Patent Application: US-201515325920-A

Abstract:
the present invention relates to perhexiline , or a pharmaceutically acceptable salt thereof , for use in the treatment of a pathology caused by trematodes .

Description:
gambogic acid , perhexiline maleate salt , praziquantel , dimethyl sulphoxide ( dmso ), percoll ( starting density 1 . 13 g / ml ) and foetal bovine serum ( fbs ) were purchased from sigma - aldrich . celltiter - glo reagent , used in the luminescent viability schistosomula assay , was purchased from promega . biowhittaker dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ) lacking phenol red but containing 4500 mg / l glucose ( lonza ), supplemented with 1 mm hepes ( lonza ), 2mm l - glutamine ( lonza ), 1 × antibiotic - antimycotic reagent ( life technologies ) and 10 % fbs was the completed tissue culture media for schistosomula . juvenile and adult worms ( s . mansoni ) were cultured in biowhittaker dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ) containing 4500 mg / l glucose ( lonza ) supplement with 2 mm l - glutamine ( lonza ), penicilline 100 u / ml , streptomycine 100 μg / ml ( lonza ), amphothericin b 0 . 5 μg / ml ( cambrex ) and 10 % heath inactivated fbs . all animals were subjected to experimental protocols as reviewed and approved by the public veterinary health department of the italian ministry of health ( rome , italy ), according to the ethical and safety rules and guidelines for the use of animals in biomedical research provided by the relevant italian laws and european union &# 39 ; s directives . a puerto - rican strain of s . mansoni was maintained by passage through albino biomphalaria glabrata , as the intermediate host , and icr ( cd - 1 ) outbred female mice ( harlan laboratories ). the snails had been individually infected with 8 - 12 miracidiae per snail . snails were kept in tanks with dechlorinated tap water in a humid room simulating a 12 hour day and night cycle . first shedding of cercariae occurred from 4 weeks post infection . approximately 100 - 200 snails ( size of snails : 6 - 11 mm ) were placed twice under a direct 2000 lux lamp for 60 min at 27 ° c . the cercarial suspension was collected and used for the preparation of schistosomula . adult parasites were harvested by reversed perfusion of the hepatic portal system of infected mice previously euthanized with peritoneal ( i . p .) injections of tiletamina / zolazepam ( 800 mg / kg )+ xylazina ( 100 mg / kg ). female icr ( cd - 1 ) outbred and c57bl / 6 , 4 - 7 weeks old mice ( harlan laboratories ) were housed under controlled conditions [( 22 ± 2 )° c . ; ( 65 ± 15 )% relative humidity ; 12 / 12 h light / dark cycle ]. the mice received standard food and water ad libitum . female icr ( cd - 1 ) outbred mice were infected transcutaneously with approximately 80 ( for mixed infection ) or 200 ( single sex ) s . mansoni cercariae for life cycle maintainance and in vitro worm assays . for in vivo experiments , c57bl / 6 mice were infected with 140 cercariae and administered with selected compounds . cercariae , shed from infected snails , were subsequently converted to schistosomula by mechanical transformation using an optimised version of the protocol of brink et al ., 1977 previously described ( protasio et al . 2013 ). briefly , the cercarial suspension ( approximately 50 . 000 cercariae ) was placed in a glass 40 ml tube on ice for 60 minutes in order to reduce parasite motility . tail detachment was obtained by shaking cercariae vigorously for approximately 30 seconds in a vortex mixer before passing these through a 22g syringe needle approximately 10 - 12 times . next , the separation of heads / schistosomula and tails was obtained by placing the heads plus tails suspension on 4 - 5 ml of ice - cold 70 % percoll in tube and centrifugating ( 600 × g ) for 10 minutes at 4 ° c . finally , the schistomula preparations were washed twice ( with dmem complete media lacking fbs ) and microscope examination was used to assess the quantity and quality of purified schistosomula ( less than 1 % tails ). schistosomula were cultured at 37 ° c . in 6 wells tissue culture plates containing 3 ml schistosomula complete media in an atmosphere of 5 % co 2 for 24 hr before any further experimental manipulations proceeded . negligible parasite death occurred in this media during the 24 hr culturing period . following this , schistosomula were aliquoted into flat - bottom 384 - well black - sided for compound assays . a compound set of 1280 molecules comprising all the fda , emea and other agencies approved drugs ( prestwick chemicals , fr ) was tested according to the following procedure . compounds are stored as solution of 100 % dmso at − 20 ° c . under inert atmosphere . intermediate storage microplates , in the 384 well / plate format , are prepared on demand and stored in the same controlled environment as the stock solutions . intermediate microplate stored compounds are transferred to assay plates by the acoustic droplet ejection technology ( ats - 100 , edc biosystems usa ) which ensures a safe , contactless , pre - dilution free delivery . the test set was transferred to 384 well , black , tissue culture treated destination plate . the initial test was carried out at a single concentration of 10 μm . a suspension of schistosomula , in dmem added with 10 % fbs , was transferred to each well of the compound containing assay plates in order to have 100 schistosomula per well in a final volume of 30 μl . the plate set was let to incubate with the compounds at 37 ° c ./ 5 % co 2 for 24 hours . at the end of the incubation period each well was filled by and equal volume ( 30 μl ) of celltiterglo ( promega corp . p / n g7570 ) and let to incubate for 30 minutes at room temperature . the light based single emitted by the reaction is proportional to the atp amount in the culture which ultimately reflects the mitochondrial function and thus the schistosomula viablity . the readout , luminescence based , was carried out by a ccd based detector ( viewlux , perkinelmer usa ). each plate contains 16 dmso treated samples as negative controls and 16 gambogic acid treated samples as positive controls . in order to select the best cytotoxic compounds , the effect of each molecule was resealed between 0 % cytotoxicity ( dmso treated samples ) and 100 % cytotoxicity ( gambogic acid treated samples ). a threshold was set to 70 % cytotoxicity that , together with other presumably cytotoxic compounds , surprisingly showed phx as positive compound . the latter molecule was purchased from the provider ( cas # 6621 - 47 - 2 ; prestwick chemicals , fr ) as fresh lot and subjected to the uplc - ms quality control . after having passed the qc , the new batch of phx was tested in a dose response manner . to this aim , a serial dilution of the compound was carried out in dmso in order to cover the concentration range between 50 μm and 20 nm . the transfer of the serially diluted compounds and the assay protocol were identical to those described in the previous paragraphs . in vitro studies with s . mansoni juvenile ( 4 weeks old ) and adult ( 8 weeks or older ) worms seven - eight male worms or 4 - 5 worm pairs were recovered under aseptic conditions from infected mice by perfusion of mesenteric veins at 28 days ( juvenile ) or 56 - days post infection ( adult pairs ) or 2 - 4 months post - infection ( adult males ). after washing , the worms were transferred into a 35 mm plates containing 3 ml of dmem complete worms media and incubated at 37 ° c . in a humid atmosphere containing 5 % co 2 with drugs overnight . next , the worms were washed 3 times with drug - free medium and were incubated for 5 days at 37 ° c ./ 5 % co 2 and monitored daily under a stereo microscope for mobility , tegumental damage , viability and egg output ( worm pairs ). the experiment was repeated at least three times . c57b / l6 inbred female mice ( 5 - 6 weeks old ) were divided in 4 groups of 7 animals each and treated with dmso ( control ), pzq or two dosages of phx . all compounds were administered orally ( vehicle is 2 . 5 % cremophor el ) 42 days post infection . the amount of phx administered ( 23 mg / kg and 70 mg / kg ) was guided by available information of therapeutic dose in use for angina treatment ( ashrafian h ., 2007 ); pzq was used at the standard dose ( 500 mg / kg ). toxicity of compounds ( e . g ., death and behavioral changes ) was assessed daily during and after treatment until the time of euthanasia . at 56 days post infection , mice were euthanized with an intraperitoneal injection of tiletamina / zolazepam ( 800 mg / kg )+ xylazina ( 100 mg / kg ) and adult worms perfused as described above . compound efficacy in vivo , measured using a number of criteria , was compared to that of the anti - schistosomal drug , pzq . the criteria include the numbers of male and female worms ( total worm count ) recovered by perfusion and hepatic egg burden ( number of eggs in the liver ). to recover eggs trapped in liver , whole livers from individual mice were excised , weighed and incubated in a 4 % koh solution over night at 37 ° c . eggs were counted under a dissecting microscope as previously described ( cheever , a . w ., 1968 ). in order to investigate the effect of phx on s . mansoni larvae viability , newly transformed schistosomula were cultured in vitro for 24 h in presence of a range of drug concentrations ( 0 . 019 - 50μm ). parasites survival was assessed by using a celltiterglo luminescent assay and the cc 50 ( calculated by fitting the four parameters equation ) is shown in table 1 . gambogic acid , previously described as a potent killer agent for schistosomes ( peak et al , 2010 ), was used as positive control and dmso ( drug vehicle ) as negative control . gambogic acid , was confirmed to be effective in killing the schistosomula within the expected concentration range . perhexiline was also shown to be effective in this validated model . efficacy of phx on different stages of s . mansoni in vitro male adult worms ( 7 - 8 worms / sample ) obtained from infected mice were cultured at 37 ° c ., 5 % co 2 in dmem complete medium ( 10 % fbs ) in presence of different concentrations of phx ( 3 - 10 μm ) and for variable time ( 12h to 5 days ). for all experiments gambogic acid ( 1 - 10 μm ) and pzq ( 1 - 10 μm ) were used as positive controls while dmso was used as negative control . overnight incubation with 10 μm phx resulted in a statistically significant decreased viability of parasites after 5 days as shown in fig1 . parasites death was assessed by optical examination each day using the following criteria : reduction of motility , tegumental damages and darker appearance . phx was also tested at the concentrations of 5 μm and 3 μm leading to a reduced viability ( around 50 %) in the first case and having no effect in the second one . juvenile worms ( 4 weeks old parasites ) were also tested at the same conditions reported above . following over night incubation of phx ( 10 μm ), viability of parasites was reduced by 80 % after 5 days of observation ( fig2 ). remarkably , pzq ( at the same concentration ) treated worms started to recover after the wash at day 1 and pzq treatment showed a 50 % survival rate at 5 days . this is in accordance with previous report on pzq showing a survival of approximately 80 % of juvenile worms at 8 days ( livia - pica et al , 2004 ). in order to test the effect of phx on egg production , adult worm pairs were treated overnight using the sub lethal dose of 5 μm . total number of eggs laid by female parasites was determined 3 and 6 days after the treatment . to do so , the medium containing the eggs was harvested and briefly centrifuged . the pellet containing the eggs was re - suspended in 500 μl of saline solution and the egg number was determined by microscopy analysis . results showed that phx treatment led to a reduction in egg production of around 60 % after both 3 and 6 days compared to the untreated control incubated with dsmo alone . efficacy of phx in vivo in mice infected with s . mansoni based on the strong in vitro effects of phx against larvae , juvenile ( 4 weeks old ) and adult worms , the in vivo efficacy of the drug was tested in mice infected with s . mansoni . based on the previous cardiovascular studies of the drug ( ashrafian h ., 2007 ), two different concentrations of phx were used : a low dose ( 23 mg / kg ) and a high dose ( 70 mg / kg ). the compound was administered orally as single dose in 2 . 5 % cremphor el to mice with patent s . mansoni infection ( 56 days post infection ). as positive control , pzq was administered orally in a single dose at the standard concentration of 500 mg / kg . two weeks after the treatment , mice were sacrificed and the total worm count and egg burden ( number of eggs in the liver ) were determined ( fig3 ). a significant decrease in total worm counts was observed after treatment with both low and high doses of phx ( 35 and 63 % respectively ). for egg burden , a reduction of 50 and 60 % was shown after treatment with low and high doses of phx , respectively . as shown by the data reported herein , perhexiline is a potent inhibitor of schistosoma worms with cc 50 in the low micromolar range . worthy of specific note is the fact that the compound is active against larvae and both juvenile and adult worms , thus overcoming one of the major limitations of pzq , the only drug currently in use for the treatment of schistosomiasis . in addition , repurposing fda or ema - 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