Crystalline insulin-conjugates

Abstract

The present disclosure provides crystalline insulin-conjugates. The present disclosure also provides formulations, methods of treatment, methods of administering, and methods of making that encompass these crystalline insulin-conjugates.

1 - 154 . (canceled) 
     
     
         155 . A method of making a crystalline insulin-conjugate of formula (I): 
       
         
           
           
               
               
           
         
         wherein: 
         each occurrence of 
       
       
         
           
           
               
               
           
         
       
       represents a potential branch within the conjugate;
 each occurrence of 
 
       
         
           
           
               
               
           
         
       
       represents a potential repeat within a branch of the conjugate:
 each occurrence of   is independently a covalent bond, a carbon atom, a heteroatom, or an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, and heterocyclic; 
 each occurrence of T is independently a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C 1-30  hydrocarbon chain wherein one or more methylene units of T are optionally and independently replaced by —O—, —S—, —N(R)—, —C(O)—, —C(O)O—, —OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O) 2 —, —N(R)SO 2 —, —SO 2 N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; 
 each occurrence of R is independently hydrogen, a suitable protecting group, or an acyl moiety, arylalkyl moiety, aliphatic moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic moiety; 
 —B is -T-L B -X; 
 each occurrence of X is independently a ligand that includes a saccharide; 
 each occurrence of L B  is independently a covalent bond or a group derived from the covalent conjugation of a T with an X; 
 -D is -T-L D -W I ; 
 W I  is an insulin molecule: 
 each occurrence of L D  is independently a covalent bond or a group derived from the covalent conjugation of a T with a W I ; 
 k is an integer from 1 to 12, inclusive: 
 each occurrence of p is independently an integer from 1 to 5, inclusive; and 
 each occurrence of n is independently an integer from 0 to 5, inclusive; and 
 each occurrence of m is independently an integer from 1 to 5, inclusive; and 
 each occurrence of v is independently an integer from 0 to 5, inclusive, with the proviso that within each k-branch at least one occurrence of n is ≧1 and at least one occurrence of v is ≧1 comprising crystallizing the insulin-conjugate from a phosphate buffer. 
 
     
     
         156 . The method of  claim 155 , wherein the buffer has a higher concentration of potassium than sodium. 
     
     
         157 . The method of  claim 155 , wherein the buffer has a higher concentration of potassium phosphate monobasic than sodium phosphate dibasic. 
     
     
         158 . The method of  claim 155 , wherein the buffer has a pH in the range of about 6 to 9. 
     
     
         159 . The method of  claim 155 , wherein the buffer has a pH of about 8. 
     
     
         160 . The method of  claim 155 , wherein the buffer has a pH of 8.2. 
     
     
         161 . The method of  claim 155 , wherein the crystalline insulin-conjugate is conjugated at Lys B29 . 
     
     
         162 . (canceled) 
     
     
         163 . The method of  claim 155 , wherein one or more additives is added, wherein the additive is selected from the group consisting of cresol, phenol, protamine, poly(arginine), glycerol, zinc chloride, or zinc acetate. 
     
     
         164 . The method of  claim 155 , wherein polyethylene glycol is added. 
     
     
         165 . The method of  claim 155 , wherein an organic solvent is added to facilitate crystallization. 
     
     
         166 . The method of  claim 155 , wherein the organic solvent is methanol, ethanol, or isopropanol. 
     
     
         167 . A method of making a crystalline insulin-conjugate of formula (I): 
       
         
           
           
               
               
           
         
         wherein: 
         each occurrence of 
       
       
         
           
           
               
               
           
         
       
       represents a potential branch within the conjugate:
 each occurrence of 
 
       
         
           
           
               
               
           
         
       
       represents a potential repeat within a branch of the conjugate;
 each occurrence of   is independently a covalent bond, a carbon atom, a heteroatom, or an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, and heterocyclic; 
 each occurrence of T is independently a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C 1-30  hydrocarbon chain wherein one or more methylene units of T are optionally and independently replaced by —O—, —S—, —N(R)—, —C(O)—, —C(O)O—, —OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O) 2 —, —N(R)SO 2 —, —SO 2 N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; 
 each occurrence of R is independently hydrogen, a suitable protecting group, or an acyl moiety, arylalkyl moiety, aliphatic moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic moiety; 
 —B is -T-L B -X; 
 each occurrence of X is independently a ligand that includes a saccharide; 
 each occurrence of L B  is independently a covalent bond or a group derived from the covalent conjugation of a T with an X; 
 -D is -T-L D -W I ; 
 W I  is an insulin molecule: 
 each occurrence of L D  is independently a covalent bond or a group derived from the covalent conjugation of a T with a W I ; 
 k is an integer from 1 to 12, inclusive; 
 each occurrence of p is independently an integer from 1 to 5, inclusive; and 
 each occurrence of n is independently an integer from 0 to 5, inclusive; and 
 each occurrence of m is independently an integer from 1 to 5, inclusive; and 
 each occurrence of v is independently an integer from 0 to 5, inclusive, with the proviso that within each k-branch at least one occurrence of n is ≧1 and at least one occurrence of v is ≧1 comprising crystallizing the insulin-conjugate from a HEPES buffer or a TRIS buffer. 
 
     
     
         168 . (canceled) 
     
     
         169 . The method of  claim 155 , wherein the insulin molecule conjugate comprises a saccharide selected from the group consisting of mannose, bimannose, and branched trimannose conjugated to the Lys B29  residue of the insulin molecule. 
     
     
         170 . The method of  claim 155 , wherein the insulin molecule conjugate comprises a saccharide selected from the group consisting of aminoethylmannose (AEM), aminoethylbimannose (ABM), and aminoethyltrimannose (AETM) conjugated to the Lys B29  residue of the insulin molecule. 
     
     
         171 . The method of  claim 167 , wherein the insulin molecule conjugate comprises a saccharide selected from the group consisting of mannose, bimannose, and branched trimannose conjugated to the Lys B29  residue of the insulin molecule. 
     
     
         172 . The method of  claim 167 , wherein the insulin molecule conjugate comprises a saccharide selected from the group consisting of aminoethylmannose (AEM), aminoethylbimannose (ABM), and aminoethyltrimannose (AETM) conjugated to the Lys B29  residue of the insulin molecule. 
     
     
         173 . The method of  claim 155 , wherein the insulin molecule is selected from the group consisting of insulin lispro, insulin aspart, insulin glulisine, insulin glargine, and insulin detemir. 
     
     
         174 . The method of  claim 167 , wherein the insulin molecule is selected from the group consisting of insulin lispro, insulin aspart, insulin glulisine, insulin glargine, and insulin detemir.