# The in vivo antimalarial activity of methylene blue combined with pyrimethamine, chloroquine and quinine

Giovanny Garavito, Stephane Bertani, Miguel Quiliano, Alexis Valentin, Ignacio Aldana, Eric Deharo

<figure><img src="image_1.png" /></figure>

## To cite this version:

Giovanny Garavito, Stephane Bertani, Miguel Quiliano, Alexis Valentin, Ignacio Aldana, et al.. The in vivo antimalarial activity of methylene blue combined with pyrimethamine, chloroquine and quinine. Memórias do Instituto Oswaldo Cruz, 2012, 107 (6), pp.820-823. 10.1590/s0074-02762012000600019 . hal-03572729

HAL Id: hal-03572729

https://ut3-toulouseinp.hal.science/hal-03572729

Submitted on 14 Feb 2022

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# The in vivo antimalarial activity of methylene blue combined with pyrimethamine, chloroquine and quinine

Giovanny Garavito<fnref n="1" />/+ , Stéphane Bertani<fnref n="2" />,<fnref n="3" /> , Miguel Quiliano<fnref n="4" /> ,
Alexis Valentin<fnref n="2" />, Ignacio Aldana<fnref n="4" />, Eric Deharo<fnref n="2" />,<fnref n="5" />

The effectiveness of methylene blue (MB) combined with pyrimethamine (PYR), chloroquine (CQ) or quinine (Q) was examined in a classical four-day suppressive test against a causative agent of rodent malaria, Plasmodium berghei. A marked potentiation was observed when MB was administered at a non-curative dose of 15 mg/kg/day in combination with PYR (0.19 mg/kg/day) or Q (25 mg/kg/day). No synergy was found between MB (15 mg/Kg) and CQ (0.75 mg/Kg). Our results suggest that the combination of MB with PYR or Q may improve the efficacy of these currently used antimalarial drugs.

Key words: antimalarials - methylene blue - drug combination

With almost 3.3 billion people living in endemic ar-
eas, malaria remains a major global health issue (WHO
2011). In response to the emergence of resistance strains
of malaria, new antimalarials that are combinable with
classical treatments are urgently needed (WHO 2001,
2010). Methylene blue (MB) is the oldest and least expen-
sive drug ever used in antimalarial therapy and has been
proven safe and effective in patients (Guttmann & Ehrlich
1891, Ferreira 1893, Meissner et al. 2005, 2006). In vitro
studies showed that the antiplasmodial activity of MB
could be enhanced when this drug was combined with ad-
ditional antimalarials (Vennerstrom et al. 1995, Akoach-
erre et al. 2005, Garavito et al. 2007a). In many countries,
MB has already been approved by regulatory agencies for
the treatment of methemoglobinaemia and for ifosfamide-
induced encephalopathy in cancer treatment (Schirmer et
al. 2003, Mandi et al. 2005). Thus, MB could easily be
reintroduced into antimalarial therapeutic schemes.

The purpose of our study was to determine wheth-
er MB in combination with the low-cost drug py-
rimethamine (PYR) is able to inhibit parasitaemia in
rodent malaria models and to compare the effectiveness
of this treatment with that of combinations of MB with
quinine (Q) or chloroquine (CQ) diphosphate.

The four-day suppressive test for the quantitative as-
sessment of blood schizontocidal activity was performed
according to the method of Peters and Robinson (1999).
Male Swiss albino mice (Bioterio DFUNC) were inocu-
lated with 10⁷ Plasmodium berghei ANKA-parasitised
red blood cells (RBCs). Batches of 10 animals per group
were then randomly distributed after infection and orally

treated (0.1 mL) at the same time daily for four consecu-
tive days beginning on the day of infection. A control
group received 0.9% saline solution and a reference group
was administered CQ orally at 3 mg/kg/day. The percent-
age of parasitised erythrocytes was determined in Giem-
sa-stained blood smears made from peripheral blood on
day four post-infection. The percent inhibition of parasi-
taemia was calculated by the following formula:

(parasitaemia in control - parasitaemia in drug-treated) x 100

parasitaemia in control

The ED₅₀ and ED₉₀ values were estimated with a non-linear regression logistic dose-response model. For the interaction study, batches of five animals (infected as described above) were orally treated for four consecutive days with the individual drugs at two different doses and with the combination of MB (15 mg/Kg) and PYR (0.19 mg/Kg), Q (25 mg/Kg) or CQ (0.75 mg/Kg). Statistical analysis was performed with GraphPad Prism 5.01 software (GraphPad Software, San Diego).

All drugs were obtained from Sigma-Aldrich (L'Isle
d'Abeau Chesnes, France) and dissolved in water. The
animals were treated according to the French and Co-
lombian legislation on laboratory animal use and care.

The results are reported in Table and Figure. PYR
was the most active compound, with an ED₅₀ less than
half that of CQ and almost 150 times lower than that of
Q. Under these conditions, Q showed an ED₉₀ close to the
116 mg/Kg found by Peters (1987). In a previous study,
we obtained an equivalent ED₅₀ for CQ against P. ber-
ghei ANKA of 0.53 mg/Kg (Garavito et al. 2008). The
ED₉₀ of CQ reported in Table was similar to the ED₉₀ of
3.3 mg/Kg previously demonstrated (Peters 1987). The
combination of 15 mg/Kg of MB with 0.75 mg/Kg of
CQ did not reveal any interaction (data not shown), in
accordance with the in vitro data recorded previously
(Garavito et al. 2007a).

<footnote n="1">¹Departamento de Farmacia, Facultad de Ciencias, Universidad Nacional de Colombia, Bogotá, Colombia</footnote>

<footnote n="2">²Pharma-Dev UMR 152, Faculté de Pharmacie, Université de Toulouse, Université Paul Sabatier, Toulouse, France</footnote>

<footnote n="3">³Pharma-Dev UMR 152, Institut de Recherche pour le Développement, Lima, Peru</footnote>

<footnote n="4">⁴Neglected Diseases Section, Drug R&D Unit, Center for Applied Pharmacobiology Research, University of Navarra, Pamplona, Spain</footnote>

<footnote n="5">⁵Pharma-Dev UMR 152, Faculté de Pharmacie, Institut de Recherche pour le Développement, Toulouse, France</footnote>

<footnote>Financial support: DIB/UNC, IRD (France)
+ Corresponding author: ggaravitoc@unal.edu.co
Received 25 January 2012
Accepted 29 May 2012</footnote>
The combination of 15 mg/Kg of MB with 0.19 mg/Kg of PYR resulted in an inhibition of parasitaemia of 78.7% ± -11.1%, displaying an activity superior to that expected if the effect of the two drugs was additive (44.1% inhibition). In vitro drug interaction experiments with the FcM29 strain of *Plasmodium falciparum* showed an antagonist effect when MB was combined with PYR and an antagonistic interaction was also described using the K1 strain of *P. falciparum* (Akoachere et al. 2005, Garavito et al. 2007a, Garavito 2007).

Combining 15 mg/Kg of MB with 25 mg/Kg of Q led to an inhibition of 78.8% ± 9.7%. This activity was greater than expected if the effect of both drugs was additive (63% inhibition). We reported a synergistic effect between MB and Q used against the CQ-resistant *P. falciparum* strain FcM29 in vitro (Garavito et al. 2007a, Garavito 2007), whereas Akoachere et al. (2005) found additivity in a CQ-resistant *P. falciparum* K1 strain.

The antimalarial activity of MB has been attributed to antioxidant defences that specifically target glutathione reductase (GR), a crucial enzyme for *P. falciparum*.

*parum* (Ginsburg & Atamna 1994, Atamna et al. 1996, Atamna & Ginsburg 1997, Färber et al. 1998), or inhibit the glutathione-dependent degradation of haeme (Garavito et al. 2007b). In a previous study, we demonstrated that MB inhibited almost 60% of parasite growth during the late ring/early trophozoite stages, when intense digestion of haemoglobin occurs; this inhibition of parasite growth correlates with a peak expression of plasmodial GR at the 24th h of the intraerythrocytic cycle (PlasmoDB 2005, Garavito et al. 2007a). MB reaches maximum plasma concentrations 1-2 h following oral administration at a dose of 100 mg in humans. Its plasmatic half-life is approximately 5-6.5 h and renal excretion is low, at approximately 20-30%. The rapid antimalarial effect of MB, combined with its pharmacokinetic properties, including a short half-life, might promote the slow development of resistance because exposure of parasites to sub-therapeutic levels of the drug would be very brief; this effect has been shown to occur with artemisinin.

Q is relatively inexpensive and remains the preferred anti-malarial drug for the treatment of severe and complicated malaria in most African countries. Although Q resistance is not yet a significant problem, drug failure may occur as a result of poor compliance with the prolonged treatment course and problems with tolerability. An alternative is the development of shorter-course treatments in combination with therapies that have the potential to improve compliance and treatment outcomes (Achan et al. 2011). To treat malaria in the first trimester of pregnancy, the WHO recommends the use of Q plus clindamycin; unfortunately, this combination is too expensive for most affected people (Achan et al. 2011). Q is quickly absorbed after oral or parenteral administration, readily crossing the placental barrier and detectable in cerebral spinal fluid. Q has a half-life of between 9.7-18 h (3.2 h in healthy paediatric subjects and 12.1 h in patients infected with malaria). We have previously shown that Q targeted the late ring/early trophozoite stages, when digestion of haemoglobin occurs, but had a greater impact on the haeme-glutathione interaction than on haemozoin formation (Garavito 2007, Garavito et al. 2007b).

Although the combination of Q with MB was effective in our experiment, as expected, this combination has some drawbacks that preclude its therapeutic use, such as the fact that both drugs target similar developmental stages of the parasite with similar mechanisms of action. Further, both drugs exhibit similarly short half-lives, requiring multiple administrations, which can lead to poor compliance. Finally, Q is approximately 10 times more expensive than PYR or MB.

PYR was discovered more than 60 years ago as a result of an intense antimalarial research program conducted by the United States army to find an alternative to Q during World War II (Plowe 2001). PYR has been used for decades because of its high therapeutic index and very low cost. Unfortunately, used alone, PYR generates a high rate of resistance and must therefore be combined with another antimalarial. It has been hypothesised that PYR's mechanism of action mainly involves its interference in folate metabolism through the inhibition of dihydrofolate reductase (DHFR) (Peterson & Wellems 1988).

<caption>TABLE</caption>

Dose of tested drugs able to inhibit 50% or 90% of *Plasmodium berghei* growth

<table><thead><tr><th>Drug</th><th><i>P. berghei</i> ED<sub>50</sub><br>(mg/kg/day)</th><th><i>P. berghei</i> ED<sub>90</sub><br>(mg/kg/day)</th></tr></thead><tbody><tr><td>Methylene blue</td><td>12.80</td><td>45.77</td></tr><tr><td>Chloroquine</td><td>0.53</td><td>2.04</td></tr><tr><td>Pyrimethamine</td><td>0.19</td><td>4.05</td></tr><tr><td>Quinine</td><td>33.57</td><td>93.55</td></tr></tbody></table>

<figure><img src="image_3.png" /></figure>

In vivo combination study of methylene blue (MB) with pyrimethamine (PYR) and quinine (Q). The upper part of the figure shows the combinations, the lower part shows the drug alone at two concentrations. Results show means standard deviation. ED<sub>50</sub>, ED<sub>90</sub>: dose that inhibits 50% or 90% of parasite growth; *: expected inhibition if both drugs were additive (white bars); **: p-value comparing MB and MB + PYR < 0.0001; ***: p-value comparing MB and MB + Q < 0.0001.
Recently, Legorreta-Herrera et al. (2010) provided new details on the effect of PYR by highlighting its production of free radicals in rodent models of malaria. These authors hypothesised that PYR induces oxidative stress in *Plasmodium yoelii*-infected RBCs; in support of this proposed mechanism, increased redox enzyme activities (superoxide dismutase and glutathione peroxidase) were demonstrated in both erythrocytes and the liver. PYR also increases nitric oxide levels in the serum and upregulates mRNA concentrations of superoxide dismutase, glutathione peroxidase, catalase and iNOS in the spleen. PYR is also well absorbed when administered orally, reaching maximum plasma concentrations 2-6 h after administration and exhibiting a plasma half-life of 80-96.5 h.

A rapid analysis of the structure-activity relationship between PYR and MB emphasises the complementary and synergic mechanisms of action of these drugs. PYR is a pyrimidine compound that interacts with the active site of the DHFR protein, establishing hydrogen bond interactions and the number of flexible bonds plays a crucial role in drug resistance (Yuvaniyama et al. 2003). MB is a phenothiazine compound that exhibits a planar geometry in its “blue form” and a partial planar geometry, uncharged and colourless, in its “Leuco form”. To date, crystallographic data for the MB-GR complex have not been reported, although due to a high number of aromatic and non-polar residues in the region, stacking-π and van der Waals interactions occur in the interdimeric cavity of GR (Sarma et al. 2003, Schirmer et al. 2011). From a chemical research standpoint, fragment-base discovery is an exciting and novel method of lead generation (Warr 2009). Fragments are small, low-molecular-weight molecules that usually form part of a drug compound. Based on fragment-base discovery, the pyrimidine-diamine system of PYR may be found to serve as an effective scaffold upon which to build further antimalarial drug development. In addition, both phenothiazine and quinoline templates have been promoted as “privileged structures” that can help in the modification and improvement of existing active molecules (Wermuth 2004).

MB has been reported not only to exhibit non-competitive inhibitor, but also as a substrate reduced by flavoenzymes that produce reactive oxygen species (Buchholz et al. 2008). The complementary mechanisms of PYR and MB seem clear; both compounds generate oxidative stress, but in different ways, whereas MB also induces indirect structural changes in the interdimeric region of GR. Both MB and PYR target the crucial redox machinery of *Plasmodium*, but at two different stages of the intraerythrocytic development: PYR acts against young schizonts (Maerki et al. 2006), whereas MB is effective against the late ring/early trophozoite stages (Garavito 2007). Therefore, in theory, the effects of these drugs should be synergistic. This premise is corroborated by the experimental data obtained herein. Moreover, the effective therapeutic half-life of MB is much shorter than that of PYR and, consequently, selection for resistance should be less intense. Finally, both compounds are affordable for afflicted populations, with the combination approximately 30 times less expensive than the combination of artesunate and PYR

(Baird 2005). These results confirm that the combination of MB and PYR, two established, reliable and effective molecules, should be considered with renewed interest as a new, low-cost antimalarial therapy.

## ACKNOWLEDGEMENTS

To Pr Irene Landau (MNHN, Paris), for providing *P. berghei* ANKA rodent malaria parasite, and to Joshua L Halford and David Parker Jr, for their critical discussions.

## REFERENCES

Achan J, Talisuna AO, Erhart A, Yeka A, Tibenderana JK, Baliraine FN, Rosenthal PJ, D'Alessandro U 2011. Quinine, an old antimalarial drug in a modern world: role in the treatment of malaria. *Malar J* 10: 144.

Akoachere M, Buchholz K, Fischer E, Burhenne J, Haefeli WE, Schirmer RH, Becker K 2005. *In vitro* assessment of methylene blue on CQ-sensitive and resistant *Plasmodium falciparum* strains reveals synergistic action with artemisinins. *Antimicrob Agents Chemother* 49: 4592-4597.

Atamna H, Ginsburg H 1997. The malaria parasite supplies glutathione to its host cell. Investigation of glutathione transport and metabolism in human erythrocytes infected with *Plasmodium falciparum*. *Eur J Biochem* 250: 670-679.

Atamna H, Krugliak M, Shalmiev G, Deharo E, Pescarmona G, Ginsburg H 1996. Mode of antimalarial effect of methylene blue and some of its analogues on *Plasmodium falciparum* in culture and their inhibition of *P. vinckei* petteri and *P. yoelii* nigeriensis in vivo. *Biochem Pharmacol* 51: 693-700.

Baird JK 2005. Effectiveness of antimalarial drugs. *N Engl J Med* 352: 1565-1577.

Buchholz K, Schirmer RH, Eubel JK, Akoachere MB, Dandekar T, Becker K, Gromer S 2008. Interactions of methylene blue with human disulfide reductases and their orthologues from *Plasmodium falciparum*. *Antimicrob Agents Chemother* 52: 183-191.

Färber PM, Arscott LD, Williams CH, Becker K, Schirmer RH 1998. Recombinant *Plasmodium falciparum* glutathione reductase is inhibited by the antimalarial dye methylene blue. *FEBS Lett* 422: 311-314.

Ferreira 1893. Treatment of malaria with methylene blue. *Lancet*: 821-822.

Garavito G 2007. *Etude pharmacologique expérimentale de l'activité antipaludique d'un composé de synthèse: le chlorure de methylthioninium*, PhD Thesis, Université Toulouse III/Paul Sabatier École Doctorale BSB, IOP Publishing Physics, Toulouse, 155 pp.

Garavito G, Bertani S, Deharo E 2008. Complementary findings on the antimalarial activity and toxicity of methylene blue. *Vitae* 15: 91-95.

Garavito G, Bertani S, Rincon J, Maurel S, Monje MC, Landau I 2007a. *Blood schizontocidal activity of methylene blue in combination with antimalarials against Plasmodium falciparum*. *Parasite* 14: 135-140.

Garavito G, Monje MC, Maurel S, Valentin A, Nepveu F, Deharo E 2007b. A non-radiolabeled heme-GSH interaction test for the screening of antimalarial compounds. *Exp Parasitol* 116: 311-313.

Ginsburg H, Atamna H 1994. The redox status of malaria-infected erythrocytes: an overview with an emphasis on unresolved problems. *Parasite* 1: 5-13.

Guttmann P, Ehrlich P 1891. Guttmann ueber die wirkung des methylenblau bei malaria. *Berlin Klin Wochenschr* 28: 953-956.
Legorreta-Herrera M, Retana-Ugalde R, Ventura-Gallegos JL, Narváez V 2010. Pyrimethamine induces oxidative stress in *Plasmodium yoelii* 17XL-infected mice: a novel immunomodulatory mechanism of action for an old antimalarial drug? *Exp Parasitol* 126: 381-388.

Maerki S, Brun R, Charman SA, Dorn A, Matile H, Wittlin S 2006. *In vitro* assessment of the pharmacodynamic properties and the partitioning of OZ277/RBx-11160 in cultures of *Plasmodium falciparum*. *J Antimicrob Chemother* 58: 52-58.

Mandi G, Witte S, Meissner P, Coulibaly B, Mansmann U, Rengelshausen J 2005. Safety of the combination of chloroquine and methylene blue in healthy adult men with G6PD deficiency from rural Burkina Faso. *Trop Med Int Health* 10: 32-38.

Meissner PE, Mandi G, Coulibaly B, Witte S, Tapsoba T, Mansmann U 2006. Methylene blue for malaria in Africa: results from a dose-finding study in combination with chloroquine. *Malar J* 5: 84.

Meissner PE, Mandi G, Witte S, Coulibaly B, Mansmann U, Rengelshausen J 2005. Safety of the methylene blue plus chloroquine combination in the treatment of uncomplicated *falciparum* malaria in young children of Burkina Faso. *Malar J* 4: 45-54.

Peters W 1987. *Chemotherapy and drug resistance in malaria*, 2nd ed., Academic Press, London, 1100 pp.

Peters W, Robinson BL 1999. *Handbook of animal models of infection*, Academic Press, New York, 1136 pp.

Peterson DW, Wellems TE 1988. Evidence that a point mutation in dihydrofolate reductase-thymidylate synthase confers resistance to pyrimethamine in *falciparum* malaria. *Proc Natl Acad Sci USA* 85: 9114-9118.

PlasmoDB 2005. [database on the Internet]. The *plasmodium* genome resource. Expression of plasmodial glutathione reductase. [cited 2005 Nov 17]. Available from: plasmodb.org/.

Plowe C 2001. Folate antagonists and mechanisms of resistance. In PJ Rosenthal, *Antimalarial chemotherapy: mechanisms of action*,

*resistance, and new directions in drug discovery*, Humana Press Inc, New Jersey, p. 173.

Sarma GN, Savvides SN, Becker K, Schirmer M, Schirmer RH, Karplus PA 2003. Glutathione reductase of the malarial parasite *Plasmodium falciparum*: crystal structure and inhibitor development. *J Mol Biol* 328: 893-907.

Schirmer RH, Adler H, Pickhardt M, Mandelkow E 2011. "Lest we forget you - methylene blue...". *Neurobiol Aging* 32: 2325.

Schirmer RH, Coulibaly B, Stich A, Scheiwein M, Merkle H, Eubel J 2003. Methylene blue as an antimalarial agent. *Redox Rep* 8: 272-275.

Vennerstrom JL, Makler MT, Angerhofer CK, Williams JA 1995. Antimalarial dyes revisited: xanthenes, azines, oxazines and thiazines. *Antimicrob Agents Chemother* 39: 2671-2677.

Warr WA 2009. Fragment-based drug discovery. *J Comput Aided Mol Des* 23: 453-458.

Wermuth C 2004. Selective optimization of side effects: another way for drug discovery. *J Med Chem* 47: 1303-1314.

WHO - World Health Organization 2001. *Antimalarial drug combination therapy. Report of a WHO technical consultation*, WHO, Geneva, 36 pp.

WHO - World Health Organization 2010. [homepage on the Internet]. WHO guidelines for the treatment of malaria [cited 2011 Jun 11]. Available from: who.int/malaria.

WHO - World Health Organization 2011. [homepage on the Internet]. Malaria. WHO-TDR Special Programme for Research and Training in Tropical Diseases. [cited 2011 March 10]. Available from: apps.who.int/tdr/svc/diseases/malaria.

Yuvaniyama J, Chitnumsub P, Kamchonwongpaisan S, Vanichtanankul J, Sirawaraporn W, Taylor P 2003. Insights into antifolate resistance from malarial DHFR-TS structures. *Nat Struct Biol* 10: 357-365.