Patent Application: US-36576406-A

Abstract:
a method for the treatment of skin fibrosis with a peptide that inhibits tgf - β , and suitable compositions for its administration . the method includes in particular , the use of peptide p144 , a compound that is a known inhibitor of tgf - β , for the treatment of skin fibrosis by topical application . the method is shown effective in an animal model of bleomycin - induced skin sclerosis , to a reduction both of the skin fibrosis and of the content of soluble collagen , without any signs of systemic toxicity being detected . this shows that p144 is effective for topical application in mammals for treating fibrotic skin diseases and pathological scarring of the skin . for the administration of this peptide , stable compositions are also supplied , with pleasant appearance without being greasy , with good spreading characteristics and with a viscosity that permits it to be processed easily in industrial plant , and which are suitable for administering the peptide to humans .

Description:
as used herein , “ consisting essentially of ” means that the composition , component or peptide may include additional ingredients or amino acids , but only if the additional ingredients or amino acids do not materially alter the basic and novel characteristics of the claimed compositions or methods . the following examples describe tests that were designed to test the antifibrotic effect of p144 in the animal model of bleomycin - induced skin sclerosis by using compositions in the form of an emulsion containing said peptide , and tests with variation of the basic formulation used for forming said emulsion to give a formulation that displays characteristics making it suitable for topical administration to humans and that could be manufactured on a large scale and dosed in commercial forms of preparation simply and efficiently . the compositions used in said tests were obtained using the products shown below in table 2 , which also shows the initial phase in which they were incorporated ( aqueous or oily ), before the two were combined to form an emulsion : weigh the components of each phase of the emulsion ( oily and aqueous phases ) separately in stainless steel vessels of suitable capacity . heat the two phases separately on a bath with thermostatic control at 70 ° c . the vessel containing the aqueous phase must be covered to prevent losses by evaporation . after approximately 20 minutes , the oily phase has melted and is homogeneous . both phases must have a clear , homogeneous appearance with temperature of approximately 70 ° c . stirring the oily phase with a propeller stirrer ( speed = 3 . 5 . ika rw16 basic stirrer ), pour the aqueous phase onto the oily phase a little at a time . this operation takes approximately 5 minutes . continue stirring the emulsion , at the same speed , for 30 - 40 minutes . at the end of the process , the temperature of the emulsion will be 20 - 25 ° c . the final stage is maturation of the emulsion . this stage is of variable duration , generally 48 - 72 hours , sufficient time for verifying its stability . with emulsions prepared in this way , tests were carried out , as described below in the examples . two emulsions or creams were prepared , an emulsion containing peptide p144 and a control emulsion with the vehicle for administration but without p144 . the control emulsion with the vehicle for administration was prepared by a procedure that starts with mixing of the components that are shown below in table 2 : the emulsion containing the p144 was prepared as previously , except that the 44 . 4 g of water was replaced with a mixture of 44 . 28 g of water plus 0 . 010 g of p144 previously dissolved in 100 μl of dimethylsulphoxide . the test was carried out using 6 - week - old female c3h mice , supplied by harlan sl ( spain ). development of skin sclerosis in the mice was induced by the administration of bleomycin ( sigma , spain ) dissolved in pbs at a concentration of 100 μg / ml and sterilized by filtration . the mice were injected with the bleomycin subcutaneously in the skin of the back , previously shaved , at daily doses of 100 μl of the solution in pbs injected with size 27 needles . the mice used as control received 100 μl of pbs without bleomycin . the injections were administered daily , at the same site , for 4 weeks . the mice treated with bleomycin were divided into groups of 10 mice . in one group , 100 μl of the preparation of the emulsion containing p144 was applied daily in the shaved area of the skin , for the 4 weeks of bleomycin injections , whereas the other group received the control emulsion with the vehicle for administration . no composition was applied to the third group . the mice that were injected with pbs without bleomycin also did not receive any composition by topical administration . the mice were sacrificed by asphyxiation by inhalation of co 2 , 24 hours after the last injection . the skin was removed from the back and biopsy specimens with diameter of 4 mm were obtained using a hole - making punch ; these were frozen for subsequent protein analysis , whereas additional samples of skin were frozen in liquid nitrogen and were embedded in the medium known as “ tissue tek ® o . c . t . compound ” ( optimal cutting temperature ), supplied by sakura , the netherlands , a medium for tissue protection by cryopreservation and then histological section , for subsequent histological and immunohistochemical investigations . the study was approved by the ethical committee of complutense university of madrid . for the histological investigations , the skin sections were stained with haematoxylin and eosin ( staining which makes the cell nuclei appear blue by reaction of the nucleic acids with the basic dye haematoxylin , whereas the cytoplasm is stained pink or red by reaction of the basic proteins present in it with the acidic dye eosin ), masson trichrome stain ( which stains collagen fibres blue , the nuclei black and the background red ) and toluidine blue for metachromatic staining of mast cells . for the histomorphometric analyses , three fields of each skin biopsy were photographed at random and were digitized using a spot rt ccd camera and spot 4 . 0 . 4 software ( diagnostic instruments , sterling heights , mich .). this provided measurement of the thickness of the dermis , and from the digitized images it was possible to count the number of myofibroblasts ( example 2 ), fibroblasts positive for phosphorylated smad2 / 3 ( example 2 ) or mast cells ( data not shown ) corresponding to each field at 400 × magnification . the results obtained are shown in the first two rows of photographs in fig1 , whereas the last row corresponds to the processing of samples obtained in the test that will be described later in example 3 . it can be seen from these that the mice treated with bleomycin ( bleo ) showed a marked increase in the collagen matrix of the dermis . in part a in the figure , corresponding to staining of the dermis with haematoxylin and eosin , it can be seen that there is an increase in thickness of the dermis in the mice treated with bleomycin which partially replaced the subcutaneous fat in comparison with the mice treated only with phosphate - buffered saline ( pbs ). part b in the figure , corresponding to staining of the hypodermic area located on the panniculus carnosus muscle with masson trichrome stain , shows that there was also an increase in the collagen matrix around the superficial fascia of the panniculus carnosus in the mice treated with bleomycin ( bleo ). in these mice we also observed an abundant inflammatory infiltrate , composed principally of mononuclear cells as well as an increase in the number of mast cells , many of which showed signs of degranulation ( data not shown ). the mice treated with peptide p144 ( photographs labelled as a - tgf ) during the weeks of the treatment with bleomycin showed a decrease in the area of dermal collagen ( part a in fig1 ) and hypodermic collagen ( part b in fig1 ) in comparison with the mice treated with the vehicle ( vehic ). the thickness of the dermis was measured on the digitized images of the sections stained with haematoxylin and eosin , and then the percentage variation in thickness of the dermis was calculated relative to the mean of the values obtained for the mice treated with pbs , adjusted to 100 %. the data obtained are shown in the graph in part a of fig2 , where each bar represents the value of the mean ± standard deviation of 10 mice per group . it can be seen in this graph that the thickness of the dermis decreased significantly in the mice treated with p144 ( bar filled with dots ) relative to the mice treated with the vehicle ( bar filled with a network of horizontal and vertical lines ), which displayed a thickness similar to that found in the mice treated with bleomycin which did not receive topical treatment ( bar with dark shading ). the other two bars of the graph correspond to samples obtained in the test described later in example 3 . to confirm the histological observation of decrease in fibrosis in mice treated with p144 , the content of pepsin - soluble collagen was determined in 4 - mm biopsies obtained with a punch . the pepsin - soluble collagen is an extractable fraction that represents the collagen recently synthesized in the tissues . to quantify it , after homogenizing the biopsies , the pepsin - soluble collagen was extracted over night using 5 mg / ml of pepsin in 0 . 5 mol / l of acetic acid . the content of soluble collagen was determined using the kit for calorimetric testing of collagen based on sircol ™ ( biocolor , northern ireland ), following the manufacturer &# 39 ; s instructions , obtaining the results shown in the graph in part b of fig2 , where each bar once again represents the mean ± standard deviation of 10 mice per group corresponding to the percentage variation of the content of soluble collagen in each sample calculated relative to the content of soluble collagen measured in the samples treated with pbs , for which the values were adjusted to 100 %. this analysis showed a significant decrease in the content of soluble collagen in the mice treated with p144 ( bar filled with dots ). no changes were observed in the density of infiltration of inflammatory cells or of infiltration of mast cells , nor morphological changes in the epidermis of the mice treated with the vehicle or with p144 when compared with the mice that only received injections of bleomycin ( data not shown ). for more extensive characterization of the cellular effects of neutralization of tgf - β1 with p144 , various immunohistochemical studies were carried out , analysing the effect of the peptide on the development of myofibroblasts positive for α - sma and the induction of smad2 / 3 in fibroblasts induced by bleomycin . for this , immunofluorescent staining was carried out ( with an anti - smooth muscle α - actin ( sma ) monoclonal antibody labelled with fluorescein isothiocyanate ( fitc ), supplied by sigma ) of skin sections obtained in the test described previously in example 1 , and the samples obtained were examined with an axioplan - 2 fluorescence microscope from zeiss ( germany ). fig3 shows photographs obtained from samples corresponding to mice that were only injected with pbs ( pbs ), mice that had received injections of bleomycin for 4 weeks ( bleo ), mice after topical administration of an emulsion that only contained the vehicle for administration during the weeks of treatment with bleomycin ( vehic ) or mice that were administered an emulsion that contained in addition the p144 peptide ( a - tgf ). the graph in the bottom part b of said fig3 shows a graph showing the results of evaluation of the number of cells that are positive for the presence of α - sma quantifiable per field , calculated from the fluorescence signal obtained ; each bar represents the mean standard deviation of the data corresponding to 10 mice per group . the results show that in the control mice , myofibroblasts that are positive for α - sma were observed very rarely , whereas an abundant number of these cells was observed after four weeks of injections of bleomycin . the mice treated with p144 showed a significant reduction in the number of myofibroblasts positive for α - sma in comparison with the mice treated only with the vehicle . in addition , we also carried out immunohistochemical detection of phosphorylated smad2 / 3 in skin sections . for this , the samples were labelled with a specific anti - phospho - smad2 / 3 polyclonal antibody , supplied by santa cruz biotechnology ( santa cruz , calif .) and a method based on biotin peroxidase ( abc , vector laboratories , burlingame , calif .). the slides were developed using a diaminobenzidine ( dab ) chromogen . the sections were contrast - stained with gill &# 39 ; s haematoxylin . the results are shown in fig4 , where in part a ( corresponding to the photographs obtained from mice submitted to each of the treatments described ) the fibroblasts labelled with the antibody are arrowed . part b of fig4 shows a graph in which each bar corresponds to the mean value ± standard deviation of the number of fibroblasts positive for presence of phospho - smad2 / 3 detectable per observation field . in this case , the data are only representative of five mice per treatment group . the data obtained in this second immunostaining indicate an increase in the number of dermal fibroblasts displaying phosphorylation of smad2 / 3 in the mice that were injected with bleomycin , which confirms earlier observations in this animal model ( takagawa et al ., 2003 ). the number of fibroblasts positive for phospho - smad2 / 3 decreased significantly in the mice treated with p144 in comparison with the mice treated with the vehicle . finally , we undertook determination of whether there is any effect on regulation of ctgf expression induced by peptide p144 in the mice treated with bleomycin . for this , immunohistochemical tests were carried out with the l - 20 polyclonal antibody ( santa cruz biotechnology , santa cruz , calif . ), a specific anti - ctgf antibody which was used for staining skin sections from mice corresponding to each of the treatment groups described in example 1 , with the presence of the antibody bound to the samples being revealed by means of a dab substrate and then performing contrast staining with gill &# 39 ; s haematoxylin . in the test described in example 2 , this l - 20 antibody specifically recognized a unique protein of 38 kd , which was induced strongly by treatment with tgf - β1 in cultured fibroblasts ( data not shown ). with respect to the samples obtained from the mice , the data shown in fig5 demonstrate that ctgf expression was induced strongly in the fibroblasts as well as in the cells of the epidermis and of the epithelial hair follicle of the mice treated with bleomycin . treatment with p144 caused a clear decrease in ctgf expression in the epidermis and the hair follicles , in comparison with mice treated with the vehicle only , whereas ctgf was still detectable in fibroblasts after treatment with p144 . to evaluate the effect of treatment of mice which already had an established fibrosis , a test was carried out similar to that described in example 1 , except that the mice did not receive any composition by the topical route in the four weeks during which they were administered injections of bleomycin . at the end of the four weeks , a topical composition was applied to the mice daily for two weeks before they were sacrificed : the emulsion containing p144 was administered to one group of mice , whereas the emulsion that only contained the vehicle for administration was administered to the other group . after the mice were sacrificed , skin samples were taken from the back in a manner similar to that described in the test in example 1 and they were examined histologically and pepsin - soluble collagen was quantified following the procedures described previously in example 1 . the photographs shown in the bottom row of fig1 correspond to the data obtained in histopathological evaluation of bleomycin - induced skin sclerosis in these mice ; part a shows the results of staining of the dermis with haematoxylin and eosin , whereas part b corresponds to staining of the hypodermic region located on the panniculus carnosus muscle ( m ) effected with masson trichrome stain . evaluations of the dermis thickness changes and the skin collagen content are shown , respectively , in the graphs in parts a and b of fig2 . the data obtained show that the fibrosis persisted in the mice treated with the composition that only contained the vehicle ( labelled vehic in the bottom part of fig1 and represented by bars filled with inclined lines in the graphs in fig2 ), whereas the mice treated with p144 for 2 weeks ( labelled a - tgf in the bottom part of fig1 and represented by the bars filled with horizontal lines in the graphs in fig2 ) showed a significant decrease in thickness of the dermis and collagen content . antifibrotic effect of p144 on fibrosis induced by high doses of bleomycin an experiment was carried out similar to that described in example 1 , but using higher doses of bleomycin . for this , two emulsions were again prepared , one containing peptide p144 and a control emulsion with the vehicle for administration but without p144 , using the same components and following the procedure described in example 1 . once again , the test was carried out using 6 - week - old female c3h mice , with body weight of 15 - 18 g , supplied by harlan s . l . ( spain ). development of skin sclerosis in the mice was induced by the administration of bleomycin ( sigma , spain ) dissolved in pbs and sterilized by filtration , but in this case the concentration of bleomycin in the solution was higher than in example 1 , 1 mg / ml . the mice were injected with the bleomycin subcutaneously in the shaved skin of the back , using size 27 needles , at a dose of 100 μl of bleomycin in pbs , so that the dose of bleomycin injected in each mouse was approximately 6 mg / kg body weight . the injections were administered at the same site , on alternate days , for 4 weeks . the mice used as control received 100 μl of pbs without bleomycin . in all , 15 mice were used for the experiment , and were divided into 3 treatment groups , each comprising 5 mice . the treatments received by each of the groups were : group pbs : 100 μl of pbs without bleomycin every 48 hours group bleo + vehic : 100 μl of pbs with bleomycin ( 1 mg / ml ) on alternate days for 4 weeks + 100 μl of emulsion without p144 daily for the same 4 weeks . group bleo + p144 : 100 μl of pbs with bleomycin ( 1 mg / ml ) on alternate days for 4 weeks + 100 μl of emulsion with p144 ( 0 . 1 mg / ml ) daily for the same 4 weeks . the mice were sacrificed by asphyxiation by inhalation of co 2 , 24 hours after the final injection or after the last topical administration of emulsion . the skin was removed from the back and biopsy specimens with a diameter of 4 mm were obtained using a punch , and were frozen for subsequent protein analysis , whereas additional skin samples were processed for later histological and immunohistochemical investigations as in example 1 . for the histological investigations , the skin sections were stained with haematoxylin and eosin . the histomorphometric analyses were also carried out as in example 1 , by randomly photographing three fields of each skin biopsy and digitizing them using a spot rt ccd camera and spot 4 . 0 . 4 software ( diagnostic instruments , sterling heights , mich . ), obtaining digitized images on which the thickness of the dermis was measured . the results are shown in fig6 ( which shows an image representative of each of the five mice forming the different groups ) and in fig7 ( showing the percentage variation in thickness of the dermis relative to the mean of the values obtained in mice treated with pbs , adjusted to 100 %). an increase in thickness of the dermis in the mice treated with bleomycin can be seen , which almost completely replaced the subcutaneous fat in the mice treated additionally with the emulsion that only contained the vehicle for administration ( samples labelled “ bleo + vehic ”); they were also observed to have an increase in the collagen matrix around the superficial fascia when compared with the mice treated only with pbs ( samples labelled “ pbs ”). the mice treated with bleomycin and peptide p144 ( samples labelled “ bleo + p144 ”) displayed a significant decrease in area of dermal and hypodermic collagen relative to the mice treated with bleomycin and the emulsion that only contained the vehicle for administration . once the effect of topical application of p144 on distinctive features of fibrosis had been demonstrated , changes were made to the formulation used for obtaining the compositions in the form of lipogel used in the previous examples , with the aim of finding formulations that are more suitable for topical application of p144 in humans . the original formulation gave rise to extremely greasy compositions , which could cause stains on clothing , and moreover had high viscosity , making it difficult to spread them on the skin , giving low fluidity at room temperature , which was a drawback for their large - scale production and their dosing in commercial presentation forms . therefore separate tests were carried out in which the composition of the original formulation was varied with the aim of finding a pharmaceutical form of lower viscosity than the original , which will flow and allow processing in machines for semisolid forms , with pleasant appearance , good spreadability and preserving their characteristics of local action . the majority of the tests described were intended to eliminate or reduce the content of cetostearyl alcohol , which is responsible for the high viscosity . the products previously mentioned in table 1 and the method of preparation described after table 2 were used for preparing the various compositions . firstly , the content of cetostearyl alcohol , which is responsible for the high viscosity , was reduced by increasing the percentage of water in the formulation . the other components in the formulation were maintained in their initial proportions . thus , the formulations shown in table 3 were tested ; all the percentages correspond to weight / weight ratios ( w / w ). as was hoped , the viscosity of the semisolid preparation decreases when the percentage of cetostearyl alcohol decreases . after 24 hours at room temperature , all the formulations remained stable . the formulation with higher content of cetostearyl alcohol ( no . 4 ) was still excessively viscous , extremely greasy and of very low fluidity . in contrast , formulations 2 and 3 were less viscous and had pleasant organoleptic characteristics ; however , their fluidity was still inadequate . finally , formulation 1 had good organoleptic characteristics , reduced viscosity and good fluidity . 400 g of each of these formulations was prepared for investigating their behaviour in the dosing machine , and it was found that formulations 1 and 2 flowed adequately . however , the other formulations were excessively viscous . in this case a proportion of the cetostearyl alcohol was replaced with liquid paraffin with the aim of obtaining a formulation that flows adequately and remains stable . another five different emulsions were prepared , combining different percentages of cetostearyl alcohol and liquid paraffin , which were selected taking into account the specific characteristics of each of them and their contribution to the fluidity and stability of the final emulsion . these emulsions were designated : 1 ′, 2 ′, 3 ′, 4 ′ and 5 ′. their composition is summarized in table 4 , where , unless stated otherwise , percentages correspond to weight / weight ratios ( w / w ). formulation 1 ′ was very fluid and of low viscosity . formulation 2 ′ had the same characteristics as 1 ′, but generated persistent foam during manufacture . 3 ′ had some characteristics similar to 2 ′, except for its higher viscosity and therefore lower fluidity than 2 ′. as for formulations 4 ′ and 5 ′, they were found to be very viscous , and with fluidity similar to the starting formulation . next , small batches of emulsions 1 ′ to 5 ′( 500 ml ) were manufactured , and were used for more extensive dosing tests . formulations 1 ′, 2 ′ and 3 ′ were the most suitable , displaying suitable fluidity for homogeneous dosing in the manufacture of large batches . with the aim of improving the viscosity characteristics of the formulation on the one hand and simplifying production of the cream on the other hand ( by eliminating or reducing preheating to permit mixing of the components in the presence of cetostearyl alcohol ), the complete replacement of the latter or its elimination was investigated . two formulations were prepared , replacing the cetostearyl alcohol with two hydrophilic emulsifiers that were incorporated in the aqueous phase : tween 80 ( 4 %) or sodium laurylsulphate ( 1 %). this gave rise to the formulations shown in table 5 , in which , unless stated otherwise , the percentages correspond to weight / weight ratios ( w / w ). once again an attempt was made to eliminate the cetostearyl alcohol from the formulation , this time by increasing the percentage of liquid paraffin ( formulation c , shown in table 6 ) and by adding stringy vaseline ( formulation d , shown in table 7 ). in both formulations , unless stated otherwise , the percentages correspond to weight / weight ratios ( w / w ). the result obtained with formulation c was that , after a period of maturation at room temperature ( less than 24 hours ), the emulsion broke , and so was discarded . although initially the emulsion of formulation d remained stable and very liquid , after two days at rest at room temperature it began to release water , a sign of poor stability of the emulsion , and therefore it too was discarded . a new formulation was tested starting from the aforesaid formulation 1 , in which half of the cetostearyl alcohol was replaced with aristoflex avc ®. this is an excipient indicated for the formulation of hydrophobic gels , and endows them with great fluidity . to eliminate the need to apply heat in the production process , a new formulation was tested without cetostearyl alcohol , as this is the only component in the formulation that needs to be heated , to melt it and enable it to be mixed with the rest of the formulation . this new modification means that tests have to be carried out for dissolving the chlorocresol in water . after various tests , it was verified that this product cannot be dissolved in water . after a brief review of the literature , ethanol was tested as cosolvent in the aqueous phase for dissolving the chlorocresol . once the emulsion was prepared , after a short period of maturation at room temperature the emulsion broke . this is due to the complete absence of cetostearyl alcohol . finally , we decided to make small changes to formulation 2 ′, selected as one of the most suitable on the basis of its characteristics of easy dosing and cutaneous adsorption , with the aim of replacing the chlorocresol with antimicrobials that are more suitable for topical formulations . in addition , peptide p144 , and dmso as solvent thereof , were added . these changes did not affect the behaviour of the emulsion . the definitive composition of the formulation that was considered to be the most suitable for the manufacture of a medicinal product intended for the treatment of skin fibrosis by topical administration is that shown in table 10 , in which , unless stated otherwise , the percentages correspond to weight / weight ratios ( w / w ). in order to assess the cutaneous penetration of peptide 144 , and of p144 in a semisolid preparation ( p144 emulsion ), a preliminary study of percutaneous absorption was carried out . for this purpose in vitro penetration tests using dermatomized pig ear skin were conducted ( santoyo et al ., 2002a and 2002b ). porcine skin , specifically the outer region of the ear , was chosen since its use for permeation experiments has been extensively documented ( bhatia and singh , 1998 ) and it is well suited for representing the human skin permeability ( simon and maibach , 2000 ). the substance of reference for the study was p144 , supplied by neomps sa ( strasbourg , france ; batch number hf 320440 ; storage conditions : − 20 ° c .). the test product was the semisolid preparation containing p144 ( p144 emulsion ; p144 concentration was 0 . 01 % w / w ). a single dose of 100 μg / ml of p144 emulsion was tested , with sampling in the diffuser compartment at 4 different timepoints ( 0 , 2 , 6 , 12 and 24 hours ). control a : solution of p144 in 1 ml acetic acid ( 100 % glacial acetic acid merck , batch number : k 34022763 447 ) diluted to the same concentration used for the test product ; and composition of test product and control preparations is shown on table 11 , where , unless stated otherwise , all the percentages correspond to weight / weight ratios ( w / w ). the permeation test was performed in a franz diffusion cell system franz 57 - 100 - 828 ( hanson research ). the franz cell apparatus consists of 12 borosilicate and teflon glass cells divided into two blocks ( a and b , with 6 cells each ). each cell in turn is composed of a receptor compartment ( volume 4 . 5 ml ) and a donor compartment ( diffusion surface 0 . 636 cm 2 ). the test product or its control is placed on the donor compartment , while in the receptor compartment we place the solution to collect the amount of drug crossing the experimental system ( pig skin in this case ). this experimental system is located at the interface between the donor and receptor compartments . in the donor compartment , in cells 1 , 2 and 3 of side a , 1 ml was added of p144 solution ( control a ; concentration of 100 μg / ml ). in cells 4 , 5 and 6 of side a , and in cells 1 , 2 and 3 of side b , we added 1 ml of p144 emulsion ( test product ; p144 concentration of 100 μg / ml ). finally , in cells 4 , 5 and 6 of side b we added 1 ml of the vehicle ( control b ) without peptide . in the first place a dermatomization step was conducted . the porcine ears are washed , manipulated and sectioned into adequate portions using a dermatome ( aesculap - wagner dermatome c . ga 630 ; b . braun surgical s . a ., barcelona , spain ) to obtain a membrane with a thickness of 1 mm . the portions of skin are stored separately at − 20 ° c ., using tissue tek ® oct compound ( sakura , ref . : 4583 ) as a cryoprotectant . secondly , the percutaneous diffusion test was conducted as follows . a 24 - hour diffusion study is made , testing the p144 emulsion containing 100 μg of peptide p144 / ml . this concentration has been defined as one of the suitable concentrations for use in humans . as diffusion membrane , use is made of pig ear skin dermatomized to 1 . 0 mm . prior to testing , the skin specimens are hydrated by immersion for 30 minutes in isotonic phosphate buffer solution ( pbs )( sigma , ref . p4417 - 100 tab ; ph 7 . 2 - 7 . 3 ). on the other hand , the receptor of each of the cells is filled with 4 . 5 ml of pbs adjusted to ph 7 . 4 ± 0 . 2 . on this receptor compartment we place the dermatomized and hydrated portion of skin and , over the experimental system , we position the donor compartment ( with a diffusion surface of 0 . 636 cm 2 ). in the donor compartment we place 1 ml of the test product or control preparations . the receptor is maintained at a temperature of 37 . 0 ± 0 . 5 ° c . and under stirring conditions ( 300 rpm ). at predetermined time intervals ( 0 h , 2 h , 6 h , 12 h and 24 h ) 1 ml samples are collected from the receptor compartment , this volume then being replaced by 1 ml of original solution to maintain the receptor volume constant . the collected samples are stored in identified cryotubes at − 80 ° c . for posterior chromatographic analysis . finally , for completing the study a quantification of peptide p144 in the collected samples was carried out using high performance liquid chromatography with mass spectrometry ( hplc / ms ). prior to injection of the different samples into the liquid chromatograph , they were centrifuged ( abbott table centrifuge ) at 10 , 800 rpm during 5 minutes . equipment : liquid chromatograph hewlett - packard 1100 column : zorbax sb c 18 3 . 5 μm 150 × 4 . 6 mm ( agilent ) precolumn : zorbax 300 sb - c 18 5 μm 12 . 5 × 4 . 6 mm ( agilent ) detection : ms / ms ionization : electrospray , positive ions ( m / z ): 1580 . 09 → 791 . 1 ; detector parameters : temperature 350 ° c . carrier gas flow rate 10 l / min nebulizer pressure 50 psig capillary voltage 4500 v mobile phase : acetonitrile : elution buffer ( 38 : 62 ) at a flow rate of 0 . 5 ml / min ( elution solution : aqueous solution of trifluoroacetic acid ( tfa ), at a concentration of 0 . 05 % ( w / v ) temperature of the column : 25 ± 3 ° c . injection volume : 10 μl duration of chromatogram run : 6 min . fig8 shows a chromatogram corresponding to the analysis of a sample of an aqueous solution with 0 . 1 % tfa at a concentration of 100 ng / ml . the retention time obtained for the peak of interest ( p144 ) is approximately 4 . 5 minutes . under the chromatographic conditions described in material and methods , it is possible to quantify the peptide in aqueous solutions over a concentrations range of between 0 . 1 and 1000 ng / ml of sample ( r & gt ; 0 . 996 ). fig9 shows the mass spectrum of the peptide , obtained after analysis of a sample with a concentration of 1000 ng / ml . fig1 shows the cutaneous permeability profile of p144 from the semisolid formulation ( test product ). in all cases , the amount of peptide p144 capable of crossing the pig skin membrane was below the detection limit of the analytical technique used . therefore , in all cases the concentration of p144 in the sample analyzed is undetectable and inferior to 30 pg / ml — the latter being the limit of detection ( lod ) established for the analytical method employed . likewise , p144 solution ( control a ) did not facilitate arrival of the drug in the receptor compartment . in this case , in the same way as from p144 emulsion , the amount of peptide p144 capable of crossing the biological membrane was below the detection limit of the analytical technique used . under the 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