Enantiomerically pure amino-alcohol quinolines: in vitro anti-malarial activity in combination with dihydroartemisinin, cytotoxicity and in vivo efficacy in a Plasmodium berghei mouse model
© Mullié et al.; licensee BioMed Central Ltd. 2014
Received: 31 July 2014
Accepted: 10 October 2014
Published: 16 October 2014
As resistance to marketed anti-malarial drugs continues to spread, the need for new molecules active on Plasmodium falciparum-resistant strains grows. Pure (S) enantiomers of amino-alcohol quinolines previously displayed a good in vitro anti-malarial activity. Therefore, a more thorough assessment of their potential clinical use through a rodent model and an in vitro evaluation of their combination with artemisinin was undertaken.
Screening on a panel of P. falciparum clones with varying resistance profiles and regional origins was performed for the (S)-pentyl and (S)-heptyl substituted quinoline derivatives, followed by an in vitro assessment of their combination with dihydroartemisinin (DHA) on the 3D7 clone and an in vivo assay in a mouse model infected with Plasmodium berghei. Their haemolytic activity was also determined.
A steady anti-malarial activity of the compounds tested was found, whatever the resistance profile or the regional origin of the strain. (S)-quinoline derivatives were at least three times more potent than mefloquine (MQ), their structurally close parent. The in vitro combination with DHA yielded an additive or synergic effect for both that was as good as that of the DHA/MQ combination. In vivo, survival rates were similar to those of MQ for the two compounds at a lower dose, despite a lack of clearance of the parasite blood stages. A 50% haemolysis was observed for concentrations at least 1,000-fold higher than the antiplasmodial IC50s.
The results obtained make those two (S)-amino-alcohol quinoline derivatives good candidates for the development of new artemisinin-based combination therapy (ACT), hopefully with fewer neurologic side effects than those currently marketed ACT, including MQ.
The latest figures on the incidence and mortality of malaria show that, despite progress in the implementation of preventive measures such as insecticide-treated mosquito nets and intermittent preventive treatments, this parasitic disease is still estimated to affect over 207 million people and to account for 627,000 deaths in 2012. The death toll is particularly high in children under five and pregnant women of the World Health Organization (WHO) African region . Even though the proportion of Plasmodium vivax cases rises in certain regions, the vast majority of malaria cases and deaths are due to Plasmodium falciparum infections . This species has elaborated resistance mechanisms against almost all anti-malarial drugs available on the market today . Even artemisinin derivatives have seen their efficacy challenged in Southeast Asia and, more recently, in South America [3, 4], leading WHO to officially recommend the use of artemisinin-based combination therapy (ACT) as first-line treatment of uncomplicated malaria as far back as in 2006 . This recommendation was reiterated in the 2011 WHO Global Plan for artemisinin resistance containment .
The test drugs included DHA, chloroquine (CQ) diphosphate, MQ hydrochloride (Sigma-Aldrich, Saint-Quentin Fallavier, France) and the 4-amino-alcohol quinolines represented in Figure 1. The 4-amino-alcohol quinolines were synthesized following the general procedure described by Jonet et al.. Only their (S) enantiomers were used as they had previously been shown to be more potent than their (R) analogues . All other chemical products were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France), unless stated otherwise.
Plasmodium falciparum culture
Parasites were cultivated in A+ human erythrocytes (2% haematocrit) suspended in RPMI 1640 medium (Invitrogen, Paisley, UK) supplemented with 10% human serum (Abcys S A, Paris, France) and buffered with 25 mM HEPES-25 mM NaHCO3 under controlled atmospheric conditions (10% O2, 5% CO2, and 85% N2) at 37°C with 95% humidity .
Drug sensitivity assay on selected Plasmodium falciparum clones
Susceptibility and geographical origin of the Plasmodium falciparum strains and clones used in this study
From NF54 African strain (MR4: Malaria Research and Reference Reagent Resource centre)
Indochina (from W2 strain)
The 50% inhibitory concentration (IC50) was evaluated using tritiated hypoxanthine . The specific activity of tritiated hypoxanthine is 1 mCi/mL (Perkin-Elmer, Courtaboeuf, France). The IC50 values were evaluated by analysing incorporation of tritiated hypoxanthine according to the concentration by a non-linear regression analysis processing on dose–response curves (RiaSmart, Packard, Meriden, USA). Results were expressed as geometrical average of IC50.
In vitro combination assay
Plasmodium falciparum parasite strain 3D7 was used for this experiment. MQ, the (S)-pentyl and (S)-heptyl derivatives were associated with DHA following the fixed-ratio method of Fivelman et al.. All compounds were solubilized in dimethyl sulphoxide (DMSO) to prepare extemporaneous stock solutions. DHA and either the (S)-pentyl, (S)-heptyl derivative or MQ were then mixed to reach final ratios of 5:0, 4:1, 3:2, 2:3, 1:4, and 0:5. The mixed stock solutions were then diluted in supplemented RPMI medium so as to reach eight times the IC50 previously determined for the 3D7 strain in the most concentrated well for the 5:0 mix of each compound. Serial two-fold dilutions were then carried out in 96-well-plates under a 50 μL final volume to generate a range of six concentrations for each mix. The final concentration of DMSO in the wells never exceeded 0.02% (v/v). A negative control with the same amount of DMSO was performed to check for any potential toxicity. Then, 200 μL of parasitized red blood cells (pRBC) (final parasitaemia of 0.5% and final haematocrit of 2%) were added. After a 48-hour incubation at 37°C with the drugs, growth inhibition was assessed and dose–response curves fitted.
The assessment of drug interaction was based on the calculation of the fractional inhibitory concentrations (FICs) of the two molecules. The FIC was calculated for each association by dividing the IC50 of the drug in the combination by the IC50 of the drug alone. The sum of these two FICs (∑FICs) was calculated to plot isobologram curves [18, 19] using the software R . ΣFICs <1 denote synergism, ΣFICs ≥1 and <2 denote additive interaction, ΣFICs ≥2 and <4 denote slight antagonism, and ΣFICs ≥4 denote marked antagonism. ΣFICs <0.5 indicate substantial synergism .
In vivo assay
The BALB/c female mice used in this assay were four weeks old and pathogen-free (Charles River Laboratories, France). They were housed under standard conditions, with unlimited access to food and water. All experiments adhered to the French guidelines for animal research and were approved by the ethical committee of the Institut de Recherche Biomédicale des Armées - Antenne de Marseille (Number 2007–02). All efforts were made to minimize animal suffering.
The P. berghei ANKA line was graciously provided by Dr Salah Méchéri (Institut Pasteur, Paris, France). Mice (average body weight 16.7 g) were infected with P. berghei parasites by intraperitoneal (IP) inoculation of donor mouse blood diluted in normal saline so as to contain 3.106 infected red blood cells (RBC). Parasitaemia was then monitored regularly with blood smears. The treatment was started when the parasitaemia of receiver mice was greater than 1% (Day 0). Five groups of seven mice were randomly distributed. These groups received a daily IP dose for five days of either MQ at 9 mg/kg or either the (S)-pentyl or (S)-heptyl derivative (hydrochloride salts) at 3 or 9 mg/kg under a volume of 100 μL per 20 g of body weight. A control group (13 mice) received an IP of the vehicle. Afterwards, the parasitic growth was evaluated daily by blood smears and mice were monitored for their survival up to Day 40.
The parasite multiplication rate (PMR) was used to normalize the activity of the studied compounds on the parasite growth (treated), to the natural parasite growth (control) .
Survival curves were used to study the likelihood of death in treated and control mice. They were fitted using GraphPad Prism® software version 5.04 (GraphPad Software Inc, 2012, La Jolla, CA). The survival analysis was performed according the method of Kaplan-Meier, which determines the probability of survival when at least one ‘death’ is recorded .
The assay was adapted from the protocol described by Taniyama et al. . Briefly, human RBC were centrifuged (1,000 g, 10 min), the pellet washed three times with TRIS buffer (TRIS 10 mmol.L−1, NaCl 150 mmol.L−1, pH 7.4) and finally diluted at 0.5% (v/v) in the same buffer. Experiments were conducted in triplicate on 96-well plates. Saponin 1% (m/v) was used as a positive haemolysis control (100% haemolysis), DMSO and TRIS buffer were used as negative controls. Fifteen μL of the tested molecule (concentration range: 120 nmol.L−1 to 500 μmol.L−1) or control solution were added to 235 μL of the 0.5% (v/v) RBC suspension. Plates were incubated at 37°C for 1 hour and then centrifuged (1,000 g, 10 min). Then, 100 μL of supernatant were retrieved from each well for optical density measurement at 405 nm. The percentage of haemolytic activity was calculated using the saponin mean absorbance at 405 nm as the 100% haemolysis value and the negative control absorbance at 405 nm as blank. The drug concentration inducing a 50% haemolysis (HC50) was then deduced from the dose–response curves.
The Pearson’s correlation coefficient between IC50s values obtained on the various strains for CQ, MQ and the (S)-pentyl derivative was calculated and the test for the statistical significance of the obtained value of r subsequently performed. A log-rank test was performed to compare the survival and the delay for parasitic recrudescence curves. A two-way analysis of variance (ANOVA) on treatment and time parameters was performed to compare PMR results, using the R software . A p-value inferior to 0.05 was considered as significant.
In vitro anti-malarial activity against selected Plasmodium falciparum strains/clones
IC 50 s expressed as average (CV%) for CQ, MQ, ( S )-pentyl and ( S )-heptyl derivatives on the selected Plasmodium falciparum strains and clones
These strains were selected to evaluate the efficacy of the (S)-pentyl substituted amino-alcohol quinoline against a variety of resistance profiles both to CQ and MQ and, to a lesser extent, the efficiency of the (S)-heptyl. The clones were also selected to allow for a variety of geographical origins. As similar results were obtained for both derivatives on the first two strains against which the molecules were tested (3D7 and W2), only the (S)-pentyl derivate was thereafter tested on the other strains. The IC50s obtained with this compound ranged from 3.7 nmol.L−1 (FCM29 clone) to 13.9 nmol.L−1 (HB3 clone) and were constantly lower than those observed for CQ and, more interestingly, than those for MQ, their structurally close parent (Table 2). These low IC50s were retained whatever the resistance profile exhibited by the strains/clones or their geographical origin. The correlations between the CQ and (S)-pentyl IC50s and the MQ and (S)-pentyl IC50s were not statistically significant (p = 0.144 and p = 0.0659, respectively). However, CQ and (S)-pentyl IC50s tended to display a negative correlation while MQ and (S)-pentyl IC50s tended to be positively correlated (r = −0.612 and r = 0.724, respectively). Nevertheless, the calculated MQ/(S)-pentyl IC50s ratios varied from 3.5 to 15.9, depending on the strains, giving a clue to an at least partially different mechanism of action for this compound as compared to MQ.
In vitro combination assay
Similar values were obtained for the substituted amino-alcohol quinolines as only the (S)-pentyl/DHA association for which the 3:2 and 2:3 ratios exhibited a ΣFIC superior to 1, as well as the 3:2 ratio for the (S)-heptyl/DHA combination. Otherwise, ΣFICs for the combinations tested fell between 0.5 and 1, similarly to the ΣFICs obtained for the MQ/DHA combination, denoting a synergistic effect at least as good as that of MQ for the new compounds tested.
In vivo anti-malarial activity
Parasite multiplication rate (PMR)
P-values obtained for post-ANOVA two-by-two comparisons on parasite multiplication rates
3 mg kg−1
9 mg kg−1
3 mg kg−1
9 mg kg−1
9 mg kg−1
3 mg kg−1
9 mg kg−1
5.64 10 −5
9 mg kg−1
9.47 10 −6
1.62 10 −7
5.02 10 −5
3.49 10 −7
P-values for differences in survival curves according to the treatment received
3 mg kg−1
9 mg kg−1
3 mg kg−1
9 mg kg−1
9 mg kg−1
3 mg kg−1
9 mg kg−1
3 mg kg−1
9 mg kg−1
As a preliminary way to assess the potential cytotoxicity of the (S)-pentyl and (S)-heptyl derivatives, as they appeared to act on the erythrocytic stage of the parasite cycle, their haemolytic activity was evaluated. The HC50 determined for the (S)-pentyl and (S)-heptyl derivatives were of 302 μmol L−1 (95% confidence interval [301.0-302.8]) and 261 μmol L−1 (95% confidence interval [235.1-287.9]), respectively. Meanwhile, under the same experimental conditions, MQ displayed an HC50 of 203 μmol L−1 (95% confidence interval [197.0-210.0]). Therefore, the least favourable HC50/IC50 ratios calculated for MQ, the (S)-pentyl and (S)-heptyl derivatives would be of 2,057, 21,726 and 18,000, respectively. As regards the haemolytic activity, the (S)-amino-alcohol quinoline derivatives would then have a ten-fold higher safety margin than their MQ parent. An additional point can be made on the safety of use for these products as, in a previous report, the cytotoxicity of MQ and its (R)-amino-alcohol derivatives was evaluated on hepatocellular carcinoma HepG2 cells using the MTT assay . Inhibitory concentrations 50, in this case defined as concentrations of products leaving 50% of viable cells as compared to the control, were calculated after a 24-hour incubation with MQ or the amino-alcohol derivatives. The (R)-heptyl derivative IC50 value was found to be of 16.6 μg mL−1 (39.3 μmol L−1). MQ gave an IC50 value of 11 μg mL−1 (26.5 μmol L−1). The (S)-heptyl derivative, but not the (S)-pentyl one, was tested at the same time and yielded an IC50 of 26.2 μg mL−1/66.5 μmol L−1 (95% confidence interval [19.9-32.6 μg mL−1]/[50.5-82.7 μmol L−1]). The safety indexes of MQ and the (S)-heptyl derivative calculated on this model were of 268 and 5,752, respectively. Based on these results, the enantiomerically pure amino-alcohol quinoline derivatives would be at least as safe to use as MQ.
The (S)-pentyl and (S)-heptyl amino-alcohol quinoline derivatives studied in this work displayed a steady activity against a panel of P. falciparum clones with varying resistance profiles to CQ and MQ and from different regional origins. Three major points in favour of a continuing development of these compounds in preclinical studies were (i) their in vitro combination with DHA that allowed for addition of the effects or synergy, depending on the ratios tested, and displayed an anti-malarial activity as good as that of the DHA/MQ combination; (ii) in vivo survival rates as good as those of MQ, in a mouse-model of P. berghei ANKA infection; and, (iii) HC50s at least 1,000-fold higher than their antiplasmodial IC50s, pointing towards a rather safe use as compared to MQ. Taken together, and even though the precise mechanism of action of these compounds remains to be elucidated, the results obtained here make those two (S)-amino-alcohol quinoline derivatives good candidates for the development of new ACT, hopefully with fewer neurologic side effects than those currently marketed ACT including MQ, their structurally close parent.
The authors thank the DGA (Direction Générale de l’Armement, Ministère de la Défense, France) and ANR Astrid (project ANR-12-ASTR-003) for financial support.
- WHO: World Malaria Report 2013. 2013, Geneva: World Health OrganizationGoogle Scholar
- Worldwide Antimalarial Resistance Network: Malaria Drug Resistance. http://www.wwarn.org/resistance/malaria (accessed July 15, 2014)
- Dondorp AM, Nosten F, Yi P, Das D, Phyo AP, Tarning J, Lwin KM, Ariey F, Hanpithakpong W, Lee SJ, Ringwald P, Silamut K, Imwong M, Chotivanich K, Lim P, Herdman T, An SS, Yeung S, Singhasivanon P, Day NP, Lindegardh N, Socheat D, White NJ: Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med. 2009, 361: 455-467. 10.1056/NEJMoa0808859.PubMed CentralView ArticlePubMedGoogle Scholar
- Pribluda VS, Evans L, Barillas E, Marmion J, Lukulay P, Chang J: Were medicine quality and pharmaceutical management contributing factors in diminishing artemisinin efficacy in Guyana and Suriname?. Malar J. 2014, 13: 77-10.1186/1475-2875-13-77.PubMed CentralView ArticlePubMedGoogle Scholar
- WHO: Guidelines for The Treatment of Malaria. 2006, Geneva: World Health OrganizationGoogle Scholar
- WHO: Global Plan for Artemisinin Resistance Containment. 2011, Geneva: World Health OrganizationGoogle Scholar
- Brocks DR, Mehvar R: Stereoselectivity in the pharmacodynamics and pharmacokinetics of the chiral antimalarial drugs. Clin Pharmacokinet. 2003, 42: 1359-1382. 10.2165/00003088-200342150-00004.View ArticlePubMedGoogle Scholar
- Douglas NM, Nosten F, Ashley EA, Phaiphun L, van Vugt M, Singhasivanon P, White NJ, Price RN: Plasmodium vivax recurrence following falciparum and mixed species malaria: risk factors and effect of antimalarial kinetics. Clin Infect Dis. 2011, 52: 612-620. 10.1093/cid/ciq249.PubMed CentralView ArticlePubMedGoogle Scholar
- Taylor WR, White NJ: Antimalarial drug toxicity: a review. Drug Saf. 2004, 27: 25-61.View ArticlePubMedGoogle Scholar
- Schlagenhauf P, Adamcova M, Regep L, Schaerer MT, Rhein HG: The position of mefloquine as a 21st century malaria chemoprophylaxis. Malar J. 2010, 9: 357-10.1186/1475-2875-9-357.PubMed CentralView ArticlePubMedGoogle Scholar
- Karle JM, Olmeda R, Gerena L, Milhous WK: Plasmodium falciparum: role of absolute stereochemistry in the antimalarial activity of synthetic amino alcohol antimalarial agents. Exp Parasitol. 1993, 76: 345-351. 10.1006/expr.1993.1042.View ArticlePubMedGoogle Scholar
- Dassonville-Klimpt A, Cézard C, Mullié C, Agnamey P, Jonet A, Da Nascimento S, Marchivie M, Guillon J, Sonnet P: Absolute configuration and antimalarial activity of erythro-mefloquine enantiomers. ChemPlusChem. 2013, 7: 642-646.View ArticleGoogle Scholar
- Shepherd J: Use of (+) Mefloquine for the Treatment of Malaria. 1998, International patent WO98/39003Google Scholar
- Jonet A, Dassonville-Klimpt A, Da Nascimento S, Léger JM, Guillon J, Sonnet P: First enantioselective synthesis of 4-aminoalcohol quinoline derivatives through a regioselective SN2 epoxide opening mechanism. Tetrahedron Asymmetry. 2011, 22: 138-148. 10.1016/j.tetasy.2011.01.003.View ArticleGoogle Scholar
- Mullié C, Jonet A, Desgrouas C, Taudon N, Sonnet P: Differences in anti-malarial activity of 4-aminoalcohol quinoline enantiomers and investigation of the presumed underlying mechanism of action. Malar J. 2012, 11: 65-10.1186/1475-2875-11-65.PubMed CentralView ArticlePubMedGoogle Scholar
- Desjardins RE, Canfield CJ, Haynes JD, Chulay JD: Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother. 1979, 16: 710-718. 10.1128/AAC.16.6.710.PubMed CentralView ArticlePubMedGoogle Scholar
- Lambros C, Vanderberg JP: Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol. 1979, 65: 418-420. 10.2307/3280287.View ArticlePubMedGoogle Scholar
- Fivelman QL, Adagu IS, Warhurst DC: Modified fixed-ratio isobologram method for studying in vitro interactions between atovaquone and proguanil or dihydroartemisinin against drug-resistant strains of Plasmodium falciparum. Antimicrob Agents Chemother. 2004, 48: 4097-4102. 10.1128/AAC.48.11.4097-4102.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Berenbaum MC: A method for testing synergy with any number of agents. J Infect Dis. 1978, 137: 122-130. 10.1093/infdis/137.2.122.View ArticlePubMedGoogle Scholar
- R Develoment Core Team: R: A Language and Environment for Statistical Computing. http://r-project.org (accessed November 2013)
- Douglas AD, Andrews L, Draper SJ, Bojang K, Milligan P, Gilbert SC, Imoukhuede EB, Hill AV: Substantially reduced pre-patent parasite multiplication rates are associated with naturally acquired immunity to Plasmodium falciparum. J Infect Dis. 2011, 203: 1337-1340. 10.1093/infdis/jir033.PubMed CentralView ArticlePubMedGoogle Scholar
- Collett D: Modelling Survival Data in Medical Research. 1994View ArticleGoogle Scholar
- Taniyama S, Arakawa O, Terada M, Nishio S, Takatani T, Mahmud Y, Noguchi T: Ostreopsis sp., a possible origin of palytoxin (PTX) in parrotfish Scarus ovifrons. Toxicon. 2003, 42: 29-33. 10.1016/S0041-0101(03)00097-7.View ArticlePubMedGoogle Scholar
- Gupta S, Thapar MM, Wernsdorfer WH, Björkman A: In vitro interactions of artemisinin with atovaquone, quinine, and mefloquine against Plasmodium falciparum. Antimicrob Agents Chemother. 2002, 46: 1510-1515. 10.1128/AAC.46.5.1510-1515.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Kerschbaumer G, Wernsdorfer G, Wiedermann U, Congpuong K, Sirichaisinthop J, Wernsdorfer WH: Synergism between mefloquine and artemisinin and its enhancement by retinol in Plasmodium falciparum in vitro. Wien Klin Wochenschr. 2010, 122 (Suppl 3): 57-60.View ArticlePubMedGoogle Scholar
- Sowunmi A, Gbotosho GO, Happi C, Okuboyejo T, Folarin O, Balogun S, Michael O: Therapeutic efficacy and effects of artesunate-mefloquine and mefloquine alone on malaria-associated anemia in children with uncomplicated Plasmodium falciparum malaria in southwest Nigeria. Am J Trop Med Hyg. 2009, 81: 979-986. 10.4269/ajtmh.2009.09-0394.View ArticlePubMedGoogle Scholar
- Cerutti C, Durlacher RR, de Alencar FE, Segurado AA, Pang LW: In vivo efficacy of mefloquine for the treatment of Falciparum malaria in Brazil. J Infect Dis. 1999, 180: 2077-2080. 10.1086/315141.View ArticlePubMedGoogle Scholar
- Jonet A, Dassonville-Klimpt A, Sonnet P, Mullié C: Side chain length is more important than stereochemistry in the antibacterial activity of enantiomerically pure 4-aminoalcohol quinoline derivatives. J Antibiot (Tokyo). 2013, 66: 683-686. 10.1038/ja.2013.71.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.