Evaluation of the safety and relative bioavailability of a new dihydroartemisinin tablet formulation in healthy Thai volunteers

Transactions of the Royal Society of Tropical Medicine and Hygiene (2007) 101, 972—979

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Evaluation of the safety and relative bioavailability of a new dihydroartemisinin tablet formulation in healthy Thai volunteers Supornchai Kongpatanakul a,∗, Somruedee Chatsiricharoenkul a, Korbtham Sathirakul b, Yupin Suputtamongkol c, Suvajana Atipas d, Suchat Watnasirichaikul e, Piyapat Pongnarin a, Polkit Sangvanich f a

Department of Pharmacology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand Department of Pharmacy, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand c Department of Medicine, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand d Department of Oto-Rhino-Laryngology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand e Research and Development Institute, Government Pharmaceutical Organization, Bangkok, Thailand f Research Centre for Bioorganic Chemistry, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand b

Received 22 August 2006; received in revised form 24 May 2007; accepted 24 May 2007 Available online 6 August 2007

KEYWORDS Dihydroartemisinin; Bioavailability; Drug toxicity; Drug formulation; Pharmacokinetics; Malaria

∗

Summary A new dihydroartemisinin (DHA) tablet formulation has been developed by the Thai Government Pharmaceutical Organization (GPO). In this report, its in vitro dissolution and in vivo pharmacokinetics as well as its safety in healthy volunteers were evaluated, using the DHA tablet made by Dafra Pharma NV as a reference. A two-period crossover clinical study design was utilised. Twenty-four volunteers were randomly allocated to two sequences (12 volunteers in each) to receive a 200 mg single oral dose of either the GPO or Dafra formulation with a wash-out period of 5—7 days. In vitro, the GPO formulation dissolved more readily. In vivo, the GPO formulation had a higher maximum plasma concentration and approximately 149% (90% CI 125—179%) greater bioavailability. Both formulations were well tolerated. Interestingly, significant decreases in haemoglobin and haematocrit values (P < 0.001) were noted following administration of one dose of DHA (decrease of 0.73 g/dl haemoglobin and 2.0% haematocrit compared with baseline) or two doses of DHA (decrease of 0.95 g/dl haemoglobin and 3.3% haematocrit compared with baseline). The second dose was associated with additional toxicity compared with one dose with regard to haematocrit (P < 0.001) but not haemoglobin. This finding

Corresponding author. Tel.: +66 2 412 9786, +66 2 419 7565; fax: +66 2 411 5026. E-mail addresses: [email protected], [email protected] (S. Kongpatanakul).

0035-9203/$ — see front matter © 2007 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.trstmh.2007.05.010

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warrants further investigation, since the drug will be used for the treatment of malaria in which anaemia is a consequence. © 2007 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

1. Introduction

2. Materials and methods

Malaria is one of the most important tropical diseases and is a leading cause of mortality and morbidity in developing areas of the world. As resistance to available drugs increases, new drugs for treatment are needed (Hyde, 2005; White, 2004; Yeung et al., 2004). Artemisinin, a peroxide-containing sesquiterpene lactone isolated from the plant Artemisia annua, has proven to be a promising antimalarial drug (Haynes, 2001; Woodrow et al., 2005). However, its effectiveness is impaired by: (1) its short plasma half-life; (2) its low solubility in water or oil; (3) the requirement for a high dosage with oral administration; and (4) a high rate of recrudescence when used as monotherapy. Several artemisinin derivatives have been investigated to increase oil or water solubility as well as efficacy. Dihydroartemisinin (DHA), a derivative of artemisinin with the C-10 lactone group replaced by hemiacetal, is found to be a more potent antimalarial in vitro than artemisinin (Klayman, 1985). DHA is the main active metabolite of a number of artemisinin derivatives (O’Neill and Posner, 2004). Based on the observation that it is well absorbed by oral administration, development of oral DHA for clinical use has been carried out. The first clinical trial of DHA for oral treatment of acute uncomplicated falciparum malaria conducted outside China showed high efficacy, with a cure rate of 90% in 49 patients treated for 7 days (Looareesuwan et al., 1996). The pharmacokinetics of various oral formulations of DHA in healthy volunteers has been well documented in previous studies (Binh et al., 2001; Hung et al., 1999; Kongthaisong et al., 2004; Na-Bangchang et al., 1997, 2004). Recently, a new DHA tablet formulation has been developed by the Thai Government Pharmaceutical Organization (GPO) for use in the National Malaria Control Programme. In the current study, as a part of the development and for registration purposes, we compared this new DHA formulation with reference DHA provided by Dafra Pharma NV. In vitro dissolution profiles of the two formulations were compared. A two-period crossover clinical trial was conducted in healthy volunteers to evaluate the safety, pharmacokinetic profile and relative bioavailability of the two DHA formulations. Recent work has demonstrated that treatment of uncomplicated malaria with an artemether/lumefantrine combination may be associated with hearing loss (Toovey and Jamieson, 2004). However, this neurotoxic effect is still controversial owing to lack of evidence in other studies (Hutagalung et al., 2006; McCall et al., 2006; Van Vugt et al., 2000). Thus, hearing loss as a consequence of DHA neurotoxicity was also investigated in this study.

2.1. Study drugs The study drug was a 100 mg DHA tablet for oral ingestion (two × 100 mg tablets per dose), newly formulated by the GPO (Bangkok, Thailand). The reference DHA was Dynamax (two × 100 mg artenimol tablets per dose; Dafra Pharma NV, Turnhout, Belgium). Dafra Pharma and artenimol were formerly known as Arenco Pharmaceutica and dihydroartemisinin, respectively.

2.2. In vitro dissolution study Dissolution profiles of the two DHA formulations were compared. Briefly, the tablet was dissolved in 0.5% sodium lauryl sulfate (SLS) dissolution medium (one × 100 mg GPO or Dafra tablet per 900 ml) using a paddle-type USP apparatus II (SRII 8-Flask Dissolution Test Station; Hanson Research Corp., Chatsworth, CA, USA). Twelve tablets were used in one series of experiments. At 0, 5, 10, 15, 30, 45 and 60 min, a small amount of medium was removed, filtered through a 10 ␮m filter and then 20 ␮l of the filtrate was assayed by HPLC for the amount of DHA dissolved (detailed below). Dissolution profiles of the two DHA formulations were compared using a model independent approach (US Food and Drug Administration, 1997). Difference (f1) and similarity (f2) factors were calculated. Acceptable f1 and f2 ranges for dissolution were 0—15 and 50—100, respectively. The HPLC determination for DHA was developed inhouse at GPO’s Research and Development Laboratory. The HPLC system consisted of a Spectra SYSTEM P1000 pump, AS3000 autosampler, UV6000 detector and PC1000 Data Station (Thermo Separation Products, Riviera Beach, FL, USA) and Luna C-18 column (250 mm × 4.6 mm, 5 ␮m particle; Phenomenex, Torrance, CA, USA). The mobile phase was a mixture of HPLC-grade acetonitrile and 0.01 M monobasic potassium phosphate buffer (55:45, v/v, pH 5.6). The injection volume was 20 ␮l. The assay was run at 2000—2500 psi at a flow rate of 1.5 ml/min. The retention times for ␣DHA and ␤-DHA were 5 min and 7 min, respectively, with a total run time of 12 min. The amount of DHA was calculated by comparing the twin-peak area (␣-DHA and ␤-DHA) on the chromatograph of the sample with that of a standard DHA preparation measured at a wavelength of 216 nm. The stock of standard DHA for HPLC was GPO DHA in a mixture of acetonitrile and 0.05 M potassium phosphate monobasic (65:35, v/v) at a concentration of 1.5 mg/ml. For construction of a calibration curve, the stock DHA was further diluted to concentrations of 0.024, 0.045, 0.060, 0.090, 0.120 and 0.150 mg/ml using 0.5% SLS solution. The assay was linear over the range 0.024—0.150 mg/ml (r = 0.9996).

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2.3. Clinical study design The study was a single randomised, two-period crossover trial in 24 healthy Thai volunteers. Based on an intrasubject variability of 20%, ␣ of 0.05 and ␤ of 0.2, the sample size of 24 was adequate for this crossover bioequivalence study (US Food and Drug Administration, 2003). The DHA tablets from GPO or Dafra were given as a single 200 mg dose (two × 100 mg tablets) with 250 ml of water on an empty stomach following an overnight fast. The dosage of 200 mg was used in accordance with WHO guidelines for artesunate (4 mg/kg body weight given once a day for 3 days). The volunteers were randomised into two study groups (12 volunteers in each) to receive either the GPO or Dafra formulation for the first period. A wash-out period of 5—7 days was allowed before the second drug administration.

2.4. Subjects Twenty-four healthy Thai volunteers (5 male and 19 female), aged between 19 years and 33 years, weighing 37.5—68.2 kg and with a body mass index of 18—24, participated in this study. Inclusion criteria included: male or female (nonlactating, non-pregnant); normal findings on history; and normal physical and laboratory examination, including normal liver and renal function tests. The volunteers had no history of antimalarial drug ingestion in the preceding 3 months, no other medications ingested in the preceding week and no history of hearing loss and/or disequilibrium during the preceding 6 weeks. Written informed consent for participation was obtained from all volunteers prior to initiation of the study.

2.5. Safety assessment Physical examination was performed at enrolment, on the day of each drug administration and at completion of the study (5—7 days after the second drug). Blood and urine samples were collected at enrolment (2—3 days before the first drug), 5—7 days after administration of the first drug (shortly before administration of the second drug) and 5—7 days after administration of the second drug. Clinical laboratory tests for blood included complete blood count, glucose, urea nitrogen, creatinine, sodium, potassium, chloride, bicarbonate, total bilirubin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, albumin and total protein. Routine urinalysis was performed. Volunteers were monitored for 8 h following each drug administration for any adverse events (AE) and were followed up until all abnormal laboratory values, if any, returned to normal.

2.6. Audiometry and posturography tests Air conduction audiometry was performed at enrolment, at 6 h and 5—7 days following each drug administration using a clinical audiometer (Madsen orbiter 922, version 2; Madsen Electronics, Taastrup, Denmark). The hearing threshold was measured for both ears at the following frequencies: 250 Hz, 500 Hz and 1, 2, 3, 4, 6 and 8 kHz. Volunteers with hearing levels more than 25 dB were considered to have hearing loss.

S. Kongpatanakul et al. Hearing loss was regarded as significant if the level changed from baseline by more than 10 dB. Vestibular function was evaluated by posturography at the same time as audiometry assessment using a Tetrax Interactive Balance System (Tetrax Ltd., Ramat Gan, Israel) (Kohen-Raz, 1991). The system is based on measurement and computerised elaboration of electronic signals emitted by four independent platforms, one for each heel and toe, respectively. General stability, synchronisations between heel and toe pressure patterns, Flourier transformations of postural sway, weight distribution and patterns of postural responses on eight standard positions were assessed. The result of the entire examination was analysed by the system software and reported according to the performance to the norm. The standard scores were reported in four categories: absent pathology; mild impairment; moderate impairment; and severe impairment.

2.7. Blood samples for pharmacokinetic study Blood samples were collected at 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6 and 8 h following each drug administration via an indwelling venous catheter. Plasma samples were separated immediately after blood collection and stored frozen at −70 ◦ C until analysis.

2.8. Determination of DHA level by liquid chromatography—tandem mass spectrometry (LC-MS/MS) The concentration of DHA in plasma was determined by LC-MS/MS. Briefly, 200 ␮l of plasma was transferred to a polypropylene microtube. Then, 50 ␮l of saturated sodium chloride solution and 1 ml of 1-chlrobutane-isooctane (55:45, v/v) were added. The mixture was mixed by vortexing for 5 min and then centrifuged at 12 418 × g for 15 min. The organic layer (0.8 ml) was transferred into a new conical polypropylene tube and the solvent was evaporated to dryness under a nitrogen stream. The residue was reconstituted with 100 ␮l of ethanol/water (50:50, v/v) and 20 ␮l was injected onto the LC-MS/MS system. The LC-MS/MS system consisted of a Waters Separations Module 2795 Xe HPLC system (Waters Corp., Milford, MA, USA) and a Micromass Quattro micro-tandem mass spectrometer, triple quadrupole (Micromass UK Ltd., Manchester, UK). The analytical column (Luna C-18 column, 150 × 2 mm, 3 ␮m material) was fitted with a guard column of the same packing material. The run time was 15 min and the flow rate was 0.2 ml/min. The positive electrospray ionisation source was operated using N2 as a nebuliser gas and desolvation gas, the flow rates were 20 l/h and 550 l/h and the desolvation and source temperatures were 300 ◦ C and 120 ◦ C. The mass analyser was operated in multiple reaction mode. Data acquisition and analysis were carried out using Masslynx version 3.1 software (Waters Corp.). Blank plasma spiked with the standard GPO DHA and artemisinin (Sigma, St Louis, MO, USA) internal standard was used to construct a calibration curve. The curve was linear over the concentration range 0.5—750 ng/ml DHA. Recovery rates for 10, 300 and 600 ng/ml DHA were 81%, 86% and 87%, respectively. The within-day and between-day accuracies at 10,

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300 and 600 ng/ml were within the acceptable limits of ±20% at the lower limit of quantitation of 0.5 ng/ml and ±15% at all other concentrations. The overall coefficient of variation (precision) was below 13.8%.

2.9. Pharmacokinetic analysis Pharmacokinetic calculations were performed using noncompartmental analysis of plasma concentration—time data. The time at which the maximum concentration occurred (tmax ) and the maximum plasma concentration (Cmax ) were obtained directly from the plasma concentration—time data. The terminal slope (␭z ) was calculated from log-linear regression of at least three of the last plasma concentration—time data. The elimination half-life (T1/2z ) was calculated as 0.693/␭z . The area under the curve from time zero to the last observable plasma concentration time (AUC0—tlast ) was calculated using the linear trapezoidal rule for ascending data points up to tmax and the logarithmic trapezoidal after this point.

2.10. Statistical analysis The percent relative bioavailability was calculated for each individual by dividing AUC[test]0—last by AUC[ref.]0—last (test = GPO DHA; ref. = Dafra DHA). The 90% CI for the ratio of the population geometric means (test/ref.) of the parameters were calculated according to the US Food and Drug Administration (FDA) guidelines (US Food and Drug Administration, 2003). For AUC and Cmax , the data were logarithmically transformed and analysed by the mixed-effect ANOVA model. Safety data were analysed using the computer package SPSS for Windows (SPSS Inc., Chicago, IL, USA). Group mean comparison was determined by Student’s t-test. Differences in the number of events occurring in study groups were analysed by McNemar’s statistic. A two-tailed P-value <0.05 was considered statistically significant.

3. Results 3.1. In vitro dissolution study The dissolution profiles of the two DHA tablet formulations are shown in Figure 1. The difference (f1) and similarity (f2) factors were calculated to be 26.17 and 35.87, respectively. Both numbers lay outside the limits recommended by the FDA (US Food and Drug Administration, 1997), indicating differences in dissolution. The GPO formulation dissolved more readily.

3.2. Volunteer profile Twenty-four healthy volunteers participated in this twoperiod crossover study. Volunteers were randomly allocated into two groups to receive either GPO DHA (n = 12) or Dafra DHA (n = 12) as the first study drug. A wash-out period of 5—7 days was allowed before administration of the second drug. The demographics, mean age, mean body weight and mean body mass index were similar in the two groups. The sex

Figure 1 In vitro dissolution profile of two dihydroartemisinin tablet formulations, from the Thai Government Pharmaceutical Organization (GPO) and Dafra Pharma NV. Twelve tablets were used for each series of experiments. Data are mean ± SD.

distribution was similar, although females were more predominant in both study groups (male:female ratios 3:9 and 2:10). Baseline laboratory results were within normal limits in all volunteers.

3.3. Safety study: laboratory data Both study drugs were given as a single 200 mg dose; the mean actual dose by weight was 3.9 mg/kg (95% CI 3.7—4.2 mg/kg). It should be noted that the wash-out period of 5—7 days was chosen on pharmacokinetic grounds; however, the toxicity effects might have still been present after 5—7 days, thus additive toxicity was possible. With this caution in mind, analyses of laboratory data were performed separately on two groups of volunteers: (1) the GPO—Dafra group (first drug = GPO and second drug = Dafra); and (2) the Dafra—GPO group (first drug = Dafra and second drug = GPO). Laboratory results comparing the values after each drug administration and their baseline values obtained at screening are shown in Table 1. It should be noted that one volunteer who received the Dafra formulation as the first drug developed mild erythematous rash on the face approximately 20 h to 6 days after drug administration, possibly due to drug allergy. This volunteer was discontinued from the study, leaving 11 volunteers in the Dafra—GPO group for further analysis. The results in Table 1 indicate some changes in laboratory values in both study groups. Mean haemoglobin and haematocrit significantly decreased from baseline in both groups after the first and second drug. Analysis of these groups in terms of haemoglobin showed no significant additive effect of two doses. Mean haematocrit after the second dose was significantly different from that after the first drug in both study groups. Additional significant changes were also detected for blood urea nitrogen, alkaline phosphatase and total protein for the GPO—Dafra group, and for alkaline

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Table 1

Laboratory data GPO—Dafra group (n = 12)

Haematologya White blood cells (×103 /␮l) Absolute neutrophils (×103 /␮l) Haemoglobin (g/dl)

Dafra—GPO group (n = 11)

Baseline

After GPO DHA

After Dafra DHA

Baseline

After Dafra DHA

After GPO DHA

6.2 (5.1—7.4) 3.1 (2.2—3.9) 12.9 (12.1—13.7)

6.8 (6.1—7.6) 3.4 (2.9—4.0) 12.2 (11.5—12.9) (P = 0.002c ) 38.4 (36.4—40.4) (P = 0.01c )

6.0 (5.4—6.7) 2.8 (2.4—3.3) 12.1 (11.2—13.0) (P = 0.007c ) 36.8 (34.3—39.3) (P < 0.001c ; P = 0.01d ) 284 (262—306)

6.5 (5.5—7.5) 3.7 (2.7—4.6) 13.0 (12.0—14.0)

7.4 (6.6—8.2) 4.3 (3.4—5.2) 12.3 (11.2—13.3) (P = 0.003c ) 37.7 (34.8—40.5) (P < 0.001c )

Haematocrit (%)

39.8 (37.2—42.4)

Platelets (×103 /␮l)

278 (250—307)

293 (267—318)

Blood biochemistrya Glucose (mg/dl) Urea nitrogen (mg/dl)

85 (82—89) 9.8 (8.2—11.5)

Creatinine (mg/dl) Sodium (mmol/l) Potassium (mmol/l) Chloride (mmol/l) Bicarbonate (mmol/l) Total bilirubin (mg/dl) AST (U/l) ALT (U/l) Alkaline phosphatase (U/l)

0.63 (0.51—0.74) 141 (139—142) 4.18 (4.00—4.35) 106 (105—107) 29.0 (27.8—30.2) 0.63 (0.41—0.84) 13.3 (11.5—15.0) 16.1 (13.2—19.0) 59.6 (52.6—66.6)

87 (84—90) 13.2 (10.9—15.4) (P = 0.003c ) 0.66 (0.55—0.76) 141 (140—142) 4.05 (3.89—4.21) 106 (105—106) 27.9 (26.3—29.6) 0.54 (0.42—0.67) 15.3 (11.6—18.9) 16.9 (12.7—21.1) 58.6 (51.3—65.9)

Albumin (g/dl)

4.18 (4.03—4.32)

4.20 (4.07—4.33)

Total protein (g/dl)

7.4 (7.2—7.7)

7.5 (7.3—7.7)

286 (252—321)

285 (249—321)

6.6 (5.6—7.6) 3.6 (2.8—4.5) 11.9 (10.8—13.0) (P < 0.001c ) 36.7 (34.0—39.4) (P < 0.001c ; P = 0.03d ) 283 (257—309)

85 (81—88) 10.9 (8.8—13.1)

83 (79—88) 10.9 (9.3—12.5)

83 (80—86) 11.8 (10.6—13.1)

82 (78—86) 11.5 (10.0—13.2)

0.66 (0.56—0.76) 141 (140—142) 4.08 (3.91—4.25) 106 (105—107) 28.0 (27.0—29.0) 0.58 (0.40—0.75) 13.3 (11.4—15.1) 16.3 (12.7—20.0) 54.9 (48.4—61.5) (P = 0.007c ) 4.17 (4.04—4.29)

0.60 (0.47—0.73) 141 (140—142) 4.22 (3.86—4.57) 105 (104—106) 29.0 (27.3—30.9) 0.59 (0.40—0.78) 13.2 (10.2—16.2) 16.2 (13.4—19.0) 58.5 (52.1—65.0)

0.62 (0.47—0.77) 141 (140—142) 3.99 (3.83—4.15) 106 (105—107) 26.9 (25.8—28.1) 0.63 (0.38—0.88) 14.7 (10.6—18.9) 17.9 (11.6—24.2) 53.0 (47.4—58.6) (P = 0.04c ) 4.2 (4.0—4.3)

7.3 (7.1—7.5) (P = 0.006d )

7.7 (7.5—7.9)

0.60 (0.49—0.71) 140 (139—142) 4.06 (3.88—4.25) 105 (104—106) 27.4 (25.9—28.8) 0.58 (0.34—0.83) 15.6 (11.3—19.9) 16.7 (10.1—23.3) 55.3 (49.4—61.2) (P = 0.04c ) 4.1 (4.0—4.2) (P = 0.03c ) 7.4 (7.2—7.7) (P = 0.02c )

40.3 (37.5—43.2)

4.2 (4.1—4.4)

7.4 (7.2—7.6) (P = 0.003c )

S. Kongpatanakul et al.

Urinalysisb Normal 10 6 10 9 9 6 Abnormal Protein positive 0 2 0 0 0 2 Blood positive 2 4 1 1 1 1 Bilirubin positive 0 0 1 0 0 1 Red blood cell >2/HPF 1 4 1 1 1 1 White blood cell >5/HPF 0 1 2 1 1 1 DHA: dihydroartemisinin; GPO: Thai Government Pharmaceutical Organization (Bangkok, Thailand); Dafra: Dafra Pharma NV (Turnhout, Belgium); AST: aspartate aminotransferase; ALT: alanine aminotransferase; HPF: high-power field. a Haematology and blood biochemistry data are presented as mean (95% CI). Data were compared using paired Student’s t-test. Only P-values of significant changes are given. b Urinalysis data are shown as the number of volunteers. Data were compared using McNemar’s test. Only P-values of significant changes are given. c Comparison between laboratory values after one or two doses of DHA vs. baseline. d Comparison between two doses vs. one dose of DHA.

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phosphatase, albumin and total protein for the Dafra—GPO group. However, these values remained within normal limits. If both study groups were combined (n = 23) and the data were analysed irrespective of the drug formulations, the mean decrease from baseline after the first dose was 0.73 g/dl for haemoglobin (P < 0.001) and 2.0% for haematocrit (P < 0.001). The mean decrease from baseline after two doses was 0.95 g/dl for haemoglobin (P < 0.001) and 3.3% for haematocrit (P < 0.001). Additive toxicity (a larger decrease after two doses compared with one) was detected for haematocrit (P < 0.001) but not haemoglobin (P = 0.15). Additional significant changes in laboratory values included white blood cell count, blood urea nitrogen and bicarbonate after the first dose and bicarbonate, alkaline phosphatase and total protein after the second dose. These values were within normal limits.

3.4. Adverse events AEs occurring within 7 days following each drug administration are shown in Table 2. Relatedness of the AEs to the study drugs were categorised as: possibly related (decreased haemoglobin, decreased haematocrit and hearing loss); unlikely to be related (impairment of vestibular function, upper respiratory tract infection, increased eosinophils); and not related (fever). All AEs were mild and resolved during follow-up. All baseline measurements of hearing and vestibular function were within normal limits. One volunteer (F1) in the Dafra—GPO group experienced hearing loss (threshold increasing to 35 dB from baseline of 20 dB) at 6 KHz frequency of the right ear 5 days after Dafra DHA administration. The hearing level returned to normal at a follow-up visit 49 days after administration of GPO DHA. Impaired vestibular function was observed in three volunteers and was mild and transient in all cases.

Table 2

Figure 2 Mean timed plasma concentration profile (±95% CI) of dihydroartemisinin (DHA) following a single 200 mg oral dose (two × 100 mg tablets) of Thai Government Pharmaceutical Organization (GPO) or Dafra Pharma NV formulation in healthy volunteers (n = 23 for GPO; n = 24 for Dafra).

3.5. Pharmacokinetics The plasma concentration—time profiles of DHA in volunteers after receiving a single oral dose of GPO or Dafra DHA formulations are illustrated in Figure 2. Analysis of pharmacokinetic data by the non-compartmental method yielded a geometric mean Cmax for GPO and Dafra DHA of 369 ng/ml and 247 ng/ml, respectively. The Cmax ratio was 148% (90% CI 122—179%). The geometric mean AUC0—8h of GPO and Dafra DHA was 1264 ng h/ml and 836 ng h/ml, respectively. The relative bioavailability of the GPO formulation to the Dafra formulation (AUC0—8h ratio) was 149% (90% CI 125—179%). The 90% CI for the ratio of geometric mean Cmax and AUC0—8h

Adverse events following dihydroartemisinin (DHA) administrationa

Adverse event

Mild anaemia (haemoglobin 10.0—12.0 g/dl) Mild anaemia (haematocrit 30—35%) Hearing loss (threshold hearing level >25 dB, or >10 dB change from baseline) Impairment of vestibular function Upper respiratory tract infection Increased eosinophils Fever

GPO—Dafra group (n = 12)

Dafra—GPO group (n = 11)

After GPO DHA

After Dafra DHA

After Dafra DHA

After GPO DHA

F8, F10, F15, F19

F6, F8, F10, F15, F19

F1, F7, F14, F18

F1, F7, F9, F14, F18, F22, F24*

F19

F6, F8, F10, F15, F19

F7, F14, F18

F7, F9, F14

—

—

F1

—

F2, F12

F2

—

F7

F6

F19

—

—

— F21

M13 —

— —

— —

GPO, Thai Government Pharmaceutical Organization (Bangkok, Thailand); Dafra, Dafra Pharma NV (Turnhout, Belgium). a Volunteer identification numbers are shown (F = female, M = male). * Significant increase in the number of volunteers compared with those at baseline screening: McNemar’s test, exact P-value = 0.02.

978 were outside the 80—125% acceptable range (US Food and Drug Administration, 2003), indicating that the two DHA formulations were not bioequivalent. Median (range) tmax of the GPO and Dafra formulations were 3 h (30 min to 6 h) and 2 h (45 min to 3 h), respectively. Median (range) T1/2z of the GPO and Dafra formulations were 1.7 h (1.0—3.0 h) and 2.0 h (1.3—4.4 h), respectively.

4. Discussion Our in vitro dissolution data indicated that the GPO formulation dissolved more readily than the Dafra formulation. The difference (f1 = 26.17) and similarity (f2 = 35.87) factors lay outside the acceptable ranges specified by the US FDA guidelines (f1 = 0—15 and f2 = 50—100) (US Food and Drug Administration, 1997), indicating a significant difference. This was likely due to the tablet formulation itself dissolving rapidly in the dissolution medium and our observation that the GPO tablet contained a higher amount of DHA than the Dafra formulation (97.79% vs. 92.05%, respectively; data not shown). In general, our pharmacokinetic data follow the same trend seen in previous clinical studies conducted in healthy volunteers, in which the DHA level rises rapidly, peaks at 1.5—3 h and declines to nadir at approximately 12 h post oral administration (Binh et al., 2001; Hung et al., 1999; Kongthaisong et al., 2004; Na-Bangchang et al., 1997, 2004). In this study, the 200 mg oral dose both of the GPO and Dafra formulations yielded Cmax and AUC values within the ranges similar to those in previous reports (dose range 100—300 mg). Nevertheless, our data indicated that the GPO formulation had higher mean Cmax and AUC0—8h compared with the reference Dafra formulation and had approximately 50% greater bioavailability. The higher AUC0—8h of the GPO formulation also correlated well with higher plasma levels of DHA for the GPO formulation at 3, 4 and 6 h post administration (Figure 2; t-test, P = 0.02, 0.001 and 0.006, respectively). The median tmax of the Dafra formulations (2 h) in this study was comparable with those reported previously (range 1.5—2.5 h), whilst tmax of the GPO formulation (3 h) was slightly longer. Of note in our study were the longer half-lives of the drugs (GPO 1.7 h and Dafra 2.0 h) compared with those in some previous reports using the same 200 mg or 300 mg DHA doses (approximately 0.8—1.3 h) (Na-Bangchang et al., 1997, 2004). A relatively longer half-life of DHA (2.0 h) was observed in one study of a 240 mg dose of Arenco (Dafra) DHA tablets in eight healthy Vietnamese volunteers (Hung et al., 1999). Previous studies in healthy volunteers (Binh et al., 2001; Hung et al., 1999; Kongthaisong et al., 2004; Na-Bangchang et al., 1997, 2004) and in patients with uncomplicated falciparum malaria (Li et al., 1999; Newton et al., 2002; Wilairatana et al., 1998) have demonstrated that DHA is well tolerated with no serious clinical AEs. Similarly, both DHA tablet formulations used in our study were relatively safe. All AEs were mild and the laboratory test values returned to baseline within 2 weeks. However, one striking observation was a high frequency of decrease in haemoglobin and haematocrit values in both study groups. In some volunteers, the values dropped below the normal limit (Table 2).

S. Kongpatanakul et al. Moreover, additive toxicity was also detected with regard to haematocrit following two doses of DHA (two doses vs. one dose, P < 0.001). Although a trend of additive toxicity was also observed for haemoglobin, the decrease was not statistically significant (P = 0.15). Significant decreases in haemoglobin values in healthy volunteers after receiving oral DHA or artesunate were also reported previously by Na-Bangchang et al. (2004); however, the issue was not further investigated. Note that in our study the majority of volunteers were female (18 of 23) whilst all or most volunteers were male in the majority of previous reports (Binh et al., 2001; Hung et al., 1999; Kongthaisong et al., 2004; Na-Bangchang et al., 1997). An association of the decreased values with sex and menstruation period in female volunteers was investigated; however, no obvious connection was observed. With respect to safety studies of other artemisinin derivatives in malaria patients, a study in healthy volunteers like ours may be more sensitive in detecting haematological toxicity owing to the lack of confounding effects by the parasite. Animal studies have demonstrated that artemisinin derivatives are associated with an unusual pattern of damage to specific brain stem nuclei, particularly those involved in auditory processing (Genovese et al., 1998; Nontprasert et al., 1998, 2002). However, no evidence of neurotoxicity (specific to auditory function) of artemisinin or its derivatives has been reported in humans (Hutagalung et al., 2006; Kissinger et al., 2000; McCall et al., 2006; Van Vugt et al., 2000). Recently, Toovey and Jamieson (2004) have reported apparent hearing loss in uncomplicated falciparum malaria patients treated with co-artemether. This report has evoked a re-evaluation of the safety and neurotoxicity of artemisinin and its derivatives. In our study, one volunteer in the Dafra—GPO group experienced transient right-sided hearing loss (35 dB at 6 KHz) 5 days after receiving the Dafra formulation. From our study, if neurotoxicity causing impaired auditory function did occur, it might be mild at this dosage. A larger sample size and more thorough evaluation are needed. In conclusion, the DHA formulation developed by Thai GPO is well tolerated in healthy volunteers. AEs were mild and consistent in both drug study groups. The GPO formulation dissolved more readily in vitro and was more bioavailable in vivo than the reference Dafra formulation. The new formulation may act well in combination with a longer half-life partner drug such as mefloquine as an effective antimalarial treatment. However, our finding of decreased haematological values, in particular haemoglobin and haematocrit, suggests that further investigations are warranted before work moves on to patients. The finding also has further safety implications for the development of other artemisinin derivatives in general. Authors’ contributions: SK, KS, YS and PS designed the study protocol; SK and SC carried out the clinical assessment and interpretation; SA performed the audiometry and posturography assessments; KS, PS and PP carried out the pharmacokinetic study and analysis; YS performed the clinical data management; SW formulated, assured the quality of and performed in vitro dissolution study of the GPO DHA; SK drafted the manuscript. All authors read and approved the final manuscript. SK is guarantor of the paper.

Evaluation of a new dihydroartemisinin tablet formulation Acknowledgements: We sincerely thank Dr Juntra Karbwang and Dr Win Gutteridge for their advice. We also thank sponsors, consultants, investigators, clinical staff and the co-ordinating team for their assistance in driving this DHA project into the clinical phase. Funding: Thailand Tropical Diseases Research Programme (T-2), under the Thailand Research Fund (TRF); National Center for Genetic Engineering and Biotechnology/National Science and Technology Development Agency (BIOTEC/NSTDA); and the Special Programme for Research and Training in Tropical Diseases/World Health Organization (TDR/WHO). Conflicts of interest: The authors have no conflicts of interest concerning the work reported in this paper. Dafra DHA used in this study was a gift from Dafra Pharma NV, Belgium; the manufacturer was not involved in the management of the study or in analysis of the data. Ethical approval: Ethics Committees of the Faculty of Medicine, Siriraj Hospital, Mahidol University, Thailand, and the Ministry of Public Health, Thailand.

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