[0001] The present invention relates to a new compound for the inhibition of the transmission of malaria in humans, a pharmaceutical composition comprising said com- pound, and a method for the inhibition of the transmission of malaria in humans compris- ing the administration of said compound.

FIELD OF THE INVENTION

[0002] The invention relates to the field of pharmaceutically active compounds, more particular to the field of anti-malaria compounds. BACKGROUND OF THE INVENTION

[0003] Malaria remains one of the world's most serious public health problems, especially in tropical and subtropical areas, with approximately 229 million cases and 409,000 deaths in 2019. Intracellular parasites of the genus Plasmodium cause the disease among which six species can infect humans. These include P. falciparum as the most virulent species and P. knowlesi, described originally as a simian parasite, which can cause severe malaria in humans. Clinical symptoms of malaria appear during the replica- tion of parasites in the erythrocytic cycle (lasting 24 h for P. knowlesi and 48 h for P. falciparum) in the human host. These symptoms can develop into life-threatening compli- cations such as severe anemia, liver and kidney failure and cerebral malaria if not treated properly.

[0004] Plasmodium parasites have a complex life cycle that includes mosquito and vertebrate hosts. During each replication cycle, a small number of asexual parasites starts a different genetic program and develops into the transmissible sexual form, the gametocytes.

[0005] Currently, the mainstay malaria treatments are artemisinin-based com- bination therapies (ACT). Artemisinin and its derivatives are fast-acting and rapidly reduce the asexual parasite load but have a short half-life. They are combined to a slow-acting partner drug with longer half-life to clear remaining parasites, and to protect artemisinin against resistances. Nonetheless, reports of P. falciparum with delayed removal after artesunate or ACT treatment are accumulating, especially from the Greater Mekong Region where also resistances to the partner drugs are present. This poses a threat to the existing malaria treatment options. In addition, the current antimalarial drugs are inactive against mature P. falciparum (stage IV and V) gametocytes, favoring the transmission of the parasite.

[0006] The interest in using antibiotics for malaria treatment appeared due to the emergence of resistant parasites to the former mainstay drug chloroquine. Tetracy- clines were the first antibiotics to treat uncomplicated malaria that dates back to the 1950s. Currently, doxycycline and the combination of sulfadoxine-pyrimethamine are used for malaria prevention of travelers and risk groups in malaria-endemic regions, respective- ly. In addition, clindamycin combined with quinine is used to treat pregnant women during the first trimester or combined with artesunate or quinine to treat uncomplicated malaria when an ACT is unavailable.

[0007] Tetracyclines and macrolides, both naturally produced by Streptomyces, have shown promising antiplasmodial activities. Most antibiotics with antiplasmodial activity target the apicoplast; a plastid-like organelle derived from endosymbiotic bacteria responsible for the biosynthesis of isoprenoid precursors. Antibiotics can be fast or slow- acting compounds, depending on whether they target the treated parasites or act on their progeny, causing the so-called delayed death effect. Commonly, tetracyclines belong to the latter group, but novel tetracyclines have shown superior antiplasmodial activity. Some macrolides such as ivermectin, borrelidin, and kitasamycin (also called leucomycin) were already described as fast-acting: they inhibit parasite growth during the first cycle in vitro and in vivo at low nanomolar concentrations but the molecular targets are not known.

[0008] Unfortunately, primaquine is the only approved drug active against ga- metocytes, therefore allowing the inhibition of the transmission of the malaria parasite between humans; see Sinden. Developing transmission-blocking strategies for malaria control. PLoS Pathog. 533 (2017) 13:1-12. However, its use is restricted mainly due to its haemolysis-inducing effect in patients with glucose-6-phosphate dehydrogenase deficien- cy (Wadi et al. Recent advances in transmission-blocking drugs for malaria elimination. Future Med Chem. (2019) 11 :3047-88), and additionally it has to be metabolized by a liver enzyme (CYP2D6) to be active. Compounds with multi-stage activities and novel modes of action are especially desired for the next generation of antimalarial compounds.

[0009] Against this background it is an object underlying the invention to pro- vide for a new compound which cannot only be used for the treatment of an acute illness from malaria but for controlling and eliminating malaria. Especially, such a compound is to be provided which allows the inhibition of the transmission of malaria and/or the malaria parasite in humans. SUMMARY OF THE INVENTION

[0010] The object is met by the provision of boromycin for use in the inhibition of the transmission of malaria in humans.

[0011] This object is also met by the provision of boromycin for use as a game- tocytocidal compound.

[0012] Furthermore, this object is also met by the provision of a pharmaceutical composition for the inhibition of transmission of malaria in humans comprising boromycin and a pharmaceutically acceptable carrier.

[0013] According to the invention, "inhibition of the transmission" means that the transfer of the malaria parasite from the human being having received said boromycin to another human being is reduced or even prevented, said reduction or prevention is in comparison to the transfer from a reference human being, not having received said boromycin, to another human being. In an embodiment of the invention the inhibition of the transmission refers to an inhibitory activity of said boromycin against the developmen- tal stage of the malaria parasite, which is responsible for the transmission to another host. In an embodiment of the invention said developmental stage is a gametocyte.

[0014] According to the invention, a "gametocytocidal compound" refers to an active agent which is destructive to gametocytes, i.e. the developmental stage of the malaria parasite which is responsible for the transmission to another host.

[0015] "Pharmaceutically acceptably carriers" are well known to the skilled per- son. They allow a proper formulation of the boromycin and serve to improve the selectivi- ty, effectiveness, and/or safety of drug administration. Pharmaceutically acceptable carriers include, without being limited thereto, solvents, fillers, binders, lubricants, stabiliz- ers, surfactants, suspensions, thickeners, emulsifiers, preserving agents, liposomes, micelles, microspheres, nanoparticles, etc. suitable for the particular form of dosage. Except for cases, when the medium of conventional carriers is incompatible with the active ingredient, for example, upon occurrence of any undesirable biological effects or other adverse interactions with any other ingredient(s) of the pharmaceutical composition, the use of such compositions falls within the scope of this invention. Materials that can serve as pharmaceutically acceptable carriers include, but are not limited to, monosaccha- rides and oligosaccharides, as well as derivatives thereof; malt, gelatin; talc; excipients such as: cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminium hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solutions. In addition, the composition may contain other non-toxic compatible lubricants, for example sodium lauryl sulfate and magnesium stearate, as well as coloring agents, parting liquids, film formers, sweeteners, flavoring additives and flavorants, preserving agents and antioxidants.

[0016] The phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or compli- cation, commensurate with a reasonable benefit/risk ratio. Suitable pharmaceutical carriers or excipients as well as pharmaceutical necessities for use in pharmaceutical formulations are described in Remington - The Science and Practice of Pharmacy, 23rd edition, 2020, a well-known reference text in this field, and in the USP/NF (United States Pharmacopeia and the National Formulary). Other sources are also available to one of ordinary skill in the art.

[0017] The pharmaceutical composition as well as the method to be explained below of the present invention may be utilized to treat a human being in need thereof.

[0018] "Boromycin" (CAS number: 34524-20-4, molecular mass: 879.89 mol ^-1 , formula: C ₄₅ H ₇ 4BNO ₁₅ ) is a bacteriocidal polyether-macrolide antibiotic. It was first isolated from a Streptomyces antibioticus found in a soil sample from Ivory Coast and was first described in 1967. It is notable for being the first natural product found to contain the element boron; see Dunitz et al. Structure of Boromycin. Helv Chim Acta. (1971) 54:1709-13.

[0019] According to the invention, boromycin includes pharmaceutically accept- able salts thereof, as well as derivatives of boromycin and/or its pharmaceutically accept- able salts.

[0020] The findings of the inventors were surprising and could not have been suspected. So far, bromomycin has been described in the art in relation with different activities.

[0021] Boromycin has so far been described a potent effect against Gram- positive bacteria in vitro; see Moreira et al. Boromycin kills Mycobacterial persisters without detectable resistance. Front in Microbiol. (2016) 7:199. It was also shown to be active against HIV; see Kohno et al. Boromycin, an Anti-HIV antibiotic. Biosci Biotechnol Biochem. (1996) 60:1036-7. Furthermore, activities against Babesia (Prelog et al. (1973). Antibiotic A 28829. U.S. patent no. 584,215. Ardsley, New York: Ciba-Geigy Corporation) and other pathogens (Abenoja et al. Boromycin has potent anti-Toxoplasma and anti- Cryptosporidium activity. Antimicrob Agents Chemother. (2021) 65:e01278-20) have been described.

[0022] U.S. patent no. 3,769,418 discloses an antibiotic named "A 28829" formed by cultivating Streptomyces antibioticus A 28829, said antibiotic is described as having an activity against Plasmodium berghei and Plasmodium gallinaceum which may infect rodents and poultry but no humans.

[0023] The inventors found that boromycin is a highly active and fast acting an- tibiotic against Plasmodium species which may cause malaria in humans. Importantly, they found that boromycin cannot only be used as a conventional anti-malaria substance but to prevent or reduce the transmission of the Plasmodium species to other subjects due to its surprising gametocytocidal activity. [0024] In the investigation of the inventors, boromycin showed a high activity and a fast onset of action, a desired property of antimalarials to quickly reduce parasite load and avoid the disease progression to the severe form. It also showed an excellent selectivity and a favorable toxicity profile.

[0025] Boromycin’s in vitro activity against laboratory strains showed superior activity as it was active at low nanomolar and even at picomolar concentrations. No cross- resistance with chloroquine/multi-resistant strains was seen.

[0026] The inventors additionally evaluated the activity of boromycin against mature gametocytes and could see a very potent activity superior to the positive control and similarly active as a reference compound. Drugs with transmission-blocking activity are of special interest and would be a great asset for controlling and eliminating malaria.

[0027] The object underlying the invention is herewith completely met.

[0028] In an embodiment of the invention said malaria is caused by Plasmodi- um falciparum.

[0029] The inventors have found in an experimental set-up that boromycin is of particular activity against a transmission of malaria caused by Plasmodium falciparum. This finding is important because Plasmodium falciparum is the most virulent intracellular malaria-causing parasite of the genus Plasmodium, which can infect humans. It is mainly found in Africa and is responsible for most malaria cases and deaths.

[0030] In another embodiment of the invention said malaria is caused by Plas- modium knowlesi.

[0031] In another experimental set-up the inventors were able to demonstrate activity against the species of Plasmodium knowlesi. Plasmodium knowlesi, described originally as a simian parasite, was recognized as a human malaria parasite only in 2000. Although Plasmodium knowlesi is prevalent only in a restricted area in South-East Asia and not responsible for many infections when regarded globally, it can cause severe malaria in humans and is phylogenetically closer to other not cultivable human Plasmodi- um species. The invention provides a remedy for this.

[0032] While in one embodiment of the pharmaceutical composition according to the invention said boromycin is the only active ingredient, in another embodiment said pharmaceutical composition comprises an additional active ingredient.

[0033] Said latter measure has the advantage that the addition of an additional active ingredient may result in an action amplification of the boromycin or even a synergis- tic effect. E.g., the additional active ingredient may be another anti-malaria compound.

[0034] Another subject-matter of the invention relates to a method for the inhibi- tion of the transmission of malaria in humans comprising the administration of the boromycin according to the invention or of the pharmaceutical composition according to the invention into a human suffering from or suspected of having malaria or of being infected by the parasite.

[0035] The feature, characteristics, advantages and embodiments disclose for boromycin according to the invention or of the pharmaceutical composition according to the invention apply to the method according to the invention mutatis mutandis.

[0036] It is to be understood that the before-mentioned features and those to be mentioned in the following cannot only be used in the combination indicated in the respec- tive case, but also in other combinations or in an isolated manner without departing from the scope of the invention.

[0037] The invention is now further explained by means of embodiments result- ing in additional features, characteristics and advantages of the invention. The embodi- ments are of pure illustrative nature and do not limit the scope or range of the invention. The features mentioned in the specific embodiments are general features of the invention which are not only applicable in the specific embodiment but also in an isolated manner and in the context of any embodiment of the invention.

[0038] The invention is now described and explained in further detail by refer- ring to the following non-limiting examples and figures.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 : Chemical structures of tetracyclines tested by the inventors.

Fig. 2: Chemical structures of macrolides tested by the inventors.

Fig. 3: Representative micrographies of Plasmodium falciparum infected erythrocytes stained with Giemsa: (A) untreated ring stage parasites, (B) strain 3D7 treated with 1 nM of boromycin for 6 h and (C) strain Dd2 treated with 1 nM of boromycin for 6 h. Arrows: parasites. Scale bar: 10 μm.

Fig. 4: Activity of boromycin, and the positive controls epoxomicin and methylene blue against mature (stage IV-V) Plasmodium falciparum gametocytes treated for 48 h. Boromycin showed a more potent activity than methylene blue and a comparable activity to epoxomicin. All experiments have been done at least 3 times in duplicate.

EXAMPLES

1. Material and methods

1.1 Chemicals

[0039] All tested drugs were first dissolved in their respective solvents and sub- sequently further diluted in complete medium to have the desired final concentration (see Table 1).

Table 1 : List of drugs tested by the inventors.

The concentration of the solvent did not interfere with parasite growth. Parasite in vitro culture Plasmodium falciparum asexual stages [0040] Plasmodium falciparum laboratory strains 3D7 (chloroquine-sensitive, provided by BEI resources, MRA-102), Dd2 (multidrug-resistant, provided by BEI re- sources, MRA-150), K1 (multidrug-resistant, provided by BEI resources, MRA-159), 7G8 (chloroquine resistant, provided by BEI resources, MRA-154) and NF-54 (chloroquine sensitive, provided by Sanaria, a vigorous gametocyte producer), and Plasmodium knowlesi laboratory strain A1-H.1 (kindly provided by Robert Moon of LSHTM, London, UK) were cultivated in vitro as described before. Briefly, the parasites were maintained in complete culture medium consisting of RPMI-1640 (Sigma-Aldrich) supplemented with 1M HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) solution (2.4% v/v) (Sigma-Aldrich), 200 mM L-glutamine (Gibco), 50 pg/ml gentamicin (Gibco), and 10% of AlbuMax II solution containing RPMI, HEPES, NaHCO ₃ , D-Glucose and 0.5% wt/vol of albumin, 0.4 g of hypoxanthine at 2.5% hematocrit. The cultures were maintained at 5% CO ₂ , 5% O ₂ , at 37°C, with a change of medium every two days. Plasmodium falciparum parasites were synchronized before assays through magnetic column separation.

1.2.2 Plasmodium knowlesi asexual stages

[0041] Plasmodium knowlesi was cultivated as described for Plasmodium falci- parum, with minor modifications. Parasites were cultivated in complete culture medium as detailed above supplemented with 5% human AB serum. Shortly before the growth inhibition assays, Plasmodium knowlesi were synchronized by density centrifugation with Nycodenz solution (Progen) to recover mature schizonts as described before. The Nyco- denz solution was prepared according to the manufacturer’s instruction and kept protected from the light at 4°C and warmed before using.

[0042] The asexual growth inhibition assays were performed with 3D7 and A1- H.1 , while NF-54 was used for gametocyte assays.

1.2.3 Plasmodium falciparum sexual stages

[0043] Plasmodium falciparum gametocyte culture was performed as described previously with minor modifications. The gametocyte culture was initiated with synchro- nized ring-stage NF54 parasites at 6% hematocrit and 0.3% parasitemia. Complete culture medium (as described above) was supplemented with 5% human serum and changed daily without parasite dilution for 14 days. The medium was doubled when parasitemia reached 5%, and 50-mM N-acetyl-d-glucosamine (MP Biomedicals GmbH) was added between day 11 and day 14 to remove asexual stages. On day 15, mature gametocytes were purified with a Nycodenz and magnetic column separation (MACS) to remove erythrocytes and concentrate the gametocyte population.

1.3 Growth inhibition assays with asexual parasites

[0044] Growth inhibition assays of asexual stages of Plasmodium falciparum were performed as described before. Briefly, the drugs (see Table 1) were distributed in a 3-fold serial dilution in 96-well plates. Synchronized ring-stage parasites were diluted to a parasitemia of 0.05% with O Rh+ erythrocytes in complete culture medium and seeded at a hematocrit of 1.5% in a total volume of 225 μl per well. Plates were subsequently incubated at 5% CO ₂ , 5% O ₂ , at 37°C before the plates were frozen and thawed three times. The growth inhibition of Plasmodium falciparum was measured through an enzyme- linked immunosorbent assay (ELISA) for histidine-rich protein 2 (HRP2) using a mi- croplate reader (CLARIOstar BMG Labtech) (excitation filter: 450 nm).

[0045] Growth inhibition assays with Plasmodium knowlesi were performed as described previously with minor modifications. Briefly, the drugs (see Table 1 ) were distributed in a 3-fold serial dilution in 96-well plates. Synchronized ring-stage parasites were diluted to a parasitemia of 0.5% with O Rh+ erythrocytes and subsequently seeded at a hematocrit of 2% and complete culture medium without human AB serum but double (20%) AlbuMax II solution in a total volume of 225 pl per well. The growth inhibition of Plasmodium knowlesi was measured with SYBR Green-I (ThermoFisher) using CLAR- IOstar (BMG Labtech) (490 nm excitation filter).

[0046] Plasmodium falciparum and Plasmodium knowlesi were incubated with the drugs for 3 and 6 days to assess the onset of activity. Both time points were assessed because many antibiotics target only the progeny of the treated parasites, causing a delayed death effect that is measured only after the second replication cycle (after 6 days). Chloroquine and clindamycin were used as positive controls for 3- and 6-day assays, respectively.

1.4 In vitro activity against mature gametocytes of Plasmodium falciparum

[0047] Drug sensitivity assays against mature (stage V) Plasmodium falciparum gametocytes were performed as described previously. Briefly, the drugs were precoated in a 3-fold dilution in 96-well plates. Epoxomicin and methylene blue (Table 1 ) were used as positive control. Subsequently, the previously purified mature gametocytes were seeded to the plates (50,000 gametocytes/well) and incubated at 37°C in 5% CO ₂ and 5% O ₂ . After 48 h, the ATP production was measured by the BacTiterGlo assay (Promega), according to the manufacturer’s instructions. Finally, the results were quantified using a luminometer (LUmo; Autobio).

1.5 Thin blood smear microscopy

[0048] Asexual Plasmodium falciparum stages, 3D7 and Dd2 were incubated with boromycin at 1 nM for 6 h for visual observation of drug effect. Then, thin blood smears were prepared, fixed in 100% methanol for 10 seconds, and stained with Giemsa (Merck) solution (5%) for 20 minutes. The slides were washed and observed under a Leica DMBL microscope, and pictures were taken using a ProgRes C10 camera and software (Jenoptik), at 100X magnification.

1.6 Cytotoxicity assay

[0049] HepG2 cells (obtained from ATCC; HB-8065), a human hepatocyte car- cinoma cell line, were maintained in DMEM medium (Sigma-Aldrich) supplemented with 10% of inactivated foetal bovine serum (Sigma-Aldrich), 200 mM L-glutamine (Gibco), 12 mL of HEPES buffer (Gibco), and 50 pg/ml penicillin/streptomycin solution (Gibco). Trypsin (Gibco) was used to detach the cells when they reached a semi-confluent layer. [0050] Cytotoxicity was evaluated using the neutral red assay as described previously. Briefly, 300,000 cells were seeded in supplemented Dulbecco's Modified Eagle's Medium (DMEM) medium as described above to 96-well plates. After 24 hours, the cells were incubated with a twofold serial dilution of the respective drug diluted in supplemented DMEM medium for an additional 24 hours. Subsequently, the drug- containing medium was replaced by a supplemented DMEM medium with 1 .5% of neutral red, and the cells were incubated for an additional 3 h at 37°C. Cells were then washed with phosphate buffered saline (pH 7.2), and 100 pL of freshly prepared lysing buffer (50% methanol, 49% distilled water, and 1% acetic acid) was added to the plates. Subsequent- ly, the cells were shaken for 10 min and the absorption was measured at a wavelength of 540 nm using CLARIOstar (BMG Labtech).

1.7 Apicoplast as the target of boromycin

[0051] Growth inhibition assays were prepared as described in section 2.2 with modifications to assess whether the apicoplast is the main target of boromycin. Briefly, the parasites (Plasmodium falciparum strain 3D7) were incubated with boromycin and clin- damycin (positive control) and supplemented with isopentenyl pyrophosphate trilithium salt (IPP) (Sigma) at 200 pM for 6 days as described before. The supplementation with IPP is well-described to rescue treated parasites when the apicoplast is the main target of the drug. The assay was done with and without IPP supplementation on the same plate and results were directly compared.

1.8 lonophoric activity assay

[0052] Previously, boromycin was described to act as a potassium ionophore in Bacillus subtilis and Mycobacterium tuberculosis. So herein, growth inhibition assays complemented with KCI or MgCI ₂ (negative control) were performed to evaluate if this is also the case in Plasmodium falciparum. To define a subtoxic concentration of MgCI ₂ and KCI for Plasmodium falciparum a standard growth inhibition assays with serial dilutions of these compounds were performed as described in section 1.2. The concentration of 220 pg/mL was chosen as no signs of toxicity were seen and first inhibitory effects were observed with 440 pg/mL. Subsequently, a growth inhibition assay was prepared with a 2- fold dilution of boromycin with the addition of the sub-toxic concentration of either MgCI ₂ (Sigma) or KCI (Sigma) to all wells. The assay was run in parallel to a standard growth inhibition assay of boromycin without supplementation of MgCI or KCI as control, and the obtained IC ₅₀ s were compared.

1.9. Analysis

[0053] All assays were performed at least three times in duplicates. Individual IC ₅₀ values were determined by nonlinear regression analysis of log concentration- response curves, using the drc v0.9.0 package of R v2.3.1 . Mean IC ₅₀ values and stan- dard deviations (SDs) were calculated for each growth inhibition assay using Excel. The chemical structures of the drugs were drawn using ChemDraw 19.0. The graphical presentation of the gametocyte inhibition was done with GraphPad Prism v7.04.

2. Results

2.1 Activity of antibiotics against asexual stages of Plasmodium falciparum and Plasmodium knowlesi

[0054] Plasmodium falciparum and Plasmodium knowlesi parasites were ex- posed to 15 antibiotics (tetracyclines, macrolides) and chloroquine (control drug) for 3 and for 6 days. This allowed assessing the drug's inhibitory effect on the first and second parasite replication cycle, respectively (see results in Table 2), that indicates a fast or slow onset of antiplasmodial activity of the antibiotics. -*■

[0055] None of the tetracyclines evaluated in this study showed a pronounced activity (in the nanomolar range) in the 3-days assay against Plasmodium falciparum and Plasmodium knowlesi, but had some delayed activity after 6 days of drug exposure (Table 2). Chlortetracycline, methacycline and lymecycline were still poorly active after 6 days. The first-time tested drugs against Plasmodium falciparum demeclocycline, sarecycline, omadacycline and the already previously tested drugs/controls doxycycline, minocycline, and eravacycline were active in the nanomolar range against both species after 6 days (Table 2). Boromycin was highly active against Plasmodium falciparum and Plasmodium knowlesi at an IC ₅₀ of 1.0 nM after 3 days of incubation (Table 2).

[0056] Most antibiotics were equipotent and displayed similar activities (within a three-fold difference) against Plasmodium falciparum and Plasmodium knowlesi especially in the 6-day assay. Differences can be seen for meclocycline, and sarecycline. In the 3- day assay Plasmodium knowlesi was more vulnerable than Plasmodium falciparum except for boromycin and oleandomycin.

2.2 Cytotoxicity of boromycin against human cells

[0057] Boromycin was cytotoxic at millimolar concentrations against human HepG2 cells with a selectivity index (SI) higher than 200,000. (Table 3). Chloroquine was used as comparator and was more cytotoxic than boromycin.

IC ₅₀ (|jM) in HepG2 cells SI 3D7 * SI A1-H.1 *

Boromycin 1250.5 ± 230 1 ,388,000 211 ,864

Chloroquine 153.4 ± 52.9 18,214 10,551

Table 3: Cytotoxicity of boromycin and the comparator chloroquine against HepG2 cells and calculated selectivity index. All experiments have been done at least 3 times in duplicate.

2.3 Characterization of the antiplasmodial activity of boromycin

2.3.1 Activity of boromycin against asexual stages of Plasmodium falciparum chloro- quine-resistant strains [0058] Different drug resistant strains of Plasmodium falciparum (Dd2, K1 and 7G8) were incubated with boromycin for 3 days to assess the potential of cross- resistance. Activities were comparable to activities against 3D7 with IC ₅₀ values in the picomolar range, and no cross-resistance of boromycin in these parasite lines was seen (Table 4) (Figure 2).

Table 4: Antiplasmodial activity of boromycin against different Plasmodium falciparum chloroquine-resistant strains in comparison to 3D7 after 3 days of incubation. All experi- ments have been done at least 3 times in duplicate.

[0059] In addition, Plasmodium falciparum parasites strain 3D7 (Figure 2 B) and Dd2 (Figure 2 C) showed the same pyknotic appearance after boromycin incubation at 1 nM for 6 h, while the untreated control showed the usual ring shape (Figure 2 A).

2.3.2 Activity of boromycin against mature gametocytes

[0060] Mature Plasmodium falciparum gametocytes (stage IV and V) were in- cubated with a serial dilution of boromycin for 48 h. Afterwards, ATP production was quantified to evaluate the gametocytocidal effect (Figure 4). The results revealed that boromycin was highly active with IC ₅₀ at 8.5 ± 3.6 nM, similar or even more active than the comparator compounds epoxomicin (15.2 ± 3.4 nM) and methylene blue (284 ± 35 nM), respectively.

2.3.3 Activity of boromycin on the apicoplast

[0061] Plasmodium falciparum parasites were incubated with boromycin sup- plemented with IPP for 6 days to assess whether boromycin targets the apicoplast as described for many antibiotics; clindamycin was used as positive control. At the same time a standard growth inhibition assay without IPP was carried out as a comparator. As expected, clindamycin-treated parasites could be rescued by the supplementation with IPP since the IC ₅₀ values of control and IPP-supplemented parasites were 6.9 nM ± 1 .3 and >18000 nM, respectively. In contrast to this, the IC ₅₀ values for boromycin remained similar at low nanomolar concentrations for control (0.9 nM ± 0.1) and in the presence of IPP (1.4 nM ± 0.8), suggesting that this drug effect cannot be rescued by IPP and has a different target from the apicoplast.

2.3.4 lonophoric activity of boromycin

[0062] P. falciparum parasites were incubated with a serial dilution of boromycin, and chloroquine (control) with or without supplementation of 220 pg/mL of MgCI ₂ or KCI, for 3 days to assess whether boromycin has the ionophoric activity on Plasmodium falciparum as described for Mycobacterium and Bacillus subtilis (Table 5).

Table 5: Activity of boromycin and the control drug chloroquine against the Plasmodium falciparum strain 3D7 in the presence/absence of KCI (220 pg/ml) and MgCI ₂ . (220 pg/ml). All experiments have been done at least 3 times in duplicate.

[0063] Boromycin and chloroquine displayed the expected IC50 value, the sup- plementation of KCI at 220 pg/mL increased IC ₅₀ of boromycin by 3-fold, while no differ- ence was observed for chloroquine. The presence of MgCI2 had a synergistic effect for both boromycin and chloroquine leading to a 3-5-fold decrease in the respective IC ₅₀ s.

3. Discussion [0064] Antibiotics for malaria treatment have been investigated since the middle of the last century, with tetracyclines playing a central role. In recent years, macrolides have also been particularly studied as antimalarials. Although the delayed death effect caused by most antibiotics limits their use as first-line chemotherapy, some antibiotics such as doxycycline, clindamycin, and the combination sulfadoxine-pyrimethamine (that does not display the delayed activity) are recommended as prophylaxis or treatment in certain populations. The need for new alternative therapies, along with the limited invest- ments in drug discovery to treat malaria, make drug repurposing a worthy alternative. By the inventors the in vitro antiplasmodial activity of tetracyclines and macrolides not previ- ously tested against Plasmodia along with comparator drugs were evaluated against blood-stages of Plasmodium falciparum and Plasmodium knowlesi, revealing boromycin, as a potent antiplasmodial drug with a fast/first cycle activity that the inventors assessed further.

[0065] All tested active tetracyclines showed a delayed death effect against both Plasmodium species: Plasmodium falciparum (strain 3D7) and Plasmodium knowlesi (strain A.1-H1 ). The newly licensed tetracyclines omadacycline (licensed for treatment of bacterial pneumonia and acute skin infections) and sarecycline (licensed for treatment of acne) showed comparable activity to doxycycline. None of the newly tested tetracyclines achieved the activity of the next generation tetracyclines tigecycline and eravacycline that suggested that modifications at the C9 position on the D ring that circumvents the riboso- mal protection resistance mechanism found in bacteria could be beneficial for the activity against Plasmodia. However, the structurally similar omadacycline did not show this improved activity. In a similar way, differences in activities for these three antibiotics were also seen for rickettsia species. The delayed death effect was also shown by the macrolide josamycin suggesting that it also targets the apicoplast. The tetracyclines chlortetracycline, methacycline, lymecycline, and the macrolides troleandomycin and oleandomycin were only poorly active with IC ₅₀ in the micromolar range. Whereas the macrolide boromycin showed a fast onset of activity at a single-digit nanomolar concentra- tion.

[0066] Plasmodium falciparum is undoubtedly responsible for most of the malaria burden, especially for most of the malaria deaths, which justifies to mainly tailor treatments to this malaria species. However, there are five additional Plasmodium species responsible for malaria in humans (Plasmodium vivax, Plasmodium ovale wallikeri, Plasmodium ovale curtisi, Plasmodium malariae, and Plasmodium knowlesi) and when elimination of malaria is the aim, all these species must be targeted. Until recently, Plasmodium falciparum was the only option for drug screening in vitro because the other human malaria parasites could not be kept in continuous in vitro culture. However, now Plasmodium knowlesi has been adapted for in vitro culture and its inclusion in drug screenings could give additional information on antiplasmodial activities of compounds as it is phylogenetically closer to the other human Plasmodium species. Even though previ- ous studies have claimed that most antiplasmodial compounds are equipotent against Plasmodium falciparum and Plasmodium knowlesi, recent studies have reported different susceptibility profiles especially for the novel compounds in development. Plasmodium knowlesi was described as less susceptible to inhibitors of the dihydrofolate reductase, dihydroorotate dehydrogenase, and ATP4. The inventors found in the short 3-day assay a higher activity of most antibiotics against Plasmodium knowlesi in comparison to Plasmodium falciparum. This is probably due to the assay conditions, as Plasmodium knowlesi has a shorter erythrocytic cycle (24 hours) compared to Plasmodium falciparum (48 hours) and the inventors performed the short assay for both species for 3 days. Different results might also be due to the different assay conditions such as parasitemia (0.05% versus 0.5%), haematocrit (1 .5% versus 2%) and mode of evaluation (protein quantification via HRP2 versus DNA quantification via 318 SYBR-Green) of the growth inhibition assays for Plasmodium falciparum and Plasmodium knowlesi, respectively. However, despite these differing conditions the results for the 6 day/multiple cycle assay were similar for eleven of the drugs indicating that most tested antibiotics are equally active against both species.

[0067] The most promising compound from the inventors' screening was the macrolide boromycin. Boromycin was isolated from a Streptomyces antibioticus found in a soil sample from Ivory coast and was first described in 1967. It has a potent effect against Gram-positive bacteria in vitro, and it was shown to be active against HIV, Babesia and some other pathogens. An activity against two animal-specific Plasmodia was once described in a patent filed in the US in 1973 where its activity in models of Plasmodium berghei in mice and Plasmodium gallinaceum in birds is given. However, no description of the evaluation of its in vitro activity, its activity against human malaria pathogens, such as Plasmodium falciparum and Plasmodium knowlesi, or its gametocytocidal activity have been described before.

[0068] In the investigation of the inventors, boromycin showed a high activity and a fast onset of action, a desired property of antimalarials to quickly reduce parasite load and avoid the disease progression to the severe form. It also showed an excellent selectivity index against HepG2 cells. A favorable toxicity profile was confirmed and encouraged a deeper investigation about the antiplasmodial activity of boromycin.

[0069] Boromycin’s in vitro activity against laboratory strains showed superior activity as it was active at low nanomolar and even at picomolar concentrations. No cross- resistance with the tested chloroquine/multi-resistant strains (Dd2, 7G8, and K1 ) was seen as the IC ₅₀ values were in same range as for the drug sensitive strain 3D7, suggesting that is not affected by the common resistance mechanisms. Morphologically, ring stage parasites showed a pyknotic appearance after 6 h treatment with boromycin at 1 nM, suggesting that it leads to a rapid death. The inventors additionally evaluated the activity of boromycin against mature gametocytes and could see a very potent activity superior to the positive control methylene blue, similarly active as the proteasome inhibitor epox- omicin. Drugs with transmission-blocking activity are of special interest and would be a great asset for controlling and eliminating malaria. Currently, primaquine is the only approved drug active against gametocytes but its use is restricted mainly due to its haemolysis-inducing effect in patients with glucose-6-phosphate dehydrogenase deficien- cy, and additionally it has to be metabolized by a liver enzyme (CYP2D6) to be active. Compounds with multi-stage activities and novel modes of action are especially desired for the next generation of antimalarial compounds.

[0070] The apicoplast is described as the main target of antibiotics that exert the delayed death effect and the fast-acting antibiotic fosmidomycin. Apicoplast-free parasites can survive and proliferate when the medium is supplemented with IPP, but this did not rescue the boromycin-treated parasites in our assay; therefore, we could rule out the apicoplast as a main target. [0071] The ionophoric activity of boromycin, especially for K ⁺ channels, was firstly described in Bacillus subtilis in addition to the impairment of protein, RNA, and DNA synthesis. This ionophoric activity was later confirmed in Mycobacterium bovis, where a rapid loss of membrane potential, reduction of intracellular ATP, and leakage of cytoplas- mic proteins were induced and the external addition of a high concentration of KCI (20 mg/mL) decreased boromycin activity from 50% to 10%. To assess whether boromycin acts as a potassium ionophore also in Plasmodium falciparum, parasites were simultane- ously incubated with boromycin together with the highest non-toxic concentration (220 pg/mL) of KCI or MgCI ₂ (negative control). The inventors could not incubate Plasmodium parasites at a similar high ion concentration as in the Mycobaterium experiments, as this concentration was toxic to the parasites. The inventors identified a three-fold IC ₅₀ increase with the addition of KCI, suggesting that ionophoric activity could play a role in the potent antiplasmodial activity of boromycin. However, it is most probably not the primary mode of action, as the activity remained in the low nanomolar range. Remarkably, the inventors saw a potentiation of the drug effect by addition of MgCL

[0072] In conclusion, the inventors presented the antiplasmodial activity of dif- ferent tetracycline and macrolide antibiotics against different Plasmodium species. The tetracyclines tested by the inventors did not show higher in vitro activities than previously tested tetracyclines and all displayed a slow/second cycle onset of action. The macrolide boromycin showed the most noticeable results as a fast acting and gametocytocidal compound. These results suggest that boromycin may provide for an alternative therapy for diseases caused by apicomplexan parasites, including Plasmodia.