New antimalarial drug combinations are been explored to overcome challenges associated with Plasmodium resistance. Clindamycin (C) has promising antiplasmodial activity. This study explored the in-vivo antiplasmodial activity of artemether/lumefantrine/clindamycin (A/L/C) on Plasmodium berghei-infected mice. Plasmodium berghei-infected adult Swiss albino mice (22-30g) were grouped and orally treated daily with A/L (2.3/13.7mg/kg), C (10 mg/kg) and A/L/C, respectively. The negative and positive controls were treated with normal saline (0.2mL) and chloroquine (CQ) (10 mg/kg), respectively. At the end of treatment, blood samples were collected and assessed for percentage parasitemia, inhibitions and hematological indices. Mice were observed for mean survival time. The curative, suppressive and prophylactic tests showed that A/L/C significantly decreased percentage parasitamia when compared to C or A/L with difference at p< 0.05. A/L/C significantly prolonged mean survival time with difference observed at p< 0.05 when compared to A/L or C. In the curative test, A/L, C, and A/L/C produced 70.3%, 64. 0% and 92.1% parasitemia inhibitions respectively when compared to CQ, which produced 83.2% inhibition. In the suppressive test, 77.2%, 70.3% and 97.9% parasitemia inhibitions were produced by A/L, C, and A/L/C, respectively when compared to CQ, which produced 90.2 % parasitemia inhibition. A/L/C significantly curtailed anemia through increased red blood cells, hemoglobin, packed cell volume and decreased white blood cells when compared to A/L or C with difference at p<0.05. Liver Plasmodium was eradicated in mice treated with A/L/C. C augmented the antiplasmodial effect of A/L. A/L/C may be clinically effective against malaria.

Keywords: Artemether, lumefantrine, clindamycin, Plasmodium, mice

Malaria is a parasite vector-borne disease that is one of the serious health predicaments in the tropical regions. The emergence and fast expansion of Plasmodium falciparum resistance to antimalarial drugs has gradually narrowed the choice and the prescription of antimalarial drugs by clinicians.^1-3 Artemether/lumefantrine (A/L) and other artemisinin-based combination therapies (ACTs) are used for the treatment of malaria.⁴ Combining antimalarial drugs increases efficacy and prevents the occurrence or development of resistant Plasmodium parasites⁵. Artemisinin derivatives are primarily combined with drugs that have long plasma elimination half-lives for prolong parasite clearance.^5-8 Despite the use of ACTs, resistance strains of Plasmodium have been reported in some Asian and African countries.⁹ Plasmodium falciparum, the primary culprit has been observed to develop resistance to most of the available antimalarial drugs, which makes the search for new antimalarial drugs or new combinations through repurposing imperative.

Drug repurposing is a research method that seeks new therapeutic applications for drugs that are already approved for other indications. This strategy reduces costs and research time line considerably.¹⁰ The traditional method of drug discovery is time-consuming, costly, laborious, and a high-risk process.¹¹ It takes about 10-15 years to develop a new drug with a success rate of 2.01%. Thus, repurposing is important, due to the numerous advantages over traditional approaches.¹² It has been used to screen existing drug with antimalarial activity. It delivers novel candidates, and also provides partner drugs for combination with artemisinins, thereby increasing activity against Plasmodium parasites.^13,14

Clindamycin (C) is a semisynthetic derivative of lincomycin, which was introduced in the 1960s as an antibiotic. In bacteria, it binds to 50S subunit of the ribosome, thereby inhibiting the synthesis of cell proteins.¹⁵ C is used for the treatment of bacterial infections, toxoplasmosis, Pneumocystis carinii pneumonia and babesiosis. In-vitro, C and its three primary metabolites have shown potent inhibitory activities on Plasmodium falciparum. C is a slow-acting drug, which accumulates slowly in parasites with a mean parasite clearance time of four to six days.¹⁶ It combination with a fast-acting drug is necessary to take advantage of its full antimalarial potential.¹⁶ C has shown promising antimalarial activity in uncomplicated falciparum malaria when combined with quinine¹⁷ and choloroquine.¹⁸ In the absence of scientific literature, this study assessed whether C can be repurposed in combination with A/L for the treatment of malaria using a mouse model infected with Plasmodium berghei.

Drugs and animals

Adult Swiss albino mice (22-30g) were sourced from Ignatius Ajuru University of Education, Rivers State, Nigeria. The mice were grouped (n=5) per cage with free access to standard diet and water. The mice were kept under natural condition (12 hour light/dark cycles) and were acclimated for 2 weeks prior to the study. Chloroquine (CQ) (Evans Pharm, Nigeria), Artemether/lumefantrine (A/L), (Artepharm Co., Ltd., China) and Clindamycin (C) (Mediplantex National Pharaceutical, Viet Nam) were used. CQ (10 mg/kg),¹⁹ A/L (2.3/13.7mg/kg)²⁰ and C (10 mg/kg)¹⁶ were used. The animals were handled according to the National Institute of Health (NIH) guide for handling experimental animals.²¹

 Plasmodium parasite

CQ sensitive Plasmodium. berghei (P. berghei) (NK65) was supplied in donor mice by the Nigerian Institute of Medical Research, Yaba, Lagos State. The parasites in the donor mice were kept alive by continuous serial intraperitoneal (ip) passage of blood samples from donor mice to uninfected mice every 5-6 days. 

Antiplasmodial test

Test for curative activity

It was performed as explained by Ryley and Peters (1970) ²². Twenty -five adult Swiss albino mice were randomized and inoculated with erythrocytes (0.2ml) containing P. berghei (1 × 10⁷) ip. After 3 days, the mice were orally treated daily for 4 days as follows: A/L, (2.3/13.7 mg/kg), C (10 mg/kg) and A/L/C respectively. The negative and positive controls; normal saline (0.2ml) and CQ (10 mg/kg), respectively. On day 5, tail blood samples were collected and stained with 10% Giemsa stain on slides. Parasitemia levels were counted with the aid of a light microscope. Percentage parasitemia and parasitamia inhibitions were calculated as shown below. 

$\text{\% Parasitemia= }\frac{\text{Number} \text{of parasitized} \text{red} \text{blood} \text{cells }\left(\text{RBCs}\right)}{\text{Total} \text{number} \text{of} \text{RBCs} \text{count}}\text{×100}$

$\text{\%Inhibition=}\frac{\text{(\%Parasitemia} \text{of} \text{negative} \text{control} \text{-\%} \text{Parasitemia of} \text{treated} \text{group)}}{\text{\%Parasitemia} \text{of} \text{negative} \text{control}}\times 100$

Test for suppressive activity

It was performed according to the method described by Knight and Peters (1980).²³ Twenty-five adult Swiss albino mice were randomized and inoculated with erythrocytes (0.2ml) containing P. berghei (1 × 10⁷) ip. After 2 hours, the mice were orally treated daily for 4 days as follows: A/L (2.3/13.7 mg/kg), C (10 mg/kg) and A/L/C, respectively. The negative and positive controls: normal saline (0.2mL) and CQ (10 mg/kg), respectively. On the 5^th day, tail blood samples were collected from the mice and stained with 10% Giemsa stain on slides. Percentage parasitemia and parasitemia inhibitions were calculated as shown above. 

Test for prophylactic activity

It was performed using the method explained by Peters.²⁴ Twenty-five Swiss albino mice were randomized and treated daily with A/L, (2.3/13.7mg/kg), C (10 mg/kg) and A/L/C for 4 days, respectively. The negative and positive controls were treated orally with normal saline (0.2mL) and CQ (10 mg/kg) daily for 4 days, respectively. On day 5, the mice were inoculated with erythrocytes (0.2ml) containing P. berghei (1 × 10⁷) ip. After, 3 days, the tail blood samples of the mice were collected and stained with 10% Gemsa stain on slides. Percentage parasitemia and parasitemia inhibitions were calculated as shown above.

Evaluation of hematological parameters

Blood samples collected in heparinized containers from the mice used for the curative test were evaluated for white blood cell (WBCs), red blood cell (RBCs), packed cell volume (PCV), and hemoglobin (Hb) using an auto analyzer (Cell-Dyn Model 331 430).

Statistical analysis

Data as mean± SEM (Standard error of mean). Data was analyzed using one way analysis of variance (ANOVA) and Tukey’s test. Significance was set at p<0.05.

Curative antiplasmodial activity of artemether/lumefantrine/clindamycin on Plasmodium berghei-infected mice

Treatment with A/L/C decreased percentage parasitemia significantly when compared to A/L or C with difference at p<0.05 (Table 1). A/L, C and A/L/C produced parasitemia inhibitions of 70.3%, 64.0% and 92.1%, respectively whereas CQ produced 84.2 % (Table 1). Treatment with A/L/C prolonged MST significantly with difference at p<0.05 when compared to A/L or C (Table 1).

Suppressive antiplasmodial activity of artemether/lumefantrine/clindamycin on Plasmodiumberghei-infected mice

Percentage parasitemia levels were decreased significant by A/L/C when compared to A/L or C with difference observed at p<0.05 (Table 2). Parasitemia inhibitions which represent 77.2%, 70.3% and 97.9% were produced by A/L, C and A/L/C, respectively compared to 90.2 % produced by CQ (Table 2). A/L/C produced significant prolongation of MST with difference observed at p<0.05 when compared to A/L or C (Table 2). 

Prophylactic antiplasmodial activity of artemether/lumefantrine/clindamycin on Plasmodiumberghei-infected mice

Treatment with A/L/C produced significant decreases in percentage parasitemia with difference observed at p<0.05 when compared to A/L or C (Table 3). Treatment with A/L, C and A/L/C produced 76.6%, 66.2% and 98.8% parasitemia inhibitions, respectively while CQ produced 91.5 % parasitemia inhibition (Table 3). A/L/C prolonged MST when compared to A/L or C with difference observed at p<0.05 (Table 3).

Antiplasmodial activity of artemether/lumefantrine/clindamycin on hematological indices of Plasmodium berghei-infected mice

Significant (p<0.01) decreases in RBCs, Hb, and PCV with significant (p<0.01) increases in WBCs were observed in P. berghei-infected mice (Table 4). However, treatment with A/L/C significantly increased RBCs, Hb, and PCV and significantly decreased WBCs when compared to A/L or C with difference observed at p<0.05 (Table 4). 

Antiplasmodial activity of artemether/lumefantrine/clindamycin on the liver of Plasmodiumberghei-infected mice

Figures A-E showed liver micrographs of mice in the control and experimental groups. A: Normal control showed normal hepatocytes and central vein. B: Negative control showed vascular congestion, merozoites, steatosis and normal hepatocytes. C: Treatment with C showed normal hepatoctyes and decreased merozoites. D: Treatment with A/L showed normal hepatoctyes and central vein congestion. E: Treatment with A/L/C showed normal hepatocytes and central vein. X 400.

Figure A-E are liver micrographs of mice in the control and treated groups. A: Normal control. B: Negative control. C: Treatment with artemether/lumefantrine, D: Treatment with clindamycin, E: Treatment with artemether/lumefantrine/clindamycin. ST: Steatosis, NCV: Normal central vine, CV: Central vein congestion, MZ: Merozoites, NH: Normal hepatocytes. X 400.

Challenges caused by Plasmodium resistance created platforms for discovering new antimalarial drugs and also experimenting on new antimalarial drugs combination using drug repurposing.²⁵ C has potential antipasmodial activity,¹ therefore, this study assessed its antiplasmodial activity in combination with A/L in mice infected with P. berghei. This study used a rodent model, because it is extensively used for the in-vivo investigations of natural and synthetic potential antimalarial agents. An in-vivo model also takes into account possible prodrug effect and the involvement of the immune system in infection eradication.²⁶ Although, rodent models may not produce the same signs and symptoms observed in the human plasmodial infection, but produce disease features similar to those of human plasmodial infection, when infected with P. berghei.^27-29 This study used suppressive model, because it is widely used for the preliminary assessment of anti-malarial drug candidates.³⁰ Curative test was used for this study, because it allows for established infection. P. berghei was used for the induction of malaria in mice, because it is widely used for preclinical antimalarial studies of drug candidates with measurable treatment outcomes.³¹ In the current study, A/L/C decreased percentage parasitemia levels and increased percentage parasitamia inhibitions in the curative and suppressive tests. A/L/C also decreased percentage parasitamia level in the prophylactic study. Comparatively, it was observed that A/L/C produced better curative, suppressive and prophylactic effects than CQ. In this study, the ability of A/L/C to prolonged MST was assessed, because MST is an important index for the assessment of potential antimalarial agents³² In this study, curative, suppressive and prophylactic tests showed decreased MST in P. berghei - infected mice. This is in agreement with observation reported by Georgewill et al.²⁵ On the other hand, in curative, suppressive and prophylactic tests, A/L/C prolonged MST in the treated mice.

Anemia is one of the features of P. berghei-infected mice, which is due to the clearance and/or destruction of infected RBCs, erythropoietic suppression and dyserythropoiesis.³³ The current study observed decreased RBCs, Hb, and PCV with increased WBCs in P. berghei-infected mice, which are signs of anemia. This supports reported observation by Georgewill et al.²⁵ Interestingly, treatment with A/L/C prevented the anemic impact of P. berghei characterized by increased RBCs, Hb, and PCV and decreased WBCs. The current study also assessed the antiplasmodial activity of A/L/C on the liver of P. berghei-infected mice. The liver of P. berghei-infected mice showed steatosis,central vein congestion and merozoites . This is consistent with reported finding by Udonkang.³⁴ But treatment with A/L/C eradicated the liver merozoites and restored liver structure. This finding showed that A/L/C may clear both blood and liver stages of Plasmodium parasite infection. A/L is among the first-line artemisinin based combination therapies recommended for the treatment of malaria.³⁵ The antiplasmodial activity of A/L is due to the release of free radicals by its artemether component, which then binds covalently to parasite proteins and heme. Also, the inhibition of malarial parasite’s calcium ATPase (sarcoplasmic endoplasmic reticulum calcium ATPase) has been suggested.³⁶ On the other hand, its lumefantrine component accumulates in parasite food vacuole where it interferes with heme polymerization through the formation of complexes leading to the production of toxic heme. The accumulation of toxic heme causes parasite death.³⁷ C is an antibiotic with high activity against Gram-positive aerobic and anaerobic bacteria, including pathogens that produce beta-lactamase. It reaches a high concentration at the infection site, reducing the virulence of bacteria and increasing the phagocytic activity of host lymphocytes.³⁸ Its antiplasmodial activity is not well defined, but it was speculated to target bacterium-derived translational machinery in the relict plastid and apicoplast, present in Plasmodium parasites.³⁹

Treatment with C augmented the antiplasmodial effect of A/L by clearing blood and liver stages of P. berghei    infection in mice best than A/L or C. A/L/C has prospect as a viable antimalarial drug combination.

The authors are grateful to Confidence Ogechi Nworgu for technical assistance.

The authors report no conflict of interest.

None.

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