comparison of pharmacokinetic parameters was compared using one way ANOVA performed using SPSS19.0 (SPSS Inc., USA). Comparison of AUC0-t and Cmax was performed after logarithmic transformation, and the comparison of Tmax was performed using the Wilcoxon signed-rank test. The acceptable level of significance was established at P < 0.05. A greater than 1.5-fold increase in AUC0-t ratio was defined as significant.

Prediction of in vivo antiplasmodial potency using PK/PD indices

To evaluate the therapeutic potency of ART-M, the commonly used PK/PD indices such as the ratio of area under the free concentration–time curve to minimum inhibitory concentration (fAUC0-t/MIC50), maximum free plasma concentration to MIC (fCmax/MIC50) and time that free plasma concentration exceeds MIC50 (T > MIC50) were employed. Plasma protein binding of ART and ART-M at the concentration of 0.6 μM was determined in pooled human plasma using ultrafiltration technique described previously [22]. A greater than 1.5-fold difference in ratios of fCmax/MIC50 and fAUC0-t/MIC50 was defined as significant.

Results

Metabolite profiling of ART in HLMs

Metabolite identification of ART in HLMs was first performed using high-resolution mass spectrometry, and detected metabolites were proposed based on the accurate MS and MS/MS spectra. ART was mainly metabolized to the hydroxylated metabolite ART-M with a trace metabolite isomer (M1) in the presence of NADPH (Fig. 1). By comparing the chromatographic and mass spectrometric behavior with the reference standard, the major hydroxylated metabolite of ART in HLMs was identified as 10β-hydroxyartemisinin (ART-M).

Fig. 1
figure 1

Selected ion chromatograms and MS/MS spectra of artemisinin (ART) and its hydroxylated metabolites, when ART (1 μM) was incubated in human liver microsomes (1 mg/mL) for 30 min. I: total ion chromatogram (TIC); II: ART (m/z 283.1540); III: hydroxylated metabolites of ART (m/z 316.1755). ART-M: 10β–hydroxyartemisinin

Full size image

Enzyme kinetics for ART hydroxylation in HLMs

The formation rate of ART-M as a function of ART concentrations (1–32 μM) followed Michaelis–Menten kinetics (Fig. 2), which showed the CLint,H of 27.6 μL/min/mg. The CLint,H derived from the substrate depletion was 30.3 μL/min/mg, with the Km value of 23.8 μM (Fig. 2).

Fig. 2
figure 2

Enzyme kinetics for ART metabolism in human liver microsomes (0.5 mg/mL) for 15 min. A substrate depletion; B formation of 10β–hydroxyartemisinin (ART-M). The experiment was performed in triplicate

Full size image

Enzyme phenotyping for ART hydroxylation

The formation of ART-M in HLMs was also evaluated in the presence of (non)-selective inhibitors, to determine the relative contribution of these CYP enzymes. The formation of ART-M was completely inhibited by 1-ABT (CYPs) or ketoconazole (CYP3A4), but not by other selective inhibitors (Fig. 3). Of the ten human cDNA-expressed recombinant CYP enzymes assessed, CYP3A4/3A5 produced ART-M as a major metabolite of ART (Fig. 3).

Fig. 3
figure 3

Enzyme phenotyping for the formation of ART-M (10β–hydroxyartemisinin) from artemisinin (ART; 1 µM) using human liver microsomes (HLM, 1 mg/mL) in the presence of (non)selective inhibitors of total P450 enzymes (1-ABT), CYP1A2 (α-naphthoflavone, NAPH), CYP2B6 (ticlopidine, TCL), CYP2C8 (quercetin, QUER), CYP2C9 (sulfaphenazole, SULF), CYP2C19 (ticlopidine, TCL), CYP2D6 (quinidine, QUIN) or CYP3A4 (ketoconazole, KTZ), or using recombinant P450 isoforms (100 pmol/mL). A ART-M in HLMs; B ART-M in rCYPs

Full size image

The metabolite ART-M showed antiplasmodial activity against Pf3D7 and PfDd2

The positive model drug CQ showed consistent antiplasmodial activity against Pf3D7 (MIC50, 10.6 nM) and PfDd2 (MIC50, 93.8 nM). ART also showed a remarkable antiplasmodial activity against Pf3D7 (MIC50, 10.1 nM) and PfDd2 (MIC50, 20.4 nM). Weaker antiplasmodial activity was observed for ART-M (MIC50, 64.1 nM against Pf3D7; MIC50, 132.9 nM against PfDd2) (Table 1, Fig. S4 and Fig. S5).

Table 1 Antiplasmodial activity of artemisinin (ART) and its metabolite 10β–hydroxyartemisinin (ART-M) against two reference Plasmodium strains (Pf3D7 and PfDd2)
Full size table

The pharmacokinetics of ART in humans

By comparing the chromatographic and mass spectrometric behaviour with the reference standard, the major hydroxylated metabolite of ART in human blood circulation was identified as 10β-hydroxyartemisinin (ART-M) (Fig. S3). After the first oral dose of ART, the maximum concentration of ART (Cmax, 206.6 nM) was reached at 1.0 h (Fig. 4). ART was rapidly eliminated, with a high total oral clearance (CL/F) of 11.0 L/h/kg and a short half-life (t1/2, 1.7 h). The hydroxylated metabolite ART-M was detected in relatively higher level (Cmax, 578.9 nM), with a similar t1/2 (1.6 h) (Table S2). The metabolic ratio of AUCART-M/AUCART was 4.1. Compared with the first dose, the second oral dose of ART resulted in a decrease in AUC0-t of ART-M (by 39.6%) in three subjects (Fig. 1), with increased CL/F (1.6-fold). The metabolic ratio, Tmax or t1/2 did not change after repeated oral doses.

Fig. 4
figure 4

Mean (+ S.D.) plasma concentration–time profiles of artemisinin (ART) and its metabolite 10β–hydroxyartemisinin (ART-M) in healthy Chinese subjects (n = 6) after a recommended two-day oral dose of Artequick (125 mg/dose of ART plus 750 mg/dose of piperaquine), and individual AUC0-t values

Full size image

In vivo antiplasmodial potency predicted using PK/PD indices

The human plasma protein binding was 74.9% and 68.9% for ART and ART-M, respectively. The therapeutic durations when the free plasma levels of ART and ART-M exceeded their respective MIC50 was 5 h (Table 2). After the first oral dose, the fAUC0-t/MIC50 was 15.4 h for ART and 12.5 h for ART-M. The fCmax of ART was 5.1-fold higher than its MIC50, and fCmax/MIC50 of ART-M was 2.8. The repeated oral dosing led to non-significantly lower fAUC0-t/MIC50 values for both ART (11.2 h) and ART-M (9.8 h).

Table 2 The pharmacokinetic/pharmacodynamic indices for in vivo anti-malarial potency of artemisinin (ART) and its metabolite 10β-hydroxyartemisinin (ART-M)
Full size table

Discussion

In this study, a major hydroxylated metabolite (ART-M) was identified for ART in human blood circulation. The metabolite profiling of ART in human liver microsomal incubates (HLMs) was first performed. The major metabolite of ART was identified as 10β-hydroxyartemisinin (ART-M), the reference of which was prepared by microbial transformation. The enzyme kinetics study of ART in HLMs showed an intrinsic hepatic clearance (CLint,H) of 27.6 μL/min/mg based on the formation of ART-M. The CLint,H determined by ART depletion was 30.3 μL/min/mg, indicating that ART-M was the predominant metabolite for ART in HLMs. Monohydroxylated metabolites have been detected in both HLMs and human plasma using mass spectrometry based on MS fragmentation patterns, without providing exact structures [9]. 10β-Hydroxyartemisinin has been found as a microbial transformation product of ART by Cunninghamella elegans [20]. The subsequent hydroxylation metabolite 9,10-dihydroartemisnin instead of 10-hydroxyartemisinin was identified in human urine [8]. The metabolites formed via loss of oxygen (e.g., deoxyartemisinin) were also found in the present study, and they were not studied in detail due to loss of antiplasmodial activity.

The depletion of ART in HLMs has been attributed to CYP2B6, with a minor role of CYP3A4 and CYP2A6 [15]. The present enzyme phenotyping study showed that the hydroxylation of ART to ART-M (10β-hydroxyartemisinin) was mediated by CYP3A4/3A5, which was supported by the inhibition study and recombinant CYPs incubation. CYP2B6 was found to be involved in the formation of another trace metabolite isomer (M1), which was inhibited completely by 1-ABT (a non-selective CYP inhibitor) or partially by preincubation with ticlopidine (a selective inhibitor of CYP2B6) (not discussed in detail) suggesting that CYP2B6 was involved in M1 formation. The results indicated that various CYP enzymes were involved in formation of metabolite isomers of ARM. Moreover, auto-induction of CYP3A4/3A5 should be more pharmacologically relevant for ART. A more rapid auto-induction of ART metabolism has been observed in children [9, 23], who own higher CYP3A activity than adults. The combination of ART with CYP3A inducers (e.g., rifampin) will probably lead to subtherapeutic concentration of ART and the final malaria treatment failure.

Due to the presence of the unique peroxide bridge essential for the anti-malarial activity [11], the antiplasmodial activity of the hydroxylated metabolite ART-M against Pf3D7 (chloroquine-sensitive) and PfDd2 (chloroquine-resistant) was investigated in this study. The susceptibility of Pf3D7 and PfDd2 to the model drug CQ were determined at 10.6 nM (MIC50) and 93.8 nM (MIC50), respectively, which were in the range of reported data (MIC50, 5.0–40.0 nM for Pf3D7, and MIC50, 62.0–135.4 nM for PfDd2) [24, 25]. ART also showed consistent antiplasmodial activity against two reference Plasmodium strains with literature data (10.0–30.0 nM for Pf3D7 and PfDd2) [26]. The metabolite ART-M was less active against two strains, with higher MIC50 values (61.4 nM vs. 10.1 nM against Pf3D7). As a reduction product of ART, the widely used artemisinin derivative dihydroartemisinin showed a higher antiplasmodial activity (MIC50, ~ 1.0 nM against PfDd2) than ART [27]. These results indicated that a slight modification of structure could lead to alteration of anti-malarial activity. As a metabolite of ART, ART-M may differentiate in its in vivo efficacy due to reabsorption and further metabolism when orally administered to infected rodents. The in vivo anti-malarial activity of ART-M against Plasmodium yoelii or Plasmodium berghei was not investigated in this study. The ring-stage susceptibility of artemisinin-resistant Plasmodium falciparum (especially with PfKelch13 mutation) to ART-M deserves further study.

Previous studies have evaluated the time-dependent pharmacokinetics of the parent drug ART in healthy subjects and malaria patients, after oral administration of ART either as monotherapy or ACTs [6, 7]. Due to unavailability of reference standards, these studies either focused on the parent drug ART or tentatively investigated the relative abundance of ART metabolites using mass spectrometry based on the calibration curve of ART [9]. There was evidence of reduced concentration levels of phase I metabolites of ART with increased phase II metabolites after repeated oral dosing of ART plus piperaquine (Artequick). However, structure modification during metabolism will inevitably vary the MS response and therefore compromise the quantitative data of metabolites. Whether the therapeutic exposure of ART-M can be achieved in humans after the recommended oral dosing of ART was finally investigated in this study. After a recommended two oral doses of ART (125 mg/dose) plus piperaquine to healthy Chinese subjects, the exposure of ART (AUC0-t and Cmax) in the present study was comparable to literature data [9]; however, there was a 4.0-fold difference in ART-M exposure between this study and MS response-based quantitation [9]. After the first oral dose of ART, the achieved free plasma Cmax of ART-M (180.0 nM) was 2.8-fold higher than its MIC50 value (64.1 nM). After repeated oral administration of ART, decreased exposure was found for both ART and ART-M in three out of six subjects, with increased oral clearance of ART (1.6-fold). However, the metabolic capability (calculated by AUCART-M/AUCART) did not change after the second dose. The present results indicated the presence of time-dependent pharmacokinetics for both ART and its metabolite ART-M. Lower concentration levels of ART and its pharmacologically active metabolites may contribute to the risk of recrudescence in some patients. ART monotherapy is not available, and an ACT containing ART and piperaquine was used in the present pharmacokinetic study. Based on the inhibition constant of CYP3A (Ki, 0.68 μM; [28]), Cmax value (0.4 μM; [21]) and plasma protein binding of piperaquine (97.4%; [29]), the value of [I]/Ki for CYP3A by piperaquine is below the cutoff value of 0.1 (a “remote” inhibitor). The effect of piperaquine on ART pharmacokinetics in the ACT (Artequick) was not expected, which has been supported by a previous clinical study [30].

The in vivo therapeutic potency of ART-M was finally predicted using PK/PD indices, including fAUC0-t/MIC50, fCmax/MIC50, and T > MIC50. The MIC50 of 10.1 nM was selected as the apparent MIC value for ART. For comparison, MIC50 of 64.1 nM was considered for deriving the PK/PD indices of ART-M. Equivalent T > MIC50 was found for ART and ART-M. ART showed a ~ 2.0-fold higher fCmax/MIC50 than ART-M. The fAUC0-t/MIC50 of ART-M was comparable to ART, due to its lower plasma protein binding and higher exposure. The PK/PD indices indicated that ART-M retains comparable antiplasmodial potency to ART.

Conclusion

What this study provided is that the major hydroxylated metabolite of ART (10β-hydroxyartemisinin) found in human blood circulation had good potency against P. falciparum (Pf3D7 and PfDd2). Therapeutic concentration levels of the metabolite 10β-hydroxyartemisinin can be achieved under recommended oral dosing of ART. The contribution of the metabolite 10β-hydroxyartemisinin should be considered when evaluation of the clinical outcomes of ART.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

ACT:

Artemisinin-based combination therapy

AUC:

Area under the plasma concentrations-time curve

fAUC:

Area under the free concentration–time curve

ART:

Artemisinin

ART-M:

10β-Hydroxyartemisinin

HLMs:

Human liver microsomes

Cmax :

Maximum plasma concentration

fCmax :

Maximum free plasma concentration

MIC50/MIC90 :

The 50% or 90% growth inhibitory concentration

PK/PD:

Pharmacokinetic/pharmacodynamic

rCYP:

Recombinant cytochrome P450

t1/2 :

Terminal elimination time

T > MIC50 :

Time that free plasma concentration exceeds MIC50

CL/F:

Total oral clearance

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Acknowledgements

We would like to than the volunteers who participated in the study.

Funding

This work was supported by the National Natural Science Foundation of China (No. 82274005 and No. 81773807).

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Author notes

  1. Fanping Zhu and Huixiu Mao have contributed equally to this work.

Authors and Affiliations

  1. School of Pharmaceutical Sciences, Shandong University, Jinan, 250012, China

    Fanping Zhu, Huixiu Mao, Shanshan Du & Jie Xing

  2. School of Environmental Science and Engineering, Shandong University, Jinan, China

    Fanping Zhu

  3. Key Laboratory of Vector Biology and Pathogen Control of Zhejiang Province, Huzhou University, Huzhou, China

    Hongchang Zhou

  4. Qilu Hospital, Shandong University, Jinan, China

    Rui Zhang & Pingli Li

Authors
  1. Fanping Zhu
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  2. Huixiu Mao
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  5. Rui Zhang
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  7. Jie Xing
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Contributions

FPZ and HXM performed the experiments and analysed the data. SSD, HCZ, RZ and PLL helped in performing the experiments. JX designed the experiments, analysed the data, and wrote the paper. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Jie Xing.

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Ethics approval and consent to participate

The study protocol was approved (KYLL-202307–025) by the Ethics Committee of Qilu Hospital (Shandong University, China). The study was conducted in accordance with applicable local law(s) and regulations(s).

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Not applicable.

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The authors declare no competing interests.

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Zhu, F., Mao, H., Du, S. et al. CYP3A4-mediated metabolism of artemisinin to 10β-hydroxyartemisinin with comparable anti-malarial potency. Malar J 23, 328 (2024). https://doi.org/10.1186/s12936-024-05163-y

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  • DOI: https://doi.org/10.1186/s12936-024-05163-y

Keywords

Malaria Journal

ISSN: 1475-2875

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