Bear derived isolate and method

Abstract

A bear derived isolate found in denning black bears or black bears from which food has been withheld for at least two weeks when injected into another mammal produces reduced heart rate, reduced respiratory rate, reduced temperature, and a wakeful tranquility not unlike the phenomena of the denning black bear. The isolate is taken from either the serum or urine of such denning bear. The method of the invention involves the steps of fractionating the serum or urine of a denning black bear to produce fractions of varying potency.

1 .- 47 . (canceled)  
   
   
       48 . A composition of matter having the following characteristics: 
 obtained from deproteinating the urine or blood of a fasting black bear which has not eaten for two weeks or more.    soluble in water, methanol, and 1-butanol,    insoluble in less polar organic solvents including ethyl acetate, chloroform, toluene, and hexane,    stable at room temperature for four days or more,    heat resistant to 65° C., and    stable when frozen in a light resistant container under nitrogen gas, and    wherein said composition, when injected in a guinea pig, produces observable conditions of reduced heart rate, reduced temperature, or wakeful tranquility.    
   
   
       49 . A composition of matter comprising the deproteinated urine or serum of a denning black bear, which denning black bear neither eats, drinks, urinates, or defecates for lengthy periods of time having the following properties: 
 soluble in water, methanol, and 1-butanol,    insoluble in less polar organic solvents including ethyl acetate, chloroform, toluene, and hexane,    stable at room temperature for four days or more,    heat resistant to 65° C., and    stable when frozen in a light resistant container under nitrogen gas which, when injected into a guinea pig, is capable of producing reduced heart rate, reduced temperature, or observable tranquility differing from normal.    
   
   
       50 . The composition of matter of  claim 49  which, when subjected to in vitro analysis, produces the following: 
 increased osteoblastic activity, or    decreased osteoclastic activity, or    increased fibroblastic activity.    
   
   
       51 . The composition of matter of  claim 49  which, when subjected to in vivo analysis with ovariectomized rats, produces the following: 
 increased osteoblastic activity,    decreased osteoclastic activity, or    both.    
   
   
       52 . (canceled)  
   
   
       53 . A pharmaceutical composition for stimulating osteoblastic activity as shown by alkaline phosphatase production, said composition comprising an active agent obtained by the steps comprising: 
 (a) obtaining the serum or urine of a fasting bear;    (b) deproteinating said serum or urine;    (c) drying said deproteinated serum or urine;    (d) separating the product of step (c) into fractions by chromatography,    (e) drying the fractions obtained in step (d);    (f) testing the fractions for alkaline phosphatase stimulating activity in an in vitro bone culture.    
   
   
       54 . A pharmaceutical composition for stimulating osteoblastic activity as shown by alkaline phosphatase production, said composition comprising an active agent obtained by the steps comprising: 
 (a) obtaining the serum or urine of a denning bear;    (b) deproteinating said serum or urine;    (c) drying said deproteinated serum or urine;    (d) separating the product of step (c) into fractions by means of countercurrent chromatography using a 1-butanol:water:acetic acid (20:20:1) mixture, wherein the organic phase of said mixture is used as a stationary phase arid the aqueous phase of said mixture is used as a mobile phase, wherein the product is eluted in 100 ml fractions and the first 100 ml eluted is Fraction I and each successive 100 ml to be eluted is a subsequent Fraction and continuing step (d) up to the collection of Fraction VI.    
   
   
       55 . A pharmaceutical composition as in  claim 54 , wherein the aqueous phase of a 1-butanol: water:acetic acid (20:20:1) mixture as a mobile phase is passed through the product of step (c) at a rate of 4 ml/minute for 25 minutes of each of Fractions I through VI.  
   
   
       56 . A pharmaceutical composition as in  claim 54 , wherein said composition is obtained by the further steps comprising: 
 (e) after collection of fraction VI, collecting Fractions VII and VIII by passing the aqueous phase of said 1-butanol:water:acetic acid (20:20:1) mixture as a mobile phase through the product of step (c) remaining after step (d) at a rate of 10 ml/minute for 10 minutes for each of Fractions VII and VIII.    
   
   
       57 . A pharmaceutical composition as in  claim 56 , wherein said composition is obtained by the further steps comprising: 
 (f) after collection of Fractions VII and VIII, collecting Fraction IX by replacing the 1-butanol:water:acetic acid (20:20:1) mixture with methanol:water (1:1) and passing the mobile phase through the product of step (c) remaining after step (e) at a rate of 10 ml/minute for 10 minutes for collection of Fraction IX.    
   
   
       58 . A pharmaceutical composition as in  claim 57 , wherein said composition is obtained by the further steps comprising: 
 (g) after collection of Fraction IX, collecting Fraction X, by replacing the methanol:water (1:1) mixture with methanol and passing the mobile phase through the product of step (c) remaining after step (I) at a rate of 10 ml/minute for 10 minutes followed by forced air for collection of Fraction X.    
   
   
       59 . (canceled)  
   
   
       60 . (canceled)  
   
   
       61 . A pharmaceutical composition containing for inhibiting osteoblastic activity as shown by inhibition of alkaline phosphatase production, said active agent obtained by the steps comprising: 
 (a) obtaining the serum or urine of a denning bear;    (b) deproteinating said serum or urine;    (c) drying said deproteinated serum or urine;    (d) separating the product of step (c) into fractions by means of countercurrent chromatography using a 1-butanol:water:acetic acid (20:26:1) mixture, wherein the organic phase of said mixture is used as a stationary phase and the aqueous phase of said mixture is used as a mobile phase, wherein the product is eluted in 100 ml fractions and the first 100 ml to be eluted is Fraction I and each successive 100 ml to be eluted is a subsequent Fraction, and continuing step (d) up to the collection of Fraction III;    (e) drying the said fractions obtained in step (d); and    (f) testing the fractions for osteoblastic inhibition in an in vitro culture.    
   
   
       62 . (canceled)  
   
   
       63 . A pharmacological composition of matter comprising the capability of enhancing bone formation in ovariectomized rats taken from a substance present in the blood or urine of fasting bears, which when fasting are unique in that they have not eaten for two weeks or more, said composition including a quantity of resorptive form of 24,25-dihydroxyvitamin D 3  which stimulates bone formation.  
   
   
       64 . A pharmacological composition of matter taken from the blood or urine of fasting beam, which bear had been fasted for two weeks or more, said composition having a molecular weight of 100 or less, which composition when injected into a mammal other than a bear, which mammal has been ovariectomized, produces by comparison to an ovariectomized mammal not treated with said composition of matter, enhanced bone growth.  
   
   
       65 . In the pharmacological composition of matter of  claim 64 , said composition being characterized by an operative and effective quantity of 24,25-dihydroxyvitamin D 3 .  
   
   
       66 . (canceled)