Compounds for the treatment of leishmaniosis (Machine-translation by Google Translate, not legally b

专利摘要:
Compounds for the treatment of leishmaniosis. The present invention relates to a family of compounds which are effective against parasites of the genus leishmania and which have a low toxicity even at high doses of the compound. The invention also relates to pharmaceutical compositions, preferably suitable for oral administration, comprising these compounds and their use as medicaments, preferably for the treatment of diseases caused by parasites of the genus leishmania. (Machine-translation by Google Translate, not legally binding)
公开号:ES2607628A1
申请号:ES201630944
申请日:2016-07-11
公开日:2017-04-03
发明作者:José Antonio GÓMEZ VIDAL；Mónica DÍAZ GAVILÁN；Francisco FRANCO MONTALBÁN；Francisco MORILLAS MÁRQUEZ；Victoriano CORPAS LÓPEZ；Joaquina MARTÍN SÁNCHEZ；Margarita LÓPEZ-VIOTA GALLARDO；Julián LÓPEZ-VIOTA GALLARDO
申请人:Universidad de Granada；
IPC主号:

专利说明:

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COMPOUNDS FOR THE TREATMENT OF LEISHMANIOSIS SECTOR OF THE TECHNIQUE
The present invention is generally framed in the field of biomedicine and in particular refers to a new compound useful as a medicament, preferably useful for the treatment of diseases caused by parasites of the genus Leishmania.
STATE OF THE TECHNIQUE
Leishmaniasis
Leishmaniasis is a vector-borne disease caused by parasitic protozoa of the genus Leishmania (Kinetoplastida, Trypanosomatidae) and transmitted by the female bite of phlebotomes (Insecta, Diptera, Phlebotomidae) belonging to the genera Phlebotomus and Lutzomyia. It is one of the most important diseases in tropical diseases and is included in the "Neglected Tropical Diseases" or forgotten tropical diseases. Despite this, it is not limited to tropical and subtropical areas, and it also occurs with endemic character in temperate zones such as our country. The climactic spectrum of leishmaniasis is broad and ranges from a strict cutaneous form that can heal spontaneously leaving a scar, to a serious and deadly form without treatment such as visceral leishmaniasis. According to the World Health Organization, up to 350 million people are at risk of leishmaniasis in 98 countries in temperate and tropical areas of the planet. Approximately 12 million people are considered to be currently infected, and every year there are 2 million new cases, of which 500,000 cases are visceral.
Leishmania species show two morphological phases during their life cycle:
• Promastigote, with an elongated and mobile shape with an anterior scourge, in the intestine of the vector.
• Amastigote, with a spherical or ovoid shape and without a visible scourge under the optical microscope, which reproduces in a mandatory manner within the cells of the vertebrate host endothelial reticulum system (man and various species of animals depending on the Leishmania species).
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Of the twenty species responsible for leishmaniosis, only two, L. donovani and L. infantum (synonym L. chagasi) cause visceral leishmaniasis. L. infantum is the only species identified that causes both visceral and cutaneous disease. In addition, it has also been identified as a causative agent of cutaneous-mucosal and mucosal leishmaniasis. While in visceral leishmaniasis the parasite proliferates in the bone marrow, the gland and the spleen, organs in which the cells of the endothelial reticulum system abound, in the cutaneous form the parasite remains at the site of the phlebotome bite, which can cause Long-lasting ulcers that end up leaving disfiguring scars. As we said before, the disease in its visceral form is fatal if it is not treated correctly. L. infantum is the species with the widest distribution being present in 61 of the 98 endemic countries of leishmaniasis. L. infantum is endemic in all the countries of the Mediterranean Basin unlike L. donovani, leishmaniasis due to L. infantum is a zoonosis, the dog being the main reservoir. In Spain, the prevalence of canine leishmaniasis in random sampling using serological techniques varies between 5 and 30%, and can reach 100% in some foci if the PCR technique is used [Martm-Sanchez J, Morales-Yuste M , Acedo-Sanchez C, Baron S, Diaz V, Morillas-Marquez F. Canine leishmaniasis in southeastern Spain. Emerg Infect Dis. 2009 May; 15 (5): 795-8. doi: 10.3201 / eid1505.080969]. These high prevalence of infection are a considerable risk factor for the emergence of the disease in humans. In the dog, L. infantum causes canine leishmaniasis which is a multisystemic syndrome of very difficult healing due to the lack of collaboration of the animal's immune system. Until now, none of the drugs used for the treatment of canine leishmaniasis (which are the same used in man) allows the complete elimination of the parasite in the animal. In Spain there can be between 6 and 13 million dogs. Although the prevalence of canine leishmaniasis varies between regions and according to the technique used, we can estimate that 30% of them are infected; This would give us 2.8 million dogs with canine leishmaniasis as potential drug recipients against leishmaniosis, only in Spain. To them we must add those existing in other endemic countries such as Portugal, France, Italy, Brazil, etc., in addition to other non-endemic ones. For example, in Germany, despite not being an endemic country of leishmaniosis, it is estimated that there are 20,000 dogs with canine leishmaniasis acquired during the holidays in the countries of the Mediterranean Basin that would also be potential recipients.
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Leishmaniosis Treatment
The drugs currently used for the treatment of leishmaniasis present various problems, including host toxicity, adverse effects, the emergence of resistance and the high cost. Among them, pentavalent antimony derivatives, such as meglumine antimoniato or methylglucamine antimoniato (Glucantime®) have been considered first choice for a long period of time, despite their high toxicity. However, meglumine antimoniato is not suitable for oral administration because it is poorly absorbed and is extremely irritating to the gastrointestinal tract, so it must be administered parenterally or by local injection.
Pentamidine, amphotericin B and paromomycin have been considered as second-choice drugs, although lately liposomal amphotericin B is replacing antimony derivatives as a drug of choice in some geographical areas. However, pentamidine and amphotericin B have a high toxicity that can be reduced with the lipid formulations of the latter but raising the price considerably. On the other hand, paromomycin is not absorbed orally, so it must be administered parenterally or topically; it is excreted by the kidney without metabolizing and induces nephrotoxicity and ototoxicity, as well as other aminoglycosides [Ramos, J. and Segovia, M. - Spanish Journal of Chemotherapy, 1997. p 26].
The last drug incorporated into the therapeutic arsenal of leishmaniasis is miltefosine that initially conceived as an antineoplastic drug has the advantage of allowing an oral administration but is not exempt from toxicity, has resistance problems [Mondelaers A, and others. "Genomic and Molecular Characterization of Miltefosine Resistance in Leishmania infantum Strains with Either Natural or Acquired Resistance through Experimental Selection of Intracellular Amastigotes”. PLoSOne. 2016 Apr 28; 11 (4): e0154101. Doi: 10.1371 / journal.pone.0154101], causes gastrointestinal adverse effects in addition to being potentially teratogenic and abortive.
HDAC inhibitors with activity against Leishmaniosis
The drug vorinostat (also called SAHA) (Formula I) is a histone deacetylase (HDAC) inhibitor that has activity against Leishmania [9, 10].
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image 1
Vorinostat has as its main disadvantage its toxicity at high concentrations
On the other hand, patent ES 2 402 252, requested by the University of Granada, describes a structure with formula II that has activity against Leishmania.
image2
As with meglumine antimonate, compound II is not suitable for oral administration since it is not chemically stable at low pH. The trityl chemical group, present in compound II, is a known unstable protective group at acidic pH (4 and less).
There is therefore a need to find a drug for the treatment of Leishmania infection, which solves the problems described in the state of the art, especially that provides greater efficacy in the elimination of the parasite, less toxicity and adverse effects for the patient ( host) and that allows the possibility of oral administration.
In addition, the use of vehiculization and release systems such as nanoparticles can solve problems of solubility of hydrophobic compounds whose use in vivo would be unfeasible, in addition to improving efficiency and reducing adverse effects.
BRIEF DESCRIPTION OF THE INVENTION
The present invention provides a family of compounds effective against parasites of the Leishmania genus that have low toxicity even at high doses of the compound.
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Thus, in a first aspect, the present invention relates to the compound of formula III:
H
OR'"
In a second aspect, the present invention relates to a pharmaceutical composition comprising the compound of formula III as described above and pharmaceutically acceptable excipients. In a particular embodiment, the invention relates to a pharmaceutical composition comprising the compound of formula III supported on pharmaceutically acceptable nanoparticles, preferably gold nanoparticles.
In a third aspect the present invention relates to the use of the compound of formula III or of the pharmaceutical compositions comprising it as a medicament. In particular, the invention relates to its use for the treatment of diseases caused by parasites of the genus Leishmania, preferably L. infantum.
image3
BRIEF DESCRIPTION OF THE FIGURES Figure 1.- Synthesis scheme of compound III.
Figure 2.- Image obtained by microscopy of the nanoparticles. In the image above, only the gold nuclei without compound are observed; in the lower one, the nanoparticles are vehiculizing the compound and a crown is observed around the gold nuclei that almost doubles the hydrodynamic size of the particles in the upper image.
DETAILED DESCRIPTION OF THE INVENTION Definitions
The term "nanoparticles" as described in the present invention refers to particles of less than 1 micrometer in any of its dimensions, preferably in a range of 10 to 900 nanometers.
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The term "treatment" or "treat" in the context of this document refers to the
administration of a compound or a composition according to the invention to improve or eliminate a disease, pathological condition or one or more symptoms associated with said disease or condition in a mammal, preferably in canids or humans, more preferably in dogs. "Treatment" also covers the improvement or elimination of the physiological sequelae of the disease. Specifically, the concept "treat" can be interpreted as:
i. Inhibit the disease or pathological condition, that is, stop its development;
ii. Relieve disease or pathological condition, that is, it causes disease regression or pathological condition;
iii. Stabilize the disease or the pathological condition.
Throughout the description and claims the term "comprises", which may also be construed as "consists of", and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will be derived partly from the description and partly from the practice of the invention.
Compounds of the invention
In a first aspect of the invention, the present invention relates to compounds ("compounds of the invention"), of formula III
H
N
image4
Ph
III
Where Ph is a phenyl of formula:
image5
Ph
Being:
R1 = H, F, Cl, Br, I, Me, OMe or NO2.
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R2 = H, F, Cl, Br, I, Me, OMe 6 NO2 R3 = H, F, Cl, Br, I, Me, OMe 6 NO2
In a particular embodiment, the compounds of the invention are presented in crystalline form as free or solvated compounds (for example hydrates) or as enantiomers, isomers, or salts of said compounds, all of these forms being within the scope of protection of the present invention. . Solvation methods are generally known in the state of the art.
The prodrugs of the compounds of the invention are also included in the scope of the present invention.
In a particular embodiment the compounds of the invention are compounds of formula III characterized in that R1 = H or R2 = H or R3 = H.
In an even more particular embodiment, the compounds of the invention are compounds of formula III characterized in that at least two of the three phenyl radicals are formed by hydrogen. That is, compounds of formula III characterized in that R1 = R2 = H or R1 = R3 = H or R2 = R3 = H.
In a preferred embodiment, the invention relates to compounds of formula IIIa consisting of compounds of formula III characterized in that the three phenyl radicals are formed by hydrogen. That is, compounds of formula III characterized in that R1 = H, R2 = H and R3 = H.
image6
N
H
image7
H
H
Pharmaceutical compositions
In a second aspect, the invention provides pharmaceutical formulations or compositions, hereinafter "formulations of the invention" or "compositions of the invention" comprising as active ingredient at least one compound of the invention, in particular a compound of those detailed in preferred embodiments
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of the first aspect of the invention. Said formulations may contain any other active ingredient in the treatment of leishmaniosis or be characterized as containing only one compound of the invention or a combination of compounds of the invention as the active principle.
The compounds of the invention, III, by their hydrophobic nature, make their utilization in vivo unfeasible unless it is vehiculized. In particular, compound III in aqueous medium adds rapidly, making parenteral administration impossible.
Thus, said pharmaceutical formulations comprise one or more pharmaceutically acceptable vehicles.
The "pharmaceutically acceptable vehicles" that can be used in the formulations of the invention are the vehicles known to those skilled in the art and commonly used in the elaboration of therapeutic compositions. An example of a pharmaceutically acceptable vehicle is a gold nanoparticle with a size suitable for administration.
Optionally the pharmaceutical composition may comprise another active ingredient. In addition to the requirement of therapeutic efficacy, which may require the use of therapeutic agents, in addition to the compounds of the invention, there may be additional fundamental reasons that compel or strongly recommend the use of a combination of a compound of the invention and other therapeutic agent, such as in the treatment of diseases or conditions that directly or indirectly modulate the function of the substance.
The formulations may also contain excipients.
The excipients and vehicles used must be pharmaceutically and pharmacologically tolerable, so that they can be combined with other components of the formulation or preparation and do not exert adverse effects on the treated organism. Pharmaceutical compositions or formulations include those that are suitable for oral or parenteral administration (including subcutaneous, intradermal, intramuscular and intravenous), although the best route of administration depends on the patient's condition. The formulations can be in
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Simple dose form. The formulations are prepared according to methods known in the field of pharmacology. The amounts of active substances to be administered may vary depending on the particularities of the therapy.
The compositions of the invention are prepared using usual methods such as those described or referred to in the Spanish and US Pharmacopoeias and similar reference texts.
Formulation in nanoparticles
In a preferred embodiment of the compositions of the invention the compound of the invention, III, is supported on pharmaceutically acceptable nanoparticles, more preferably on gold nanoparticles.
In a particular embodiment, the gold nanoparticles used as transport vehicles of the compound of the invention are coated by sodium citrate.
In another particular embodiment, the gold nanoparticles used as transport vehicles of the compound of the invention have an approximate size of between 20 and 30 nm, preferably between 22 and 26 nm.
Compound III thus functionalized has been shown to have much better solubility characteristics than compound alone, without diminishing its effectiveness.
Use of the compounds of the invention
A third aspect of the invention consists in the use of the compounds or compositions of the invention as a medicament. In a particular embodiment, the compounds or compositions of the invention are used as a medicament for the treatment of Leishmaniosis, preferably Leishmaniosis caused by L. infantum.
Similarly, the invention is also directed to the use of the compounds or compositions of the invention in the preparation of a medicament. In a particular embodiment, the compounds or compositions of the invention are employed in
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the preparation of a medication for the treatment of Leishmaniosis, preferably Leishmaniosis caused by L. infantum.
In another aspect, the present invention relates to a method for the treatment of patients affected by Leishmaniosis through the use of the compounds or compositions of the invention. The effects of this method of treatment include, but are not limited to, the effects of disease elimination, the increase in disease progression time and the survival rate. The effects of treatment include longer term disease control.
This treatment consists in the administration to the individuals affected by these diseases of therapeutically effective amounts of a compound of the invention, or a pharmaceutical composition that includes it.
In the sense used in this description, the expression "therapeutically effective amount" refers to that amount of a compound of the invention that when administered to a mammal, preferably canids and humans, more preferably dogs, is sufficient to produce the treatment, as defined below, of a disease or pathological condition of interest in the mammal, preferably canids and humans, more preferably dogs. The amount of a compound of the invention that constitutes a therapeutically effective amount will vary, for example, according to the activity of the specific compound employed; the metabolic stability and duration of the action of the compound; the species (preferably human or canine), the chemical form of the human disease (preferably visceral, cutaneous or mucous), age, body weight, general state of health, sex and diet of the patient; the route of administration, given the possibility of oral or systematic administration; the mode and time of administration; the rate of excretion, the combination of drugs; the severity of the particular disorder or pathological condition; and the subject who undergoes therapy, but can be determined by a specialist in the technique according to his own knowledge and that description.
The administration of the compounds of the invention, in pure form or in an appropriate pharmaceutical composition, can be carried out by means of the administration modes of agents accepted to serve similar utilities.
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The method used to synthesize the compounds of the invention comprises [Figure 1] reacting in an solution an acid of formula IV, with benzylhydroxyl amine of formula V.
image8
Where Ph is a phenyl of formula
image9
Ph
Being:
R1 = H, F, Cl, Br, I, Me, OMe or NO2
R2 = H, F, Cl, Br, I, Me, OMe or NO2
R3 = H, F, Cl, Br, I, Me, OMe or NO2
Preferably the solution will be carried out in anhydrous methanol / THF keeping the reaction in an argon atmosphere.
In a preferred embodiment, the compound of general formula III is obtained from a solution with the carboxylic acid derivative of general formula IV (1 equiv.) Ethyl chloroformate (1.35 equiv.), Triethylamine (3.5 equiv.) And benzylhydroxyl. amine (1.7 equiv.) in THF / methanol. The reaction should be maintained at 0 ° C and argon atmosphere.
Functionalization of III with gold nanoparticles
In a preferred embodiment, the compositions of the invention comprise compound III functionalized with gold nanoparticles.
To carry out the functionalization of the gold particles with compound III, it is
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Add the gold particle suspension to the solution of the dimethylsulfoxide compound (DMSO), preferably 0.5%, and Triton X100, preferably 0.1%, dropwise. Then, the suspension obtained is added dropwise onto a solution of Bobino Serum Albumine (Bovine Serum Albumina or BSA), preferably at 1%. After incubation for at least 2 hours, the particles are concentrated by centrifugation and resuspended in phosphate buffered saline or phosphate buffered saline (Phosphate Buffered Saline or PBS) pH 7.2.
MODES OF REALIZATION
The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention:
Synthesis Example of Compounds of the Invention
The compound O-benzylhydroxamate, IIIa, was obtained according to a reaction scheme (Figure 1) that derives from the procedure for synthesis of hydroxamic acids. Thus, the compound of formula IIIa is obtained from a solution with the carboxylic acid derivative of formula IV (1 equiv.), In anhydrous THF under argon. The solution is cooled and ethyl chloroformate (1.35 equiv.) Is added followed by triethylamine (3.5 equiv.). The mixture is stirred at low temperature in argon atmosphere. The precipitated solid obtained is filtered and the filtered liquid is added to a solution containing the compound of formula Va (1.7 equiv.) Dissolved in anhydrous methanol.
image10
It is kept under stirring for 1.5 h at room temperature, after which time the reaction is diluted with ethyl acetate and washed with distilled water (3x 15 mL). The resulting organic phase is dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue obtained is purified by flash chromatography using a mixture of dichloromethane and methanol as eluent to obtain IIIa (white solid, 28 mg, 40% yield).
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Ilia
H
N
image11
image12
H
H
Mp 130-131 ° C. 1H NMR (500 MHz, CD3OD) 5 7.54 (m, 2H), 7.33-7.25 (m, 7H), 7.08 (m, 1H), 4.75 (s, 2H), 2.52 (m, 2H), 2.36 (m, 2H), 1.70-1.65 (m, 4H), 1.41 (m, 4H); 13C NMR (125 MHz, CD3OD) 5 176.1, 174.6, 139.9, 138.0, 129.7, 129.5, 129.3, 128.5, 125.1, 121.3, 52.9, 37.9, 33.1, 30.1, 30.0, 26.8, 25.8; HRMS (ESI) m / z calculated for C21H27N2O3 [M + H] + 355.2022, found 355.2025. Purity according to HPLC (A = 254): 100%, tR = 11.14 min.
HPLC Method: Solution A (H2O: ACN 70:30 + 0.1% HCOOH), solution B (100% ACN + 0.1% HCOOH); isocratic solution 2 min .; gradient from A to B 13 min; isocratic solution B 5 min.
Conservation of the compounds
The compound Ilia can be stored at 4 ° C for at least 6 months without apparent changes in its physical-chemical characteristics or loss of activity.
On the other hand, the compound Ilia is chemically stable at pH 2 and pH 5, which allows oral administration.
Synthesis of inorganic gold-drug particles of the compound Ilia
Initially, gold nanoparticles were synthesized according to the method described by Turkevich [J. Turkevich, P.C. Stevenson, J. Hillier, Discuss. Trans. Faraday Soc. 11 (1951) 55], based on the reduction of gold salts in the presence of citrate at high temperatures.
Accordingly, a solution of 4.5mg of sodium tetrachlorouraurate (III) dihydrate in a volume of 25 mL was prepared. Under constant magnetic stirring, the solution was subjected to a temperature of 180 ° C under reflux. Once this condition was reached, 1mL of a 1% solution of sodium citrate was added to the gold solution. The particles were allowed to grow in the presence of citrate ions for a time never less than 20
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minutes Subsequently, the particle suspension was allowed to reach room temperature before being used. The result is a stable suspension of gold, wine red particles.
To carry out the functionalization of the gold particles with the Ilia compound, the compound was first dissolved at a concentration of 0.25% in a solution composed of 0.5% Dimethylsulfoxide (DMSO) and 0.1% Triton X100. Once the Ilia compound has dissolved, 20 mL of gold particle suspension is added dropwise. Immediately afterwards, the suspension of gold particles functionalized with the compound Ilia on a 1% BSA solution is added dropwise. It is allowed to incubate for a minimum of 2 hours in the presence of BSA, and then afterwards, it is centrifuged at 14000 rpm to remove excess BSA present in the middle of the conjugated particles. Once the particles have been centrifuged, the supernatant is removed and resuspended in PBS pH 7.2 prior to use.
It has been found that the initial suspensions that have a pinkish color tend to change color towards blue, when the volume in which the particles are resuspended is low. For this reason, in order to avoid the aggregation of the particles, we proceed to resuspend in volumes greater than 5 mL.
By means of measurements carried out by light scattering techniques and by micro-micrographs obtained by electron transmission microscopy such as those shown in the image below, an approximate size has been determined for the gold particles used as transport vehicles of the Ilia drug, of (24 ± 2) nm. The presence of IIIa and BSA is clearly observed by light scattering techniques, such as a crown around the gold nuclei, reaching the hydrodynamic size of the particles (47 ± 8) nm [Figure 2]. The suspensions have a pinkish color.
The adsorption of the drug IIIa on the surface of the gold particles shows changes in the surface electric charge of the gold particles. Gold particles at physiological pH show surface electric charge (-36 ± 7) mV, while gold particles coated with IIIa and BSA under the same pH conditions show a surface electric charge of (-10 ± 4) mV . From the electrokinetic point of view, any difference determined on the surface electric charge is attributed to the adsorption of any entity on the surface of the particles, in this case, the drug IIIa and the BSA.
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Materials and methods
To carry out the in vitro and in vivo assays described below, the parasite L. infantum has been used as a model.
The tests for obtaining CC50 have been performed on macrophages derived from bone marrow of mice (BMDM) and on mouse fibroblasts L929. In vivo assays have been performed in mice. Both are explained below.
In vitro evaluation: In vitro tests in intracellular amastigotes
To test the percentage of infection by L infantum macrophages derived from bone marrow (BMDM) of Swiss ICR (CD-1) mice were used. These cells were removed as established by [Zamboni, D, S,; Rabinovitch, M. Nitric oxide partially controls Coxiella burnetii phase II infection in mouse primary macrophages. Infect Immun 2003, 71 (3), 1225-1233]. BMDMs were grown on coverslips in wells of 24-well microtiter plates, at a concentration of 4x105 BMDM in each well containing RPMI-1640 medium with 10% fetal bovine serum (FCS) and 5% L929 cell medium. The BMDM plates were left overnight at a temperature of 37oC at 5% CO2 for the cells to adhere. Infections were performed as established by [Zauli-Nascimento, R C., Miguel, D. C., Yokoyama-Yasunaka, J. K .; Pereira, L. I., PeIIi de Oliveira, M. A., Ribeiro-Dias, F., Doria, M. L. Uliana, S. R. In vitro sensitivity of Leishmania (Viannia) braziliensis isolates to meglumine antimoniate and amphotericin B. Trop. Med. Int. Health 2010 15 (1) 68-76] adjusted to the proportion of 5 promastigotes in the stationary phase of L. infantum for each macrophage. Promastigotes were added to the wells by keeping them for 2 hours at 37 ° C and 5% CO2 in RPMI-1640 with 10% FCS. After this time the medium was removed and the macrophages were washed with RPMI-1640 medium to remove the promastigotes.
A new RPMI-1640 medium enriched with 10% SBF and 5% conditioned medium of L929 cells and containing the drug in its different concentrations was added. After 48 h of incubation at 37oC, 5% CO2, the covers were fixed with methanol and tineron with 20% Giemsa. 200 macrophages distributed by all were counted
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the fields of the cover and the percentage of infected and non-infected macrophages were calculated. Macrophages containing at least one amastigote were considered infected. Each experiment was performed in triplicate. After treatment at various concentrations of each compound, the response observed in the form of% infection becomes% infection reduction. Finally, a multiple linear regression analysis (SPSS 150 software) is performed to calculate the IC50 (concentration of the compound that reduces the parasite load by 50%) and CC50 (concentration of the compound that kills 50% of the mammalian cells) of the product evaluated. The activity of compound IIIa against the parasite responds to a double inverse pattern with the following formula: RP = 1 / (0.0118124 + 0.00044687 / C) (R2 = 97%), where RP is the reduction in the percentage of infection and C It is the concentration of compound IIIa.
The results obtained for compound IIIa are shown in Table 1, after their in vitro evaluation against intracellular L. infantum (IC50) and its cytotoxicity in mammalian cells (CC50). As control vorinostat (I), pentamidine (PI) and meglumine (MA) or Glucantime ® antimoniato drugs were used.
 Compound  IC50 (MM) CC50 (MM) to CC50 (MM) b
 II  3.21 ± 1.8> 100> 100
 IIIa  0.69 ± 0.1> 100> 100
 Vorinostat (I)  6.3 ± 3.8 5-20 NRc
 PI  1.50 ± 0.1 5-20 6.08 ± 0.2
 MA  144.40 ± 41.0 200-500 273.08 ± 12.2
Table 1.CI50 and CC50 of compound IIIa: Activity against amastigotes of L. infantum in macrophages and cytotoxicity in mammalian cells.
(a) CC50 on macrophages derived from bone marrow of mice (BMDM)
(b) CC50 on L929 mouse fibroblasts
(c) NR: Not performed
Efficacy and toxicity in vitro
Compound IIIa showed an IC50 against intracellular amastigotes of L. infantum approximately 5 times higher than that of compound (II) and also showing an activity superior to the drugs used as control. This fact, together with its low cytotoxicity (CC50> 100 ^ iM), gives it a selectivity index greater than 100. Like compound II, compound IIIa showed no activity against promastigotes of L. infantum in the concentration range 1-100 ^ M.
In vivo evaluation: Evaluation of the in vivo activity of the drug (IIIa) in a
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animal model infected with L. infantum
Subacute toxicity in vivo
The compound (IIIa) vehiculized on gold nanoparticles was administered to a group of 10 6-week-old Swiss ICR (CD-1) mice at a dose of 500 mg / kgMa for 10 days. After the treatment, an additional observation of the animals was carried out for two weeks. The appearance of signs of pain, suffering, diarrhea, weight loss, hepato or splenomegaly was evaluated. During treatment and subsequent follow-up, no animal died or any of the signs indicated above appeared, indicating that the compound lacks acute or subacute toxicity (lethal dose 50% (LD50)> 500 mg / kg).
Infection procedure and treatment
35 female 6-week-old female Balb / c mice were infected with 10 million promastigotes of L. infantum in a stationary phase of intraperitoneal growth in a volume of 0.4 mL of isotonic saline solution. After 28 days post-infection, the animals were divided into five groups of 7 mice each that underwent different daily treatments intraperitoneally for 10 days.
The treatments used in vivo are as follows:
- Control group treated with gold nanoparticles without drug; group treated with compound (IIIa) at 10 mg / kg;
- Group treated with compound (IIIa) vehiculized in gold nanoparticles at 25 mg / kg;
- Reference control group treated with meglumine antimonate at 104 mg SbV / kg;
- Group treated with a combination of meglumine antimonate at 104 mg SbV / kg and compound (IIIa) vehiculized in gold nanoparticles at 10 mg / kg.
For the quantification of the parasitic load in the organs of the mice sacrificed after treatment, 20 mg of tissue is weighed and the DNA is extracted with a suitable commercial kit, this being resuspended in a volume of 20 microL. Subsequently, a quantitative PCR is performed (in real time). This PCR uses TaqMan probes and is specific to L. infantum. The number of parasites present in each sample is calculated by interpolating the Ct obtained in a previously established standard curve using serial dilutions of parasites. From the number of parasites present in the sample and knowing the weight of the organ used for DNA extraction, the number of parasites / mg of organ is estimated.
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Results obtained in vivo
The compound Ilia vehiculized in gold nanoparticles was very effective at 25 mg / kg, reducing parasitic loads by 99% in liver and 93% in spleen, compared to reductions of 83% in liver and 52% in spleen using as Control drug meglumine antimoniato.
Table 2 shows a comparison, in terms of reduction of parasitic load, of compounds II and Ilia, both vehiculized in gold nanoparticles.
 Compound II Compound IIIa MA
 Reduction of the parasitic load in spleen  96% 93% 52%
 Reduction of parasitic load in liver  68% 99% 83%
Table 2. Reduction in the percentage of infection by L. infantum with respect to the control without treatment after 14 days of treatment at a dose of 25 mg / kg / day of compound II or 10 days of treatment at the same dose of compound IIIa. The dose of meglumine antimony (MA) was 390 mg / kg (equivalent to 104 mg / kg / day of SbV antimony) and the treatment lasted 14 days.
Assessment of toxicity in infected animals
In the treatment with compound IIIa of animals experimentally infected with L. infantum no gastrointestinal adverse effects, weight loss, pain or other general symptoms were observed. Nor were alterations in the biochemical and enzymatic values evaluated in serum or blood, such as transaminases (GPT and GOT), urea, alkaline phosphatase and creatinine. Nor was hepato or splenomegaly associated with the treatment. Therefore, the drug also shows no toxicity in infected mice.
Compound IIIa has proven to be a potent drug against the amastigotes of L. infantum, the dose-dependent activity being. There is a positive relationship between the concentration administered and the percentage of infection reduction found. In addition, it has not shown at the doses evaluated activity on the promastigote form. The dose at which cytotoxic effects are evidenced is much higher than the antiparasitic dose. The Selectivity Index is also higher than that found for pentamidine isetinate (IS = 4) and megumin antimoniato (IS = 2) that constitute the control drugs (drugs commonly used in the treatment of leishmaniasis).

权利要求:
Claims (14)
[1]
5
10
fifteen
twenty
25
1. A compound of general formula III
image 1
III
as well as racemic compounds, stereoisomers, salts or solvates thereof; where Ph is a phenyl of formula
R3
image2
And where
R1 = H, F, Cl, Br, I, Me, OMe or NO2.
R2 = H, F, Cl, Br, I, Me, OMe or NO2
R3 = H, F, Cl, Br, I, Me, OMe or NO2
[2]
2. Compound according to previous claim characterized in that R1 = H or R2 = H or R3 = H.
[3]
3. Compound according to previous claim characterized in that at least two of the three phenyl radicals are formed by hydrogen
[4]
4. Compound according to previous claim characterized in that R1 = H, R2 = H and R3 = H (Compound IIIa).
H
N
image3
H
H
IIIa
5
10
fifteen
twenty
25
30
35
[5]
5. Pharmaceutical composition comprising at least one compound according to any one of claims 1 to 4 as an active ingredient and a pharmaceutically acceptable carrier.
[6]
6. Pharmaceutical composition comprising as the only active ingredient a compound, or a combination of compounds, according to any one of claims 1 to 4, together with a pharmaceutically acceptable carrier.
[7]
7. Pharmaceutical composition according to any of claims 5 or 6 characterized in that the compounds according to any of claims 1 to 4 are functionalized in gold nanoparticles.
[8]
8. Composition according to previous claim characterized in that the gold nanoparticles are composed of gold core and coated with sodium citrate.
[9]
9. Composition according to any of claims 7 or 8 characterized in that the nanoparticles have a diameter between 20 and 30 nm, preferably between 22 and 26 nm.
[10]
10. Composition according to claims 5 to 9 suitable for oral administration.
[11]
11. Use of a compound according to any one of claims 1 to 4 or composition according to any of claims 5 to 10 for the preparation of a medicament
[12]
12. Use according to the previous claim for the preparation of a medicament for the treatment of Leishmaniosis.
[13]
13. Use according to the previous claim characterized in that the Leishmaniosis is caused by L. Infantum.
xz
image4
OR
image5
IV
1. EtCO2Cl, THF anh.
[2]
2. H2N
Or Ph MeOH anh.
image6
Figure 1
image7
50 nm VJOTA-0021 120000X _ 21 Oi'12 / 2014
A. VIOTA -J21 Au-Attract NPs'
«
image8
50 nm
VIOTA-0027 120000X 27 01'12'2014
± VIOTA -J27 ‘Ab citrate NPs UDG-BSA '
Figure 2

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公开号 | 公开日

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ES2607628B1|2018-02-28|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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WO2013045734A1|2011-09-26|2013-04-04|Universidad De Granada|Compounds having antileishmanial activity|

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申请号 | 申请日 | 专利标题

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ES201630944A|ES2607628B1|2016-07-11|2016-07-11|COMPOUNDS FOR THE TREATMENT OF LEISHMANIOSIS|ES201630944A| ES2607628B1|2016-07-11|2016-07-11|COMPOUNDS FOR THE TREATMENT OF LEISHMANIOSIS|

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PCT/ES2017/070502| WO2018015595A2|2016-07-11|2017-07-11|Compounds for the treatment of diseases caused by leishmania genus parasites|