Griseofulvin as an adjunct drug for the treatment of cerebral and severe malaria

Abstract

Malaria remains a major concern of morbidity and mortality. Despite treating infected individuals with artemisinin-based combination therapies (ACTs), cerebral and severe malaria cause enormous mortality in children and adults. The only treatment option is parenteral administration of artemisinin or quinine with supportive symptomatic therapies. However, the fatality rate remains high despite parasite clearance emphasizing the need of adjunct therapy to prevent malaria mortality. In this invention, heme synthesized in the malaria parasite is shown to be associated with parasite virulence, disease severity and cerebral pathogenesis. Parasite heme enhances the formation of hemozoin - a parasite molecule associated with cerebral and severe malaria. Griseofulvin prevents experimental cerebral malaria (ECM) by inhibiting parasite heme synthesis and hemozoin formation, without affecting parasite growth. Griseofulvin exhibits synergy with arteether - the principal component of ACTs and prevents ECM. Griseofulvin is claimed as an adjunct drug for ACTs and other antimalarials to prevent malaria mortality.

Description

The following descriptions of embodiments and examples are offered by way of illustration and not by way of limitation. Unless contraindicated or noted otherwise, throughout this specification, the terms “a” and “an” mean one or more, and the term “or” means and / or. As used in the description herein and throughout the claims that follow, the meaning of “a.” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise. Further, it may also be noted that throughout the specification, some spelling of words may be put in American English and also in British English, for example “Fibre” may also be written as “Fiber”, and the likes. No confusion may be there regarding the actual meaning of any such word, differed only in spelling owing to different practices. Mortality in cerebral malaria (CM) and severe malaria remains high, despite parenteral administration of artemisinin derivatives or quinine with supportive symptomatic therapies. The fatality rates do not correlate with parasite clearance and targeting parasite virulence becomes important. The present studies carried out in C57BL / 6 mice using the rodent parasite P. berghei ANKA, a well-established in vivo animal model to study human CM have shown that de novo heme synthesis of malaria parasite is required to develop CM pathogenesis by enhancing hemozoin (Hz) formation. Mice infected with heme pathway knockout (KO) parasites are devoid of cerebral complications and are completely protected from CM. The death due to anemia that happens in the absence of any therapeutic intervention is also delayed in the mice infected with KO parasites. Mice infected with KO parasites show an overall decrease in systemic and neuronal inflammation, and T cell infiltration in the brain milieu. Therefore, inhibitors of the parasite heme-biosynthetic pathway have the potential to prevent cerebral and severe malaria. The technical advance in this invention is that griseofulvin, an FDA-approved antifungal drug, can inhibit parasite heme synthesis and prevent onset of cerebral pathology, although parasite growth is not inhibited. Thus, griseofulvin exhibits synergy with artemisinin derivative - a,P-arteether in terms of protecting mice from cerebral malaria. Therefore, a combination of artemisininbased combination therapies or other antimalarials used as frontline therapies for malaria to inhibit parasite growth, together with griseofulvin as an adjunct drug would serve as a new product and therapeutic combination for preventing cerebral malaria and severe malaria in humans. The present invention explored the possibility of including griseofulvin in the treatment protocol in affected patient population. The only inclusion will be griseofulvin for three days to prevent the development of cerebral and severe malaria. In humans, griseofulvin dosage is given to the extent of 500-1000 mg / day in adults and 5-10 mg / kg / day in children for several weeks. The conversion of 5-10 mg / kg / day into animal dose is carried out by multiplying human dose with 12.3. Taking the average weight of 7-8 weeks mouse as 25 g, this comes to 1.5-3.0 mg per mouse. Therefore, the dose used in this invention for griseofulvin (2 mg per mouse per day) is matching to human dose that has been routinely used for the treatment of fungal infections. It may especially be noted that griseofulvin is not an antimalarial drug, and has no known direct effect against the parasitic growth. However, the present invention suggests prevention of cerebral malaria and disease severity, and a consequent measure to avert mortality caused by malaria that occurs despite treating the patients with ACTs. Further, it must be noted that the existing WHO-approved treatment for malaria to be followed and no changes has been suggested in the dosage or treatment regimen of ACTs. Griseofulvin will be included in the treatment protocol for three days at the same dosage (500-1000 mg / day in adults and 5-10 mg / kg / day for children) that has been used for fungal diseases along with the existing ACTs to prevent cerebral and severe malaria. The treatment protocol suggested in this invention will be the standard ACT that has been given as per the WHO recommendation for malaria patients along with griseofulvin for three days starting from the first day of ACT treatment. There will not be any change in the standard malaria therapy except that the griseofulvin will be included for three days. The following are the ACTs recommended by WHO: 1) artemether / arteether + lumefantrine 2) artesunate + amodiaquine 3) artesunate + mefloquine 4) dihydroartemisinin + piperaquine 5) artesunate + sulfadoxine-pyrimethamine for malaria. Any one of these will be used in malaria endemic regions based on national policies, resistance pattern in that particular region etc. This ACT treatment is given for 3 days. Mice infected with heme pathway KO parasites are protected from CM Growth analyses for the P. berghei KO parasites in CM-susceptible C57BL / 6 inbred mouse strain was carried out by injecting 105 asexual stage parasites. Assessment of the peripheral blood parasitemia showed 2-3 days delay in the growth of KO parasites with respect to the wild-type (WT) parasites. Importantly, about 80% of the WT-infected mice succumbed to CM within day 10 when the blood parasitemia was around 10-30% showing typical symptoms of CM. The WT-infected mice that could escape from CM died of anemia on day 12-16 post-infection. In contrast, all the mice infected with ALASKO and FCKO parasites were protected from CM and they died because of anemia on day 20-30. The delay in the growth of KO parasites was associated with an early increase in the spleen weight of the infected mice suggesting a better splenic clearance. To rule out the possibility of protection from CM is because of a delay in the blood parasitemia increase, growth analyses in C57BL / 6 mice infected with 107 ALASKO and FCKO parasites were performed. While the growth of KO parasites in mice injected with 107 parasites was comparable with 105 WT parasites, mice infected with KO parasites were once again completely protected from CM. The mortality of the KO-infected mice due to anemia was also delayed by almost 6-10 days, and the KO-infected mice could sustain a higher blood parasitemia for a prolonged period. There were no significant differences in the reticulocyte versus mature RBC preference between WT and KO parasites in the first 9 days of infection, the duration in which the CM mortality occurred in WT-infected mice. However, in comparison with WT, KO parasites showed significantly increased reticulocyte preference and multiple infections in the reticulocytes during the later course of infections that represent the anaemic phase. The rapid murine coma and behavioural scale (RMCBS) score of 105 WT parasite-infected mice that succumbed to CM was below 5 on day 7 whereas, RMCBS score of 107 KO parasite-infected mice was around 17 and 14 on day 7 and 14, respectively (Figure 1). These results were confirmed with another set of independent KO parasite lines wherein, ALAS and FC genes were replaced individually with GFP-luciferase (Luc)-expressing cassette containing m-cherry. The successful replacement of ALAS and FC was confirmed by PCR analyses performed with DNA and RNA isolated from the respective KO parasites, and by examining GFP and m-cherry fluorescence. For control, c / d ssurRNA locus in the WT parasite was replaced with GFP-Luc-expressing cassette. In vivo bioluminescence studies carried out for mice infected with Luc-expressing WT and KO parasites showed the accumulation of pRBCs in the brain of WT-infected mice, but not in the KO-infected mice. This was confirmed by performing ex vivo imaging for the perfused brain collected from WT- and KO-infected mice. The mice infected with Luc-expressing KO parasites were also completely protected from CM (Figure 2). Absence of cerebral pathology in heme pathway KO-infected mice To evaluate the integrity of BBB and assess vascular leakage, Evans blue extravasation analyses were carried out. While the brain collected from WT-infected mice on day 7 / 8 stained intensely with the intravenously injected Evans blue, the extravasation of Evans blue into the brain was barely detectable in KO-infected mice on day 7 and 14. Quantification of Evans blue in the brain extracts confirmed this observation. Histopathological assessment of hematoxylin and eosin (H&E) stained brain sections of WT-infected mice on day 7 showed intracerebral hemorrhages with extravasation of erythrocytes into the perivascular space, petechial hemorrhages, thrombosed and leukocyte-packed vessels, gross demyelination and myelin pallor. No such hallmark features of CM could be detected in the brain sections of KO-infected mice. Immunohistochemical studies carried out with the brain sections of WT-infected mice showed the extravasation of IgG in cerebral parenchyma and the presence of IgG in occluded vessels and hemorrhages, but not in the KO-infected mice. The luminal and abluminal leukocytes, and parasite-derived Hz could also be detected in the occluded vasculature of WT-infected mice. Immunofluorescence analyses of the brain sections using Pb glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and mouse CD31 antibodies showed the accumulation of parasites in CD31+ vasculature and extravascular parasites in the hemorrhages of WT-infected mice, but not in the KO-infected mice. Similarly, antibodies specific for CD3 and P-amyloid precursor protein (P-APP) showed the accumulation of CD3+ T cells in the cerebral vasculature and axonal injury in the brain sections of WT-infected mice, but not in the KO-infected mice. All these data indicated that the CM-associated brain lesions are absent in the mice infected with heme pathway KO parasites (Figure 3). Decreased levels of inflammatory parameters in heme pathway KO-infected mice Multiplex assays carried out for plasma cytokines and chemokines in mice infected with KO parasites on day 8 showed a significant decrease in the levels of IL-6, TNFa, IFNy, G-CSF, CCL3 (MIP-la) and CCL5 (RANTES). There was also a significant increase in the levels of anti-inflammatory cytokines - IL-4, IL-10 and IL-13. Quantitative RT-PCR analyses carried out to examine the expression of cytokines, chemokines, chemokine receptors and other key mediators of cerebral pathogenesis in the brain samples of KO-infected mice showed a substantial reduction of more than 1.5-fold in the transcript levels of TNFa, IFNy, CXCL9, CXCL10, CCL2 (MCP-1), CCL5, CCL19, perforin, granzyme B, ICAM-1, p-selectin and HO-1. In particular, the decrease in the expression levels of IFNy, CXCL10, CCL2, CCL5, granzyme B, ICAM-1 and HO-1 in FCKO was greater than 4-fold and the observed decrease was more in case of FCKO than ALASKO. It is known that the infiltration of CD8+ T cells expressing early activation marker CD69, and proinflammatory and cytotoxic effector molecules such as TNFa+, IFNy+, CXCR3+, perforin and granzyme B leads to endothelial leakage, BBB disruption and neuronal damage in CM. Flow cytometry analyses of the leukocytes isolated form the perfused brain samples of KO-infected mice showed significant reduction in CD3+ CD4+ and CD3+ CD8+ double positive cells, and CD3+ CD8+ CD69+, CD3+ CD8+ CXCR3+, CD3+ CD8+ perforin+, CD3+ CD8+ granzyme B+, CD3+ CD8+ TNFa+ and CD3+ CD8+ IFNy+ triple positive cells. These data indicate an overall decrease in systemic inflammation, inflammation-associated pathogenesis and T cell infiltration in the brain milieu of KO-infected mice. Western analyses for the brain homogenates of KO-infected mice showed reduction in phospho-NLRP3, phospho-NF-KB, cleaved caspase-1 and IL-10 that are key mediators of neuroinflammation, cerebrovascular dysfunction, endothelial activation, and endothelial and neuronal apoptosis. All these evidences suggested that the cerebral pathology is impeded in mice infected with heme pathway KO parasites (Figure 4). Decreased Hz formation in the KO parasites Light microscopy analyses of the Giemsa stained peripheral blood smears indicated a substantial decrease with Hz formation in the asexual stages (trophozoites and schizonts) of KO parasites in comparison to WT parasites. The observed decrease in the Hz formation was more prominent in the asexual stages than gametocytes. Such decreased levels of Hz could be readily detected in almost 60-70% of the pRBCs containing asexual stages. These findings were verified by examining the Hz content in paraformaldehyde-fixed pRBCs. To further confirm these findings, total Hz content of the parasites normalized with respect to the protein was examined. The total Hz content of the KO parasites was only 20-25% of the WT. It is known that Hb degradation and Hz formation in the FV results in the leaching of heme into the parasite cytosol. Free heme in the parasite lysates of the KO parasites prepared by hypotonic lysis showed around 55% decrease when compared with WT. In addition, there was also a significant decrease of around 55-60% in the plasma free heme and heme / hemopexin ratio of the KO-infected mice. However, there were no significant differences between the plasma hemopexin levels of WT- and KO-infected mice, and there was only a marginal 15-20% decrease in the plasma Hb levels that turned out to be significant in case of FCKO, but not in ALASKO. The quantification of Hz load in the spleen and liver showed 40-50% decrease in the KO-infected mice. These results suggested an overall decrease in the Hz synthesis of KO parasites (Figure 5). Griseofulvin treatment protects mice from CM The present invention evaluated the potential of griseofulvin in preventing CM and treated WT-infected C57BL / 6 mice from day 4 when the blood parasitemia was around 2%, and examined for CM pathogenesis. A single dose of 2 mg / day administered from day 4 and continued until day 8 showed the best protection from CM, a dosage that is very much comparable with the dosage of humans. While -80% of the control mice succumbed to CM within day 10, more than 80% of the griseofulvin-treated mice were protected from CM. Similar protection was also observed for the mice treated with 1 mg dose, twice a day from day 4 to day 8. In addition to CM protection, there was a significant delay in the mortality of the treated mice that occurred due to anemia. While the anemia mortality in CM-escaped control mice occurred within day 17, more than 80% of the griseofulvin-treated mice could survive beyond day 20 with almost 50% of them surviving even beyond day 24. Interestingly, the growth curves of WT parasites in treated and untreated mice were very much comparable, suggesting that CM protection is due to the inhibition of parasite heme synthesis, and not because of mitosis and parasite growth. This is also in agreement with the clinical trial performed in humans infected with Plasmodium falciparum wherein, griseofulvin treatment did not inhibit the in vivo parasite growth (34). To confirm that the CM protection is due to inhibition of parasite heme synthesis, the present invention analyzed heme synthesis in griseofulvin-treated and -untreated WT parasites by incubating the in vzvo-treated pRBCs in vitro with 14C-ALA (committed precursor of heme synthesis) for 9 h. There was around 60% decrease in the 14C-labelling of free heme in griseofulvin-treated parasites. Further, griseofulvin-treated mice showed less Evans blue extravasation in the brain, suggesting the preservation of BBB integrity. Histochemical and immunofluorescence analyses of the brain sections indicated the absence of intracerebral hemorrhages, and lack of accumulation of parasites and CD3+ T cells in the cerebral vasculature. This was also the case for axonal injury examined by P-APP staining. Light microscopy analyses of Giemsa-stained peripheral smears and paraformaldehyde-fixed pRBCs showed less Hz content. As observed for the KO parasites, the total Hz content in the griseofulvin-treated parasites was around 50-60% less when compared with untreated parasites and there was also close to 60% decrease in the free heme levels. Further, the plasma levels of heme, hemopexin and Hb together with heme / hemopexin ratio of the griseofulvin-treated WT-infected mice were found to be comparable to that of the KO-infected mice. Altogether, these results indicated the potential of griseofulvin in preventing CM and disease severity through the inhibition of parasite heme synthesis (Figure 6). Griseofulvin exhibits synergy with a,P-arteether in preventing CM in mice The present invention examined the potential of griseofulvin as an adjunct drug with a,P-arteether (primary artemisinin component of ACTs) in preventing CM in mice. The dose optimization studies performed with a,P-arteether indicated that a single intramuscular dose of 0.25 mg / mouse on day 6 when the parasitemia was around 10% resulted in CM in -60-70% of WT-infected mice, despite the clearance of peripheral parasitemia in blood smears. However, the inclusion of two doses of 2 mg griseofulvin at 24 h interval on day 6 and 7, post-a,P-arteether treatment, led to a complete protection of CM. The synergistic outcome of CM protection in mice treated with the combination of a,P-arteether and griseofulvin in comparison with a,P-arteether alone was observed with not much significant differences in day 7 parasite load and in the mortality due to anemia caused by recrudescence. While the two drugs act differently - a,P-arteether being responsible for clearing / reducing the parasite load and griseofulvin responsible for preventing cerebral pathogenesis, the combination is effective in bringing out a synergistic outcome in terms of preventing CM mortality. (Figure 7). Now, the invention will be described through illustrative examples. The examples given are merely illustrative of the uses, processes and products claimed in this invention, and the practice of the invention itself is not restricted to or by the examples described. EXAMPLE 1 Routine propagation of P. berghei in mice and CM experiments. P. berghei ANKA WT and KO parasites were propagated in C57BL / 6 male / female mice of 7-8 weeks old. Peripheral blood parasitemia was monitored by performing light microscopy for Giemsa stained thin blood smears prepared from tail vein blood. When the blood parasitemia was around 10%, 105 P. berghei ANKA WT or 105 / 107 ALAS / FC KO parasites were collected and injected intraperitoneally in 7-8 weeks old C57BL / 6 male / female naive mice to initiate CM experiments. Growth curve analysis was carried out by monitoring the blood parasitemia. The development and progression of ECM were monitored by examining RMCBS for neurological symptoms. To assess BBB integrity, Evans blue uptake assays were carried out by injecting 200 pl of 2% Evans blue in PBS intravenously and examining the extravasation of dye after one hour in the brain of the infected mice that were transcardially perfused with PBS. The extent of BBB damage was quantified by incubating the brain samples in formamide at 37°C for 48 h, extracting the Evans blue and measuring the absorbance at 620 nm. EXAMPLE 2 Histological and immunofluorescence analyses of cerebral pathology in the brain of infected mice. For H&E staining to assess vascular blockage, hemorrhages and demyelination, brain samples were fixed with formalin for 72 hours at room temperature. After dehydrating with ethanol and treating with xylene, paraffin embedded blocks were made and sections of 7 pm thickness were prepared using rotary microtome. The sections were then processed and stained with H&E using standard protocols. Immunohistochemical analysis of IgG extravasation in the brain sections was carried out as described. Brain sections of 30 pm thickness was antigen retrieved by treating them at 95°C for 30 min in sodium citrate buffer pH 6.0, followed by blocking with 3% H2O2 at room temperature for 30 min to prevent endogenous peroxidase activity. The sections were then incubated with HRP-conjugated goat antimouse IgG at 1:250 dilution in PBS containing 0.3% Triton X-100 and 0.1% BSA, followed by developing with diaminobenzidine tertrahydrochloride and counterstaining with hematoxilin. Immunoflourescence analysis of brain sections for parasite sequestration was carried out by fixing the brain samples in 4% paraformaldehyde in PBS containing 20% sucrose for 24 h at 4°C and cryoprotecting them for 48 h in PBS containing 20% sucrose. Coronal sections of 30 pm thickness was prepared using cryostat microtome and antigen retrieval was carried out by treating them at 95°C for 30 min in sodium citrate buffer pH 9.0. After blocking with 1% BSA, the sections were incubated with anti-CD31 mouse monoclonal antibody conjugated with Alexa Fluor 594 and anti-PhG APDH rabbit polyclonal serum or anti-mouse CD3 rat monoclonal antibody for 16 h at 4°C. The sections were then treated with FITC-conjugated donkey anti-rabbit IgG or FITC-conjugated goat anti-rat IgG (1:200 dilution), followed by 4',6-diamidino-2-phenylindole (DAPI) staining. Anti-mouse 0-APP rabbit polyclonal antibody was used in 1:200 dilution. EXAMPLE 3 Heme, Hz, hemoglobin and hemopexin estimations. Free heme levels in the plasma samples of WT- and KO-infected mice were quantified using Hemin colorimetric assays. The assay is specific for free heme and it utilizes peroxidase activity of hemin to facilitate the conversion of a colorless probe to a strongly colored compound with absorbance at 570 nm. The quantification of free heme in the parasite lysates was carried out by resuspending the parasite pellets in 5 volumes of hypotonic lysis buffer containing 5 mM Tris pH 7.5 with protease inhibitors and incubating them in ice for 30 min. The lysates were then centrifuged at 20,000 g for 20 min, 4°C, and the supernatants obtained were used for free heme estimation as mentioned above for the plasma samples. The Hz content of the WT and KO parasite pellets was estimated as described. The parasite pellet was resuspended in 1 ml of 100 mM sodium acetate buffer, pH 5.0 and left at 37°C for overnight, followed by centrifugation at 10,000 g for 5 min. The resultant pellet was resuspended in 100 mM Tris buffer pH 8.0 containing 2.5% SDS and incubated at 37°C for 30 min, followed by centrifugation at 10,000 g for 5 min. The pellet obtained was washed once with 100 mM alkaline bicarbonate pH 9.2 and then with distilled water. The final Hz pellet was dissolved in 100 mM NaOH containing 2.5% SDS, and the absorbance was measured at 405 nm. The supernatants of sodium acetate and Tris SDS steps were collected to estimate the protein content by Micro BCA protein assay kit and heme content of the Hz was expressed per mg of total protein. To estimate the Hz content in the spleen and liver of the WT- and KO-infected mice, 50 mg tissue of the respective organs was homogenized in 50 mM Tris pH 8.0 containing 50 mM NaCl, 5 mM CaCh and 1% Triton X-100, and incubated for 12 h at 37°C in the presence of proteinase K. The lysates were sonicated and centrifuged at 15,000 g for 30 min. The pellets obtained were resuspended with 100 mM sodium bicarbonate containing 2% SDS and sonicated, followed by centrifugation at 15,000 g for 15 min. After repeating this step thrice, the pellets were solubilized in 100 mM NaOH containing 2% SDS and 3 mM EDTA, and the absorbance was measured at 405 nm. In parallel, the parasite load in the organs was examined by quantitative PCR (qPCR) analysis carried out with Pbl8SrRNA primers for the total RNA isolated from 30 mg tissue of the organs. After normalizing with respect to the parasite load, the total heme content of the Hz isolated was expressed per mg weight of the organ. Hemoglobin and hemopexin levels in the plasma samples of WT- and KO-infected mice were measured by ELISA. EXAMPLE 4 In vivo bioluminescence imaging. In vivo bioluminescence imaging for mice infected with WT and KO parasite lines expressing luciferase was carried out as described. Luc-expressing ALASKO and FCKO parasites were generated by transfecting WT parasites with GOMO-GFP-Luc plasmid containing GFP-Luc-expressing cassette with m-cherry flanked on either side by 5’- and 3’-UTR regions of the respective genes. For whole body imaging, 7-8 weeks old C57BL / 6 mice were injected with Luc-expressing 105 WT or 107 KO parasites. On day 8 post-infection, mice were injected intraperitoneally with D-luciferin substrate (100 mg / kg animal weight in 200 pl of PBS), and imaged after 5 min using in vivo Imaging System with medium binning, 10 sec exposure and 12.5 FOV, under gas anesthesia system. For ex vivo imaging, transcardial perfusion was carried out for infected mice with cold PBS after injecting D-luciferin, and the organs were dissected out and imaged. EXAMPLE 5 Analyses of cytokines, chemokines, chemokine receptors and other key mediators of cerebral pathogenesis. Bio-Plex assays for cytokines and chemokines were carried out. The plasma samples utilized for the assays were prepared from the infected mouse blood collected on day7 / 8 post-infection. For transcript levels, total RNA was isolated from the brain samples of WT and KO-infected mice that were collected after a thorough perfusion with cold PBS and qPCR analyses were performed. Expression levels were normalized with GAPDH and fold changes for the transcripts of KO-infected mice with respect to WT were calculated using 2^^-1 method. EXAMPLE 6 Flow cytometry. Flow cytometry analyses of T cells in the brain samples of WT- and KO-infected mice were carried out as described. Mice were anesthetized and transcardially perfused with PBS, and the brain samples were dissected out and harvested in RPMI-1640 medium containing 10% FBS. For preparing single cell suspensions, the samples were minced and digested in RPMI-1640 medium containing 0.05% Collagenase D and 2U / ml DNase I for 30 minutes at room temperature, and passed through 70 pm nylon cell strainer, followed by 5 minutes of incubation on ice. Brain homogenates were then overlaid on 30% Percoll cushion and centrifuged at 400 g for 20 minutes at room temperature. The leukocyte pellets obtained were resuspended in 1ml of RBC lysis buffer (155 mM NH4C1, 10 mM NaHCO3 and 0.1 mM EDTA; pH 7.3) and incubated on ice for 5 minutes to remove any residual RBCs. The pellets were then washed with RPMI-1640, counted and stained for various markers. For intracellular markers like TNFa, IFNy, perforin and granzyme B, staining was carried out after fixing the cells with 4% paraformaldehyde. EXAMPLE 7 Labelling studies with 14C-ALA. The infected blood samples were collected, centrifuged at 1,000 g for 5 min to remove plasma and buffy coat, and washed twice with RPMI-1640 medium containing 10% FBS. The washed cells were resuspended in 10 volumes of RPMI-1640 medium containing 10% FBS and then incubated at 37°C in a CO2 incubator with the respective radioactive compounds. For 14C-ALA labelling, blood samples were collected from griseofulvin treated and control WT-infected mice around 16:00 h and the labelling was carried out for 9 h at a radioactivity of 1 pCi / ml. The infected RBCs were then centrifuged, washed with PBS and the parasites were isolated by saponin treatment. After washing the parasite pellet with PBS for four times, free heme present in the parasites was extracted using ethylacetate:glacial acetate (4:1) followed by 1.5 N HC1 and water washes to remove porphyrins and ALA. The upper phase was separated, dried under nitrogen stream, dissolved in methanol and analysed by TLC on silica gel using the mobile phase 2,6-lutidine and water (5:3 v / v) in ammonia atmosphere. The intensity of radiolabelling was scanned using Biomolecular Imager by exposing the TLC sheets to phosphorimager screen and the radioactive counts were measured. EXAMPLE 8 Griseofulvin treatment in mice. Griseofulvin was prepared by dissolving 1 or 2 mg in 40 pl DMSO and then making up the volume to 200 pl with 10% solutol HS 15 in saline. The mixture was vortexed thoroughly for 10 min to form an emulsion and injected intraperitoneally into the mice. All single dose injections were carried out at 06:00 h and double dose injections were carried out at 06:00 h and 18:00 h for the respective days. Control mice were injected with the solvent. a,P-arteether (E MAL) was administered intramuscularly. For experiments involving the combination of a,P-arteether and griseofulvin, a,P-arteether was administered intramuscularly at 06:00 h on day 6, followed by two doses of griseofulvin at 24 h interval (at 09:00 h on day 6 and 7). The present invention explored the possibility of including griseofulvin in the treatment protocol in affected patient population. The only inclusion will be griseofulvin for three days to prevent the development of cerebral and severe malaria. In humans, griseofulvin dosage is given to the extent of 500-1000 mg / day in adults and 5-10 mg / kg / day in children for several weeks. The conversion of 5-10 mg / kg / day into animal dose is carried out by multiplying human dose with 12.3. Taking the average weight of 7-8 weeks mouse as 25 g, this comes to 1.5-3.0 mg per mouse. Therefore, the dose used for griseofulvin (2 mg per mouse per day) in this invention is matching to human dose that has been routinely used for the treatment of fungal infections. Griseofulvin would be included in the treatment protocol for three days at the same dosage (500-1000 mg / day in adults and 5-10 mg / kg / day for children) that has been used for fungal diseases along with the existing ACTs to prevent cerebral and severe malaria. The treatment protocol suggested in this invention will be the standard ACT that has been given as per the WHO recommendation for malaria patients along with griseofulvin for three days starting from the first day of ACT treatment. There will not be any change in the standard malaria therapy except that the griseofulvin will be included for three days. The ACTs recommended by WHO are a,0-arteether / artemether in combination with lumefantrine or artesunate in combination with amodiaquine or artesunate in combination with mefloquine or dihydroartemisinin in combination with piperaquine or artesunate in combination with sulfadoxine-pyrimethamine. This ACT treatment is given for 3 days. Now, the crux of the invention is claimed implicitly and explicitly through the following claims. Each of the appended claims defines a separate invention, which for infringement purposes is recognized as including equivalents to the various elements or limitations specified in the claims. Depending on the context, all references below to the “invention” may in some cases refer to certain specific embodiments only. In 5 other cases, it will be recognized that references to the “invention” will refer to subject matter recited in one or more, but not necessarily all, of the claims. Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to a claimed individually or in any combination with other members of the group or other elements found herein. 10 One or more members of a group can be included in or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all groups used in the appended claims. It is to be noted that the present invention is susceptible to modifications, adaptations and changes by those 15 skilled in the art. Such variant embodiments employing the concepts and features of this invention are intended to be within the scope of the present invention, which is further set forth under the following claims.

Claims

I. A pharmaceutical composition for use in preventing and treating cerebral and severe malaria comprising artemisinin-based combination therapy (ACT), in combination with griseofulvin, for three days.

2. The composition as claimed in claim 1, wherein 500-1000 mg / day of griseofulvin is used in adults and 5-10 mg / kg / day of griseofulvin in children, in combination with ACT.

3. The composition as claimed in claim 1, characterized in that, the ACTs are a,P~ arteether in combination with lumefantrine or artemether in combination with lumefantrine or artesunate in combination with amodiaquine or artesunate in combination with mefloquine or dihydroartemisinin in combination with piperaquine or artesunate in combination with sulfadoxine-pyrimethamine.

4. Griseofulvin for use in a method of prevention or treatment of cerebral or severe malaria, in combination with artemisinin-based combination therapy (ACT).

5. Griseofulvin for use in a method of prevention or treatment of cerebral or severe malariaas claimed in claim 4, wherein 500-1000 mg / day of griseofulvin is used in adults and 5-10 mg / kg / day of griseofulvin in children in combination with ACTs.

6. Griseofulvin for use in a method of prevention or treatment of cerebral or severe malaria as claimed in claim 4 and 5, wherein the ACTs are a,P~arteether in combination with lumefantrine or artemether in combination with lumefantrine or artesunate in combination with amodiaquine or artesunate in combination with mefloquine or dihydroartemisinin in combination with piperaquine or artesunate in combination with sulfadoxine-pyrimethamine.

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