Patent Publication Number: US-5156847-A

Title: Wound-healing composition

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to compositions useful for the healing of wounds and the like. 
     2. Description of the Background Art 
     The indigenous population of the upper Amazon basin of South America have traditionally used plants and plant products for medicinal purposes. Knowledge gained from such traditional uses of medicinal plants has provided valuable contributions to Western medicine, including the benefits of both curare and quinine. 
     One material which is utilized medicinally by the Jivaro people of north-central Peru is the sap of the tree Croton lechleri, which is used for treating external cuts, wounds, various abrasions and the like, and internally to treat, for example, ulcers. The red sap of this species has been found to contain the alkaloid, taspine. The hydrochloride salt of taspine is reported to have anti-inflammatory activity. Persinos-Purdue et al., Journal of Pharmaceutical Sciences 68: 124-126 (1979). 
     U.S. Pat. No. 3,694,557 to Persinos points out that taspine is insoluble in a great many conventional vehicles, such as water, alcohol, alkaline solution and the like, but that acid salts of taspine are partially soluble in such vehicles. The Persinos patent also describes the anti-inflammatory effects of taspine salts. 
     Vaisberg et al., Planta Medica 55: 140-143 (1988), reported that the hydrochloride salt of taspine is responsible for the wound-healing activity of Croton lechleri sap. However, the levels of significance for taspine hydrochloride as a wound healer, as reported by Vaisberg et al., are low. 
     Other materials, such as growth hormones, have been proposed for promoting the healing of wounds. However, such materials are generally prohibitively expensive, and there is no wound healing agent recognized medically that is available commercially. There thus remains a need in the art for effective and economical medicaments which can be utilized to accelerate the healing rate of wounds. 
     SUMMARY OF THE INVENTION 
     In accordance with the present invention, a composition is provided which comprises a pharmaceutical dosage unit form including a wound-healing amount of an aporphinoid alkaloid dissolved in a physiologically tolerable, non-aqueous solvent. The invention is further directed to a wound dressing made up of a sterile bandage including an aporphinoid alkaloid, and to a method of treating a wound involving application to the wound of an aporphinoid alkaloid dissolved in a physiologically tolerable, non-aqueous solvent. 
    
    
     BRIEF DESCRIPTION OF THE DRAWING 
     FIG. 1 is an elevational view, partly schematic, of a pre-packaged wound dressing with a sterile bandage including an aporphinoid alkaloid in accordance with the present invention. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     According to one aspect of the present invention, an aporphinoid alkaloid is dissolved in a non-aqueous solvent delivery system to provide a wound-healing composition. 
     Aporphinoid alkaloids contain a tetracyclic ring system formed by phenol oxidative coupling of a benzylisoquinoline precursor. Included within this group are simple aporphines such as glaucine, dehydro derivatives of such simple aporphines, oxidative derivatives of such simple aporphines, aporphines with an aromatic isoquinoline ring and a carbonyl group such as liriodenine (so-called oxoaporphines), and miscellaneous aporphines. Dictionary of Alkaloids, Southan and Buckingham (eds.), Chapman and Hall, London and New York (1989). The aporphinoid alkaloids to which the present invention is applicable include isomers, analogs and derivatives thereof. 
     Aporphinoid alkaloids having an intact hetero ring include magnofluorine, corydine, corytuberine, danguyeeline, hernovine, arosinine, glaunine, glaunidine and bulbodione. 
     Miscellaneous aporphines in which the hetero ring is opened to give phenanthrene derivatives include taspine. Other miscellaneous aporphinoid alkaloids include thalphenine and its analogs, santiagonamine, sampangine, pentauregine, imerubrine, grandirubrine and duguenaine. Still other miscellaneous aporphines have lost a carbon from the opened hetero ring, mostly containing a 5-membered lactam ring, some even in which the nitrogen of the opened hetero ring has been oxidized to a nitro group, as in aristolochic acid-A. Aporphinoid alkaloids also include miscellaneous derivatives of benzylisoquinoline, such as rufescine. 
     Compositions in accordance with particularly preferred embodiments of the present invention include taspine in its free acid form. It has surprisingly been discovered that taspine free acid has much greater wound healing activity than taspine hydrochloride, stated as being responsible for the wound healing activity of Croton lechleri sap by Vaisberg et al., supra. However, because of the insolubility of taspine free acid in water, the outstanding efficacy of taspine free acid was not discovered until tests were performed with taspine free acid dissolved in suitable non-aqueous solvent delivery systems. 
     One physiologically tolerable non-aqueous solvent that works extremely well with taspine free acid is dimethylsulfoxide (DMSO). The levels of significance of healing using taspine free acid dissolved in DMSO have been up to P=0.0001. Furthermore, no effects have been found, either irritating and hence negative, or healing and thus positive, of DMSO alone when compared to controls. 
     Other non-aqueous solvent delivery systems include N,N-dimethylformamide (DMF) and tetrahydrofuran (THF). 
     In wound-healing compositions in accordance with the present invention, the aporphinoid alkaloid is dissolved in the solvent to a concentration of from about 0.1-10 mg/ml. In preferred embodiments, the alkaloid concentration is from about 1-5 mg/ml, more preferably about 2-3 mg/ml, most preferably about 2.5 mg/ml. Additionally, the alkaloid can be present in a form of a cream, salve or ointment at the above-noted concentrations. 
     In a method of treating a wound in accordance with the present invention, a pharmaceutical composition is applied to the wound, the composition containing a wound-healing amount of an aporphinoid alkaloid dissolved in a physiologically tolerable, non-aqueous solvent, as described above, which composition can be in the form of a cream, salve or ointment. In preferred embodiments, the pharmaceutical composition is applied to the wound so as to provide the wound with from about 1 μg/cm 2  to about 10 mg/cmz of the aporphinoid alkaloid. In more preferred embodiments, the composition is applied to the wound so as to provide the wound with from about 50-500 μg/cm 2 , even more preferably about 200-300 μg/cm 2 , most preferably about 250 μg/cm 2 . 
     Advantageously, the inventive composition is applied to the wound site after cleansing thereof, and as soon as possible after the wounding. The inventive composition can be applied as a single dose, or three or more times a day over a period of time during healing. In preferred embodiments, the inventive composition is applied at least once a day for up to 10 or more days after wounding. 
     The present invention is also applicable to a wound dressing, and preparation thereof. In accordance with this aspect of the invention, a wound dressing is provided which includes a sterile bandage 10 onto which an aporphinoid alkaloid 12 has been deposited or applied (See FIG. 1). The aporphinoid alkaloid 12 can be applied by contacting a bandage material with a solution containing the aporphinoid alkaloid and thereafter evaporating the solution from the bandage, resulting in a dried bandage containing the alkaloid. As shown in FIG. 1, the alkaloid-containing bandage can be provided sealed within a package 14. 
     The invention is illustrated by the following examples, which are not intended to be limiting. 
     EXAMPLE I 
     Isolation of Taspine 
     The viscous sap of Croton lechleri was mixed with four times its volume of acidic water (3.7% HCL, pH 3-4) and gently swirled in a separatory funnel with an equal amount of ethyl acetate. While rinsing the funnel with acidified water, the acidic sap mixture was drained off and the ethyl acetate discarded. This procedure was repeated three times. 
     To the saved acidic sap mixture, 5% NaOH solution was pipeted to achieve a pH of 9-11, and the mixture was returned to a separatory funnel with about an equal amount of ethyl acetate, rinsed and drained. This extraction was repeated three times. The basic sap mixture was discarded and the ethyl acetate solution retained. The ethyl acetate collections were combined and an equal amount of anhydrous NaSO 4  was added to absorb water soluble material. The ethyl acetate collection was drawn off, and the ethyl acetate was removed by rotary evaporation, leaving a whitish precipitate. 
     Taspine was detected using thin layer chromatography (TLC) (silica gel with fluorescent indicator UV 254 ) ascending in the solution chloroform/methanol 3% NH 4  OH in the ratio 25 ml/5 ml/0.25 ml, and observed on the TLC plates by its inhibition of the short wavelength induced influorescence of the fluophore present in the plates and by its reaction to Dragendorf&#39;s reagent (orange spot). 
     Once the presence of taspine was verified, the taspine mixture was dissolved in chloroform, the solute placed in a silica gel flash chromatography column, and taspine isolated using a series of gradient solutions as follows: chloroform, 10% methanol/90% chloroform, 20% methanol/80% chloroform, 20% methanol/0.01% ammonium hydroxide/79.99% chloroform, 40% methanol methanol/0.01% ammonium hydroxide/59.99% chloroform, and 99.99% methanol/0.01% ammonium hydroxide. Each solution was poured through the column and collected in separately labeled flasks. Taspine crystallized in highest concentrations in the 20% and 40% methanol solutions as verified by TLC tests, and NMR spectral data. 
     EXAMPLE II 
     Wound Healing with Tasgine Free Acid in DMSO 
     Taspine free acid dissolved in DMSO to a concentration of 2.5 mg/ml was tested on rat wounds against control (DMSO alone), which in turn was tested against untreated rat wounds. 
     Tensile strength of the wounds 5, 7 and 12 days after administration are shown in Tables 1, 2 and 3 respectively for taspine free acid in DMSO (alkaloid +solvent) as compared to DMSO alone (solvent), and in Table 4 below for DMSO alone (solvent) as compared to no treatment. 
     
                       TABLE 1                                                     
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Tensile strength studies 5 days after administration.                     
              Dose                                                        
              Alkaloid +                                                  
                        Solvent-                                          
              Solvent   Control                                           
Rat No.       (newton)  (newton)                                          
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1             3.738     1.958                                             
2             1.907     1.872                                             
3             2.967     1.747                                             
4             2.778     1.574                                             
5             2.480     2.340                                             
7             2.242     2.198                                             
8             2.400     1.235                                             
9             2.662     1.170                                             
10            1.873     1.656                                             
______________________________________                                    
 Alkaloid mean (X) = 2.522 ± 0.176 SEM                                 
 Solvent mean (Y) = 1.755 ± 0.118 SEM                                  
 Paired T test = 0.005                                                    
 Dose: 0.25 mg alkaloid dissolved in 0.1 ml solvent                       
 Control: 0.1 ml solvent                                                  
 
    
     
                       TABLE 2                                                     
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Tensile strength studies 7 days after administration.                     
              Dose                                                        
              Alkaloid +                                                  
                        Solvent-                                          
              Solvent   Control                                           
Rat No.       (newton)  (newton)                                          
______________________________________                                    
1             3.149     3.059                                             
2             5.016     3.171                                             
3             4.014     3.237                                             
4             3.865     2.493                                             
5             3.837     2.469                                             
6             3.526     2.911                                             
7             3.192     2.747                                             
8             3.306     2.747                                             
9             3.836     2.714                                             
10            3.878     2.796                                             
______________________________________                                    
 Alkaloid mean (X) = 3.760 ± 0.17 SEM                                  
 Solvent mean (Y) = 2.810 ± 0.09 SEM                                   
 Paired T test = 0.0001                                                   
 Dose: 0.25 mg alkaloid dissolved in 0.1 ml solvent                       
 Control: 0.1 ml solvent                                                  
 
    
     
                       TABLE 3                                                     
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Tensile strength studies 12 days after administration.                    
              Dose                                                        
              Alkaloid +                                                  
                        Solvent-                                          
              Solvent   Control                                           
Rat No.       (newton)  (newton)                                          
______________________________________                                    
1             5.325     7.778                                             
2             7.314     5.841                                             
3             5.516     7.638                                             
4             8.259     6.412                                             
5             7.018     7.094                                             
6             5.597     4.982                                             
7             8.440     9.341                                             
8             7.993     7.056                                             
9             7.050     9.038                                             
10            9.059     8.375                                             
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 Alkaloid mean (X) = 7.167 ± 0.42 SEM                                  
 Solvent mean (Y) = 7.355 ± 0.44 SEM                                   
 Paired T test = 0.72                                                     
 Dose: 0.25 mg alkaloid dissolved in 0.1 ml solvent                       
 Control: 0.1 ml solvent                                                  
 
    
     
                       TABLE 4                                                     
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Tensile strength studies 7 days after administration.                     
              Dose     No Treatment-                                      
              Solvent  Control                                            
Rat No.       (newton) (newton)                                           
______________________________________                                    
1             3.388    3.142                                              
2             4.221    5.035                                              
3             2.909    3.466                                              
4             3.937    3.695                                              
5             3.706    4.225                                              
6             2.451    2.045                                              
7             3.286    2.750                                              
8             2.972    2.886                                              
9             3.230    2.077                                              
10            3.237    2.798                                              
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 Solvent mean (X) = 3.333                                                 
 Nothing mean (Y) = 3.212                                                 
 Paired T test =not significant                                           
 Dose: solvent 0.1 ml                                                     
 Control: nothing                                                         
 
    
     The above results indicate that taspine free acid in DMSO heals wounds significantly faster than DMSO alone or no treatment. 
     EXAMPLE III 
     Example II was repeated in all essential details, but utilizing an aqueous solution of taspine hydrochloride salt instead of taspine free acid in DMSO. The results of these experiments indicated that there was no significant difference between taspine hydrochloride salt solution and controls in wound healing rate.