TREATMENT OF MALARIA"

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Abstract of the Invention

Object of the present invention is the use of inhibitor compounds of the human GTPase Racl in the treatment of malaria.

Technical field and background art

Malaria is an infectious disease causing vast mortality worldwide (WHO, 2015). In 2015, malaria caused about 214 million cases and 438.000 deaths, mainly among children below 5 years of age.

Malaria is a parasitosis, a disease caused by protozoan parasites of the genus Plasmodium (Kingdom Protista, Phylum Apicomplexa, Class Sporozoea, Order Eucoccidiida). Among the 4 species of Plasmodium which infect humans, the most dangerous is Plasmodium falciparum, with the highest mortality rate among infected subjects. The reservoir of the parasite is represented by chronically infected subjects. Vectors are mosquitoes of the genus Anopheles.

The clinical symptoms are caused by the development of the malaria parasites within erythrocytes.

Malaria is the most common parasitosis, being an acute fever disease with different severity levels depending on the infecting species. Its actual diffusion is not limited to tropical areas of South America, Africa and Asia, but sporadically also affects USA and other industrialized countries, where clinical cases of the disease can appear following the transit of people getting infected in endemic areas .

It is known since the '60s that malaria parasites can develop antimalarial drug resistance. Resistance to any class of antimalarial drugs progressively emerged, including the artemisinin derivatives, which have been the last-line treatments for malaria for decades.

To date there are no known classes of antimalarial drugs, for which resistant parasites have not emerged..

The identification of new classes of antimalarial drugs is therefore a scientific topic of extreme urgency, considering the absence of an effective vaccine. Objects of the Invention

Object of the present invention is therefore to provide a compound that could be used in the treatment and/or prevention of malaria.

Another object of the present invention is to provide a compound proving to be effective against different strains of malaria parasite.

Further object of the present invention is to provide a compound that minimizes the possibility that the malaria parasite will develop a resistance.

Still object of the present invention is to provide a compound that could be used alone or in combination with other therapies in the treatment of malaria.

Description of the Invention

The above mentioned objects and still other objects that will be better clarified in the following, are achieved by the present invention, concerning the use of one or more inhibitor compounds of the human GTPase Racl for use in the treatment of malaria. As a matter of fact it has been surprisingly observed that the compounds able to inhibit the human Racl enzyme can hinder the invasion process of the erythrocyte by the P. falciparum malaria parasite.

The human Racl protein, also known as“Ras-related C3 botulinum toxin substrate 1”, is a GTPase ubiquitously expressed in the tissues, which regulates several cell events among which, e.g., the organization of actine cytoskeleton, and which is involved in the development of several types of cancers, e.g. the breast cancer.

According to the present invention, the inhibitor compounds of the GTPase Racl act on an enzyme of the human host, namely Racl, rather than acting on the malaria parasite. Molecules directed towards the proteins of the human host, i.e. the inhibitor compounds of Racl according to the invention, have several advantages with respect to known compounds acting on the parasite. For example, advantageously, the inhibitor compounds of Racl, object of the present invention, prove to be active against different strains of the malaria parasite and thus usable at broad-spectrum in the treatment of different forms of malaria, which are caused by different Plasmodium strains. Moreover, advantageously, such compounds result to be less susceptible to induce the development of resistances in the malaria parasite.

In embodiments of the present invention, the inhibitor compound of the human GTPase Racl for use in the treatment and/or prevention of malaria is selected among Aza-l, MLS532223, NSC23766, EHT1864 and EHor-016, and mixtures thereof.

The term "Aza-l" shortly identifies the compound (N*2*,N*4*-Bis-(2-methyl-lH- indol-5-yl)-pyrimidin-2, 4-diamine).

The term “MLS532223” shortly identifies the compound l-(3-Nitrophenyl)-3- phenyl-2-propyn- 1 -one.

The term“NSC23766” shortly identifies the compound N6-[2-[[4-(Diethylamine)-l- methylbutyl]amine]-6-methyl-4-pyrimidinyl]-2-methyl-4,6-quin olinediamine trihydrochloride.

The term “EHT1864” shortly identifies the compound 5-(5-(7- (Trifluoromethyl)quinolin-4-ylthio)pentyloxy)-2-(morpholinem ethyl)-4H-pyran-4- one dihydrochloride.

The term“EHop-0l6” shortly identifies the compound N4-(9-Ethyl-9H-carbazol-3- yl)-N2-(3-morpholin-4-yl-propyl)-pyrimidin-2, 4-diamine.

Advantageously, the compounds Aza-l, MLS532223, NSC23766, EHT1864 and EHor-016 showed IC50 values indicating particular efficiency of such compounds as antiparasitics.

The“IC50” value, or“inhibitory concentration", is the concentration of an enzyme inhibitor (drug, toxin or poison, etc.) required to inhibit the 50% of the target under examination (enzyme, cell, receptor or microorganism). In the case of the present invention, the IC50 value is the concentration of inhibitor compound of Racl required to halve the amount of erythrocytes infected by the malaria parasite.

Such values are for example calculated by treating a mixed culture for 72 hours. Preferably, the inhibitor compound is selected from Aza-l, EHor-016, and mixtures thereof. As a matter of fact such compounds showed particularly low IC50 values, i.e. about 250 nM and about 140 nM, respectively. As it will be described in detail later, such IC50 values have been determined by using asexual parasites of the Chloroquine-sensitive D10 strain.

The pg/kg unit of measure denotes that the dosage of the inhibitor compound is calibrated to the body weight of the subj ect or animal to which such compound will be administered; in particular, the amount of the inhibitor (as weight, expressed in mg) to be administered for each kg of weight of the patient or animal model is defined.

According to an embodiment of the present invention, the inhibitor compounds of the human GTPase Racl can be administered alone or in combination between them. Another object of the present invention is a composition comprising at least one inhibitor compound of the human GTPase Racl for use in the treatment of malaria. According to an embodiment, the composition of the present invention comprises at least one inhibitor compound of Racl selected among Aza-l, MLS532223, NSC23766, EHT1864 and EHor-016, and mixtures thereof. Preferably, the inhibitor compound is selected from Aza- 1 , EHop-016, and mixtures thereof.

In embodiments, the composition of the present invention further comprises excipients and/or additives of pharmaceutical use.

Object of the invention, according to another of the aspects thereof, is a method for the treatment and/or the prevention of malaria, which comprises the administration of an effective amount of at least one inhibitor compound of the human GTPase Racl to a subject in need thereof. In the present description, the preferred embodiments discussed with reference to the inhibitor compounds of the Racl, are applied mutatis mutandis also to the compositions comprising such inhibitor compounds and to the treatment method of the invention.

The carried out experiments, which will be discussed in detail later, confirmed the surprising efficiency in inhibiting both erythrocyte invasion by the malaria parasite and its subsequent development, and thus confirmed that such compounds can be effectively used as antimalarial drugs.

The following experimental evidences are reported as further support of the present invention.

The experimental results are associated with the appended Figures.

Figure 1 shows an immunofluorescence assay showing the localization of the GTPase Racl in erythrocytes infected by P. falciparum and in non-infected erythrocytes. The figure highlights that the GTPase Racl is recruited by the parasite to the parasitophorous vacuole membrane .

Figure 2 shows the percentage of infected erythrocytes with respect to the untreated control as a function of the concentration of the Racl inhibitor compound EHT1864, in an invasion assay wherein the number of infected erythrocytes is measured after 6 hours of exposure to synchronous invasive stages. The graph shows the amount of compound required to specifically inhibit the invasion process of the erythrocyte by the malaria parasite. The parasites are synchronized and placed to invade erythrocytes for 6 hours.

Figure 3A shows the invasion percentage, with respect to the control, in the absence of inhibitor, of erythrocytes pre-treated with 100 mM EHT1864.

Figure 3B shows the percentage of infected erythrocytes with respect to the control at increasing treatment times with 100 mM EHT1864.

Figure 4 shows the percentage of infected erythrocytes with respect to the control after treatingerythrocytes with the Racl inhibiting compound ML141 at 50 mM concentration.

Figure 5 A shows the molecular structure of the Racl -inhibiting compound EHor-016; Figure 5B shows the molecular structure of the Racl -inhibiting compound AZA- 1.

Experimental Section

In the present experimental section the results obtained by using different inhibitor compounds of the GTPase Racl will be showed and discussed. These must be considered merely exemplary of the principle of the invention and not limiting the scope of the invention.

Localization of Racl in infected and non-infected erythrocytes

Experiment 1

The subcellular localization of Racl in erythrocytes infected by P. falciparum and in non-infected erythrocytes has been determined by immunofluorescence assays with monoclonal antibodies directed against such enzyme.

Figure 1 shows the results of the immunofluorescence assay: the "A" series of images shows an erythrocyte invaded by a very young parasite (about 2 hours after the invasion); the "B" series of images shows an erythrocyte invaded by a more mature parasite (about 6 hours after the invasion). The immunofluorescence assay was carried out by using anti -Racl antibodies coupled to a green fluorescent marker, and anti- expl antibodies (a marker of the parasitophorous vacuole) coupled to a red fluorescent marker; the parasite nuclei were stained with DAPI.

These experiments showed that the GTPase Racl is localized on the cell membrane of non-infected erythrocytes, while it is recruited to the parasitophorous vacuole (the membrane containing the parasite within the erythrocyte) immediately after parasite invasion (Figure 1).

Effect of Racl inhibition by means of the compound EHT1864 on erythrocyte invasion by the malaria parasite

Experiment 2

In order to evaluate the effect of the human enzyme Racl inhibition on erythrocyte invasion by the malaria parasite, we tested the effect of a specific Racl inhibitor, EHT1864 (which inserts itself into the catalytic site of the Racl protein thus blocking its GTPase activity), on invasion efficiencies of the host erythrocyte by the P. falciparum malaria parasite.

The inhibitor compound EHT1864 was added at increasing concentrations to P. falciparum cultures in invasion assays. The parasite nuclei were stained with the Hoechst 33342 vital dye and the number of infected. erythrocytes was estimated by means of FACS analysis.

The FACS technique, i.e. “Fluorescence-activated cell sorting”, is a known methodology which allows to sort, within a group of cells, the ones marked with a selected fluorochrome. In this case, the parasite nuclei have been stained with the Hoechst 33342 dye. The erythrocytes infected by the malaria parasite, are then identifiable with respect to the non-infected erythrocytes on the basis of the signal produced by the Hoechst 33342 dye, which indicates the presence of a parasite within the erythrocyte.

By means of these experiments it was possible to observe that the Racl -inhibiting compound EHT1864 can significantly inhibit the erythrocyte invasion process by the malaria parasites in a dose-dependent way (Figure 2).

Figure 2 shows the percentage of infected erythrocytes with respect to the untreated control, as a function of EHT1864 concentration (1 mM to 100 mM). The shown values are the average of 3 biological replicates, each carried out in 3 technical replicates, and have been normalized over the untreated control.

In order to demonstrate that the inhibitory effect on the invasion process of erythrocytes by malaria parasites is due to an action on the host cell and not to a toxic effect on the parasites, two further experiments have been carried out, herein in the following described in the sections "Experiment 3" and "Experiment 4".

Experiment 3

Erythrocytes were pre-treated with the inhibitor EHT1864 and then infected with P. falciparum in a medium free from the inhibitor. Also in this case a significant reduction of the invasion efficiency was observed (Figure 3 A).

Figure 3 A shows the percentage of infected erythrocytes compared to the control after a pre-treatment with 100 mM EHT1684; the invasion assay of the erythrocytes by P. falciparum has been carried out both in the presence and in the absence of the inhibitor compound EHT1684. The values are the average of 3 biological replicates. Experiment 4

In this experiment, erythrocytes were pre-incubated in EHT1864 for increasing times and subsequently infected with P. falciparum in the presence of such inhibitor. The invasion efficiency resulted to be inversely proportional to the incubation time, thus demonstrating that EHT1864 inhibits the invasion process by specifically acting on the host cell (Figure 3B).

Figure 3B shows the percentage of erythrocytes infected by P. falciparum with respect to the untreated control, at increasing treatment times (0, 4, 8 and 24 hours) with EHT1864 (100 mM).

Moreover, the effect of EHT1864 on parasite growth, once they are within the erythrocyte, has been evaluated (data not shown). As a result, an inhibitory effect on parasite growth at the trophozoite stage was observed.

Overall, on a culture of non-synchronous parasites, the inhibitor EHT1864 showed an IC ₅₀ of 3.9 mM, after a 72 hour treatment . The expression“non-synchronous parasites” indicates a mixed culture of parasites, containing different stages of the life cycle. In these experiments it is possible to determine the overall effect on the parasites in their entirety. A parasite culture is taken and aliquoted on a 96-well plate. The inhibitor is then added to each well at increasing concentrations (serial dilutions 1 :2) and after 72 hours the amount of still alive parasites is measured by means of an enzyme assay. A curve is then plotted and the IC ₅₀ is determined.

Effect of the Racl -inhibition by means of the compound ML141 on the erythrocyte invasion by the malaria parasite

Experiment 5

A second Racl inhibitor , the compound ML 141, has been subsequently tested in invasion assays, with the same methodology described above for experiment 2, giving results comparable to EHT1864 (Figure 4).

The term “ML141” shortly identifies the compound 4-[4,5-Dihydro-5-(4- methoxyphenyl)-3 -phenyl- 1 H-pyrazol- 1 -yljbenzenesulfonamide.

Figure 4 shows the percentage of erythrocytes infected by P. falciparum with respect to the control after a treatment with 50 mM ML141. The methodology is the same as described above for Experiment 2, with the difference that in the previous case the erythrocytes are pre-treated and then washed before the infection. The values shown are the average of 3 biological replicates, each carried out in 3 technical replicates, and have been normalized on the untreated control.

Overall, the ML141 inhibitor showed an IC50 of around 10.7 mM.

Other tested compounds

In addition to the two already discussed compounds, EHT1864 and ML141, other 9 Racl- inhibiting compounds have been tested according to the methods described above, in order to evaluate their effect on non-synchronous cultures of P. falciparum. The IC50 values obtained by testing the different compounds on different strains of P. falciparum are reported in Table 1.

Among the tested compounds, 5 compounds (Aza-l, MLS532223, NSC23766, 553511 and EHor-016) showed significant efficiency as antiparasitics, and showed an IC50 comparable to that of the compound ML 141, or even less (see the data in Table 1). The most effective compounds resulted to be the compound EHor-016 (whose molecular structure is shown in Figure 5A) with an IC50 of 138 nM and the compound Aza-l (whose molecular structure is shown in Figure 5B), with an IC50 equal to 253 nM.

The effect of the compound EHor-016 has been evaluated also on immature and mature sexual stages of the malaria parasite, by using mature and immature gametocytes of a transgenic 3D7 strain of P. falciparum. The results reported in Table 1 show that the compound EHor-016 is effective also against immature and mature sexual stages of P. falciparum, with an IC50 equal to 630 nM on immature gametocytes and 4.7 mM on mature gametocytes. The compounds NSC23766 and EHT1864 have been tested on gametocytes as well, and both resulted to be nearly totally (IC50 about 30 mM) or totally (ICso>50 mM) inactive on mature gametocytes.

In view of performing in vivo assays, the effect of the compound EHor-016 has been evaluated also on cultures of the murine parasite Plasmodium berghei. Such compound showed antiparasitic properties against this species as well. The mouse Racl is almost identical to the human one. As a matter of fact the human anti-Racl antibodies bind the murine one as well. The murine parasite has a life cycle of 24 hours and cannot re-invade in vitro, therefore it cannot be kept in culture for more than 24 hours.

The efficiency against the inhibitor EHorOIb on P. berghei has been observed on a culture of non-synchronous parasites. The culture has been treated overnight with EHop0l6 at the concentration of 10 uM and the following day no evidence of parasites was observed .

Table 1 reported below shows the IC50 values of the different Racl -inhibiting compounds on asexual parasites of the CQ-sensitive D10 and CQ-resistant W2 strains and on mature and immature gametocytes of a transgenic 3D7 strain.

In Table 1, the acronyms "CQ" and "MB" refer, respectively, to Chloroquine (CQ) and Methylene Blue (MB), which are compounds known to act against the asexual and sexual stages of the malaria parasite, respectively, and which were used as positive controls. The data are the mean of three different experiments in duplicate ± standard deviation. The asterisk means that for the assays on the sexual stages, a transgenic strain of the 3D7 line expressing the luciferase only in the gametocytes was used.

The experiments discussed above confirm that the inhibitor compounds of the human Racl enzyme can be used in the treatment of malaria.

The compound EHor-016 showed a particularly surprising efficiency. Among the many advantages of the present invention with respect to the known art, it is of particular relevance the fact that the use of molecules directed towards host proteins, in this case the GTPase Racl, proved to be advantageously applicable against different strains of the malaria parasite, thus allowing the broad-spectrum use of such molecules in the different forms of malaria, which are caused by different Plasmodium strains. A further advantage of the compounds mentioned in the invention is that such compounds are expected to be less susceptible to induce the development of resistances in the malaria parasite.