Patent Application: US-201415103374-A

Abstract:
the invention relates to the use of pharmaceutical compositions containing as the active agent a compound of formula i in the treatment of dermal wounds . the compositions may be used for the promotion of healing of wounds affecting the deeper layers under the epithelium , including dermal - and sub dermal connective tissue , being cut , punctured , sliced wounds or originating in insufficient tissue perfusion , chronic diseases or other harms . the compositions are formulated for local application and may comprise other active agents contributing to wound healing in addition to a compound of formula .

Description:
the human skin consists of three layers : epidermis , dermis and subcutis . the epidermis is a multi - layer stratified squamous epithelium in which cells are continuously replenished from the stratum basale . melanocytes are also found in the epidermis . the base of the dermis is a loose connective tissue containing vessels , nerve endings and skin appendages ( e . g . hair follicles , sebaceous glands and sweat glands ). fatty tissue can be found among the tissue fibres of the subcutis . herein , we define the top layer of the skin as the epidermis or the upper cell layers of the epidermis . dermal wounds , extending into the upper layers of the skin , preferably the layers under the epidermis , are frequent and according to the state of the art there is a need for further therapeutic approaches . dermal wounds , treated according to the method of the invention , may be superficial wounds affecting the epidermis and the directly underlying layer or moderately deep wounds penetrating to the dermis . complex or deep dermal wounds affect not only the skin but may also extend to the muscle layer , nerves , bones and vessels under the skin . concerning etiology , dermal wounds may originate in abrasion , slice , cutting , puncturing , crushing , laceration , biting , inadequate blood supply ( e . g . atherosclerosis related ulcus ), chronic diseases ( e . g . foot ulcer due to diabetes ), necrosis ( e . g . decubitus ), thermic harms ( e . g . burns ), and chemical challenges ( e . g . acids ) or other impacts . according to certain preferred embodiments , the dermal wound is not a thermic ( e . g . burn ) wound or a wound of the same mechanism or a wound requiring the same treatment as a burn wound . the term dermal wound as used herein may refer to both acute and chronic ( difficult - to - heal ) wounds . preferably , the wound is a full thickness dermal wound . full thickness wounds affect each layer of the skin , including connective tissue of the dermis and , optionally , that of the subcutis . to our best knowledge , the background art does not offer any information regarding the effects of dantrolene or those of other compounds acting as antagonist of ryanodine receptors , described in the present invention , in the skin layers under the uppermost layer of the epidermis , and preferably in layers under the epidermis and tissue layers under the skin . it has not yet been clarified what kind of ryanodine receptors occur in the deeper layers of the skin , and therefore , no information is available on their distribution and physiological role in these tissues . one skilled in the art can test the activity of an agent as a ryanodine receptor antagonist without undue difficulties . appropriate methods are described by jaggar et al ., 1998 , arendshorst & amp ; thai , 2009 , denda et al ., 2012 and other publications cited by the the authors . it is known that ryanodine receptors play different roles in different organs and tissues . although ryanodine receptors were found first in muscle tissue , it has been recognized that they appear in other tissues , as well . their most extensively studied role is the connection of excitation and contraction , but they can be found in non - excitatory cells , too . ryanodine receptor type 1 ( ryr - 1 ) is characteristic of striated muscle , type 2 ( ryr - 2 ) can be found in cardiac muscle but has been described in langerhans islets , too . ryanodine receptors type 1 and 3 were proven to be present in leukocytes . ryanodine receptor type 3 ( ryr - 3 ) was first identified in the mammalian brain , but according to the present knowledge its occurrence may be considered universal , it can be found in the skin , striated muscle , smooth muscle and leukocytes although at lower quantities and its role is not fully clarified . ryr - 1 and ryr - 2 are also expressed in the brain . however , the pattern of their expression is different . ryr - 1 appears abundantly in purkinje cells of the cerebellum , ryr - 2 is expressed predominantly in the dentate gyrus while ryr - 3 in the pyramid cells of hippocampus ( ca1 region ), the basal ganglia and the olfactory bulb . all isoforms are present in smooth muscle cells ( kushnir et al ., 2010 ). ryanodine affects the mechanism of contraction differently in striated muscle and cardiac muscle . in striated muscle , it induces contractions leading to tetanic spasms , while it may result in cardiac arrest via decreasing the contractility in the heart . although both effects are based on calcium release , the activity of calcium - scavenging mechanisms are different in striated muscle and cardiac muscle . it has been described that ryanodine receptors can be found in vessels of different caliber in several organs , e . g . in the kidney ( arendshorst & amp ; thai , 2009 ), urether , spermatic duct , mesenteric artery ( borisova et al ., 2009 ), striated muscle ( cremaster muscle ) ( westcott & amp ; jackson , 2011 ) and the brain ( dabertrand et al ., 2012 ). ryanodine receptors influence the intracellular calcium level in the vascular smooth muscle hereby regulating vascular tonus . however , they may play a different role in different organs . inhibition of ryanodine receptors was found to lead to vasoconstriction ( jaggar et al ., 1998 ). however , inhibition of ryanodine receptors may soothe the existing vasoconstriction in the kidney ( arendshorst & amp ; thai , 2009 ). caffeine , which activates the ryanodine receptors , leads to vasoconstriction in cerebral and meningeal vessels ( dabertrand et al ., 2012 , knot et al ., 1998 ). nutritive arteries of the muscle tissue display vasodilatation due to inhibition of ryanodine receptors ( westcott & amp ; jackson , 2011 ). a paper published by denda et al . ( denda et al ., 2012 ) and patent application jp 2011250728 reveal that dantrolene and 1 , 1 - diheptyl - 4 , 4bipyridinium - dibromide ( dhbp ), another known antagonist of ryanodine receptors , ameliorate transepidermal water loss in mice in which barrier function is impaired after tape stripping of the upper epidermis . the authors have shown by means of immunohistochemistry that ryanodine receptors are present on the keratinocytes . furthermore , expression of all isotypes ( at mrna level ) is more pronounced in differentiated keratinocytes . ryr - 1 and ryr - 2 are localized in the upper layer of the epidermis . accordingly , the authors presumed that the promotion of barrier reconstruction induced by dantrolene and dhbp originates in the inhibition of ryanodine receptors . as far as we know , no data are available concerning the role of ryanodine receptors on dermal circulation and their impact on dermal injuries affecting layers under the upper part of epidermis . in our experiments , we have studied the effects of dantrolene on different parameters of wound healing . skh - 1 hairless male mice were used in the experiments . full - thickness wound was made in a dorsal skin fold . the effects of dantrolene or saline ( control group ) on wound healing were monitored by means of photo documentation , intravital videomicroscopy ( ivm ) and laser doppler flowmetry . the experimental setup was in accordance with the model described by sorg et al . ( sorg et al ., 2007 ). wounds were treated daily either with dantrolene or with saline . unexpectedly , our findings have shown that dantrolene accelerated the wound closure and decreased the number of leukocyte - endothelial interactions . another unexpected effect was that dantrolene increased blood flow in the wound edges , evaluation of ivm records revealed significantly higher vessel diameters in dantrolene - treated animals than in control ones . higher red blood cell velocities were measured in the capillaries . laser doppler flowmetry unraveled considerably higher blood flow as compared to baseline values . this microcirculation - improving effect of dantrolene was detected in two different tissue layers which , in view of the above mentioned published data , was not predictable . ivm visualized the dermal vessels of the wound edges and showed that application of dantrolene increases vessel diameters , while laser doppler sensor was placed onto the basis of the wound and blood flow in the muscle layer of the skin fold &# 39 ; s opposite side was monitored . this examination showed elevated blood flow in the muscle . macroscopic wound closure was assessed by evaluation of photographs with a special software . histology was used for characterization of epithelialization and dermal regeneration . macroscopic wound closure reached nearly 100 % in both groups . rate of wound closure in the dantrolene - treated group was significantly higher on days 4 , 8 and 12 than in the control group . faster dermal regeneration induced by dantrolene is an unexpected result , because no information was available previously on regenerative or any other effects of dantrolene and / or ryanodine receptors in the lower layers of the skin . ryanodine receptors mediate diverse effects in different tissues and the same ligands may induce distinct effects on different isoforms . thus , effects of dantrolene on wounds penetrating into the deeper layers of the skin were not predictable for one skilled in the art prior to the experiments . based on our experiments , dantrolene exerts its beneficial effects through the vasculature and microcirculation of the dermis and the muscle layer . although based on the findings described in patent application jp 2011250728 and of denda and co - workers ( denda et al ., 2012 ), the effect of dantrolene to the barrier function and regeneration of the epidermis could have been speculated , no patent ( application ) or manuscript describes the effect of dantrolene — or any other ryanodin - antagonist — on the circulation of neighboring vessels of wounds . hence , this effect of dantrolene and its role in wound healing are novel and unforeseen results of our experiments . the composition useful for wound healing referred to in patent application jp 2011250728 contains dantrolene , but based on its description the skilled artisan would have not deducted the conclusion which is described herein . the composition described in patent application jp 2011250728 is a muscle relaxant , which is used against inflammation , but not directly for the purpose of wound healing , although it was applied on skin wounds , the healing of which was depending on inflammatory processes , too . dantrolene — like other muscle relaxants described in patent application jp 2011250728 — reduces inflammation through its muscle - relaxant effect by inhibiting the post - injury muscle control . however , in the present invention healing of skin wounds where the muscle control has no significance , is facilitated , since in the experimental model we used not only the upper and middle layer of skin tissue but the muscle layer below were removed as well . therefore , according to the present invention , the effect of dantrolene on wound healing cannot be explained by its muscle relaxant effect . in our experiments , excised wound was formed , the healing tendency of which differs from combustion wounds disclosed in jp 2007308403 . reducing inflammation , such as by the use of steroids , is recommended in case of minor combustion wounds , while in wounds of other origin steroids worsen healing tendency . accordingly , reducing inflammation does not obviously result in improved wound healing . based on our experimental results , we may rightly assume that dantrolene will exert the same effect on wound healing in case of wounds similar to those in the above described experiments , such as diabetes mellitus and cardiovascular failure - related slow healing and chronic wounds . in support of our hypothesis we have designed the following experiment : application of dantrolene to promote wound healing in a skin fold chamber model after inducing diabetes by streptozotocin in mice . diabetes is induced with intraperitoneal ( i . p .) injections of streptozotocin ( 5 × 50 mg / kg ) according to literature data ( lee et al ., 2014 , langer et al ., 2002 ). after 5 weeks , the blood glucose content is measured and animals with 16 . 6 and 33 . 2 mmol / l glucose level are enrolled to the experiment . wounds of standard size are made on the dorsal part of the ear . wound healing will be investigated according to the methods described in the examples and mentioned above . the pharmaceutical composition according to the invention may be used as a topical formulation . the formulation may be e . g . an ointment , cream , gel , talcum , foam composition , patch , wound dressing ( such as a hydrogel , film bandage , mesh combined with alginate , impregnated with a cream ), solution or suspension . administration may be as usual to the applied formulation or different ; e . g . an ointment may be spread to the skin in a thin layer once or multiple times daily , a solution may be dripped into the wound once or multiple times a day to wet the wound , a patch or wound dressing may be changed to cover the wound multiple times a day or week . the concentration of the active agent in the pharmaceutical composition according to the invention depends on the formulation and the indication for application . it might be e . g . 1 to 100 mg / kg for a cream , 1 - 100 mg / ml or 1 to 1000 μm or preferably 10 to 500 μm for a solution . treating dermal wounds and determining the method of treatment in the light of the active agent and its effect is implicit in the knowledge of one skilled in the art . further information regarding the treatment of dermal wounds is to be found in cathy thomas hess “ clinical guide to skin and wound care ” 7 . ed . 2012 , and avi shai and howard i . maibach “ wound healing and ulcers of the skin — diagnosis and therapy — the practical approach ” 2005 , and : “ a nem gyógyuló ( krónikus ) börsebek ellátásának elöirányzott irányelve .” a börgyógyász szakmai kollégium jóváhagyásával készült irányelv 2011 . átdolgozott formája . (“ guidelines to the therapy of non - healing ( chronic ) dermal wounds ”. revised version of the guidelines ( 2011 ) approved by the hungarian college of dermatology . example 1 . the effects of dantrolene on the healing of full - thickness dermal wounds in mice the experiments were performed on male skh - 1 hairless mice of our own rearing ( body weight : 36 - 44 g ). procedures and protocols were approved by the competent authorities ( license number : v ./ 145 / 2013 .). only healthy animals free of any type of injury were used , mice with any sign of disorders were discarded . prior to the interventions the animals were anesthetized with a mixture of ketamine ( sigma ) ( 90 mg / kg ) and xylazine ( sigma ) ( 25 mg / kg ) administered intraperitoneally . a skin fold was formed in the dorsal region and was fixed with two symmetrical , fenestrated titanium plates ( irola gmbh , schonach , germany ) according to the description of sorg et al . ( sorg et al ., 2007 ). in the midline of the dorsal region two holding stitches were placed in order to form a skin fold . this skin fold was sandwiched by two symmetrical , fenestrated titanium plates . the plates were fixed with sutures , bolts and nuts . on one side of the skin fold , a circular full - thickness wound was made by removing the tissue down to muscle layer of the other side ( the diameter of the wound was 4 mm while those of used for laser - doppler flowmetry came to 11 mm ). the wound was covered with a glass coverslip incorporated into the window of the titanium frame . the glass was removed for the duration of treatments and measurements only . in the control group ( n = 6 ), the wounds were treated with 100 μl of sterile saline once a day while in the dantrolene - treated group ( n = 6 ) 100 μl of a solution containing the sodium salt of dantrolene ( 100 μm ) was applied to the wound once a day ( the salt was dissolved in sterile saline ). the observation period took 20 days ( according to our previous results this is the time period needed for approximately 100 % closure of wounds of 4 mm ). macroscopic wound closure . photographs of the wounds were taken immediately after surgery and on days 4 , 8 , 12 , 16 and 20 . circumstances of photographing were standardized ( same light sources and distances ). the images were evaluated with a software developed by our working group ( dermassess ©). this software is an accessory function to the imagej software and enables the determination of color intensity ( eros et al ., 2014 ) and the area measurement after calibration . decrease in the wound area relative to the baseline value is given as percentage . intravital videomicroscopy ( ivm ). microcirculation of the wound edges was monitored by means of ivm on days 4 , 8 and 12 . after anesthesia , 80 μl of a solution of fluorescein - isothyocyanate ( fitc ) labeled dextrane ( 2 %) and also 80 μl of a solution of rhodamine - 6g was administered retrobulbar . these contrast materials allow us to visualize microcirculation and leukocyte - endothelial interactions at the appropriate wavelengths . video records of the microcirculation were evaluated off - line with pictron ivm software . red blood cell velocity ( rbcv ) was determined in the capillaries , the diameter of each vessel in the evaluated fields - of - view was measured ( only diameters above 6 μm were considered ) and sticking leukocytes were counted in the postcapillary venules ( this parameter is relative to the area of the vessel wall and is given as cell / μm2 ). laser - doppler flowmetry . in a further group ( n = 6 ), the animals were anesthetized on day 1 after wound creation . the sensor of the laser - doppler equipment was placed onto the wound . the sensor was removed after 10 minutes of measurement and 200 μl of dantrolene solution was applied to the wound . another measurement was performed 10 minutes later . histology . on days 4 and 20 groups of control - and dantrolene - treated animals were sacrificed , the wounds were then excised , fixed in a solution of formaldehyde , embedded in paraffin and stained with hematoxylin and eosin . the pannoramic viewer software was used for the evaluation . zone of epithelialization was defined as the extension of epithelial regeneration measured from the wound edges and given as percentage relative to the initial diameter of the wound . dermal regeneration was determined , too ( thickness of dermal connective tissue was measured within the wound area and was given as percentage relative to that of determined in the intact skin ). control - and dantrolene - treated groups were compared with the mann - whitney test and p & lt ; 0 . 05 was considered statistically significant . macroscopic wound closure . this parameter reached nearly 100 % in both groups by day 20 . on days 4 , 8 , and 12 , the rate of wound closure in dantrolene - treated groups was significantly higher than in control group . on day 16 , no difference was found between the groups ( fig1 ). rbcv in the capillaries . rbcv of dantrolene - treated animals was found be higher at all times of measurements ( days 4 , 8 and 12 ) than in control group ( fig2 ). vessel diameters at the wound edges . vessel diameters , determined at the wound edges of mice exposed to dantrolene , were considerably higher than those in the control group at corresponding times ( fig3 ). sticking leukocytes in the postcapillary venules . concerning leukocyte - endothelial interactions , said parameter was assessed as an indicator of inflammation . our results revealed that application of dantrolene significantly decreased the number of sticking leukocytes on days 4 and 8 . the difference on day 12 was not statistically significant ( fig4 ). blood flow in the wound area . laser doppler flowmetry showed significant elevation in blood flow after dantrolene treatment ( fig5 ). extension of epithelialization zone . epithelialization determined with routine histology ( zone of epithelialization ) was more extended in dantrolene - treated mice than in control ones on day 4 ( fig6 ). dermal regeneration . on day 4 , said parameter was more expressed in animals receiving dantrolene than in control group ( fig7 ). the experiments will be performed on 12 - 15 - week - old skh - 1 hairless mice of our own rearing ( minimal weight : 30 g ). procedures and protocols were approved by the competent authorities ( license number : v ./ 145 / 2013 .). appropriate temperature ( 24 + 2 ° c . ), 12 h dark - light cycles and free access to standard laboratory chow and water will be provided . only healthy animals , free of any type of injury will be used , mice with any sign of disorders ( except diabetes mellitus ) will be discarded . according to recent data , diabetes mellitus can be induced with streptozotocin ( 50 mg / kg intraperitoneally injected daily for 5 days ) ( lee et al ., 2014 ). 5 weeks after the induction of diabetes , the blood glucose level will be determined . animals with blood glucose levels between 16 . 6 and 33 . 2 mmol / l will be accepted for the study ( langer et al ., 2002 ). prior to the interventions the animals will be anesthetized with an intraperitoneally administered mixture of ketamine ( 90 mg / kg ) and xylazine ( 25 mg / kg ). the ear will then be gently stretched . on the dorsal side of the ear , a circular area with a diameter of 2 . 5 mm will be marked . using this mark , a full - thickness dermal layer will be excised down to the underlying cartilage by means of microsurgical scissors under a surgical microscope ( langer et al ., 2002 ). the wound will be treated with a dantrolene - containing formulation ( 100 μm ), while the control group will receive the vehicle , without the active agent . the observation period will take 15 days . photographs will be taken of the wounds immediately after surgery and on days 4 , 7 , 12 , and 15 . circumstances of photographing will be standardized ( same light sources and distances ). the images will be evaluated with a software developed by our working group ( dermassess ©). this software is an accessory function to the imagej software and enables the determination of color intensity ( eros et al ., 2014 ) and the area measurement after calibration . intravital videomicroscopy ( ivm ): microcirculation of the wound edges will be monitored by means of ivm on days 4 , 7 and 12 . after anesthesia , 80 μl of a solution of fluorescein - isothyocyanate ( fitc ) labeled dextrane ( 2 %) and also 80 μl of a solution of rhodamine - 6g will be administered retrobulbar . these contrast materials allow us to visualize microcirculation and leukocyte - endothelial interactions at the appropriate wavelengths . video records of the microcirculation will be evaluated off - line with pictron ivm software . red blood cell velocity ( rbcv ) will be determined in the capillaries , the diameter of each vessel in the evaluated fields - of - view will be measured ( only diameters above 6 μm will be considered ) and sticking leukocytes will be counted in the postcapillary venules ( this parameter is relative to the area of the vessel wall and is given as cell / μm2 ). histology : on day 15 animals will be sacrificed , the wounds will then be excised , in order to be fixed in a solution of formaldehyde , embedded in paraffin and stained with hematoxylin and eosin . the pannoramic viewer software will be used for the evaluation . zone of epithelialization will be defined as the extension of epithelial regeneration measured from the wound edges and given as percentage relative to the initial diameter of the wound . daróczy j . és mtsai . a nem gyógyuló ( krónikus ) börsebek ellátásának elöirányzott irányelve . a börgyógyász szakmai kollégium jóváhagyásával készült irányelv 2011 . átdolgozott formája . http :// sebkezeles . hu / download / iranyelv . pdf denda s , kumamoto j , takei k , tsutsumi m , aoki h , denda m : ryanodine receptors are expressed in epidermal keratinocytes and associated with keratinocyte differentiation and epidermal permeability barrier homeostasis . j invest dermatol 2012 ; 132 : 69 - 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