PBPK-led assessment of antimalarial drugs as candidates for Covid-19: Simulating concentrations at the site of action to inform repurposing strategies.

The urgent need for safe, efficacious, and accessible drug treatments to treat coronavirus disease 2019 (COVID-19) prompted a global effort to evaluate drug repurposing opportunities. Pyronaridine and amodiaquine are both components of approved antimalarials with in vitro activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In vitro activity does not always translate to clinical efficacy across a therapeutic dose range. This study applied available, verified, physiologically based pharmacokinetic (PBPK) models for pyronaridine, amodiaquine, and its active metabolite N-desethylamodiaquine (DEAQ) to predict drug concentrations in lung tissue relative to plasma or blood in the default healthy virtual population. Lung exposures were compared to published data across the reported range of in vitro EC50 values against SARS-CoV-2. In the multicompartment permeability-limited PBPK model, the predicted total Cmax in lung mass for pyronaridine was 34.2 µM on Day 3, 30.5-fold greater than in blood (1.12 µM) and for amodiaquine was 0.530 µM, 8.83-fold greater than in plasma (0.060 µM). In the perfusion-limited PBPK model, the DEAQ predicted total Cmax on Day 3 in lung mass (30.2 µM) was 21.4-fold greater than for plasma (1.41 µM). Based on the available in vitro data, predicted drug concentrations in lung tissue for pyronaridine and DEAQ, but not amodiaquine, appeared sufficient to inhibit SARS-CoV-2 replication. Simulations indicated standard dosing regimens of pyronaridine-artesunate and artesunate-amodiaquine have potential to treat COVID-19. These findings informed repurposing strategies to select the most relevant compounds for clinical investigation in COVID-19. Clinical data for model verification may become available from ongoing clinical studies.

High-throughput quantitation method for amodiaquine and desethylamodiaquine in plasma using supported liquid extraction technology.

Amodiaquine is a drug used for treatment of malaria and is often used in combination with artesunate in areas where malaria parasites are still susceptible to amodiaquine. Liquid chromatography tandem-mass spectrometry was used to quantify amodiaquine and its active metabolite, desethylamodiaquine, in plasma samples. A low sample volume of 100 µl, and high-throughput extraction technique using a supported liquid extraction (SLE+) technique on an automated liquid handler platform for faster sample processing are some of the advantages of this method. Separation of amodiaquine from desethylamodiaquine was achieved using a reversed phase Zorbax SB-CN 50 mm × 4.6 mm, I.D. 3.5 µm column with acetonitrile and 20 mM ammonium formate with 1% formic acid pH ~ 2.6 (15-85, v/v) as mobile phase. The absolute recoveries of amodiaquine and desethylamodiaquine were 66% to 76%, and their isotope label internal standard were in the range of 73% to 85%. Validation results of the developed method demonstrated intra-batch and inter-batch precisions within the acceptance criteria range of ± 15.0%. There were no matrix or carry-over effects observed. The lower limit of quantification was 1.08 ng/ml for amodiaquine and 1.41 ng/ml for desethylamodiaquine. The method showed robust and accurate performance with high sensitivity. Thus, the validated method was successfully implemented and applied in the evaluation of a clinical trial where participants received artemether-lumefantrine plus amodiaquine twice daily for three days (amodiaquine dose of 10 mg base/kg/day).

Pharmacokinetic profile of amodiaquine and its active metabolite desethylamodiaquine in Ghanaian patients with uncomplicated falciparum malaria.

BACKGROUND: Accurate measurement of anti-malarial drug concentrations in therapeutic efficacy studies is essential to distinguish between inadequate drug exposure and anti-malarial drug resistance, and to inform optimal anti-malarial dosing in key target population groups. METHODS: A sensitive and selective LC-MS/MS method was developed and validated for the simultaneous determination of amodiaquine and its active metabolite, desethylamodiaquine, and used to describe their pharmacokinetic parameters in Ghanaian patients with uncomplicated falciparum malaria treated with the fixed-dose combination, artesunate-amodiaquine. RESULTS: The day-28 genotype-adjusted adequate clinical and parasitological response rate in 308 patients studied was > 97% by both intention-to-treat and per-protocol analysis. After excluding 64 patients with quantifiable amodiaquine concentrations pre-treatment and 17 with too few quantifiable concentrations, the pharmacokinetic analysis included 227 patients (9 infants, 127 aged 1-4 years, 91 aged ≥ 5 years). Increased median day-3 amodiaquine concentrations were associated with a lower risk of treatment failure [HR 0.87 (95% CI 0.78-0.98), p = 0.021]. Amodiaquine exposure (median AUC0-∞) was significantly higher in infants (4201 ng h/mL) and children aged 1-5 years (1994 ng h/mL) compared to older children and adults (875 ng h/mL, p = 0.001), even though infants received a lower mg/kg amodiaquine dose (median 25.3 versus 33.8 mg/kg in older patients). Desethylamodiaquine AUC0-∞ was not significantly associated with age. No significant safety concerns were identified. CONCLUSIONS: Efficacy of artesunate-amodiaquine at currently recommended dosage regimens was high across all age groups. Reassuringly, amodiaquine and desethylamodiaquine exposure was not reduced in underweight-for-age young children or those with high parasitaemia, two of the most vulnerable target populations. A larger pharmacokinetic study with close monitoring of safety, including full blood counts and liver function tests, is needed to confirm the higher amodiaquine exposure in infants, understand any safety implications and assess whether dose optimization in this vulnerable, understudied population is needed.

Clinical Probe of Cyp2C8*2 Mutants in a Malaria Hyperendemic Zone: Evidence from North-Central, Nigeria.

BACKGROUND: A tremendous level of success has been achieved since the introduction of chloroquine and the combination of amodiaquine and artemisinin for the treatment of both complicated and uncomplicated malaria infections in sub-Saharan Africa. However, the recent discovery of drug resistant strains of Plasmodium falciparum (P.f.) and the ability of the parasite to ingest CYP2C8 into its digestive vacuole is of great public health concern. This study probes the occurrence of CYP2C8*2 allelic mutant amongst malaria patients in North-Central Nigeria. METHODS: Three hundred and eighty five (385) unrelated study participants were screened for current malaria episodes using routine microscopy and/or rapid diagnostic test strips (RDTs). Chelex extraction method was used for single nucleotide polymorphisms (SNPs) and identification of CYP2C8*2 (805A > T) variant respectively. Wild-type (A) and the defective allele (T) were differentiated with the use of Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP). The results obtained were further validated with Sanger sequencing of a few samples and thereafter, the genotype data were statistically processed. All alleles obtained were in Hardy Weinberg equilibrium. RESULTS: Out of the 385 participants (45.5% Male and 54.5% Female) genotyped for SNPs, 75 (19.5%) had the autosomal recessive mutant trait. Occurrence of mutant traits was gender and ethnic independent (p > 0.05). Yoruba ethnic group recorded a reduction in proportion of genotypic defective CYP2C8*2 allele (T) (1 in every 8 persons) with a carrier percentage of 13.3% compared with Hausa (26.62%); Igbo (25.37%) and other minority ethnic groups (17.6%). CONCLUSIONS: A remarkable inter-ethnic differences in autosomal recessive CYP2C8*2 allele was observed. By implication, there is a gradual incursion of genetic drift for poor CQ and AQ-Artemisinin metabolizers among the inhabitants.

Pharmacokinetics and Ex Vivo Antimalarial Activity of Artesunate-Amodiaquine plus Methylene Blue in Healthy Volunteers.

High rates of artemisinin-based combination therapy (ACT) failures in the treatment of Plasmodium falciparum malaria in Southeast Asia have led to triple-drug strategies to extend the useful life of ACTs. In this study, we determined whether methylene blue [MB; 3,7-bis(dimethylamino)phenothiazin-5-ium chloride hydrate] alters the pharmacokinetics of artesunate-amodiaquine (ASAQ) and enhances the ex vivo antimalarial activity of ASAQ. In an open-label, randomized crossover design, a single oral dose of ASAQ (200 mg AS/540 mg AQ) alone or with MB (325 mg) was administered to 15 healthy Vietnamese volunteers. Serial blood samples were collected up to 28 days after dosing. Pharmacokinetic properties of the drugs were determined by noncompartmental analysis. After drug administration, plasma samples from seven participants were assessed for ex vivo antimalarial activity against the artemisinin-sensitive MRA1239 and the artemisinin-resistant MRA1240 P. falciparum lines, in vitro MB significantly increased the mean area under the curve of the active metabolite of AS, dihydroartemisinin (1,246 ± 473 versus 917 ± 405 ng·h/ml, P = 0.009) but did not alter the pharmacokinetics of AQ, AS, or desethylamodiaquine. Comparing the antimalarial activities of the plasma samples from the participants collected up to 48 h after ASAQ plus MB (ASAQ+MB) and ASAQ dosing against the MRA1239 and MRA1240 lines, MB significantly enhanced the blood schizontocidal activity of ASAQ by 2.0-fold and 1.9-fold, respectively. The ring-stage survival assay also confirmed that MB enhanced the ex vivo antimalarial activity of ASAQ against MRA1240 by 2.9-fold to 3.8-fold, suggesting that the triple-drug combination has the potential to treat artemisinin-resistant malaria and for malaria elimination. (This study has been registered in the Australian New Zealand Clinical Trials Registry [https://anzctr.org.au/] under registration number ACTRN12612001298808.).

Influence of MAMA decoction, an Herbal Antimalarial, on the Pharmacokinetics of Amodiaquine in Mice.

BACKGROUND AND OBJECTIVE: MAMA decoction (MD) is an antimalarial product prepared from the leaves of Mangifera indica L. (Anacardiaceae), Alstonia boonei De Wild (Apocynaceae), Morinda lucida Benth (Rubiaceae) and Azadirachta indica A. Juss (Meliaceae). A previous report showed that MD enhanced the efficacy of amodiaquine (AQ) in malaria-infected mice, thus suggesting a herb-drug interaction. The present study hence evaluated the effect of MD on the disposition of AQ in mice with a view to investigating a possible pharmacokinetic interaction. METHODS: In a 3-period study design, three groups of mice (n = 72) were administered oral doses of AQ (10 mg/kg/day) alone, concurrently with MD (120 mg/kg/day), and in the 3rd period, mice were given AQ after a 3-day pre-treatment with MD. Blood samples were collected between 0 and 96 h for quantification of AQ and its major metabolite, desethylamodiaquine, by a validated high-performance liquid chromatography method. RESULTS: Maximum concentrations of AQ increased by 12% with the concurrent dosing of MD and by 85% in the group of mice pre-treated with MD. The exposure and half-life of desethylamodiaquine increased by approximately 11% and 21%, respectively, with concurrent administration. Corresponding increases of approximately 20% and 33% of desethylamodiaquine were also observed in mice pre-treated with MD. CONCLUSION: MD influenced the pharmacokinetics of AQ and desethylamodiaquine, its major metabolite. The increase in the half-life and systemic exposure of AQ following its co-administration with MD may provide a basis for the enhanced pharmacological effect of the combination in an earlier study in Plasmodium-infected mice.

Adherence and Population Pharmacokinetic Properties of Amodiaquine When Used for Seasonal Malaria Chemoprevention in African Children.

Poor adherence to seasonal malaria chemoprevention (SMC) might affect the protective effectiveness of SMC. Here, we evaluated the population pharmacokinetic properties of amodiaquine and its active metabolite, desethylamodiaquine, in children receiving SMC under directly observed ideal conditions (n = 136), and the adherence of SMC at an implementation phase in children participating in a case-control study to evaluate SMC effectiveness (n = 869). Amodiaquine and desethylamodiaquine concentration-time profiles were described simultaneously by two-compartment and three-compartment disposition models, respectively. The developed methodology to evaluate adherence showed a sensitivity of 65-71% when the first dose of SMC was directly observed and 71-73% when no doses were observed in a routine programmatic setting. Adherence simulations and measured desethylamodiaquine concentrations in the case-control children showed complete adherence (all doses taken) in < 20% of children. This result suggests that more efforts are needed urgently to improve the adherence to SMC among children in this area.

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. The dolutegravir/artemether-lumefantrine interaction was evaluated in a two-way crossover study and measured artemether, dihydroartemisinin, lumefantrine, and desbutyl-lumefantrine over 264 h. The dolutegravir/artesunate-amodiaquine interaction was investigated using a parallel study design due to long half-life of the amodiaquine metabolite, desethylamodiaquine and measured artesunate, amodiaquine, and desethylamodiaquine over 624 h. Noncompartmental analysis was performed, and geometric mean ratios and 90% confidence intervals were generated for evaluation of both interactions. Dolutegravir did not significantly change the maximum concentration in plasma, the time to maximum concentration, and the area under the concentration-time curve (AUC) for artemether, dihydroartemisinin, lumefantrine, and desbutyl-lumefantrine, nor did it significantly alter the AUC for artesunate, dihydroartemisinin, amodiaquine, and desethylamodiaquine. Coadministration of dolutegravir with artemether-lumefantrine resulted in a 37% decrease in DTG trough concentrations. Coadministration of dolutegravir with artesunate-amodiaquine resulted in 42 and 24% approximate decreases in the DTG trough concentrations and the AUC, respectively. The significant decreases in DTG trough concentrations with artemether-lumefantrine and artesunate-amodiaquine and dolutegravir exposure with artesunate-amodiaquine are unlikely to be of clinical significance since the DTG trough concentrations were above dolutegravir target concentrations of 300 ng/ml. Study drugs were well tolerated with no serious adverse events. Standard doses of artemether-lumefantrine and artesunate-amodiaquine should be used in patients receiving dolutegravir. (This study has been registered at ClinicalTrials.gov under identifier NCT02242799.).

Population Pharmacokinetics of the Antimalarial Amodiaquine: a Pooled Analysis To Optimize Dosing.

Amodiaquine plus artesunate is the recommended antimalarial treatment in many countries where malaria is endemic. However, pediatric doses are largely based on a linear extrapolation from adult doses. We pooled data from previously published studies on the pharmacokinetics of amodiaquine, to optimize the dose across all age groups. Adults and children with uncomplicated malaria received daily weight-based doses of amodiaquine or artesunate-amodiaquine over 3 days. Plasma concentration-time profiles for both the parent drug and the metabolite were characterized using nonlinear mixed-effects modeling. Amodiaquine pharmacokinetics were adequately described by a two-compartment disposition model, with first-order elimination leading to the formation of desethylamodiaquine, which was best described by a three-compartment disposition model. Body size and age were the main covariates affecting amodiaquine clearance. After adjusting for the effect of weight, clearance rates for amodiaquine and desethylamodiaquine reached 50% of adult maturation at 2.8 months (95% confidence interval [CI], 1.5 to 3.7 months) and 3.9 months (95% CI, 2.6 to 5.3 months) after birth, assuming that the baby was born at term. Bioavailability was 22.4% (95% CI, 15.6 to 31.9%) lower at the start of treatment than during convalescence, which suggests a malaria disease effect. Neither the drug formulation nor the hemoglobin concentration had an effect on any pharmacokinetic parameters. Results from simulations showed that current manufacturer dosing recommendations resulted in low desethylamodiaquine exposure in patients weighing 8 kg, 15 to 17 kg, 33 to 35 kg, and >62 kg compared to that in a typical 50-kg patient. We propose possible optimized dosing regimens to achieve similar drug exposures among all age groups, which require further validation.

Moringa oleifera leaf powder alters the pharmacokinetics of amodiaquine in healthy human volunteers.

WHAT IS KNOWN AND OBJECTIVE: Moringa oleifera (MO) Lam (Moringaceae) is commonly used as food supplement and as medicine in most African countries where malaria is also endemic. Therefore, co-administration of MO with antimalarials is a possibility. This study investigated the effects of MO leaves powder on the pharmacokinetics of amodiaquine (AQ) in human subjects. METHODS: Twenty healthy volunteers were recruited for the 3-period study. In the first period, a single dose of AQ tablet (10 mg/kg) was administered orally after an overnight fast. After a 7-day washout period, AQ was co-administered with MO. For the third period, each subject took 3 g MO once daily for 7 days and on the 8th day, MO was co-administered with AQ. The plasma concentrations of amodiaquine and desethylamodiaquine (DEAQ) were simultaneously determined using a validated HPLC method. RESULTS AND DISCUSSION: The results showed a significant decrease (P = .037) in the Cmax of AQ after concurrent administration (CA) with MO, whereas after pretreatment (PT), there was a 32% decrease in the Cmax of AQ. For the metabolite, DEAQ, Cmax increased significantly (P = .006) by 79.36%, and Cmax in PT was significantly higher than (P = .001) that of the CA arm of the study. AUC of DEAQ increased significantly by 40.4% (P = .006) and by 188% (P = .001) after CA and PT, respectively. WHAT IS NEW AND CONCLUSION: The study established pharmacokinetic interaction between AQ and MO when given together or following a long period of ingestion of MO. This may have clinical implications for malaria therapy.

Comparison of the Pharmacokinetics and Ex Vivo Antimalarial Activities of Artesunate-Amodiaquine and Artemisinin-Piperaquine in Healthy Volunteers for Preselection Malaria Therapy.

The pharmacokinetics (PK) and ex vivo activity (pharmacodynamics [PD]) of two artemisinin combination therapies (ACTs) (artemisinin-piperaquine [ARN-PPQ] [Artequick®] and artesunate-amodiaquine [ARS-AQ] [Coarsucam™]) in healthy Vietnamese volunteers were compared following 3-day courses of the ACTs for the preselection of the drugs for falciparum malaria therapy. For PK analysis, serial plasma samples were collected from two separate groups of 22 volunteers after ACT administration. Of these volunteers, ex vivo activity was assessed in plasma samples from seven volunteers who received both ACTs. The area under the concentration-time curve (AUC0-∞) was 3.6-fold higher for dihydroartemisinin (active metabolite of ARS) than that for ARN, whereas the AUC0-∞ of desethylamodiaquine (active metabolite of AQ) was 2.0-fold lower than that of PPQ. Based on the 50% inhibitory dilution values of the volunteers' plasma samples collected from 0.25 to 3 hours after the last dose, the ex vivo activity of ARS-AQ was 2.9- to 16.2-fold more potent than that of ARN-PPQ against the drug-sensitive D6 Plasmodium falciparum line. In addition, at 1.5, 4.0, and 24 hours after the last dose, the ex vivo activity of ARS-AQ was 20.8-, 3.5-, and 8.5-fold more potent than that of ARN-PPQ against the ARN-sensitive MRA1239 line. By contrast, at 1.5 hours, the ex vivo activity of ARS-AQ was 5.4-fold more active than that of ARN-PPQ but had similar activities at 4 and 24 hours against the ARN-resistant MRA1240 line. The PK-PD data suggest that ARS-AQ possesses superior antimalarial activity than that of ARN-PPQ and would be the preferred ACT for further in vivo efficacy testing in multidrug-resistant falciparum malaria areas.

Pharmacokinetics and Safety Profile of Artesunate-Amodiaquine Coadministered with Antiretroviral Therapy in Malaria-Uninfected HIV-Positive Malawian Adults.

There are limited data on the pharmacokinetic and safety profiles of artesunate-amodiaquine in human immnunodeficiency virus-infected (HIV+) individuals receiving antiretroviral therapy. In a two-step intensive sampling pharmacokinetic trial, we compared the area under the concentration-time curve from 0 to 28 days (AUC0-28) of an active metabolite of amodiaquine, desethylamodiaquine, and treatment-emergent adverse events between antiretroviral therapy-naive HIV+ adults and those taking nevirapine and ritonavir-boosted lopinavir-based antiretroviral therapy. In step 1, malaria-uninfected adults (n = 6/arm) received half the standard adult treatment regimen of artesunate-amodiaquine. In step 2, another cohort (n = 25/arm) received the full regimen. In step 1, there were no safety signals or significant differences in desethylamodiaquine AUC0-28 among participants in the ritonavir-boosted lopinavir, nevirapine, and antiretroviral therapy-naive arms. In step 2, compared with those in the antiretroviral therapy-naive arm, participants in the ritonavir-boosted lopinavir arm had 51% lower desethylamodiaquine AUC0-28, with the following geometric means (95% confidence intervals [CIs]): 23,822 (17,458 to 32,506) versus 48,617 (40,787 to 57,950) ng · h/ml (P < 0.001). No significant differences in AUC0-28 were observed between nevirapine and antiretroviral therapy-naive arms. Treatment-emergent transaminitis was higher in the nevirapine (20% [5/25]) than the antiretroviral therapy-naive (0.0% [0/25]) arm (risk difference, 20% [95% CI, 4.3 to 35.7]; P = 0.018). The ritonavir-boosted lopinavir antiretroviral regimen was associated with reduced desethylamodiaquine exposure, which may compromise artesunate-amodiaquine's efficacy. Coadministration of nevirapine and artesunate-amodiaquine may be associated with hepatoxicity.

Assessment of formulated amodiaquine microparticles in Leishmania donovani infected rats.

The aim of this study was to formulate, characterise and evaluate the activity of amodiaquine microparticles against Leishmania donovani. Microparticles were formulated by encapsulating the drug in bovine serum albumin using the spray-dryer method. The microparticles were evaluated for size, zeta potential, drug content, encapsulation efficiency and in vitro release profile. The size range of the microparticles formulated was between 1.9 and 10 µm with an average zeta potential of -25.5 mV. Of the expected 20% drug loading, an average of 18.27% was obtained giving an encapsulation efficiency of 91.35%. Pharmacokinetic profile of amodiaquine improved with microencapsulation of the drug with Cmax, AUC0→48 and t1//2 all significantly higher than amodiaquine solution. Amodiaquine microparticles showed an overall higher bioavailability and hence were more effective in eliminating intra-tissue parasites than the drug solution. It would therefore be expected that the formulated microparticles will be more effective in treating visceral leishmaniasis.

Characterization of human cytochrome P450 mediated bioactivation of amodiaquine and its major metabolite N-desethylamodiaquine.

AIMS: Oxidative bioactivation of amodiaquine (AQ) by cytochrome P450s to a reactive quinoneimine is considered as an important mechanism underlying its idiosyncratic hepatotoxicity. However, because internal exposure to its major metabolite N-desethylamodiaquine (DEAQ) is up to 240-fold higher than AQ, bioactivation of DEAQ might significantly contribute to covalent binding. The aim of the present study was to compare the kinetics of bioactivation of AQ and DEAQ by human liver microsomes (HLM) and to characterize the CYPs involved in bioactivation of AQ and DEAQ. METHODS: Glutathione was used to trap reactive metabolites formed in incubations of AQ and DEAQ with HLM and recombinant human cytochrome P450s (hCYPs). Kinetics of bioactivation of AQ and DEAQ in HLM and involvement of hCYPs were characterized by measuring corresponding glutathione conjugates (AQ-SG and DEAQ-SG) using a high-performance liquid chromatography method. RESULTS: Bioactivation of AQ and DEAQ in HLM both exhibited Michaelis-Menten kinetics. For AQ bioactivation, enzyme kinetical parameters were Km , 11.5 ± 2.0 µmol l-1 , Vmax , 59.2 ± 3.2 pmol min-1  mg-1 and CLint , 5.15 µl min-1  mg-1 . For DEAQ, parameters for bioactivation were Km , 6.1 ± 1.3 µmol l-1 , Vmax , 5.5 ± 0.4 pmol min-1  mg-1 and CLint 0.90 µl min-1  mg-1 . Recombinant hCYPs and inhibition studies with HLM showed involvement of CYP3A4, CYP2C8, CYP2C9 and CYP2D6 in bioactivation. CONCLUSIONS: The major metabolite DEAQ is likely to be quantitatively more important than AQ with respect to hepatic exposure to reactive metabolites in vivo. High expression of CYP3A4, CYP2C8, CYP2C9, and CYP2D6 may be risk factors for hepatotoxicity caused by AQ-therapy.

Application of an LC-MS/MS method for reliable determination of amodiaquine, N-desethylamodiaquine, artesunate and dihydroartemisinin in human plasma for a bioequivalence study in healthy Indian subjects.

A sensitive and high throughput bioanalytical method has been developed for reliable determination of amodiaquine (AQ), N-desethylamodiaquine (DEAQ), artesunate (AS) and dihydroartemisinin (DHA) in human plasma by LC-MS/MS. The method employs a solid phase extraction procedure without an evaporation step and with optimum use of organic solvents to circumvent degradation of artemisinin derivatives. The analytes and their deuterated internal standards (ISs) were analyzed on Hypersil Gold (100 mm × 4.6mm, 5 µm) column using acetonitrile and 2.0mM ammonium formate (pH 2.50) in 80:20 (v/v) ratio as the mobile phase. A triple quadrupole mass spectrometer equipped with an electrospray ionization interface was used to detect and quantify the analytes. The method was established over the concentration range of 0.250-30.0 ng/mL, 1.50-180 ng/mL, 2.00-600 ng/mL and 5.00-1400 ng/mL for AQ, DEAQ, AS and DHA respectively using 250 µL human plasma. The intra-day and inter-day accuracy and precision (% CV) across quality controls varied from 93.3-105.0% and 1.7-8.3 respectively for all the analytes. The stability was assessed in whole blood as well as in plasma samples under different conditions. All four analytes were stable in whole blood up to 2h on melting ice. The long term stability in plasma was ascertained up to 90 days. IS-normalized matrix factors ranged from 0.988-1.023 for all the analytes. The method was successfully applied to a bioequivalence study using 50mg artesunate and 135 mg amodiaquine fixed dose formulation in 14 healthy subjects.

Functional characterization of 12 allelic variants of CYP2C8 by assessment of paclitaxel 6α-hydroxylation and amodiaquine N-deethylation.

Cytochrome P450 2C8 (CYP2C8) is one of the enzymes primarily responsible for the metabolism of many drugs, including paclitaxel and amodiaquine. CYP2C8 genetic variants contribute to interindividual variations in the therapeutic efficacy and toxicity of paclitaxel. Although it is difficult to investigate the enzymatic function of most CYP2C8 variants in vivo, this can be investigated in vitro using recombinant CYP2C8 protein variants. The present study used paclitaxel to evaluate 6α-hydroxylase activity and amodiaquine for the N-deethylase activity of wild-type and 11 CYP2C8 variants resulting in amino acid substitutions in vitro. The wild-type and variant CYP2C8 proteins were heterologously expressed in COS-7 cells. Paclitaxel 6α-hydroxylation and amodiaquine N-deethylation activities were determined by measuring the concentrations of 6α-hydroxypaclitaxel and N-desethylamodiaquine, respectively, and the kinetic parameters were calculated. Compared to the wild-type enzyme (CYP2C8.1), CYP2C8.11 and CYP2C8.14 showed little or no activity with either substrate. In addition, the intrinsic clearance values of CYP2C8.8 and CYP2C8.13 for paclitaxel were 68% and 67% that of CYP2C8.1, respectively. In contrast, the CLint values of CYP2C8.2 and CYP2C8.12 were 1.4 and 1.9 times higher than that of CYP2C8.1. These comprehensive findings could inform for further genotype-phenotype studies on interindividual differences in CYP2C8-mediated drug metabolism.

Role of cytochrome P450 2C8*3 (CYP2C8*3) in paclitaxel metabolism and paclitaxel-induced neurotoxicity.

AIM: The CYP2C8*3 allele has been suggested as a risk factor for paclitaxel-induced neuropathy but the data hitherto published are conflicting. MATERIALS & METHODS: In total 435 patients were investigated with respect to maximum neuropathy grade and accumulated paclitaxel dose. The enzymatic properties of CYP2C8.3 variant were analyzed using heterologous mammalian HEK293 cell expression system. RESULTS: No significant association between CYP2C8*3 allele and neuropathy was found, although a trend was observed. The paclitaxel and amodiaquine metabolism by CYP2C8.3 were found similar to CYP2C8.1, whereas CYP2C8.3 was more efficient in the metabolism of rosiglitazone. CONCLUSION: These results indicate a difference in substrate specificity between CYP2C8.1 and CYP2C8.3; however, the CYP2C8*3 allele has no major impact on paclitaxel metabolism in vitro or of paclitaxel-induced neuropathy in vivo. Original submitted on 6 February 2015; revision submitted on 9 April 2015.

Validation and pharmacokinetic application of a high-performance liquid chromatographic technique for determining the concentrations of amodiaquine and its metabolite in plasma of patients treated with oral fixed-dose amodiaquine-artesunate combination in areas of malaria endemicity.

Artemisinin-based combination therapies (ACTs) have been adopted by most African countries, including Nigeria, as first-line treatments for uncomplicated falciparum malaria. Fixed-dose combinations of these ACTs, amodiaquine-artesunate (FDC AQAS) and artemether-lumefantrine (AL), were introduced in Nigeria to improve compliance and achieve positive outcomes of malaria treatment. In order to achieve clinical success with AQAS, we developed and validated a simple and sensitive high-performance liquid chromatography (HPLC) method with UV detection for determination of amodiaquine (AQ) and desethylamodiaquine (DAQ) in plasma using liquid-liquid extraction of the drugs with diethyl ether following protein precipitation with acetonitrile. Chromatographic separation was achieved using an Agilent Zorbax C18 column and a mobile phase consisting of distilled water-methanol (80:20 [vol/vol]) with 2% (vol/vol) triethylamine, pH 2.2, at a flow rate of 1 ml/min. Calibration curves in spiked plasma were linear from 100 to 1,000 ng/ml (r > 0.99) for both AQ and DAQ. The limit of detection was 1 ng (sample size, 20 µl). The intra- and interday coefficients of variation at 150, 300, and 900 ng/ml ranged from 1.3 to 4.8%, and the biases were between 6.4 and 9.5%. The mean extraction recoveries of AQ and DAQ were 80.0% and 68.9%, respectively. The results for the pharmacokinetic parameters of DAQ following oral administration of FDC AQAS (612/200 mg) for 3 days in female and male patients with uncomplicated falciparum malaria showed that the maximum plasma concentrations (C max) (740 ± 197 versus 767 ± 185 ng/ml), areas under the plasma concentration-time curve (AUC) (185,080 ± 20,813 versus 184,940 ± 16,370 h · ng/ml), and elimination half-life values (T 1/2) (212 ± 1.14 versus 214 ± 0.84 h) were similar (P > 0.05).

Fixed dose artesunate amodiaquine - a phase IIb, randomized comparative trial with non-fixed artesunate amodiaquine.

BACKGROUND: Pharmacokinetic (PK) and pharmacodynamic (PD) data are limited for artesunate (AS) and amodiaquine (AQ) in uncomplicated Plasmodium falciparum. METHODS: From 2007-8, 54 P. falciparum-infected, Kenyan adults were assigned randomly fixed dose (FD) ASAQ (n = 26) or non-fixed (NF) ASAQ (n = 28). Total doses were 600 mg AS (both arms) + 1,620 mg (FD) or 1,836 mg (NF)AQ. Follow-up extended over 28 days. PK data were collected for AS, dihydroartemisinin (DHA), AS + DHA combined as DHA equivalents (DHAeq), AQ, desethylamodiaquine (DAQ),and their relationships assessed against the PD collected data consisting of parasitological efficacy, adverse events (AEs), and the Bazett's corrected QTinterval (QTcB). RESULTS: Mean AUC 0-72 of dihydroartemisinin equivalents (DHAeq) when administered as a fixed dose (FD) compared to NF dose were similar: 24.2 ±4.6 vs 26.4±6.9 µmol*h/L (p = 0.68) Parasite clearance rates were also similar after 24 hrs: 17/25 (68%) vs 18/28(64.3%) (p = 0.86),as well as at 48 hrs: 25/8 (100%)vs 26 (92.9%)/28 (p = 0.49). Mean FD vs NF DAQ AUC0-28 were 27.6±3.19 vs 32.7±5.53 mg*h/L (p = 0.0005). Two PCR-proven new infections occurred on Day (D) 28 for estimated, in vivo, DAQ minimum inhibitory concentrations of 15.2 and 27.5 ng/mL. Combining the FD and NF arms, the mean QTcB at D2+4 hrs increased significantly (p = 0.0059) vs baseline: 420 vs410 ms (∆ = 9.02 (95% confidence interval 2.72-15.31 ms), explained by falling heart rates, increasing DAQ concentrations and female sex in a general linear mixed effects model. Ten of 108 (9.26%) AEs (5/arm) reported by 37/54 (68.5%) patients were possibly or probably drug related. Severe, asymptomatic neutropaenia developed in 2/47 (4.25%) patients on D28: 574/µL (vsD0: 5,075/µL), and 777/µL (vsD0: 3,778/µL). CONCLUSIONS: Tolerability of both formulations was good. For QTcB, a parameter for ECG modifications, increases were modest and due to rising DAQ concentrations and falling heart rates as malaria resolved. Rapid parasite clearance rates and no resistant infections suggest effective pharmacokinetics of both formulations.

A replicate designed bioequivalence study to compare two fixed-dose combination products of artesunate and amodiaquine in healthy chinese volunteers.

Artesun-Plus is a fixed-dose combination antimalarial agent containing artesunate and amodiaquine. The current study was conducted to compare the pharmacokinetic and safety profiles of Artesun-Plus and the WHO-designated comparator product Artesunate Amodiaquine Winthrop. To overcome the high intrasubject variability of artesunate, the study applied a two-sequence and four-period crossover (2 by 4), replicate study design to assess bioequivalence between the two products in 31 healthy male Chinese volunteers under fasting conditions. The results showed that the values of the geometric mean ratios of maximum concentration of drug in plasma (Cmax) and area under the concentration-time curve from time zero to the last blood sample collection (AUC0-last) for the artesunate component in the test and reference products were 95.9% and 93.9%, respectively, and that the corresponding 90% confidence intervals were 84.5% to 108.7% and 87.2% to 101.1%, while the geometric mean ratios for the amodiaquine component in the test and reference products were 95.0% and 100.0%, respectively, and the corresponding 90% confidence intervals were 86.7% to 104.1% and 93.5% to 107.0%. In conclusion, bioequivalence between the two artesunate and amodiaquine fixed-dose combination products was demonstrated for both components. The study also confirmed high intrasubject variability, especially for artesunate: the coefficients of variation (CV) of Cmax values for the test and reference products were 39.2% and 43.7%, respectively, while those for amodiaquine were 30.6% and 30.2%, respectively.