Use of chemical compounds that can inhibit the toxic activity of sphingomyelinase D from venoms of Loxosceles spiders and pharmaceutical composition comprising said compounds
09833444 · 2017-12-05
Assignee
Inventors
- Denise Vilarinho Tambourgi (São Paulo, BR)
- Priscila Hess Lopes (São Paulo, BR)
- Mario Tyago Murakami (São Paulo, BR)
- Fernanda Calheta Vieira Portaro (São Paulo, BR)
Cpc classification
A61K31/443
HUMAN NECESSITIES
International classification
Abstract
The present invention relates preferably to the use of 4-bromo-N-[(E)-(2-methyl-1H-indol-3-yl)methyleneamino]benzenesulphonamide and 4-methyl-3-oxo-2-(3-pyridylmethylene) benzo[3,4-b]furan-6-yl-4-chlorobenzenesulphonate (compounds 5 and 6, respectively), which are compounds that can inhibit the toxic activity of sphingomyelinase D from Loxosceles venom, controlling the development of cutaneous and systemic loxoscelism; reducing haemolysis; inhibiting the formation of skin lesions; inhibiting skin necrosis; inhibiting intracellular signaling pathways and the production of reactive oxygen species. In addition to the therapeutic potential thereof, said inhibitors can be used to study the activity of sphingomyelinases and phospholipases D. The present invention also relates to a pharmaceutical composition for treating loxoscelism, reducing haemolysis, inhibiting the formation of skin lesions, inhibiting skin necrosis, inhibiting intracellular signaling pathways and the production of reactive oxygen species, comprising said compounds and a pharmaceutically acceptable carrier.
Claims
1. A pharmaceutical composition comprising (1) a compound selected from the group consisting of 4-bromo-N-[(E)-(2-methyl-1H-indol-3-yl)methylene amino]benzenesulfonamide and 4-methyl-3-oxo-2-(3-pyridylmethylene)benzo[3,4-b]furan-6-yl-4-chlorobenzenesulfonate and (2) a pharmaceutically acceptable vehicle.
2. The pharmaceutical composition, of claim 1, wherein the compound is 4-bromo-N-[(E)-(2-methyl-1H-indol-3-yl)methylene amino]benzenesulfonamide.
3. The pharmaceutical composition, of claim 1, wherein the compound is 4-methyl-3-oxo-2-(3-pyridylmethylene)benzo[3,4-b]furan-6-yl-4-chlorobenzenesulfonate.
4. The pharmaceutical composition of claim 1, which inhibits the toxicity of D sphingomyelinases in the venom of Loxosceles spiders.
5. The pharmaceutical composition of claim 4, wherein the compound is the benzenesulfonamide compound and wherein the benzenesulfonamide compound is 4-bromo-N-[(E)-(2-methyl-1H-indol-3-yl)methyleneamino]benzenosulfonamide.
6. The pharmaceutical composition of claim 4, wherein the compound is the benzenesulphonate compound, wherein the benzenesulphonate compound is 4-methyl-3-oxo-2-(3-pyridylmethylene)benzo[3,4-b]furan-6-yl-4-chlorobenzenesulfonate.
7. The pharmaceutical composition of claim 4, wherein the compound acts on the hydrolytic activity of recombinant toxin sphingomyelinase D (D SMase) and of venom of brown recluse spider.
8. The pharmaceutical composition of claim 4, wherein the compound controls the development of cutaneous and systemic loxoscelism.
9. The pharmaceutical composition of claim 5, wherein the 4-bromo-N-[(E)-(2-methyl-1H-indol-3-yl)methylene amino]benzenesulfonamide: (1) inhibits by 45.7% the activity of recombinant toxin D SMase on a sphingomyelinase (SM) substrate, (2) inhibits by 53% the activity of the venom of L. laeta on SM substrate, (3) reduces by 38.6% the activity of recombinant toxin D SMase in relation to lysophosphatidylcholine (LPC) substrate, (4) reduces by 44.4% the venom activity in relation to LPC substrate, (5) has an IC.sub.50 value of 45.4±1.2 μM, based on action of the compound on the activity of recombinant toxin on SM substrate, (6) reduces by 92.6% the removal of glycophorins from the surface of red blood cells, (7) reduces by 96.8% the removal of glycophorin C from the surface of red blood cells, (8) increases from 20.23% to 55.75% the viability of cells treated with the recombinant toxin D SMase, (9) increases from 36.7% to 45.9% the viability of cells treated with the venom, (10) reduces by 81% secretion of extracellular matrix metalloproteinases 2 (MMP-2) in keratinocytes treated with recombinant toxin D SMase, (11) completely inhibits secretion of extracellular matrix metalloproteinases 9 (MMP-9) in keratinocytes treated with recombinant toxin D SMase, (12) reduces disorganization of dermis collagen fibers, bleeding, inflammatory infiltrate, and injury to adjacent muscle layer in skin contacted with venom, (13) inhibits by 70.7% superoxide production by keratinocytes treated with recombinant toxin D SMase, (14) reverses by 27.7% the removal of TNF receptor from the surface of keratinocytes treated with recombinant toxin D SMase, (15) reverses by 73.9% the production of TNF-α in keratinocytes treated with recombinant toxin D SMase, (16) reduces by 88.3% the production of TGF-β1 in keratinocytes treated with recombinant toxin D SMase, or (17) reduces by 65.8% phosphorylated ERK1/2 in keratinocytes treated with recombinant toxin D SMase.
10. The pharmaceutical composition of claim 6, wherein the 4-bromo-N-[(E)-(2-methyl-1H-indol-3-yl)methylene amino]benzenesulfonamide: (1) inhibits by 51% the activity of recombinant toxin D SMase on SM substrate, (2) inhibits by 22.7% the activity of the venom of L. laeta on SM substrate, (3) reduces by 34% the activity of recombinant toxin D SMase in relation to LPC substrate, (4) reduces by 16% the activity of the venom in relation to substrate LPC, (5) has an IC.sub.50 value is 63.4±1.1 μM, based on the action of the compound on the activity of recombinant toxin on SM substrate, (6) reduces by 88.2% the removal of glycophorins from the surface of red blood cells, (7) reduces by 84% the removal of glycophorin C from the surface of red blood cells, (8) increases from 20.23% to 53.55% the viability of cells treated with recombinant toxin D SMase, (9) increases from 36.7 to 59.26% the viability of cells treated with the venom, (10) reduces by 98.4% the secretion of extracellular MMP-2 in keratinocytes treated with recombinant toxin D SMase, (11) completely inhibits the secretion of extracellular MMP-9 in keratinocytes treated with recombinant toxin D SMase, (12) reduces disorganization of dermis collagen fibers, bleeding, inflammatory infiltrate, and injury to adjacent muscle layer in skin contacted with venom, (13) inhibits by 92.7% superoxide production by keratinocytes treated with recombinant toxin D SMase, (14) reverses by 61.1% the production of TNF-α from the surface of keratinocytes treated with recombinant toxin D SMase, (15) reduces by 89.2% the production of TGF-β1 from the surface of keratinocytes treated with recombinant toxin D SMase, (16) reduces by 80.2% phosphorylated ERK1/2 in keratinocytes treated with recombinant toxin D SMase.
11. The pharmaceutical composition of claim 4, which controls the effects of sphingomyelinases from bacteria and from arthropods, wherein the arthropods are selected from the group consisting of (spiders, scorpions and ticks).
12. The pharmaceutical composition of claim 11, wherein the sphingomyelinases are from bacteria selected from the group consisting of Coryneumbacterium pseudotuberculosis, Arcanobacterium haemoliticum, and Bacillus cereus.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The structure and operation of the invention, along with the additional advantages thereof, can be better understood by reference to the attached drawings and the description that follows:
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(20) Annex 1 shows microphotographs of histopathological analysis of the skin of rabbits inoculated with the venom of L. laeta or with the venom incubated with compounds 5 and 6.
DETAILED DESCRIPTION OF THE INVENTION
(21) Although this invention can be susceptible to different embodiments, a preferred embodiment is shown in the drawings and in the following detailed discussion, with the understanding that this embodiment should be considered an example of the principles of the invention without the intention of limiting the present invention to what has been illustrated and described herein.
(22) This invention relates to the use of inhibitors compounds belonging to the class of benzenesulfonamides and benzenesulphonate with inhibitory activity on sphingomyelinases D of the venom of Loxosceles spiders for preparing a medicine to act on the hydrolytic activity of recombinant sphingomyelinase D toxin (SMase D) and of the venom of brown recluse spider Loxosceles laeta.
(23) The present invention preferably relates to the use of 4-bromo-N-[(E)-(2-methyl-1H-indol-3-yl) methyleneamino]benzenosulfonamide, named compound 5 (
(24) The inhibitor compounds of the present invention can be used as tools in the study of the action of sphingomyelinases and phospholipases D, and these inhibitors have therapeutic potential to venoming by Loxosceles spiders and infections by SMases producer microorganisms.
(25) Selection of Compounds
(26) The binders used in molecular Docking studies were selected from a free database of commercially available molecules, the ZINC Database (www.zinc.docking.org). Many ligands in this database are available in a variety of 3D formats immediately usable by many popular docking programs.
(27) In addition to the important physical-chemical properties, the bank contains supplier information and numbers of the original catalogue for each commercial source of this compound. It is also possible to find information about the function or activities already studied for the compound, when available.
(28) Molecular Docking Studies
(29) Molecular docking studies for selecting SMases D inhibitors were conducted at the Laboratório Nacional de Luz Síncroton, Campinas—Sao Paulo, with the aid of ICM software—Molsoft (based on Monte Carlo algorithm) Molegro Virtual Docker, (based on a heuristic search algorithm that combines differential evolution with a cavity prediction algorithm) and GOLD (based on a genetic algorithm). The docking analysis were carried out based on the 3D structure of SMase I of the venom of L. laeta, according to article of MURAKAMI, M. T.; FERNANDES-PEDROSA, M. F.; TAMBOURGI, D. V.; ARNI, R. K. and entitled “Structural basis for metal ion coordination and the catalytic mechanism of sphingomyelinases D”, published in the J. Biol. Chem., v. 280, No. 14, page 13658-13664, 2005, and incorporated herein by reference.
(30) The following topics describe the action of compounds 4-bromo-N-[(E)-(2-methyl-1H-indol-3-yl)methyleneamino]benzenosulfonamide (
(31) Analysis of the Inhibitory Action of Compounds 5 and 6 on the Hydrolytic Activity of the Toxin and Venom.
(32) Compounds 5 and 6 of this invention were diluted in dimethyl sulfoxide (DMSO) and prepared in a stock solution of high concentration, which was diluted in sterile saline solution to the concentrations used in each assay.
(33) In the fluorimetric assays, the basal fluorescence of the compounds was measured and deducted from these assays.
(34) The ability of the compounds in the concentration of 40 μM of inhibiting the hydrolytic activity of the recombinant toxin sphingomyelinase D (SMase D) and of the venom of Loxosceles laeta on substrates sphingomyelin (SM) and lysophosphatidylcholine (LPC), by fluorimetry assays, was analyzed.
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(36) The results showed that compound 5 of the present invention inhibits 45.7% and 53% of the activity of recombinant toxin and the venom of L. laeta, respectively, on the SM substrate. Compound 6 of the present invention inhibited 51% and 22.7% of the activity of the recombinant toxicity and the venom, respectively.
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(38) In relation to substrate LPC, compound 5 of the present invention reduces 38.6% and 44.4% of the activity of the recombinant toxin and the venom, respectively. As to compound 6 of this invention, it reduces 34 and 16% of this activity.
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(40) Based on said dose-response curves of the action of the compounds on the activity of the recombinant toxin on the substrate SM, the IC.sub.50 values were obtained, which were 45.4±1.2 μM to compound 5 of this invention and 63.4±1.1 μM to compound 6 of this invention.
(41) The analysis of the inhibition mechanism showed that compounds 5 and 6 of the present invention may be classified as uncompetitive action inhibitors with K.sub.i values of 1.63 and 1.73, respectively.
(42) Analysis of the Action of Compounds 5 and 6 of the Present Invention on Hemolysis Induced by the Venom of Loxosceles
(43) The removal of glycophorins from the surface of red blood cells by indirect action of the toxin is a crucial event for the development of intravascular hemolysis that occurs in accidents.
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(45) The analysis of the expression of glycophorin C on the surface of human red blood cells by flow cytometry showed that, in the presence of compounds 5 and 6 of the present invention (40 μM), the removal of these molecules from the surface of cells is reduced by 92.6 and 88.2%, respectively.
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(47) This event of glycophorin C removal is associated to the binding of the toxin on the red blood cell membrane. Thus, the binding of the toxin to the cell surface was analyzed by flow cytometry and the results indicated a reduction of 96.8 and 84% in the presence of the compounds 5 and 6 of the present invention, respectively.
(48) Analysis of the Action of Compounds 5 and 6 of the Present Invention on the Mechanisms of Cutaneous Loxoscelism
(49) The development of cutaneous lesion observed in the loxoscelism is closely related to the cell death of keratinocytes induced by SMase D.
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(52) As well as in erythrocytes, keratinocyte cell death is associated with the binding of the toxin to this cell membrane.
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(54) Other event associated with keratinocyte death during the development of skin lesion is the production of extracellular matrix metalloproteinases 2 and 9 (MMPs). Thus, the culture supernatant of keratinocytes treated with the venom of L. laeta was investigated by ELISA as for the presence of MMP-2 and 9. In the presence of compounds 5 and 6 of the present invention (10 μM), the MMP-2 secretion is reduced by 81 and 98.4%, respectively. In relation to MMP-9, both compounds completely inhibit the secretion of this MMP.
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(56) After analyzing the aspects involving the development of skin lesion, the ability of the compounds in inhibiting dermonecrosis in vivo was verified, using a model in rabbits.
(57) After 24 hours of venom inoculation, the injury was reduced by 61.8 and 36% in the presence of compounds 5 and 6 of the present invention, respectively. In 48 hours, the inhibition was 60 and 45%, and in 72 hours, it was 56 and 49% in the presence of these two compounds.
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(59) The histopathological analysis of the skin of rabbits inoculated with the venom in the presence or absence of these compounds demonstrates that, in the presence of compounds 5 and 6 of the present invention, there is a reduction in the disorganization of the collagen fibers in the dermis, absence of hemorrhage and inflammatory infiltrate, as well as injury to the adjacent muscle layer in relation to that inoculated only with venom.
(60) Annex 1 shows microphotographs of the histopathological analysis of the skin of rabbits inoculated only with the venom of L. laeta or with the venom incubated with compounds 5 and 6.
(61) Compounds 5 and 6 of this invention were used at a concentration corresponding to three times the value of IC.sub.50 (136.2 μM to compound 5 and 190.2 μM to compound 6).
(62) Action of Compounds 5 and 6 of the Present Invention on Other Mechanisms Involved in the Toxicity of Sphingomyelinases D of the Venom of Loxosceles
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(64) Another aspect analyzed was the production of reactive oxygen species by keratinocytes treated with the toxin, using the flow cytometry technique.
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(66) The results indicate that, in the presence of compounds 5 and 6 of the present invention (40 μM), superoxide production by these cells was inhibited by 70.7 and 92.7%, respectively.
(67) Additionally, the expression of the TNF receptor on the surface of keratinocytes treated with the toxin was analyzed by flow cytometry. The treatment with the toxin leads to a removal of the cell surface receptor, which is reversed by 27.7% in the presence of compound 5 of this invention (40 μM).
(68) The production of cytokines by keratinocytes can be an important aspect in the development of skin lesion of loxoscelism.
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(70) TGF-β1, which was reduced by 88.3 and 89.2% in the presence of the compounds.
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(72) Thus, taking into consideration all the aspects analyzed, compound 5 of the present invention is 61.1% efficient, while compound 6 of this invention is 54.1% efficient in relation to the mechanisms involved in the toxicity of SMases D in the development of loxoscelism.
(73) Action of Inhibitor Compounds 5 and 6 on the Hydrolytic Activity on Substrate Sphingomyelin, of Bacterial Sphingomyelinases.
(74) Phospholipases are important toxic components present in the venom of animals and bacterial toxins. They promote the hydrolysis of ester bonds of phospholipids, and they are classified in phospholipases A1, A2, C and D, by the position of hydrolyzed ester bond (VAN DEN BOSCH, 1980).
(75) Contrary to other phospholipases, those found in the venom of Loxosceles (SMases D) and in certain bacteria have an unusual substrate specificity, since of the four major phospholipids present in mammal cell membranes, only sphingomyelin is hydrolyzed by bacterial LDP, while those of the venom of Loxosceles spiders cleave sphingomyelin, generating ceramide-1-phosphate as the hydrolysis product and are also able to hydrolyze lysophosphatidylcholine, generating lysophosphatidic acid (Bernheimer; Campbell; Forrester, 1985; Tambourgi et al., 1998; Van Meeteren et al., 2004). Researches on sequence databases showed that the toxins of the bacteria Corynebacterium pseudotuberculosis and Arcanobacterium haemolyticum are also sphingomyelinases and have between 24 and 34% of similarity with the first 30 amino acids of the Loxosceles toxins (Tambourgi et al., 1998), as well as similar molecular weight and isoelectric point (Bernheimer; Campbell; Forrester, 1985); moreover, the toxins of these bacteria induce biological effects also similar to those induced by the venom of Loxosceles (Bernheimer; Campbell; Forrester, 1985; Forrester; Barrett; Campbell, 1978; McNamara; Cuevas; Songer, 1995; Rees et al., 1984; Tambourgi et al., 1998, 2002, 2007).
(76) Corynebacterium pseudotuberculosis, Corynebacterium ulcerans and Arcanobacterium haemolyticum are pathogens of pets and humans (McNAMARA et al., 1995).
(77) C. pseudotuberculosis is a gram positive Bacillus widely distributed in animal populations, causing caseous lymphadenitis in sheep, goats and both, ulcerative inflammation of the wall of the lymphatic vessels and pectoral, abdominal and inguinal abscesses in horses (Soger et al., 1990; Huerta et al., 2013). Infections also occur in cattle and humans. These infections lead to reduced production and milk quality in cattle and goats and to a low yield of wool in sheep (Hoelzle et al., 2013). In the mechanism of pathogenesis of bacteria C. pseudotuberculosis, sphingomyelinase (PLD) has been shown as an essential determinant of virulence, which contributes to bacteria spreading from the initial site of infection to secondary host sites (Nairn et al., 1977). Knockout PLD strains have shown reduced virulence, emphasizing the importance of this toxin in the pathogenesis (Hodgson et al., 1992; McNamara et al., 1994).
(78) Arcanobacterium haemolyticum are gram-positive bacteria responsible for many respiratory infections in healthy people. Most cases involve pharyngitis and/or tonsillitis, and approximately 50% are exsudative. Throat infections are often accompanied by cervical lymphadenopathy (GREEN et al., 1981; MACKENZIE et al., 1995). This bacterium produces a PLD which acts preferentially on sphingomyelin, generating ceramide-1-phosphate in the target membrane and being closely related to the PLD produced by C. pseudotuberculosis (Linder, 1997).
(79) Corynebacterium ulcerans has its main reservoir in cattle herd, in which it induces mastitis, however, cases in human patients have been reported (DIAS et al., 2009). This bacterium can produce diphtheria toxin (DT) and/or PLD, and can cause infections in humans, mimicking the cutaneous and the classic respiratory diphtheria with pseudomembranas (Dewinter et al., 2005; Tiwari et al., 2008). Furthermore, the C. ulcerans strains that produce PLD, but not DT, also affect the lower respiratory tract and are capable of causing severe disease in humans, such as pneumonia (Mane-Guaraldi et al., 2008) and granulomatous nodules in lungs (Deseau et al., 1995).
(80) Similarities in physical properties and activities of PLDs produced by C. pseudotuberculosis, C. ulcerans and Arcanobacterium haemolyticum suggest that this group of enzymes is important in the pathogenesis of diseases caused by these organisms. Thus, considering such similarities in PLD activities and clinical conditions induced by these bacteria and by Loxosceles toxins, it is important to test the compounds with activity on the Loxosceles SMases D on the PLD activity of some of these bacteria in order to possibly obtain compounds that help in the treatment of these diseases that affect farm animals and humans.
(81) for this, 50 μg of PLD of C. pseudotuberculosis incubated with 40 μM of inhibitor compounds 5 and 6, and the residual hydrolytic activity on sphingomyelin were evaluated by fluorimetry. The results showed that the PLD activity of C. pseudotuberculosis was reduced about 70% by compound 5 and about 68% by compound 6 (
(82) Another class of phospholipases produced by bacteria and related to the virulence of the same are phospholipases C (PLC). Bacteria such as Staphylococcus aureus, Bacillus cereus, Leptospira and Listeria, among others, are some of the PLC producers.
(83) Bacillus cereus is well known for its role as a mediator of foodborne diseases (Taylor & Gilbert, 1975; Gianella & Brasile, 1979; Stenfors et al., 2008; Bottone, 2010). This organism form spores and is widely distributed in dust, air and water, and therefore, it is ubiquitous in hospital environments, infecting clothings and intravenous catheters, thus providing an opportunity for infection in immunocompromised patients (Drobniewski, 1993). Generally, the infection caused by this organism leads to mild gastroenteritis, however, outbreaks of deadly food poisoning by B. cereus (Lund et al., 2000; DIERICK et al., 2005) and local and systemic infections out of the gastrointestinal tract (endophthalmitis, pneumonia and sepsis) in humans have been reported (Bottone, 2010).
(84) B. cereus secrets several toxins that target cell membranes, including Bc-SMases. Evidences point out that, in Listeria ivanovii, PLC can act on the exit of the bacteria inside the phagocytic vacuoles, thus promoting the intracellular survival and spread of the pathogen (Gonzalez-Zorn et al., 1999). SMase derived from Helicobacter pylori directly contributes to the cytotoxicity on gastric cells (Tseng et al., 2004) and toxins with SMase activity are expressed by 91% of the Staphylococcus aureus strains of high toxicity (Collins et al., 2008).
(85) Recent studies have shown that Bc-SMases lyse sheep red blood cells containing large amounts of sphingomyelin in the membrane (Oda et al., 2010), that these belong to the same group of SMases from H. pylori and L. ivanovii (Openshaw et al., 2005) and that the hydrolysis of sphingomyelin in the membrane of macrophages generates ceramide, which mitigates the fluidity of the membrane, leading to an unsuccessful phagocytosis. Therefore, PLC or Bc-SMase plays a key role in the bypass of the bacteria on immune mechanisms in macrophages (Oda et al., 2012).
(86) S. aureus produces and secretes a large number of cell surface virulence factors that enable this organism to cause a variety of human diseases ranging from relatively mild eruptions and subcutaneous abscesses to severe septic shock and necrotizing pneumonia (Lowy, 1998; McCormick et al., 2001), moreover, S. aureus was also found in cases of bovine mastitis (Aarestrup et al., 1999). One of the exotoxins produced is a beta-toxin with mass about 35 kDa which seems to work like a SMase, being classified as a PLC (Huseby et al., 2007). This toxin causes lysis of red blood cells and other cells, such as lymphocytes and neutrophils (Marshall et al., 2000), being found in 72% of the cases of bovine mastitis, 11% in bacteria isolated from nostrils of healthy people and in 13% of cases of septic shock (Aarestrup et al., 1999). In general, studies suggest that this toxin can also contribute to immunomodulation of the host in the presence of other virulence factors (HUSEBY et al., 2007).
(87) Thus, knowing the importance of PLCs in infections by some bacteria, compounds 5 and 6 (40 μM) were tested in face of the hydrolytic activity of SMases of B. cereus and Stahylococcus aureus. The results showed that the activities of SMases from both bacteria were efficiently reduced by incubation with compounds 5 and 6 (
(88) Besides of the cited bacteria, other bacteria highly pathogenic to animals and to men, such as Leptospira interrogans (Kasarov & Addamiano, 1969; Nayaranavari et al., 2012), Listeria ivanovii (Gonzalez-Zorn et al., 1999), Helicobacter pylori (Tseng et al., 2004) and Pseudomonas sp (Sueyoshi et al., 2002), among others, also produce PLDs or PLCs, which usually are part of the virulence mechanisms. In addition to bacteria, parasites such as Clonorchis sinensis (Huang et al., 2013), ticks of the genus Ixodes (Alarcon-Chaidez et al., 2009), and fungi of the genus Aspergillus and Coccidioides (Fry et al., 2009) also produce such molecules.
(89) Therefore, based on the results presented herein and on the similarity of the physical properties and biological activities between SMases of Loxosceles and of bacteria and other organisms, we propose here that the compounds 5 and 6, selected through molecular docking studies on the tertiary structure of SMase D I of Loxosceles laeta, can be used as tools to study the virulence mechanisms of these organisms, as well as a possible basis for the development of complementary therapeutic strategies for the control of diseases caused by these organisms.
(90) Therefore, compounds 5 and 6 of the present invention can be used: For preparing a medicine to act on the hydrolytic activity of recombinant toxin sphingomyelinase D (SMase D) and of the venom of brown recluse spider Loxosceles laeta; For preparing a medicine to act on hemolysis reduction, For preparing a medicine to inhibit skin lesion, For preparing a medicine to inhibit dermonecrosis, For preparing a medicine to inhibit mechanisms involved in the toxicity of SMases D in the development of loxoscelism; For preparing a medicine to inhibit intracellular signaling pathways and the production of reactive oxygen species
(91) Besides of the fact that compounds 5 and 6 of the present invention act as inhibitors of sphingomyelinases D of the venom of spiders of the genus Loxosceles, they can also act as a complimentary therapeutic drug for accidents and possibly as compounds for controlling the effects of bacterial sphingomyelinases (Coryneumbacterium pseudotuberculosis, Arcanobacterium haemoliticum, Bacillus cereus) and some arthropods (spiders, scorpions and ticks).
(92) In a second aspect, this invention relates to a pharmaceutical composition for treating loxoscelism, hemolysis reduction, inhibition of cutaneous lesion, inhibition of dermonecrosis, inhibition of intracellular signaling pathways and production of reactive oxygen species, which comprises a benzenesulfonamide compound and a pharmaceutically acceptable vehicle. The benzenesulfonamide compound preferably being the compound 4-bromo-N-[(E)-(2-methyl-1H-indol-3-yl)methyleneamine]benzenesulfonamide.
(93) A third aspect of this invention relates to a pharmaceutical composition for treating loxoscelism, hemolysis reduction, inhibition of cutaneous lesion, inhibition of dermonecrosis, inhibition of intracellular signaling pathways and production of reactive oxygen species, which comprises a benzenesulphonate compound and a pharmaceutically acceptable vehicle. The benzenesulphonate compound preferably being compound 4-methyl-3-oxo-2-(3-pyridylmethylene)benzo[3,4-b]furan-6-yl-4-chlorobenzenesulfonate.
(94) It is understood, by “pharmaceutically acceptable vehicle” any acceptable vehicle, excipient or diluent in the pharmaceutical field.
(95) Thus, although only some details of the present invention have been shown, it will be understood that several omissions, substitutions and changes to classes of benzenesulfonamide and benzenesuphonate compounds can be made by a person skilled in the art, without departing from the spirit and scope of this invention.
(96) It is expressly provided that all combinations of the elements that perform the same function substantially the same way to achieve the same results are within the scope of the invention. Substitution of elements in an embodiment described to another are also fully comprised and contemplated.
(97) It should be also understood that the drawings are not necessarily in scale, and are only conceptual in nature. The intention is, therefore, to be limited, as indicated by the scope of the attached claims.
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