INTRODUCTION
Multi-drug resistant malaria is fast emerging in Southeast Asia, Africa and other continents. The national malaria control programme in tropical countries have reported high levels of resistance to chloroquine, amodiaquine, MFQ, quinine, halofantrine and combinations including pyrimethamine- sulfadoxin and LAPDAP. According to World Health Organization reports (2008), there are approximately 275 million malaria cases throughout the world with nearly 1·1million deaths occurring annually. In a recent report of World Health Organization (2010a), malaria causes about 2414 deaths a day, over 90% of those are in Sub-Sahara Africa. The mortality among the children and infants is highest in Africa with 5 children dying every minute.
During the last 2 decades MFQ (amino alcohol) alone or its combinations with artemisinin derivatives had been preferred over artemether, artesunate monotherapy because of higher cure rates with artesunate + MFQ and artemether + MFQ (Looareesuwan et al. Reference Looareesuwan, Wilariatana, Viravan, Vanijanonta, Pitisutithum and Kyle1997; Price et al. Reference Price, Luxemburger, van Vugt, Simpson, Phaipun, Chongsuphajaisiddhi, Nosten and White1998) but now the resistance against MFQ has attained serious dimensions reaching a level of nearly 50% in Thailand (Nosten et al. Reference Nosten, Kuile, Chongsaphajaisiddhi, White, Kuile, Luxemburger, Webster, Edstein, Phajpun, Thew and White1991). Five fixed-dose ACT's combinations have been identified, namely artemether with lumefantrine, artesunate with MFQ, artesunate with pyronaridine, artesunate with amodiaquine and DHA with piperaquine for the control of uncomplicated P. falciparum (World Health Organization, 2010b, Sinclair et al Reference Sinclair, Olliaro and Garner2010). Because of the emergence of resistance the efficacy of MFQ is being compromised and overall cure rates are declining.
CLTR, a new macrolide, is specifically used for the treatment of Mycobacterium avium and M. intracellulare (atypical mycobacteria) (Malhotra-Kumar et al. Reference Malhotra-Kumar, Lammens, Coenen, Herck and Goossens2007). This macrolide has also been claimed to be effective against helminth Echinococcus multilocularis by virtue of its inhibition of mitochondrial ribosomes. Further the mitochondrial ribosome in Acanthamoeba castellanii has also been found to be a target for CLTR, resulting in inhibition of encystment of amoebae (Mathis et al. Reference Mathis, Wild, Deplazes and Boettger2004). In vitro anti-malarial activity of CLTR against schizont maturation had been studied in 2 strains of P. falciparum (chloroquine-sensitive P.f.3D7 and chloroquine-resistant P.f.K1) (Wisedpanichkij et al. Reference Wisedpanichkij, Chaijaroenkul, Sangsuwan, Tantisawat, Boonprasert and Bangchang2009) but the IC50 values were very high i.e. >10 μ m.
Being the cytochrome P450 inhibitor (Pinto et al. Reference Pinto, Wanq, Chalasani, Skaar, Kolwankar, Gorski, Lianqpunsaqul, Hamman, Arefayene and Hall2005), CLTR in the present communication has been used in combination with MFQ to reverse MFQ resistance of plasmodia.
MATERIALS AND METHODS
Animals and parasites
Outbred Swiss mice of either sex weighing 22–25 g were procured from the animal facilities at the institute and maintained on commercial pellet diet and water ad libitum under standard housing conditions. Ethical guidelines on handling and use of experimental animals were followed during the conduct of the study.
The rodent malaria parasite P. yoelii nigeriensis multi-drug resistant (MDR) was used in the study, which is resistant to chloroquine (250 mg/kg×4 d), MFQ (128 mg/kg×4 d) and quinine (400 mg/kg×4 d). These are the maximum tested doses for the particular anti-malarials. The parasite was maintained in the animals through sequential passages from the blood of infected mice, obtained by cardiac puncture.
Curative dose of CLTR
To determine the curative dose of CLTR, it was given alone to groups of Swiss mice inoculated with 2×105 multi-drug-resistant rodent malaria parasite P. yoelii nigeriensis-infected erythrocytes. The concentrations at which the drug was administered were 150, 200, 250, and 400 mg/kg×7 days. These doses were given every day in 2 divided doses for 7 days. The route of drug administration was oral. The drug was given twice in a day due to its short elimination half-life. Parasitaemia was monitored by Giemsa-stained thin blood smears on pre-determined days and the rate of survival was duly recorded.
Combination of MFQ and CLTR
To study the effect of the MFQ and CLTR combination, mice were inoculated with 2×105 P. yoelii nigeriensis MDR-infected erythrocytes. At 2–4 h after infection, they were treated with daily doses of MFQ alone (given orally once a day), CLTR alone (given orally twice a day) and with various combination groups of MFQ+CLTR (32+225 mg/kg, 48/32/16+150/75 mg/kg). For therapeutic efficacy of MFQ+CLTR different doses of this combination (64/32+300/150 mg/kg×4 d) were administrated to infected mice with 0·9–2·5% established infection. Mice in the control group were injected with the parasites only. Parasitaemia was monitored by Giemsa-stained thin blood smears on pre-determined days and survival of the mice was duly recorded.
Assessment of anti-malarial interaction between mefloquine and clarithromycin
To obtain numeric values for the type of interactions, results were expressed as the sum of the fractional inhibitory concentrations (sum FIC) at the given effective concentrations by the formula (ECx of agent A in the mixture/ECx of agent A alone)+(ECx of agent B in the mixture/ECx of agent B alone) (Gupta et al. Reference Gupta, Thapar, Wernsdorfer and Bjorkman2002). Sum FIC values indicate the type of anti-malarial interaction as follows: ‘synergistic’ if sum FIC<1; ‘fully additive’ if sum FIC=1; partially additive if sum FIC<2 provided that both contributory FICs<1; ‘antagonistic’ if any FIC>1.
RESULTS
In the present communication anti-malarial assessment of MFQ with CLTR administered orally, has been evaluated against P. yoelii nigeriensis a multi-drug-resistant rodent infection, using random-bred Swiss mice following administration of individual drugs and their combinations. The parasite used in the study was resistant to orally administered chloroquine (250 mg/kg×4 days), MFQ (128 mg/kg×4 days) and quinine (400 mg/kg×4 days). By oral administration of CLTR at doses of 150, 200, 250 and 400 mg/kg×7days it was shown that the antibiotic was fully curative at 400 mg/kg×7 days (Table 1).
Table 1. Curative dose of clarithromycin in Swiss mice infected with Plasmodium yoelii nigeriensis MDR
For further studies MFQ dosing was evaluated at 16, 32 and 48 mg/kg×4 days and these doses were found to be suppressive only, resulting in survival of 5 out of 5 mice each at 32 and 48 mg/kg while at 16 mg/kg only 2 mice survived beyond 28 days of observation. CLTR was also administered at 75, 150 and 225 mg/kg×7days, these doses were also suppressive. At the highest dose of CLTR i.e. 225 mg/kg, 5 mice survived beyond 28 days, at 75 and 150 mg/kg doses, only 2 and 4 mice survived. Control untreated mice died of high parasitaemia by day 7.
When the MFQ and CLTR combinations were used at 32 mg/kg and 225 mg/kg respectively, there was 100% cure and none of the treated mice developed parasitaemia up to 28 days of observation, while drugs alone produced only 0 and 40% cure respectively during the observation period of 28 days. In another batch when CLTR dose was reduced to 150 mg/kg with MFQ 48 mg/kg, there was no parasitaemia until 24 days but 2 out of 5 mice developed a low level of parasitaemia on day 28. In the drug groups given MFQ or CLTR, most of the treated mice had developed infection on day 4. Further, lower doses of CLTR i.e.75 and 150 mg/kg combined with MFQ at 16, 32 and 48 mg/kg showed complete suppression of parasitaemia up to day 10 while from day 14 onwards, a low level of parasitaemia appeared, as shown in Table 2. Nevertheless, judging from percentage cure on day 28 in MFQ alone groups which was very low (ranging from 0 to 40%) while the combination groups of MFQ and CLTR showed better protection which ranged from 40 to 100%.
Table 2. Prophylactic response of mefloquine and clarithromycin against MDR Plasmodium yoelii nigeriensis in Swiss mice
Number of positive mice/number of total live mice on particular day is given in parentheses.
* Clarithromycin was given in 2 divided doses.
Therapeutic anti-malarial effect
Mice were infected with P. yoelii nigeriensis and treatment was started on day 1 after confirming the average parasitaemia which ranged from 0·9–2·5%. There was an increasing trend of parasitaemia up to 60 h in all the treated batches. In MFQ/CLTR-treated groups parasitaemia started declining after 60 h. In the combination groups (32+300 and 64+300 mg/kg doses), mice showed clearance of parasitaemia on day 10 whereas at the doses of 150+32 and 64 mg/kg, parasitaemia was cleared on day 14. Parasitaemia in single treatments with CLTR or MFQ showed considerable suppression. Follow-up of parasitaemia in the treated mice showed complete clearance in all the combination-treated groups. Amongst the monotherapy with CLTR at 300 mg/kg, parasitaemia was cleared by day 28. Since this parasite is resistant to MFQ, a low level of parasitaemia persisted in the MFQ alone treatment group beyond 28 days. Finally all 4 batches treated with the CLTR and MFQ combination showed enhanced anti-malarial action as reflected by their 100% clearance of parasitaemia (Table 3).
Table 3. Therapeutic response of mefloquine and clarithromycin against MDR Plasmodium yoelii nigeriensis in Swiss mice
Treatment was started at 24 h post-infection.
**Clarithromycin was given in 2 divided doses.
Anti-malarial interaction between MFQ and CLTR
The anti-malarial interaction of MFQ in combination with CLTR was assessed against P. yoelii nigeriensis infection in Swiss mice. The median (range) sums of the FICs for the anti-malarial interaction between MFQ and CLTR were <0·66 for prophylactic treatment and for therapeutic treatment it was <0·85. The sums of the FICs of <1·0 in both the experiments indicated synergistic interaction between the two drugs (Table 4).
Table 4. Median (range) sum of FICs for the interaction of mefloquine – clarithromycin against Plasmodium yoelii nigeriensis MDR.
Maximum tolerated dose i.e. 300 mg/kg×4 days and 400 mg/kg×7days were given for mefloquine and clarithromycin respectively. FICs- Synergy<1; addtivity 1; antagonism>1.
The present study also reveals that the doses of both drugs administered orally were safe and did not produce any apparent side effects.
DISCUSSION
Our present study has shown the enhanced anti-malarial effect of a conventional anti-malarial MFQ with a new macrolide CLTR.
CLTR has recently been reported to exhibit in vitro anti-malarial activity against the chloroquine-sensitive strain of P. falciparum (3D7) and the chloroquine-resistant strain P. falciparum K1, and the IC50 was reported to be >10μ m for both the strains (Wisedpanichkij et al. Reference Wisedpanichkij, Chaijaroenkul, Sangsuwan, Tantisawat, Boonprasert and Bangchang2009). This antibiotic is specifically useful for treatment of Mycobacterium avium and M. intracellulare (atypical mycobacteria) with a dose schedule of 500 mg twice daily for 7 days. Its bioavailability is nearly 50% by the oral route and is the preferred treatment for children. This is preferred for oral use because of its basic stability in the stomach and its ability to achieve high therapeutic levels (Pai et al. Reference Pai, Graci and Amesden2000). CLTR is reported to be metabolized to 4-hydroxy CLTR, which has twice as active anti-bacterial profile compared to its parent drug. The half- life of CLTR has been reported to be 3–7 h while that of its active metabolite is nearly 7 h (Malhotra-Kumar et al. Reference Malhotra-Kumar, Lammens, Coenen, Herck and Goossens2007). This antibiotic has activity against the helminths (Echinococcus multilocularis) and Acanthamoeba castellanii (Mathis et al. Reference Mathis, Wild, Deplazes and Boettger2004, Reference Mathis, Wild, Boettger, Kapel and Deplazes2005).
After developing resistance against chloroquine and fansidar, MFQ was the only mainstay for P. falciparum treatment. During the last 2 decades there have been numerous reports of emergence of MFQ resistance in Southeast Asia (Price et al. Reference Price, Nosten, Luxemburger, Kham, Brockman, Chongsuphajaisiddhi and White1995) and Nosten et al. Reference Nosten, Kuile, Chongsaphajaisiddhi, White, Kuile, Luxemburger, Webster, Edstein, Phajpun, Thew and White1991 had reported nearly 50% MFQ resistance in P. falciparum in Thailand. This drug was considered as the backbone for anti-malarial operation because of its high level of activity and prolonged period of plasma clearance (more than 42 days). In view of the emergence of resistance towards this drug the World Health Organization (2004, 2006) proposed new drug combinations to prolong the protective life of this drug but MFQ in combination with artesunate or artemether could not produce 100% cure (Price et al. Reference Price, Nosten, Luxemburger, Kham, Brockman, Chongsuphajaisiddhi and White1995; Looareesuwan et al. Reference Looareesuwan, Wilariatana, Viravan, Vanijanonta, Pitisutithum and Kyle1997).
Preliminary studies reported earlier suggested that it could be possible to reverse MFQ resistance of P. falciparum. Kyle et al. (Reference Kyle, Milhous and Odula1988) and Odoula et al. (1993) reported that MFQ resistance of P. falciparum could be partly reversed in vitro by reversal agents such as penfluridol. Peters and Robinson (Reference Peters and Robinson1991) reported that MFQ resistance could be reversed in rodents through penfluridol. Cyproheptadine and its derivatives had also been reported to reverse this resistance of P. falciparum in vitro (Baldwin et al. Reference Baldwin, Gabriel, Friedman and Remy1991). Further studies on MFQ resistance reversal by Frederich et al. (Reference Frederich, Hayette, Tits, Mol and Angenot2001) showed the resistance reversal action of icajine against MFQ-resistant P. falciparum in vitro. Icajine was isolated as a menoindole alkaloid from the plant Strychnous myrtoides.
Ketoconazole pharmacokinetic studies had indicated that this anti-fungal had shown inhibition of cytochrome P450 3A, which inhibited the biotransformation of MFQ into 2 major metabolites namely carboxy MFQ and hydroxyl MFQ. The authors had proposed that this inhibition led to the enhanced bioavailability of MFQ resulting in increasing the plasma levels of active drug MFQ through slowing down its conversion to its metabolites. Cytochrome 3A4 inhibitor ketoconazole was reported to potentiate the action against MFQ resistance of the malaria parasite (Awasthi et al. Reference Awasthi, Dutta, Bhakuni and Tripathi2004).
Earlier studies had also reported that a combination of γ-interferon and MFQ was able to partially reverse the MFQ resistance of MDR P. yoelii nigeriensis, and this combination was reported to give 62% protection compared to MFQ alone which protected 25% in mice (Dhawan et al. Reference Dhawan, Awasthi, Tripathi, Puri and Dutta2000). The resistance reversal effect of NP30 (an uncharged poly-ethoxylated non-phenol surfactant) against MFQ resistance of P. falciparum had been documented in an in vitro study (Ciach et al. Reference Ciach, Zong, Kain and Crandall2003). In a subsequent study, the resistance reversal action of ketoconazole, a cytochrome P450 inhibitor, against MFQ-resistant simian malaria P. knowlesi strain W1 had been established (Tripathi et al. Reference Tripathi, Awasthi and Dutta2005). The strain W1 of P. knowlesi has innate resistance against MFQ up to a total dose of 80–160 mg/kg administered over 3–4 days. Combined treatment with 75 mg/kg ketoconazole for 10 days, together with a low dose of MFQ (20 mg/kg×4 days), cured the infection in rhesus monkeys. It was reported that ketoconazole besides having a suppressive anti-malarial action against P. knowlesi, was also effective in reversing the MFQ resistance of the parasite resulting in the curative effect of the combination.
Wisedpanichkij et al. (Reference Wisedpanichkij, Chaijaroenkul, Sangsuwan, Tantisawat, Boonprasert and Bangchang2009) studied the in vitro anti-malarial activity of Cyp 3A4 inhibitors namely ketoconazole and CLTR against P. falciparum K1 and 3D7 strains and demonstrated that both the Cyp 3A4 inhibitors showed anti-malarial activity as demonstrated by their IC50 of 3·8 and 4·8μ m for ketoconazole and >10 μ m for CLTR. As compared to these Cyp inhibitors, MFQ tested in vitro showed IC50 8·6 and 12·1 nm against P. falciparum K1 and 3D7 isolates respectively. They provided preliminary in vitro evidence to support the concept that Cyp inhibitors could have a role in reversing MFQ resistance of P. falciparum also. They supported our earlier finding that ketoconazole – a Cyp 3A4 inhibitor could provide a higher concentration of MFQ in plasma due to inhibition of metabolism of MFQ to carboxy MFQ (inactive metabolite) (Fontaine et al. Reference Fontaine, Sousa, Burcham, Duchene and Rahmani2000). Ridtitid et al. (Reference Ridtitid, Wongnawa, Mahatthanatrakul, Raungsri and Sunbhanich2005) had also reported an increased plasma concentration of MFQ in healthy human volunteers administered MFQ together with ketoconazole.
The present paper deals with the anti-malarial action of a new macrolide antibiotic CLTR, an inhibitor of hepatic microsomal Cyp450 enzyme (Brophy et al. Reference Brophy, Isreal, Pastor, Gillotin, Chittick, Symonds, Lou, Sadler and Polk2000; Westphal, Reference Westphal2000; Suzuki et al. Reference Suzuki, Ilda, Hirota, Akimoto, Higuchi, Suwa, Tani, Ishizaki and Chiba2003; Zhou, Reference Zhou2008) that was evaluated in combination with MFQ to assess the reversal of MFQ resistance in P. yoelii nigeriensis in vivo. The anti-malarial action of CLTR has been reported earlier by Wisedpanichkij et al. (Reference Wisedpanichkij, Chaijaroenkul, Sangsuwan, Tantisawat, Boonprasert and Bangchang2009) against P. falciparum with IC50 of >10 μ m. Based on in vitro results of the combination of MFQ and ketoconazole (Wisedpanichkij et al. Reference Wisedpanichkij, Chaijaroenkul, Sangsuwan, Tantisawat, Boonprasert and Bangchang2009) and in vivo results of P. yoelii nigeriensis (Awasthi et al. Reference Awasthi, Dutta, Bhakuni and Tripathi2004) and P. knowlesi (Tripathi et al. Reference Tripathi, Awasthi and Dutta2005), Wisedpanichkij et al. (Reference Wisedpanichkij, Chaijaroenkul, Sangsuwan, Tantisawat, Boonprasert and Bangchang2009) speculated that being a cyp inhibitor ketoconazole could exert a synergistic effect when tested in combination with MFQ. They also postulated that cyp 3A4 inhibitors, in general, could have a role in reversing MFQ resistance.
The present study also reports that CLTR can exert a curative action against MDR/MFQ-resistant P. yoelii nigeriensis at 400 mg/kg dose×7days schedule. The lower dose 250 mg/kg×7 days produces a suppressive effect with 80% cure. When MFQ was combined with CLTR, a synergistic anti-malarial effect and complete suppression of parasitaemia were observed up to 10 days. Further studies with MFQ 48 mg/kg combined with CLTR 150 mg/kg showed synergistic action as these combinations completely eliminated the P. yoelii nigeriensis parasitaemia beyond 24 days, though trace parasitaemia was observed. When a higher dose of CLTR (225 mg/kg) was combined with a lower dose of MFQ (32 mg/kg), there was complete cure beyond 28 days, indicating synergistic and curative actions of both these drugs in combination.
In order to monitor the therapeutic response of the MFQ and CLTR combination, the treatment was started 24 h after infection in mice, and doses of CLTR 300 mg/kg combined with MFQ at 32 and 64 mg/kg×4 days, the parasitaemia was completely suppressed by day 10 post-infection, and this combination completely cured P. yoelii.nigeriensis infection in both groups beyond 28 days. Even the lower doses of CLTR 150 mg/kg with 32 or 64 mg/kg of MFQ resulted in complete suppression of parasitaemia beyond 14 days of observation.
This study has established that MFQ and CLTR alone exert only suppressive effects on MFQ-resistant P. yoelii.nigeriensis. However, a combination of CLTR 150–300 mg/kg with MFQ 32–64 mg/kg exerts a synergistic effect, resulting in clearing parasitaemia beyond 14 days, and survival of all the treated mice.
The new macrolide CLTR exerts a resistance reversal effect on the MFQ-resistant P. yoelii nigeriensis infection. The data establish that CLTR, being a Cyp3A4 inhibitor, exerts an inhibitory effect on MFQ metabolism to carboxyMFQ. The MFQ-CLTR combination seems to produce significant pharmacokinetic (metabolic) drug interaction leading to a higher serum concentration of MFQ (Ridtitid et al. Reference Ridtitid, Wongnawa, Mahatthanatrakul, Raungsri and Sunbhanich2005). The CLTR–MFQ combination seems to slow down the metabolic conversion of MFQ to carboxy MFQ (inactive), thus helping to maintain a higher concentration of MFQ. These findings suggest that MFQ + CLTR combination therapy could provide elevated levels of MFQ in the blood and it may benefit human anti-malarial treatment of MFQ-resistant P. falciparum infection.
ACKNOWLEDGEMENTS
Authors are thankful to the Director, C.D.R.I. and Head, Division of Parasitology, C.D.R.I. for their continuous support throughout the study. Amber Rizvi and Swaroop K. Pandey acknowledge the financial support from CSIR, New Delhi. The funding for the present study was provided by CSIR-CDRI, India. CDRI communication No. 8065.
