Ravidasvir, velpatasvir, and elbasvir for blocking transmission of plasmodium gametocytes

Abstract

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Description

[0001] RAVIDASVIR, VELPATASVIR, AND ELBASVIR FOR BLOCKING TRANSMISSION OF PLASMODIUM GAMETOCYTES

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of medicine, in particular parasitology.

[0004] BACKGROUND OF THE INVENTION:

[0005] Protozoan parasites of the genus Plasmodium cause diseases (malaria) in humans and in many animal species. In humans, Plasmodium falciparum is the most common cause of malaria and is responsible for about 80% of all malaria cases, and is also responsible for about 90% of the deaths from malaria in humans. Plasmodium falciparum initially infects the liver, but then moves into the blood, where it multiplies and persists through an asexual replication cycle in red blood cells (also known as RBCs, haematids or erythrocytes). The RBC is thus the main host cell for Plasmodium falciparum (asexual and sexual erythrocytic stage). The release and persistence in bloodstream are prerequisites for mature gametocytes to be taken up by mosquitoes and ensure parasite transmission. It has been shown that release into the blood circulation is concomitant with an increase in infected RBC deformability that allows mature gametocytes to circulate through the spleen. This prompts the interest in developing new reagents that block gametocyte transmission. In said context, it has been shown that short exposures to TD-6450, an orally-administered NS5A hepatitis C virus inhibitor, stiffened transmission parasite stages and killed asexual stages in vitro at high nanomolar concentrations (Carucci, Mario, et al. "Safe drugs with high potential to block malaria transmission revealed by a spleen-mimetic screening. "Nature Communications 14.1 (2023): 1951).

[0006] SUMMARY OF THE INVENTION:

[0007] The present invention is defined by the claims. In particular, the present invention relates to the use of ravidasvir, velpatasvir, and elbasvir for blocking transmission of Plasmodium gametocytes.

[0008] DETAILED DESCRIPTION OF THE INVENTION:

[0009] The inventors showed that nine NS5A inhibitors display measurable IC50 on asexual stage of Plasmodium falciparum. Four of them have IC50 lower than 2 pM (ravidasvir, daclatasvir, velpatasvir, elbasvir). Ravidasvir, velpatasvir, and elbasvir are as effective on either P. falciparum artemisinin-sensitive (F-32TEM, NF54) and artemisinin-resistant (F32-ART) strains. Ravidasvir exhibits the widest therapeutic window: serum peak after a single dose of 200-300 mg (3.33 - 7.16 pM) compared to the IC50 (1.08-1.4 pM) (Wu et al. AAC 2021;65(10):e600-21 ; Andrieux-Meyer et al. lancet GastroEnterol 2021;6:448-58). Finally, the inventors showed a stiffening effect of ravidasvir on mature gametocytes of P. falciparum with an IC50 value= 0.99 pM

[0010] Thus, the present invention relates to a method of blocking transmission of a Plasmodium parasite by an infected subject comprising administering to the subject a therapeutically effective amount of a drug selected from the group consisting of ravidasvir, velpatasvir, and elbasvir.

[0011] As used herein, the terms "subject" and "patient" are used interchangeably herein and will be understood to refer to warm blooded animals, for example, mammals and birds, particularly mammals. Non-limiting examples of animals within the scope and meaning of this term include dogs, cats, rats, mice, guinea pigs, chinchillas, horses, goats, cattle, sheep, zoo animals, Old and New World monkeys, non-human primates, and humans, and any other animal susceptible to malaria.

[0012] In some embodiments, the subject can be human or any other animal (e.g., birds and mammals) susceptible to infection by plasmodium parasites (e.g. domestic animals such as cats and dogs; livestock and farm animals such as horses, cows, pigs, chickens, etc.). Typically said subject is a mammal including a non-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, and mouse) and a primate (e.g., a monkey, chimpanzee, and a human). In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a farm animal or pet. In some embodiments, the subject is a human. In some embodiments, the subject is a human infant. In some embodiments, the subject is a human child. In some embodiments, the subject is a human adult. In some embodiments, the subject is a pregnant woman.

[0013] In some embodiments, the subject is infected by a Plasmodium parasite selected from the group consisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium knowlesi, Plasmodium inui, Plasmodium cynomolgi, Plasmodium simiovale, Plasmodium brazilianum, Plasmodium schwetzi and Plasmodium simium, and more preferably from the group consisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium knowlesi and Plasmodium malariae, and more preferably from the group consisting of Plasmodium falciparum and Plasmodium vivax. In some embodiments, said parasite is Plasmodium falciparum, in particular the Palo Alto I strain of Plasmodium falciparum.

[0014] The method of the present invention is particularly suitable for inducing the spleen-dependent clearance of mature gametocytes-hosting RBCs from the circulating blood. In particular the drug of the present invention indeed makes gametocytes unavailable to Anopheles sp. (i.e. mosquitos) thereby removing them from the transmission cycle. More particularly, the drug of the present invention, especially ravidasvir, is capable of increasing the rigidity of iRBCs (i.e. stiffening effect). The method of the present invention is thus particularly suitable for blocking transmission of malaria.

[0015] As used herein, the term “iRBCs”, or “Plasmodium-infected-iRBCs”, it is meant herein ring- RBCs (or ring-hosting RBCs) and / or gametocytes-hosting RBCs, in particular mature gametocytes-hosting RBCs.

[0016] As used herein, the expression “capable of increasing rigidity of iRBCs” or “stiffening effect” means that the drug is capable of increasing rigidity of iRBCs by at least 5%, preferably at least 10% and more preferably at least 15%. The ability of the drug to increase rigidity of iRBCs can be in particular assessed by measuring the deformability of iRBCs cultured in the presence and in the absence of said drug. Thus, as used herein the expression “increase rigidity” means “decrease deformability” and in particular “decrease deformability by at least 5%, preferably at least 10% and more preferably at least 15%”.

[0017] As used herein, the term “ravidasvir” has its general meaning in the art and refers to the compound having the IUPAC name: methyl N-[(2S)-l-[(2S)-2-[5-[6-[2-[(2S)-l-[(2S)-2- (methoxycarbonylamino)-3-methylbutanoyl]pyrrolidin-2-yl]-3H-benzimidazol-5- yl]naphthalen-2-yl]-lH-imidazol-2-yl]pyrrolidin-l-yl]-3-methyl-l-oxobutan-2-yl]carbamate.

[0018] As used herein, the term “velpatasvir” has its general meaning in the art and refers to the compound having the IUPAC name: methyl N-[(lR)-2-[(2S,4S)-2-[5-[6-[(2S,5S)-l-[(2S)-2- (methoxycarbonylamino)-3-methylbutanoyl]-5-methylpyrrolidin-2-yl]-21-oxa-5,7- diazapentacyclo[11.8.0.03,11.04,8.014,19]henicosa-l(13),2,4(8),5,9,ll,14(19),15,17-nonaen- 17-yl]-lH-imidazol-2-yl]-4-(methoxymethyl)pyrrolidin-l-yl]-2-oxo-l-phenylethyl]carbamate. As used herein, the term “elbasvir” has its general meaning in the art and refers to the compound having the IUPAC name: methyl N-[(2S)-l-[(2S)-2-[5-[(6S)-3-[2-[(2S)-l-[(2S)-2- (methoxycarbonylamino)-3-methylbutanoyl]pyrrolidin-2-yl]-lH-imidazol-5-yl]-6-phenyl-6H- indolo[l,2-c][l,3]benzoxazin-10-yl]-lH-imidazol-2-yl]pyrrolidin-l-yl]-3-methyl-l-oxobutan- 2-yl]carbamate.

[0019] In some embodiments, the method of the present invention further comprises administering to the mammal at least one additional antimalarial compound. Any suitable antimalarial compound can be used, many of which are well known in the art. Non-limiting examples of suitable antimalarial compounds include primaquine, bulaquine, artemisinin and derivatives thereof, chloroquine, hydroxychloroquine, mefloquine, amodiaquine, piperaquine, pyronaridine, atovaquone, tafenoquine, methylene blue, trioxaquines, endoperoxides such as OZ 439 and OZ 277, decoquinate, 9-anilinoacridines, doxycycline, azithromycine, erythromycine, spiramycine, pyrimethamine, sulfadiazine, sulfamethoxazole, HIV-protease inhibitors, and natural products such as neem, epoxomicin, harmonine, and riboflavin.

[0020] In some embodiments, the drug can be administered at the beginning or at the end of a conventional curative treatment of a malaria attack by a known anti-malarial agent, as primaquine is added to an artemisnin-based combination therapy for its transmission-blocking potential, as recommended by the WHO in areas of low transmission of malaria.

[0021] As used herein, the term "therapeutically effective amount" of the drug of the present invention is meant a sufficient amount of the compound to block the transmission of the Plasmodium parasite at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated, the potential positive impact of treatment for the local or general human community, and the severity of the disorder or impact of Plasmodium carriage by the subject on the transmission of malaria and health of the surrounding population; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0022] Typically, the drug of the present invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Galenic adaptations may be done for specific delivery in the small intestine or colon. Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile inj ectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising the compound of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The compound of the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifusoluble agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. The compound of the invention may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered. In addition to compound formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations; time release capsules ; and any other form currently used.

[0023] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0024] FIGURES:

[0025] Figure 1. Effect of NS5A inhibitors on NF54 strain.

[0026] Figure 2. Effect of NS5A inhibitors on NF54, F32-TEM and F32-ART strains. Figure 3. Effect of NS5A inhibitors on resistance emergence. A). NF54 strain first drug pulse. B) NF54 strain_Second drug pulse. C) F32-TEM strain_First drug pulse. D) F32-TEM strain_Second drug pulse. E) F32-TEM strain_third drug pulse.

[0027] Figure 4. The stiffening effect of NS5A inhibitor ravidasvir on P. falciparum stage V gametocyte.

[0028] Figure 5. The effect of NS5A inhibitor ravidasvir on P. falciparum liver stage.

[0029] EXAMPLE 1: ACTIVITY OF NS5A INHIBITORS OF HEPATITIS C VIRUS ON ASEXUAL STAGES OF PLASMODIUM FALCIPARUM.

[0030] Methods;

[0031] Plasmodium falciparum NF54, F32-TEM and F32-ART strains were sorbitol-synchronized to the ring stage, diluted to 0.5% parasitemia and loaded into 96-well plate. The ring suspension was then exposed to serial dilutions of each NS5A inhibitor drug ranging from 20 pM to 0,097 pM for 48h. After incubation, the plates were centrifugated and RBC pellets were incubated for 30 min with Hoechst 34580 (1 / 1000 diluted in PBS). After PBS washing twice (centrifuge at 445 g, 5min), pellets were resuspended in 200 pL of PBS and parasitemia was quantified by FACSCanto cytometer and analyzed with FlowJo V10.9.0. IC50s were determined using GraphPad Prism 8.0.2.

[0032] Results:

[0033] The results are depicted in Figures 1, 2 and Table 1. Nine NS5A inhibitors display measurable IC50 on asexual stage of Plasmodium falciparum .

[0034] ■C Ranging from 0.13 to 6.37 pM

[0035] ■C 4 / 9 have IC50 lower than 2 pM (ravidasvir, daclatasvir, velpatasvir, elbasvir).

[0036] Ravidasvir, velpatasvir, and elbasvir are as effective on either P. falciparum artemisininsensitive (F-32TEM, NF54) and artemisinin-resistant (F32-ART) strains. Table 1: summary table of C max (from literature) and IC50 values.

[0037] ★ Carucci et al., Nature Communications 2023

[0038] EXAMPLE 2: GENERATING RAVIDASVIR RESISTANT STRAINS

[0039] Methods:

[0040] Synchronized ring-stage cultures of NF54 and F-32 strains were exposed to 3 pM ravidasvir at 2% hematocrit and incubated for 72 h. The drug was then removed by media change and the cultures were kept at 4% hematocrit. Once parasites appear (>1%), new IC50s were performed (as described in I) to check if resistance emerged.

[0041] An alternative method for generating ravidasvir-resistant strain was tested. Asexual stage cultures (1% parasitaemia) of NF54 strains were exposed to 3 and 10 pM ravidasvir at 2.5% hematocrit for 30 days. Media was changed twice a week and thin blood smears were read once a week. If parasites appeared (> 1% parasitaemia), new IC50s were performed (as described in I) to check for the emergence of resistance.

[0042] Results:

[0043] The results are depicted in Figure 3 and Tables 2 and 3. Moreover, no parasite appeared during the first 30 days of culture under continuous pressure of ravidasvir. Table 2: Resistance emergence on NF54 strain.

[0044] Table 3: Resistance emergence on F32-TEM strain.

[0045] EXAMPLE 3: THE STIFFENING EFFECT OF NS5A INHIBITOR RA VIDAS VIR ON

[0046] P. FALCIPARUM ST AGE V GAMETOCYTE.

[0047] Methods:

[0048] The production of gametocytes was induced from a 0.5% ring-stage parasitemia (day 0). Medium was changed daily to induce gametocytes differentiation. Once differentiated stage 2- 3 gametocytes were visible (around day 7-8 post-induction), the remaining asexual parasites were eliminated by 50mM N-acetyl-D-glucosamine treatment. At day 17, gametocytes were purified using a density-gradient purification method (Duez et al., Nat. Protoc 2018). The gametocytes were loaded into 96-well plate at 2% hematocrit, then exposed for 24 hours to serial dilutions of ravidasvir that ranged from 20 pM to 0.02 pM. Finally, spleen-mimetic microsphiltration was performed on ravidasvir-exposed gametocytes as described. The upstream and downstream samples were collected and smeared into glass slides for staining with Giemsa reagent. Gametocytemia was assessed upstream and downstream and retention rate was determined.

[0049] Results:

[0050] We showed stiffening effect of ravidasvir on mature gametocytes of P. falciparum with an IC50 value= 0.99 pM (Figure 4). EXAMPLE 5: THE EFFECT OF NS5 INHIBITOR RAVIDASVIR ON P.

[0051] FALCIPARUM LIVER STAGE.

[0052] Methods:

[0053] Primary human hepatocytes purchased from BioIVT were seeded on collagen-micropatterned 96-well plates (March, Sandra, et al. "Micropattemed coculture of primary human hepatocytes and supportive cells for the study of hepatotropic pathogens. "Nature protocols 10.12 (2015): 2027-2053). Hepatocytes were then infected with freshly dissected P. falciparum sporozoites (60,000 / well). Ravidasvir and KDU were added 3 hours later at 10 pM and 1 pM final concentrations, respectively.

[0054] Results:

[0055] Ravidasvir shows promising activity against liver stage parasites by reducing about 50% of P. falciparum-infected primary human hepatocytes (Figure 5).

[0056] REFERENCES:

[0057] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:

1. A method of blocking transmission of a Plasmodium parasite by an infected subject comprising administering to the subject a therapeutically effective amount of a drug selected from the group consisting of ravidasvir, velpatasvir, and elbasvir.

2. The method of claim 1 wherein the subject is infected by & Plasmodium parasite selected from the group consisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium knowlesi, Plasmodium inui, Plasmodium cynomolgi, Plasmodium simiovale, Plasmodium brazilianum, Plasmodium schwetzi and Plasmodium simium.

3. The method of claim 1 wherein the subject is infected by Plasmodium falciparum.

4. The method of claim Ithat further comprises administering to the mammal at least one additional antimalarial compound.

5. The method of claim 4 wherein the antimalarial compound includes primaquine, bulaquine, artemisinin and derivatives thereof, chloroquine, hydroxychloroquine, mefloquine, amodiaquine, piperaquine, pyronaridine, atovaquone, tafenoquine, methylene blue, trioxaquines, endoperoxides such as OZ 439 and OZ 277, decoquinate, 9-anilinoacridines, doxycycline, azithromycine, erythromycine, spiramycine, pyrimethamine, sulfadiazine, sulfamethoxazole, HIV-protease inhibitors, and natural products such as neem, epoxomicin, harmonine, and riboflavin.

6. The method of claim 4 wherein the drug is administered at the beginning or at the end of a conventional curative treatment of a malaria attack by a known anti-malarial compound.