The study centre was the University of Benin Teaching Hospital, Benin City, Nigeria. Ethical approval (EC/FP/015/05) was obtained prior to study. The ACTs were procured directly from the registered pharmacies in Benin City, Nigeria. Total sample size was adapted for common toxicity studies [8]. The patients recruited were those that had uncomplicated malaria as specified by systematic randomization [6,9]. These patients were randomly allotted into five groups according to the five ACTs resulting in (n=5) for each group. The ACTs were given freely to all the participants to be administered orally according to three days dosing regimen [10]. Insecticide Treated Nets and ACTs were also given freely as incentive to participants. Before and after the completion of therapy, about 5-7 ml was collected from the ante-cubital vein of each patient. These were Day 0 prior to drug administration, Days 1, 2, 3 and 4 after drug administration. All information about the patients that participated was kept confidential.

Collected blood samples introduced into plain and lithium heparin bottles were assayed for biochemical and hematological parameters respectively according to specifications [11]. Samples in the plain bottles were allowed to clot at room temperature before centrifugation using the Hettich centrifuge (Rototix 32A, Germany) at 4000 rpm for 10 min. The sera were further withdrawn into plain containers using a sterile syringe. Standard diagnostic kits specified [12]. were used and Automated Clinical System (VIS-7220G, Biotech Engineering Management Company Limited, UK; Analyzer ISE 4000 SFRI, France) was used to assay for the following biochemical parameters: Pancreatic index (Blood Glucose), Renal indices (creatinine, sodium, potassium, urea, bicarbonate), Liver indices (aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, conjugated bilirubin, total bilirubin, albumin and total protein) and Lipids (Total cholesterol, low-density lipoprotein, high-density lipoprotein and total triglycerides). The samples in lithium heparin bottles were assessed for hematological parameters using the automated hematological multichannel analyzer (ERMA PCE 210, Japan).

DATA ANALYSES

The data were first entered into Microsoft Excel and SPSS version 11.0 (SPSS, Inc. Chicago, IL). They are presented as mean ± SEM. Inferential analysis was done using ANOVA with Tukey’s and Fisher’s post hoc tests, odds ratio and relative risks (GraphPrism Version 6, San Diego, USA). p<0.05 was regarded as significant.

RESULTS

Out of the thirty-seven toxicity markers evaluated, seven of the parameters were significantly altered (p<0.05). Weight did not change significantly due to the ACTs. Blood glucose was significant increased by AL and DHP but decreased by AA, AM, ASP (p<0.05). Total cholesterol significantly increased due to ASP but decreased due to DHP. Potassium ion decreased significantly due to AA but increased due to AM. Creatinine decreased significantly due to ASP but increased due to AM. Conjugated bilirubin increased significantly due to AL but decreased due to ASP. Percentage lymphocytes decreased significantly due to AL and AA, increased due to DHP. Percentage monocytes increased significantly due to DHP but decreased due to AL, AA, ASP. None of the participants died and none degenerated to severe malaria in the study. Relative risk and odd ratio of the ACTs was found to be 0.75 and 0.70, respectively. Rank order of safety of ACTs was AL ≥ AA>AM>ASP>DHP.

DISCUSSION

The study has shown a wide variation in the safety profile ACTs in uncomplicated malaria. Following the introduction of ACTs into the malaria treatment policy in 2005 there has been an increase in their prescription [1]. This initially necessitated the initiative of National Agency for Food Drug administration and Control (NAFDAC) in collaboration with the Federal Ministry of Health [4]. to conduct a pilot program of Cohort Event Monitoring (CEM) program in the six geopolitical zones of Nigeria on patients using AL and AA to assess safety in the treatment of uncomplicated malaria [5]. This study represents a gold standard in guiding the selection of ACTs in the treatment of uncomplicated malaria in human. This study has also validated the safety profile of the conventional doses as recommended in humans [10]. In other words, the safety of recommended doses has been confirmed in humans and these doses can be extrapolated in other future studies. It is interesting to note that some of these agents have been assessed singly during pre-clinical studies and have proven to have one or two peculiar adverse effects during acute and chronic toxicity testing [3]. This study has shown a wider scope, thus providing more reliable information of the five commonly used ACTs.

Little or no renal derangement due to ACTs was seen in the study. Malarial infection in man can potentiate adverse effects of ACTs [20]. It is worthy to note that malaria pathogens induced can cause oxidative stress on the kidney [21]; thus potentiating the effect of ACTs in the renal system as seen with AM increasing creatinine levels. The rare ionic derangement as characterized by fatigue, muscle weakness, diarrhea, and dehydration that are known features of hypokalemic and hypochloremic effects may be linked with fewer side effects seen with AL subjectively [5,6,22]. Since ASP decreased creatinine level, it may be an essential tool in the management of malaria where increase renal creatinine has been observed. On the other hand, AM can also mask serum creatinine level in individual whose levels are normal.

Total cholesterol level decreased proportionally in DHP but increased in ASP combinations. From the foregoing, it can be deduced that ASP may cause more cardiovascular risk. Therefore caution needs to be taken in ASP combinations in subjects that have high cholesterol. In situation of comorbidity; such as malaria co-existing with cardiovascular disease, choice of adequate DHP combination may be beneficial rather than ASP. Meanwhile, artemether, dihydroartemisinin and artesunate as representatives of artemisinin derivative have been reported to cause dose dependent increase in TG [23], although significant changes were not observed. Despite no significant changes in other lipid parameters, it is of importance to note that High-density lipoprotein (HDL) levels are negatively correlated with risk of CVD while low-density lipoprotein (LDL) levels are positively correlated with CVD risk [24] when treating malaria with ACTs. Meanwhile, for individuals that may be dose sensitive to any of the ACTs, low or high levels of high-density lipoprotein cholesterol (HDL-C), triglycerides and lipoproteins levels may be seen; thus cardiovascular morbidity and mortality as influencing markers [25].

It was observed that essential liver enzymes (ALT, ALP and AST) were not significantly altered. Meanwhile, there had been reported opposite effect of artemisinin in elevating the activities of serum aspartate aminotransferase, alanine aminotrasferase and serum alkaline phosphatase significantly at higher doses, this represent possible hepatic toxicity [3,26,27]. The reports of no significant changes in enzyme level in this study showed a good safety profile in co-morbid hepatocellular damage. Note that, chronic administrations of the drugs can cause negative changes than short duration administration. It was also reported that dihydroartemisinin had no effect on serum level of alanine aminotransferase, serum alkaline phosphatase and serum aspartate aminotransferase activities [3]. Amodiaquine is a 4-aminoquinoline that has been identified to generate free radicals in form of amodiaquine immine and semi quinine immine and it is implicated in lipid peroxidation of the membranes of the hepatocyte cells [28]. Total bilirubin (TB) is also a biomarker associated with altered bile homeostasis and/or hepatobiliary injury [29]. The increase in level of total bilirubin with most ACTs except AL is a clear indication that bile homeostasis may have been altered. Its increase observed in the ACTs may be related to the individual drug in the combination. It has been documented that total bilirubin (TB) is a composite of unconjugated (extrahepatic) and conjugated (hepatic) bilirubin [30]. Since there were no significant changes in liver enzymes, it is a clear proof of safety of ACTs when used in conjunction with patients that may have hepatocellular disorders or drugs that may have the potential to alter liver enzymes. There can be dose sensitivity of the enzymes due to ACTs even in a short period. It should also be noted that some ACTs can mask the enzyme level in individuals that may be dose sensitive with existing hepatocellular damage.

Previous studies have described artemisinin and its derivatives alone to be generally safe and well tolerated [33]. Therefore changes observed in artemisinin combination treatment may be due to the partner agents such as amodiaquine, lumefantrine, mefloquine and piperaquine. Hematologic abnormalities as features in P. falciparum infection causing anemia are inevitable in most malaria cases. This study has further proven the safety of ACTs in a confirmed P. falciparum infection as proposed [34]. Conventional doses of milligram per kilogram body weight as recommended. WHO [10] suggest a good safety range; since no subject died in the course of the study. The order of safety was confirmed as AL ≥ AA>AM>ASP>DHP; meaning AL was most safe while ASP combination was least safe in patients that presented with uncomplicated malaria.

CONCLUSION

The commonly used ACTs in the treatment of uncomplicated malaria have been compared in malaria patients by assessing the common toxicity markers as gold standard in safety research. Following the results, discrete selection of these drugs should be ensued in patients with co-existing disorders with malarial infection to avoid adverse synergy.

ACKNOWLEDGEMENT

We wish to appreciate all that assistance during the course of the work; most especially staff of the Department of Pharmacology and Toxicology, University of Benin, Benin City, Nigeria. Staff of the Department of Pharmacy, University of Benin Teaching Hospital, Benin City, Nigeria. Physicians in the General Practice Clinic, Family Medicine Unit, University of Benin Teaching Hospital, Benin City, Nigeria.

CONFLICT OF INTEREST DECLARATION