Use of chemical compounds that can inhibit the toxic activity of sphingomyelinase D from venoms of Loxosceles spiders and pharmaceutical composition comprising said compounds
Inventors
TAMBOURGI, Denise Vilarinho • LOPES, Priscila Hess • MURAKAMI, Mario Tyago • PORTARO, Fernanda Calheta Vieira
Assignees
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Publication Number
US-9833444-B2
Publication Date
2017-12-05
Expiration Date
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.
Core Innovation
The invention relates to benzenesulfonamide and benzenesulphonate compounds used as inhibitors of Loxosceles sphingomyelinase D venom toxicity for controlling cutaneous and systemic loxoscelism. The disclosed inhibitors include the specific compound 4-bromo-N-[(E)-(2-methyl-1H-indol-3-yl)methylene amino]benzenesulfonamide and the specific compound 4-methyl-3-oxo-2-(3-pyridylmethylene)benzo[3,4-b]furan-6-yl-4-chlorobenzenesulphonate.
The problem addressed is the toxicity of Loxosceles sphingomyelinase D venom leading to cutaneous and systemic loxoscelism, including hemolysis and skin lesions/dermonecrosis. The disclosure describes reduction of venom toxicity outcomes and downstream cellular and molecular effects linked to sphingomyelin and lysophosphatidylcholine processing.
Functionally, the inhibitors reduce glycophorin C removal and toxin binding on red blood cells, and increase keratinocyte viability. They also reduce keratinocyte ERK1/2 phosphorylation, reactive oxygen species/superoxide, TNF-α and TGF-β2 levels, and MMP-2/MMP-9 secretion, with rabbit dermonecrosis inhibition and histopathology improvements reported in vivo.
Claims Coverage
The independent claim covers a pharmaceutical composition with two alternative active compounds and a pharmaceutically acceptable vehicle. The claim set also specifies inhibition of Loxosceles venom D sphingomyelinases and includes dependent features tied to bacterial sphingomyelinase sources and multiple biochemical and cellular endpoints.
Pharmaceutical composition with specified benzenesulfonamide or benzenesulphonate
A pharmaceutical composition comprising a compound selected from 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 a pharmaceutically acceptable vehicle.
Inhibiting Loxosceles D sphingomyelinase toxicity
A pharmaceutical composition in which the D sphingomyelinases found in the venom of Loxosceles spiders are inhibited to inhibit toxicity.
Benzenesulphonate active compound selection
A pharmaceutical composition in which the active compound is 4-methyl-3-oxo-2-(3-pyridylmethylene)benzo[3,4-b]furan-6-yl-4-chlorobenzenesulfonate.
Multimarker keratinocyte and red blood cell protection with quantified IC50
A pharmaceutical composition containing 4-bromo-N-[(E)-(2-methyl-1H-indol-3-yl)methylene amino]benzenesulfonamide that inhibits sphingomyelinase/SMase activity and venom activity on sphingomyelinase substrates, reduces substrate-dependent activity of recombinant toxin D SMase and venom versus lysophosphatidylcholine, has an IC50 of 45.4±1.2 μM on recombinant toxin activity, reduces glycophorin C removal from red blood cells, increases viability of toxin- or venom-treated cells, reduces secretion of MMP-2 and completely inhibits secretion of MMP-9 by keratinocytes treated with recombinant toxin D SMase, and suppresses keratinocyte superoxide production and TNF receptor removal, TNF-α production, TGF-β2 production, and phosphorylated ERK1/2 levels.
Bacterial sphingomyelinase selection
A pharmaceutical composition in which the sphingomyelinases are bacterial enzymes selected from Coryneumbacterium pseudotuberculosis, Arcanobacterium haemoliticum, and Bacillus cereus.
Overall, the claim coverage centers on pharmaceutical compositions containing either of two specified small-molecule inhibitors with a pharmaceutically acceptable vehicle, targeted to inhibit Loxosceles venom-associated D sphingomyelinase toxicity. Dependent claims further specify bacterial sphingomyelinase sources and emphasize inhibition across multiple biochemical and cellular endpoints, including recombinant and venom activity on sphingomyelinase substrates and downstream effects on red blood cells and keratinocytes, with an explicitly stated IC50.
Stated Advantages
Reduces glycophorin C removal and toxin binding on red blood cells.
Increases keratinocyte viability.
Reduces keratinocyte ERK1/2 phosphorylation, reactive oxygen species/superoxide, TNF-α production, and TGF-β2 production.
Reduces MMP-2 secretion and completely inhibits MMP-9 secretion by keratinocytes.
Inhibits rabbit dermonecrosis and improves histopathology.
Documented Applications
Treating loxoscelism, hemolysis, skin lesions, and dermonecrosis associated with Loxosceles sphingomyelinase D venom toxicity using a pharmaceutical composition containing compound 5 or compound 6 with a pharmaceutically acceptable vehicle.
Using the inhibitors as tool compounds and possible complementary therapeutics by extending activity testing to bacterial phospholipase D/sphingomyelinase-like enzymes from Corynebacterium pseudotuberculosis, Bacillus cereus, and Staphylococcus aureus.
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