Ndubuisi N. Nwobodo¹* and Paul O. Okonkwo²

¹Department of Pharmacology and Therapeutics, Ebonyi State University, Abakaliki, Nigeria.

²Department of Pharmacology and Therapeutics, University of Nigeria.

DOI : https://dx.doi.org/10.13005/bpj/509

Abstract

The occurrence of parasitological resistance to amodiaquine has been reported globally. The 3-HMG CoA reductase inhibitors, otherwise known as statins have been shown to inhibit the growth of malaria parasite.This study was aimed at evaluating the effects of simvastatin in modulating parasitological response to amodiaquine in the chemotherapy of malaria. Subjects with frank malaria (n=60) diagnosed by thick blood film and and confirmed using immunological tests were nominated for the study. Informed written content was obtained and subjects randomized into amodiaquine plus simvastatin (test) and amodiaquine alone (control) groups. The ethical clearance certificate was obtained from the University of Nigeria Teaching Hospital Research Ethics Committee (NHREC/05/01/2008B). The assessment of parasitological response was done in line with WHO criteria and patients followed up on days D0, D3, D7, D14 and D28 post-treatment. The GraphPad Prism 4.0 was employed in the analysis of data which was presented as tables and graphs. Revealed a statistically significant difference in parasitological response (p<0.05) between test and control groups. The mean value of low level resistance, RI was given as 1.3

Keywords

Amodiaquine; Falciparum malaria; HMG-CoA reductase inhibitor; Parasitological failure; Parasitological resistance; Simvastatin

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Copy the following to cite this article: Nwobodo N. N, Okonkwo P. O. 23-HMG-Coa Reductase Inhibitor Modulates Parasitological Response in Malaria Patients Treated with Amodiaquine. Biomed Pharmacol J 2014;7(2)

Copy the following to cite this URL: Nwobodo N. N, Okonkwo P. O. 23-HMG-Coa Reductase Inhibitor Modulates Parasitological Response in Malaria Patients Treated with Amodiaquine. Biomed Pharmacol J 2014;7(2). Available from: http://biomedpharmajournal.org/?p=3034

Introduction

A review recommended the continued use of amodiaquine in the treatment of uncomplicated malaria, stressing the need to take into consideration local drug resistance patterns¹. The occurrence of parasitological resistance to amodiaquinehas been reported across the globe^2-4. Cross resistance to antimalarial drugs may derive from single nucleotide polymorphisms (SNPs) in the Pfmdr and Pfcrt genes of Plasmodium falciparum. The Pfmdr1 86Y and haplotypes at Pfcrt 72-76 have been lnked to amodiaquine resistance. A study observed  rapid  but steady percent increase in wild-type parasites with regard to both Pfmdr1  and Pfcrt pointing to a significant change in parasite response⁵. The 3-HMG CoA reductase inhibitors, otherwise known as statins are lipid lowering agents and their clinical benefits could be related to reduction in anti-inflammatory responses. Statins have been shown to regulate inflammatory cell adhesion and endothelial function.  Statins prevent lipopolysaccharide (LPS)-induced intracellular adhesion molecule-1 (ICAM-1)  expression in endothelial cells via inhibition of Rho activity⁶. Statins have been reported to inhibit growth of  Plasmodium.falciparum in vitro^7,8. We hypothesize that a statistically significant difference (p<0.05) exists in parasitological response between simvastatin treated  subjects in combination with amodiaquine and subjects treated with amodiaquine alone.

Materials and Methods

Subjects: Subjects with acute malaria (n=60) in attendance at eight primary health facilities were selected for this study. Malaria infection was diagnosed using thick blood films and confirmed by immunological test (Paracheck PI^®) . Paracheck PI^®, a rapid qualitative two site sandwich immunochromatographic dipstick assay, was employed for the determination of Plasmodium falciparum specific histidine rich protein-2 (PfHRP-2) in whole blood samples.  This was in view of  the fact that  the classical method of diagnosis by microscopy involving examination of thin and thick blood smears was prone to false negative readings and time consuming.

Study Design: Informed consent was obtained after adequate explanation of the purpose of study, formal written documentation , type of treatment to be administered and clarification of any likely adverse effects or complication that may arise in the course of treatment. Patients enrolled for this study were within the age range 16 to 65 years inclusive, in attendance at eight primary health facilities within Asu Nkanu Local Health Authority in Nkanu East Local Government Area of Enugu State, Nigeria. Routine clinical clerkship and examination including body weight measurement and axillary temperature were  carried out to confirm the enrollee’s physical condition and ascertain presence of any confounding ailment. Subjects were randomised into test and control groups using a table of random numbers statistically generated. No member of the research team including the principal investigator, microscopist, field supervisor, field assistants, medical officer and nurses involved in the study had any prior knowledge of the patients’ medical records nor the treatment group to which any enrollee was assigned. The ethical clearance certification was given by the Institutional Research Ethics Review Committee of the University of Nigeria Teaching Hospital, Ituku-Ozalla, Nigeria (Ref: NHREC/05/01/2008B) in line with principles guiding human experimentation as enumerated in the Declaration of Helsinki by the World Medical Association General Assembly as last amended (Seoul 2008); while approval for the study was obtained from Enugu State Ministry of Health, Enugu-Nigeria. Amodiaquine (Camoquin^® from Pfizer West-Africa, Dakar-Nigeria) was given as 15mg/kg at initial presentation D0, then 10mg/kg daily for D1 and D2.  Simvastatin (Simvor^® from Ranbaxy Laboratories, Dewas-India) was given orally in the dosage 0.6mg/kg/d only in the evening for 3 consecutive days. The control group received Amodiaquine only  in same dose as test group. Artemether-Lumefantrine (Coartem^®from Novartis Pharma AG, Basel-Switzerland) was used to salvage subjects who presented with recrudescence or parasitological failure and eventually withdrawn from the study. The Artemether component was given as 3.2mg/kg/d while the Lumefantrine as 19.2 mg/kg/d respectively in two divided doses for 3 days. Baseline monitoring of liver function tests was done before commencement and in the course of therapy. The discontinuation of simvastatin would be indicated  following elevation of serum transaminase activity up to three times normal level.

Assessment of Response: The patients were followed up on days D0, D3, D7, D14 and D28. The World Health Organisation (WHO) criteria were applied in the categorization of parasitological response. Parasitological response is classified as low to high level parasitological resistance (RI, RII, RIII) and defined as:

High level resistance III (RIII) is parasitemia on day 3, D3 higher or 25% of parasitemia on D0.

Mid-level resistance II (RII) is parasitemia on day 3, D3 ≤ 25% of parasitemia on D0; but positive parasitemia between D4 and D7.

Low level resistance I (RI) is a negative blood smear on day 3, D3 and a positive blood smear on any day between D7 and D14.

Statistical Analysis: Graphpad Prism version 4.0 (GraphPad Software, Inc., La Jolla, CA, USA)  statistical software was employed and data presented in the form of tables and graph. Test of significance statistically determined using  two-tailed Student t-test, at 95% confidence interval, p<0.05 considered significant.

Results

Table 1 depicts the baseline characteristics of subjects in the test and control groups at presentation. A statistically significant difference (p<0.05) in the low, mid and high level parasitological resistance (RI, RII, RIII) between the test and control groups was  depicted in Table 2 and Figure 1. A statistically significant difference (p<0.05) in late parasitological failure was also reported between the test and  control groups as depicted in Table 2.

Table 1: Baseline Characteristics of Test and Control Groups Treated 

Characteristics	Test	Control	p-Value
Number of Patients	30	30	–
Male: Female Ratio	2:3	2:3	–
Mean Age (Range: 16-65 years)	38.7±2.6	39.4±3.2	p>0.05
Mean Weight (Range: 43–92 kg)	62.5±4.8	61.8±3.6	p>0.05
Mean Temperature (Range: 37.8–39.2oC)	38.8±1.4	37.9±1.1	p>0.05
Mean Parasite Density (Range: 1260-21500/µL)	9168±932	10723±821	p>0.05
Mean Hemogram (Range: 4.2 – 11.5g/dL)	9.1±1.2	8.8±1.4	p>0.05
Mean WBC Total (Range: 3000 – 11700 x 109/L)	6720±457	7700±453	p>0.05
Mean Alanine Transaminase (Range: 7.8-31.2U/L)	13.4±3.1	15.7 ±4.1	p>0.05
Mean Aspartate Transaminase (Range: 13.7-28.4U/L)	16.7±5.1	17.3±5.4	p>0.05
Mean Alkaline Phosphatase (Range: 45.2-110.7U/L)	88.7±8.4	92.4±8.1	p>0.05
Mean Total  Bilirubin (Range 4.3-13.8µmol/L)	6.4±1.2	7.2±1.2	p>0.05

Table 2: Mean Parasitological Response in the Test and Control Groups

Parasitological Resistance	Test (%)	Control (%)	p-Value
Low Level Resistance (RI)	1.3±0.14	8.7±0.42	p<0.05
Mid Level Resistance (RII)	2.7±0.15	12.8±0.49	p<0.05
High Level Resistance III (RIII)	2.4±0.17	4.6±0.17	p<0.05
Late Parasitological Failure (LPF)	3.3±0.26	6.7±0.21	p<0.05

 

Figure 1 Click here to View figure

Discussion

The current study revealed statistically significant difference (p<0.05) in the cumulative low to high level parasitological resistance (RI + RII + RIII) and late parasitological failure as depicted in Table 2 and Figure 1. Hence, the consideration of late parasitological failure (LPF) alongside sums up to an overall parasitological resistance of 9.7% and 32.8% in the test and control groups respectively. The underlying resistant mechanism to amodiaquine could be related to accumulation within the infected parasite to high levels. This highly specific accumulation causes significant drug depletion from sensitivity assay plates, leading to an under-estimation of drug activity (the innoculum effect). The net effect is to under-estimate the differences in the amodiaquine dose-response of resistant isolates. The accumulation-related resistance to amodiaquine is totally insensitive to the effects of verapamil.  The ability of the classic reverser of multi-drug resistance, verapamil to increase chemo-sensitivity to chloroquine in resistant isolates of Plasmodium falciparum in vitro has been documented. However, the verapamil insensitive component confers resistance to amodiaquine.

Studies have indicated consensus on the thresholds to define resistance to N-desethylamodiaquine, the active metabolite of amodiaquine^9-12. The differences in the reported thresholds to define amodiaquine resistance in vitro could be partially explained by: variations in the in vitro methodology such as incubation time of the parasite with the drug^13,14, the final hematocrit¹⁵ and the percentage of red blood cell parasitized¹⁶; the most important probably is the hematocrit, since amodiaquine has the tendency to concentrate inside erythrocytes¹⁷. The in vitro tests are performed with desethylamodiaquine but conclusions refer to amodiaquine^10,12,16.The use of different commercial presentation of amodiaquine without taking into account their different molecular weights; and finally, the use of parasites adapted to cultures in vitro and with incubation time >24h, were likely to show different results from those obtained with fresh isolate. A study emphasized that it is more relevant to monitor in vitro resistance to desethylamodiaquine instead of amodiaquine, since desethylamodiaquine exerts the major anti-malarial activity in vivo¹⁸. The same study maintains that understanding the mechanism of resistance to amodiaquine is useful in the design of new drugs, particularly 4-aminoquinoline derivatives.

A receptor Known as SR-BI (class B, type I scavenger receptor) mediates the selective uptake of cholesterol from both high and low density lipoproteins. The SR-BI plays a crucial role in Plasmodium hepatocyte infection¹⁹. A reduction in SR-BI expression in HUH7 hepatoma cells led to a significant reduction in Plasmodium infection rates and in vivo use of SR-BI si-RNAs also significantly reduced liver infection in Plasmodium infected mice²⁰. It is postulated that the malaria parasites may have originally selected the SR-BI for an evolutionary reason; since SR-BI plays a direct or indirect role in providing cholesterol for the parasites to build up their cell membranes. The HDL fraction has been implicated as a major substrate for the growth of infective stages of the malaria parasite and used to support growth of Plasmodium falciparum with results comparable to those obtained using human serum²¹. Scientific evidence suggest that the parasitophorous vacuole membrane lipids in malaria infected erythrocytes are derived from the host cells^22,23.  An enzyme capable of activating fatty acids which is necessary for incorporation into lipids has been localized to membrane structures found within the cytoplasm of the infected erythrocyte²⁴. Hence, the molecular link between malaria infection and cholesterol uptake pathway has been  well established. It could be deduced that simvastatin might protect against intrahepatic development of malaria parasites, thereby blocking erythrocyte invasion with its elaboration of toxins associated with increase in morbidity and mortality.  Consequently, the outcome of this study suggests that the 3-HMG-CoA reductase inhibitor, simvastatin, is implicated in modulating parasitological response to amodiaquine in the chemotherapy of malaria.

Acknowledgments

I wish to acknowledge the sacrificial and tremendous assistance of the former executive secretary, Mr. M.O. Offu and entire staff of  the 8 primary health facilities in the study site at Asu-Nkanu Local Health Authority, Enugu State, Nigeria. The selfless contribution of Mr. E.A. Ahaotu and Mr. B.C. Ezeagwoma, both of whom are chief medical laboratory scientists at University of Nigeria Teaching Hospital, is highly appreciated. My immense gratitude also goes toDr. Nick C. Obitte, Lecturer, Department of Pharmaceutical Technology, University of Nigeria, Nsukka for his assistance and useful advice. I sincerely acknowledge the contribution of Dr. G.P.I. Oluka, formerly Health Administrator, Enugu State Health Board and Pharm. P.O. Otegbulu, Director Pharmaceutical Services, Enugu State Ministry of Health.

Declarations

Authors’ contributions: The conception and design of this study was carried out by both NNN and POO. Data acquisition and conduct of the study was by NNN. Analysis and interpretation of data were carried out by both NNN and POO. The manuscript was drafted by NNN and meticulously reviewed by POO for intellectual content. NNN and POO read, scrutinized and approved the final draft of the manuscript prior to submission.

Conflict of Interest

None disclosed.

Funding

None

Ethical Clearance

Obtained from University of Nigeria Teaching Hospital, Health Research Ethics Committee (Ref: NHREC/05/01/2008B)

References

1. Oliaro P, Mussano P. Amodiaquine for treating malaria. Cochrane Database Syst Rev 2003; 2: CD000016
2. Khaliq AA, Fox E, Sarwar M, Strickland GT. Amodiaquine fails to cure chloroquine resistant Plasmodium falciparum in the Punjab. Trans R Soc Trop Med Hyg 1987; 81: 157-159.
3. Kremsner PG, Zotter GM, Feldmeier H, Grainger W, Rocha RM, Wiedermann G. A comparative trial of three regimens for uncomplicated falciparum malaria in Acre-Brazil. J Infect Dis 1998; 158: 1368-1371.
4. Mutabiugwa TK, Anthony D, Heller A, Hallet R, Ahmed J, Drakeley C, Greenwood BM, Whitty CJM. Amodiaquine alone, amodiaquine plus sulfadoxine-pyrimethamine, amodiaquine plus artesunate and artemether-lumefantrine for outpatient treatment in Tanzanian children: a four-arm randomised effectiveness trial. Lancet 2005; 365: 1474-1480.
5. Eyase FL, Akala HM, Ingasia L, Cheruiyot A, Omondi A, Okudo C, Juma D, Yeda R, Andagalu B, Wanja E, Kamau E, Schrabel D, Bulima W, Waters NC, Walsh DS, Johnson JD. The role of Pfmdr1 and Pfcrt in changing chloroquine, amodiaquine and lumefantrine susceptibility ln Western Kenya Plasmodium falciparum samples during 2008-2011. PLoS ONE 2013; e64299.
6. Takeuchi S, Kawashima S, Rikitake Y, Ueyama T, Inoue N, Hirata K, Yokotama M. Cerivastatin suppresses lipopolysacharide-induced ICAM-1 expression through inhibition o f Rho GTPase in BAEC. Biochem Biophys Res Commun 2000; 269(1): 97-102.
7. Couto AS, Kimura EA, Peres VJ, Uhrig ML, Katzin AM. Active isoprenoid pathway in the intra-erythrocytic stages of Plasmodium falciparum: presence of dolichols of 11 and 12 isoprene units. BiochemJ 1999; 341: 629-637.
8. Naik RS, Venkatesan M, Gowda DC. Plasmodium falciparum: the lethal effects of tunicamycin and mevastatin on the parasite are not mediated by inhibition of N-linked oligosaccharide biosynthesis. Exp  Parasitol  2001; 98: 110-114.
9. Ringwald P, Bickiz J, Basco LK. In vitro activity of anti-malarials against clinical isolates of Plasmodium falciparum in Yaounde, Cameroon. Am J Trop  Med  Hyg 1996; 55: 254-258.
10. Ringwald P, Keundijian A, Same Ekobo A, Basco LK. Chemoresistance of Plasmodium falciparum in the urban region of Yaounde, Cameroon. Part 2: evaluation of the efficacy of amodiaquine and sulfadoxine-pyrimethamine combination in the treatment of uncomplicated Plasmodium falciparum malaria in Yaounde, Cameroon. Trop Med Int Health 2000; 5: 620-627.
11. Brasseur P, Guiguemde R, Diallo S, Guiyedi V, Kombila M, Ringwald P, Olliaro P. Amodiaquine remains effective for treating uncomplicated malaria in West and Central Africa. Trans  R Soc  Trop Med Hyg 1999; 93: 645-650.
12. Anbony A, Majombo J, Kaundjuan A, Bakeny M, Le Brass J, Delorom P. Short report: lack of prediction of amodiaquine efficacy in treating Plasmodiumfalciparum malaria by in vitro Am J Trop Med Hyg 2002; 71: 294-296.
13. Pradines B, Tall A, Parzy D, Spregel A, Fusai T, Hienne R, Trape TJ, Donry JC. In vitro activity of pyronaridine and amodiaquine against African isolates of Plasmodium falciparum in comparison with standard anti-malarial agents. J Antimicrob Chemother   1998;. 42: 333-339.
14. Rason MA, Ariey F, Rafidimanantosa L, Andrianantemia BH, Sahondre-Harisoa JL, Randrianarivelopsia. Monitoring the drug sensitivity of Plasmodium falciparum in coastal towns in Madagascar by use of in vitro chemosensitivity and mutation deletion tests. Parasite 2002;. 9: 247-253.
15. Child SGE., Bondreau EF, Milhous WK, Wimonwattratee T, Pooyindee N, Pany I., Davidson Jr. DE. A comparison of the in vitro activities of amodaiquine and desethylamodiaquine against isolates of Plasmodium falciparum.Am J TropMed Hyg 1989; 40: 7-11.
16. Basco I.K., Ndounga M, Tejiokem M, Ngane VF, Youmba JC, Ringwald P, Soula G. Molecular epidemiology of malaria in Cameroon: geographic distribution of Plasmodium falciparum isolates with dihydrofolate reductase gene mutations in Southern and Central Cameroon. Am J Trop Med Hyg 2002; 67: 378-382.
17. Pussard E, Verdier F, Faurisson J, Schermann JM, Le Bras J, Blayo MC. Disposition of monodesethylamodiaquine after a single oral dose of amodiaquine and three regimens for prophylaxis against Plasmodium falciparum Eur JClin Pharmacol 1987; 33: 409-414..
18. Cheveny DF, Nurillo C, Restrepo PP, Osorio L. Susceptibility of Colombian Plasmodium falciparum isolates to 4-aminoquinolines and the definition of amodiaquine resistance in vitro. Inst. Oswaldo Cruz 2006;. 101(3): 341-344.
19. Rodrigues CD, Hannus M., Prudencio M., Martin C., Goncalves LA, Portugal S, Epiphanio S, Akinc A, Hadwiger P, Jahn-Hofmann K, Rohl I, van Gemert GJ, Franetich JF, Luty AJ, Sauerwein R, Mazier D, Koteliansky V, Vornlocher HP, Echeveni CJ, Mota MM. Host scavengerreceptor SR-BI plays a dual role in the establishment of malaria parasite liver infection. Cell Host Microbe 2008;. 4(3): 271-282.
20. Yalaoui S, Huby T, Franetich JF, Gego A, Rametti A, Moreau M, Collet X, Siau A, van Gemert GJ, Sauerwein R, Luty AJ, Vaillant JC, Hannoun L, Chapman J, Mazier D, Froissard P. Scavenger receptor BI boosts hepatocyte permissiveness to Plasmodium infection. Cell Host Microbe 2008; 4: 283-292.
21. Grellier P, Rigomier D, Clavey V, Fruchart JC, Schrevel Lipid traffic between   high density lipoprotein and plasmodium infected red blood cell. J Cell Biol 1999; 112(2): 26-77.
22. Ward GE, Miller LH, Dvorak JA. The origin of parasitophorous vacuole membrane lipids in malaria infected erythrocytes. J Cell Sci 1993;  106: 237-148.
23. Miller LH, Good MF, Milon G. Malaria pathogenesis. Science 1994; 264: 1878-1883.
24. Metasanz F, Duran-Chica I, Alcina A. The cloning and expression of pfACS1, a Plasmodium falciparum fatty acyl coenzyme A syntethase targeted to host erythrocyte cytoplasm. J Mol Biol 1999; 291: 59-70.

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