Insulin compositions and method of making a composition

Abstract

The invention is related to insulin compositions with a high content of zinc atoms per six molecules of acylated insulin. The insulin is an acylated insulin and may be mixed with a further insulin analogue such as the rapid acting insulin Asp B28 human insulin.

1 . A soluble pharmaceutical composition comprising an acylated insulin and further comprising more than 4 zinc atoms per 6 molecules of acylated insulin. 
   
   
       2 . The pharmaceutical composition according to  claim 1  comprising up to about 12 zinc atoms per 6 molecules of acylated insulin. 
   
   
       3 . The pharmaceutical composition according to  claim 1  comprising between about 4.3 and about 12 zinc atoms per 6 molecules of acylated insulin. 
   
   
       4 . The pharmaceutical composition according to  claim 1  comprising between about 4.5 and about 12 zinc atoms per 6 molecules of acylated insulin. 
   
   
       5 . The pharmaceutical composition according to  claim 1 , wherein at least 85% of the acylated insulin is present as complexes which are acylated insulin dodecamers or complexes with a higher molecular weight than acylated insulin dodecamer. 
   
   
       6 . The pharmaceutical composition according to  claim 1 , wherein the composition comprises a surfactant. 
   
   
       7 . The pharmaceutical composition according to  claim 1 , wherein the insulin molecule has a side chain attached either to the α-amino group of the N-terminal amino acid residue of the B chain or to an Eε-amino group of a Lys residue present in the B chain of the parent insulin moiety via an amide bond, which side chain comprises at least one free carboxylic acid group or a group which is negatively charged at neutral pH, a fatty acid moiety with about 4 to about 32 carbon atoms in the carbon chain; and possible one or more linkers linking the individual components in the side chain together via amide bonds. 
   
   
       8 . The pharmaceutical composition according to  claim 1 , wherein the side chain comprises at least one aromatic group. 
   
   
       9 . The pharmaceutical composition according to  claim 1 , wherein the side chain comprises at least one difunctional PEG group. 
   
   
       10 . The pharmaceutical composition according to  claim 1 , wherein the insulin molecule has a side chain attached to the Eε-amino group of a Lys residue present in the B chain of the parent insulin, the side chain being of the general formula:
   —W—X—Y-Z 2      wherein W is:
 an α-amino acid residue having a carboxylic acid group in the side chain which residue forms, with one of its carboxylic acid groups, an amide group together with ε-amino group of a Lys residue present in the B chain of the parent insulin; 
 a chain composed of two, three or four α-amino acid residues linked together via amide carbonyl bonds, which chain—via an amide bond—is linked to an ε-amino group of a Lys residue present in the B chain of the parent insulin, the amino acid residues of W being selected from the group of amino acid residues having a neutral side chain and amino acid residues having a carboxylic acid group in the side chain so that W has at least one amino acid residue which has a carboxylic acid group in the side chain; or 
 a covalent bond from X to an ε-amino group of a Lys residue present in the B chain of the parent insulin; 
   X is:
 — C O—; 
 —CH(COOH) C O—; 
 —CO—N(CH 2 COOH)CH 2   C O—; 
 —CO—N(CH 2 COOH)CH 2 CON(CH 2 COOH)CH 2   C O—; 
 —CO—N(CH 2 CH 2 COOH)CH 2 CH 2   C O—; 
 —CO—N(CH 2 CH 2 COOH)CH 2 CH 2 CON(CH 2 CH 2 COOH)CH 2 CH 2   C O—; 
 —CO—NHCH(COOH)(CH 2 ) 4 NH C O—; 
 —CO—N(CH 2 CH 2 COOH)CH 2   C O—; or 
 —CO—N(CH 2 COOH)CH 2 CH 2   C O—. 
   that   a) when W is an amino acid residue or a chain of amino acid residues, via a bond from the underscored carbon forms an amide bond with an amino group in W, or   b) when W is a covalent bond, via a bond from the underscored carbonyl carbon forms an amide bond with an ε-amino group of a Lys residue present in the B chain of the parent insulin;   Y is:
 —(CH 2 ) m — where m is an integer in the range of 6 to 32; 
 a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH 2 — groups sufficient to give a total number of carbon atoms in the chain in the range of 10 to 32; and 
   Z 2  is:
 —COOH; 
 —CO-Asp; 
 —CO-Glu; 
 —CO-Gly; 
 —CO-Sar; 
 —CH(COOH) 2 ; 
 —N(CH 2 COOH) 2 ; 
 —SO 3 H; or 
 —PO 3 H 
   
     and any Zn 2+  complexes thereof, provided that when W is a covalent bond and X is —CO—, then Z is different from —COOH. 
   
   
       11 . The pharmaceutical composition according to  claim 1 , wherein the acylated insulin is having a formula 
     
       
         
         
             
             
         
       
     
     wherein Ins is the parent insulin moiety which via the α-amino group of the N-terminal amino acid residue of the B chain or an ε-amino group of a Lys residue present in the B chain of the insulin moiety is bound to the CO— group in the side chain via an amide bond;
 X 4  is
 —(CH 2 ) n  where n is 1, 2, 3, 4, 5 or 6; 
 NR, where R is hydrogen or —(CH 2 ) p —COOH; —(CH 2 ) p —SO 3 H; —(CH 2 ) p —PO 3 H 2 ; —(CH 2 ) p —O—SO 3 H 2 ; —(CH 2 ) p —O—PO 3 H 2 ; arylene substituted with 1 or 2 —(CH 2 ) p —O—COOH groups; —(CH 2 ) p -tetrazolyl, where p is an integer in the range of 1 to 6; 
 —(CR 1 R 2 ) q —NR—CO—, where R 1  and R 2  independently of each other and independently for each value of q can be H, —COOH, or OH, q is 1-6 and R is defined as above; 
 —((CR 3 R 4 ) q1 —NR—CO) 2-4 —, where R 3  and R 4  independently of each other and independently for each value of q 1  can be H, —COOH, or OH, q 1  is 1-6 and R is defined as above; or 
 a bond 
 
 W 1  is arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of —COOH, —SO 3 H, and —PO 3 H 2  and tetrazolyl, or W 1  is a bond; 
 m is 0, 1, 2, 3, 4, 5 or 6; 
 X 5  is 
 
     
       
         
         
             
             
         
       
       where R is defined as above; or
 a bond; 
 
       Y 1  is
 —(CR 1 R 2 ) q —NR—CO—, where R 1  and R 2  independently of each other and independently for each value of q can be H, —COOH, a bond or OH, q is 1-6; and R is defined as above; 
 NR where R is defined as above; 
 —((CR 3 R 4 ) q1 —NR—CO) 2-4 —, where R 3  and R 4  independently of each other and independently for each value of q 1  can be H, —COOH, or OH, q 1  is 1-6 and R is defined as above; or 
 a bond; 
 
       Q 7  is
 —(CH 2 ) r — where r is an integer from 4 to 22; 
 a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH 2 — groups sufficient to give a total number of carbon atoms in the chain in the range of 4 to 22; or 
 a divalent hydrocarbon chain of the formula
   —(CH 2 ) s -Q 8 -(C 6 H 4 ) v1 -Q 9 -(CH 2 ) W -Q 10 -(C 6 H 4 ) v2 -Q 11 -(CH 2 ) t -Q 12 -(C 6 H 4 ) v3 -Q 13 -(CH 2 ) z — 
 
 
     
     wherein Q 8 -Q 13  independently of each other can be O; S or a bond; where s, w, t and z independently of each other are zero or an integer from 1 to 10 so that the sum of s, w, t and z is in the range from 4 to 22, and v 1 , v 2 , and v 3  independently of each other can be zero or  1 , provided that when W 1  is a bond then Q 7  is not a divalent hydrocarbon chain of the formula —(CH 2 ) v4 C 6 H 4 (CH 2 ) W1 — wherein v 4  and w 1  are integers or one of them is zero so that the sum of v 4  and w 1  is in the range of 6 to 22; and
 Z 1  is: 
 —COOH; 
 —CO-Asp; 
 —CO-Glu; 
 —CO-Gly; 
 —CO-Sar; 
 —CH(COOH) 2 ; 
 —N(CH 2 COOH) 2 ; 
 —SO 3 H 
 —PO 3 H 2 ; 
 —O—SO 3 H; 
 —O—PO 3 H 2 ; 
 -tetrazolyl or
 —O—W 2 , 
 where W 2  is arylene or heteroarylene substituted with one or two groups selected from —COOH, —SO 3 H, and —PO 3 H 2  and tetrazolyl; 
 provided that if W 1  is a bond and v 1 , v 2  and v 3  are all zero and Q 8 -13 are all a bonds, then Z 1  is O—W 2    
 
 
     and any Zn 2+  complex thereof. 
   
   
       12 . The pharmaceutical composition to  claim 1 , wherein the acylated insulin is having a formula 
     
       
         
         
             
             
         
       
     
     wherein Ins is the parent insulin moiety which via the α-amino group of the N-terminal amino acid residue of the B chain or an ε-amino group of a Lys residue present in the B chain of the insulin moiety is bound to the CO— group in the side chain via an amide bond;
 each n is independently 0, 1, 2, 3, 4, 5 or 6; 
 Q 1 , Q 2 , Q 3 , and Q 4  independently of each other can be
 (CH 2 CH 2 O) s —; (CH 2 CH 2 CH 2 O) s —; (CH 2 CH 2 CH 2 CH 2 O) s —; (CH 2 CH 2 OCH 2 CH 2 CH 2 CH 2 O) s — or (CH 2 CH 2 CH 2 OCH 2 CH 2 CH 2 CH 2 O) s — where s is 1-20 
 —(CH 2 ) r — where r is an integer from 4 to 22; or a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH 2 — groups sufficient to give a total number of carbon atoms in the chain in the range of 4 to 22; 
 —(CH 2 ) t — or —(CH 2 OCH 2 ) t —, where t is an integer from 1 to 6; 
 —(CR 1 R 2 ) q —, where R 1  and R 2  independently of each other can be H, —COOH, (CH 2 ) 1-6 COOH and R 1  and R 2  can be different at each carbon, and q is 1-6, 
 —((CR 3 R 4 ) q1 ) 1 —(NHCO—(CR 3 R 4 ) q1 —NHCO) 1-2 —((CR 3 R 4 ) q1 ) 1  or ((CR 3 R 4 ) q1 ) 1 —(CONH—(CR 3 R 4 ) q1 —CONH) 1-2 —((CR 3 R 4 ) q1 —)—, —((CR 3 R 4 ) q1 ) 1 —(NHCO—(CR 3 R 4 ) q1 —CONH) 1-2 —((CR 3 R 4 ) q1 ) 1  or —((CR 3 R 4 ) q1 ) 1 —(CONH—(CR 3 R 4 ) q1 —NHCO) 1-2 —((CR 3 R 4 ) q1 ) 1  where R 3  and R 4  independently of each other can be H, —COOH, and R 3  and R 4  can be different at each carbon, and q 1  is 1-6-, or 
 a bond; 
 with the proviso that Q 1 -Q 4  are different; 
 
 X 1 , X 2  and X 3  are independently
 O; 
 a bond; or 
 
 
     
       
         
         
             
             
         
       
     
     where R is hydrogen or —(CH 2 ) p —COOH, —(CH 2 ) p —SO 3 H, —(CH 2 ) p —PO 3 H 2 , —(CH 2 ) p —O—SO 3 H; —(CH 2 ) p —O—PO 3 H 2 ; or —(CH 2 ) p -tetrazol-5-yl, where each p independently of the other p's is an integer in the range of 1 to 6; and
 Z is: 
 —COOH; 
 —CO-Asp; 
 —CO-Glu; 
 —CO-Gly; 
 —CO-Sar; 
 —CH(COOH) 2 ; 
 —N(CH 2 COOH) 2 ; 
 —SO 3 H 
 —OSO 3 H 
 —OPO 3 H 2    
 —PO 3 H 2  or 
 -tetrazol-5-yl 
 
     and any Zn 2+  complex thereof. 
   
   
       13 . The pharmaceutical composition according to  claim 1 , wherein the parent insulin is a desB30 human insulin analogue. 
   
   
       14 . The pharmaceutical composition according to  claim 1  further comprising a rapid acting insulin 
   
   
       15 . The pharmaceutical composition according to  claim 1 , wherein at least 85% of the rapid acting insulin is present as rapid acting insulin hexamer or complexes with a smaller molecular weight than rapid acting insulin hexamers. 
   
   
       16 . The pharmaceutical composition according to  claim 1 , wherein the rapid acting insulin is AspB28 human insulin, LysB3 GluB29 human insulin and/or LysB28 ProB29 human insulin. 
   
   
       17 . A method for producing a pharmaceutical composition comprising an acylated insulin wherein more than about 4 zinc atoms per 6 molecules of acylated insulin are added to the composition. 
   
   
       18 . A method according to  claim 17  wherein up to about 12 zinc atoms per 6 molecules of acylated insulin are added to the composition. 
   
   
       19 . A method according to  claim 17  wherein between about 4.3 and about 12 zinc atoms per 6 molecules of acylated insulin are added to the composition. 
   
   
       20 . A method according to  claim 17  wherein the zinc is added to the composition before addition of a preservative. 
   
   
       21 . A method according to  claim 17  wherein the zinc is added to the composition after addition of a preservative. 
   
   
       22 . A method according to  claim 17 , wherein part of the zinc is added before addition of a preservative and part of the zinc is added after addition of a preservative 
   
   
       23 . A method according to  claim 17 , wherein the preservative is phenol and/or m-cresol. 
   
   
       24 . A method according to  claim 17 , wherein a surfactant is mixed with the pharmaceutical composition. 
   
   
       25 . A method according to  claim 17 , wherein acylated insulin has a side chain attached to the ε-amino group of a Lys residue present in the B chain of the parent insulin, the side chain being of the general formula:
   —W—X—Y-Z 2      wherein W is:
 an α-amino acid residue having a carboxylic acid group in the side chain which residue forms, with one of its carboxylic acid groups, an amide group together with ε-amino group of a Lys residue present in the B chain of the parent insulin; 
 a chain composed of two, three or four α-amino acid residues linked together via amide carbonyl bonds, which chain—via an amide bond—is linked to an ε-amino group of a Lys residue present in the B chain of the parent insulin, the amino acid residues of W being selected from the group of amino acid residues having a neutral side chain and amino acid residues having a carboxylic acid group in the side chain so that W has at least one amino acid residue which has a carboxylic acid group in the side chain; or 
 a covalent bond from X to an ε-amino group of a Lys residue present in the B chain of the parent insulin; 
   X is:
 — C O—; 
 —CH(COOH) C O—; 
 —CO—N(CH 2 COOH)CH 2   C O—; 
 —CO—N(CH 2 COOH)CH 2 CON(CH 2 COOH)CH 2   C O—; 
 —CO—N(CH 2 CH 2 COOH)CH 2 CH 2   C O—; 
 —CO—N(CH 2 CH 2 COOH)CH 2 CH 2 CON(CH 2 CH 2 COOH)CH 2 CH 2   C O—; 
 —CO—NHCH(COOH)(CH 2 ) 4 NH C O—; 
 —CO—N(CH 2 CH 2 COOH)CH 2   C O—; or 
 —CO—N(CH 2 COOH)CH 2 CH 2   C O—. 
   that   a) when W is an amino acid residue or a chain of amino acid residues, via a bond from the underscored carbon forms an amide bond with an amino group in W, or   b) when W is a covalent bond, via a bond from the underscored carbonyl carbon forms an amide bond with an ε-amino group of a Lys residue present in the B chain of the parent insulin;   Y is:
 —(CH 2 ) m — where m is an integer in the range of 6 to 32; 
 a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH 2 — groups sufficient to give a total number of carbon atoms in the chain in the range of 10 to 32; and 
   Z 2  is:
 —COOH; 
 —CO-Asp; 
 —CO-Glu; 
 —CO-Gly; 
 —CO-Sar; 
 —CH(COOH) 2 ; 
 —N(CH 2 COOH) 2 ; 
 —SO 3 H; or 
 —PO 3 H 
   and any Zn 2+  complexes thereof, provided that when W is a covalent bond and X is —CO—, then Z is different from —COOH.   
   
   
       26 . A method according to  claim 17 , wherein the acylated insulin is having a formula 
     
       
         
         
             
             
         
       
       wherein Ins is the parent insulin moiety which via the α-amino group of the N-terminal amino acid residue of the B chain or an ε-amino group of a Lys residue present in the B chain of the insulin moiety is bound to the CO— group in the side chain via an amide bond; 
       X 4  is
 —(CH 2 ) n  where n is 1, 2, 3, 4, 5 or 6; 
 NR, where R is hydrogen or —(CH 2 ) p —COOH; —(CH 2 ) p —SO 3 H; —(CH 2 ) p —PO 3 H 2 ; —(CH 2 ) p —O—SO 3 H 2 ; —(CH 2 ) p —O—PO 3 H 2 ; arylene substituted with 1 or 2 —(CH 2 ) p —O—COOH groups; —(CH 2 ) p -tetrazolyl, where p is an integer in the range of 1 to 6; 
 —(CR 1 R 2 ) q —NR—CO—, where R 1  and R 4  independently of each other and independently for each value of q can be H, —COOH, or OH, q is 1-6 and R is defined as above; 
 —((CR 3 R 4 ) q1 —NR—CO) 2-4 —, where R 3  and R 4  independently of each other and independently for each value of q 1  can be H, —COOH, or OH, q 1  is 1-6 and R is defined as above; or 
 a bond 
 
       W 1  is arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of —COOH, —SO 3 H, and —PO 3 H 2  and tetrazolyl, or W 1  is a bond; 
       m is 0, 1, 2, 3, 4, 5 or 6; 
       X 5  is 
     
     
       
         
         
             
             
         
       
       where R is defined as above; or
 a bond; 
 
       Y 1  is
 —(CR 1 R 2 ) q —NR—CO—, where R 1  and R 2  independently of each other and independently for each value of q can be H, —COOH, a bond or OH, q is 1-6; and R is defined as above; 
 NR where R is defined as above; 
 —((CR 3 R 4 ) q1 —NR—CO) 2-4 —, where R 3  and R 4  independently of each other and independently for each value of q 1  can be H, —COOH, or OH, q 1  is 1-6 and R is defined as above; or 
 a bond; 
 
       Q 7  is
 —(CH 2 ) r — where r is an integer from 4 to 22; 
 a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH 2 — groups sufficient to give a total number of carbon atoms in the chain in the range of 4 to 22; or 
 a divalent hydrocarbon chain of the formula
   —(CH 2 ) s -Q 8 -(C 6 H 4 ) v1 -Q 9 -(CH 2 ) W -Q 10 -(C 6 H 4 ) v2 -Q 11 -(CH 2 ) t -Q 12 -(C 6 H 4 ) v3 -Q 13 -(CH 2 ) z — 
 
 
       wherein Q 8 -Q 13  independently of each other can be O; S or a bond; where s, w, t and z independently of each other are zero or an integer from 1 to 10 so that the sum of s, w, t and z is in the range from 4 to 22, and v 1 , v 2 , and v 3  independently of each other can be zero or 1, provided that when W 1  is a bond then Q 7  is not a divalent hydrocarbon chain of the formula —(CH 2 ) v4 C 6 H 4 (CH 2 ) W1 — wherein v 4  and w 1  are integers or one of them is zero so that the sum of v 4  and w 1  is in the range of 6 to 22; and 
       Z 1  is: 
       —COOH; 
       —CO-Asp; 
       —CO-Glu; 
       —CO-Gly; 
       —CO-Sar; 
       —CH(COOH) 2 ; 
       —N(CH 2 COOH) 2 ; 
       —SO 3 H 
       —PO 3 H 2 ; 
       O—SO 3 H; 
       O—PO 3 H 2 ; 
       -tetrazolyl or
 —O—W 2 , 
 where W 2  is arylene or heteroarylene substituted with one or two groups selected from —COOH, —SO 3 H, and —PO 3 H 2  and tetrazolyl; 
 provided that if W 1  is a bond and v 1 , v 2  and v 3  are all zero and Q 8 -13 are all a bonds, then Z 1  is O—W 2    
 
     
     and any Zn 2+  complex thereof. 
   
   
       27 . A method according to  claim 17 , wherein the acylated insulin is having a formula 
     
       
         
         
             
             
         
       
       wherein Ins is the parent insulin moiety which via the α-amino group of the N-terminal amino acid residue of the B chain or an ε-amino group of a Lys residue present in the B chain of the insulin moiety is bound to the CO— group in the side chain via an amide bond; 
       each n is independently 0, 1, 2, 3, 4, 5 or 6; 
       Q 1 , Q 2 , Q 3 , and Q 4  independently of each other can be
 (CH 2 CH 2 O) s —; (CH 2 CH 2 CH 2 O) s —; (CH 2 CH 2 CH 2 CH 2 O) s —; (CH 2 CH 2 OCH 2 CH 2 CH 2 CH 2 O) s — or (CH 2 CH 2 CH 2 OCH 2 CH 2 CH 2 CH 2 O) s — where s is 1-20 
 —(CH 2 ) r — where r is an integer from 4 to 22; or a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH 2 — groups sufficient to give a total number of carbon atoms in the chain in the range of 4 to 22; 
 —(CH 2 ) t — or —(CH 2 OCH 2 ) t —, where t is an integer from 1 to 6; 
 —(CR 1 R 2 ) q —, where R 1  and R 2  independently of each other can be H, —COOH, (CH 2 ) 16 COOH and R 1  and R 2  can be different at each carbon, and q is 1-6, 
 —((CR 3 R 4 ) q1 ) 1 —(NHCO—(CR 3 R 4 ) q1 —NHCO) 1-2 —((CR 3 R 4 ) q1 ) 1  or —((CR 3 R 4 ) q1 ) 1 —(CONH—(CR 3 R 4 ) q1 —CONH) 1-2 —((CR 3 R 4 ) q1 —)—, —((CR 3 R 4 ) q1 ) 1 —(NHCO—(CR 3 R 4 ) q1 —CONH) 1-2 —((CR 3 R 4 ) q1 ) 1  or —((CR 3 R 4 ) q1 ) 1 —(CONH—(CR 3 R 4 ) q1 —NHCO) 1-2 —((CR 3 R 4 ) q1 ) 1  where R 3  and R 4  independently of each other can be H, —COOH, and R 3  and R 4  can be different at each carbon, and q 1  is 1-6-, or 
 a bond; 
 with the proviso that Q 1 -Q 4  are different; 
 
       X 1 , X 2  and X 3  are independently
 O; 
 a bond; or 
 
     
     
       
         
         
             
             
         
       
       where R is hydrogen or —(CH 2 ) p —COOH, —(CH 2 ) p —SO 3 H, —(CH 2 ) p —PO 3 H 2 , —(CH 2 ) p —O—SO 3 H; —(CH 2 ) p —O—PO 3 H 2 ; or —(CH 2 ) p -tetrazol-5-yl, where each p independently of the other p's is an integer in the range of 1 to 6; and 
       Z is: 
       —COOH; 
       —CO-Asp; 
       —CO-Glu; 
       —CO-Gly; 
       —CO-Sar; 
       —CH(COOH) 2 ; 
       —N(CH 2 COOH) 2 ; 
       —SO 3 H 
       —OSO 3 H 
       —OPO 3 H 2    
       —PO 3 H 2  or 
       -tetrazol-5-yl 
     
     and any Zn 2+  complex thereof. 
   
   
       28 . A method according to  claim 17 , wherein the parent insulin is a desB30 human insulin analogue. 
   
   
       29 . A method according to  claim 17 , wherein a rapid acting insulin is mixed with the composition. 
   
   
       30 . A method according to  claim 29 , wherein the rapid acting insulin is AspB28 human insulin, LysB3 GluB29 human insulin and/or LysB28 ProB29 human insulin. 
   
   
       31 . (canceled) 
   
   
       32 . A pharmaceutical composition for the treatment of diabetes in a patient in need of such treatment, comprising a therapeutically effective amount of a pharmaceutical composition according to  claim 1  together with a pharmaceutically acceptable carrier. 
   
   
       33 . A method of treating diabetes in a patient in need of such a treatment, comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition according to  claim 1  together with a pharmaceutically acceptable carrier. 
   
   
       34 . A method according to  claim 33  for pulmonary treatment of diabetes. 
   
   
       35 . (canceled) 
   
   
       36 . (canceled)