Abstract:
Use of a glycoprotein produced by  Pseudoalteromonas antartica  and consisting of 14% carbohydrates and 86% protein in the preparation of pharmaceutical, veterinary and cosmetic compositions for topical or mucous application in the treatment and re-epithelialization of wounds and in the preparation of cosmetic compositions for treatment of sin, hair or nails.

Description:
OBJECT OF THE INVENTION  
         [0001]    The present invention relates to the use of a glycoprotein known as Antarticine-NF3 for preparing pharmaceutical, veterinarian and cosmetic compositions for treatment and re-epithelialization of wounds.  
         STATE OF THE ART  
         [0002]    Antarticine-NF3 is a glycoprotein produced by  Pseudoalteromonas antartica , a Gram-negative bacteria isolated from the Antarctic Ocean. This aerobic heterotropic bacterium has a single polar flagellum and produces a hexapolymeric glycoprotein (Antarticine-NF3) composed of 14% carbohydrate and 86% protein that completely surround it.  Pseudoalteromonas antartica  is perfectly adapted to the low temperatures of the Antarctic environment and withstands high salt concentrations.  
           [0003]    Antarticine-NF3 in solution forms regular aggregates by self-assembly. A gradual increase in the concentration of a solution of this glycoprotein causes a gradual reduction of the surface tension of the solution. In addition, Antarticine-NF3 is known for its great affinity to phosphatidylcholine liposome, coating them and protecting them from the permeabilisation effect of surfactants such as SDS, as described in WO98/42731. In addition, Antarticine-NF3 is known for inducing nucleation of ice and preventing the growth of large hexagonal ice crystals. These data suggest that Antarticine-NF3 is responsible for the adaptation of  Pseudoalteromonas antartica  to the environmental conditions of its Antarctic habitat.  
           [0004]    Furthermore, keratinocytes are a type of cell found in the epidermis, forming its deepest layer. Keratinocytes generate the outer skin layer (corneal stratum) by multiplication and modification of their structure until forming the layer of mature, keratinocytised cells on the surface of the skin. The speed of healing of wounds is influenced, among other factors, by the rate of proliferation and migration of keratinocytes. Any factor that enhances one of these processes will favour the healing and scarring process for wounds. 
       
    
    
     DESCRIPTION OF THE INVENTION  
       [0005]    As a result of our investigations we have found that, surprisingly, the soluble glycoprotein from  Pseudoalteromonas antartica  known as Antarticine-NF3, which consists of 14% carbohydrates and 86% protein, selectively improves the adhesion of human dermal fibroblasts and promotes the cellular growth of human epidermal keratinocytes. Thus, Antarticine-NF3 has surprisingly revealed itself as a good agent for improving scar formation in wounds.  
         [0006]    Thus, the object of the present invention is the use of Antarticine-NF3 in solutions or acceptable galenical formulations for use in the treatment and re-epithelialization of wounds in the pharmaceutical and veterinary fields, as well as for cosmetic regeneration treatments. The concentrations used of Antarticine-NF3 are between 100 mg/l and 1 fg/ml, and more specifically between 10 mg/ml and 0.1 ng/ml.  
         [0007]    As a complement of the description being made and in order to aid a better understanding of the characteristics of the invention, a few examples are provided below for purposes of illustration only and in a non-limiting sense. This invention should not be considered as limited by the realizations described.  
       EXAMPLE 1  
     In Vitro Cytotoxicity  
       [0008]    Experiments were carried out with the WST-1 test, which measures the activity of mitochondrial dehydrogenases in a non-radioactive calorimeter test using formazan salts. Different concentrations of glycoprotein were tested (between 1 mg/ml and 0.01 μg/ml). NaCl 0.01M was used as a negative control and SDS 0.02% was used as a positive control.  
         [0009]    The results obtained are shown in the following table:  
                                                                                                             Concentration   Result               tested   on HDF   Result on HEK                                Control           0.46   0.92       Negative control (NaCl)   0.01   M   0.43   0.7            Positive control (SDS)   0.02%   0.17   0.2                1   fg   —   0.78           0.1   pg   —   0.92           0.0   ng1   0.44   0.92           1   ng   0.43   1.02           0.1   μg   0.41   0.97           10   μg   0.42   0.8           1   mg   0.44   0.81                  
 
         [0010]    As can be seen in the above table, the cytotoxicity tests revealed that Antarticine-NF3 does not show toxicity in human epidermal keratinocytes (HEK), nor in human dermal fibroblasts (HDF).  
       EXAMPLE 2  
     Effect on Cellular Adhesion  
       [0011]    Cellular adhesion tests in vitro revealed that Antarticine-NF3 has a specific effect on the adhesion of HDF. HDF cells showed a significant growth in cellular adhesion when growing on substrates coated with 1 mg/ml of Antarticine-NF3 after 2 and 5 hours of culture.  
         [0012]    The results obtained for HDF are shown in the following table:  
                                                                                     Concentration                       tested   Time 2 hours   Time 5 hours                                        Control       0.1   0.13           Glycoprotein   0.001 mg/ml   0.11   0.24                  1 mg/ml   0.24   0.28                      
 
         [0013]    The results obtained for HEK are shown in the following table:  
                                                               Concentration                       tested   Time 2 hours   Time 5 hours                           Control       0.09   0.15           Glycoprotein   0.001 mg/ml   0.11   0.17                  1 mg/ml   0.15   0.19                      
 
         [0014]    As may be seen, in similar culture conditions the effect seen on HEK is not statistically significant.  
       EXAMPLE 3  
     Effect on Cell Growth  
       [0015]    Experiments on in vitro cell growth conducted with the crystal violet elution method revealed that Antarticine-NF3 at different concentrations, in the presence or absence of a minimal amount of foetal cow serum (2% FCS) stimulates the growth of HEK cells, as shown in the following table:  
                                                   Concentration   Time 0   Time 24   Time 48           tested   hours   hours   hours                   Control       0.2   0.16   0.58       Glycoprotein     10 μg/mL       0.15   0.78            0.1 μg/mL       0.16   0.77           0.001 μg/mL       0.18   0.77                  
 
         [0016]    While similar experiments conducted with HDF cells did not show relevant results, as shown on the following table:  
                                                                         Concentration   Time 0   Time 24   Time 48           tested   hours   hours   hours                                Control       0.21   0.16   0.25       Glycoprotein     10 μg/mL       0.175   0.26            0.1 μg/mL       0.17   0.248           0.001 μg/mL       0.175   0.249                  
 
       EXAMPLE 4  
     Effect on In Vitro Wound Regeneration  
       [0017]    Experiments for regeneration of wounds in vitro were conducted in cultures of differentiated HEK cells, in the presence and absence of FCS. The duration of the tests was seven days.  
         [0018]    Concentrations of Antarticine-NF3 above 0.1 ng/ml showed a positive effect on wound regeneration when compared to control cultures; this effect of Antarticine-NF3 on wound regeneration in vitro can be seen in FIG. 1 for a concentration of 0.1 μg/mL.