Synthesis of insulin derivatives

Details Synthesis of insulin derivatives Synthesis of insulin derivatives

1999-09-22

2001-11-27

Russel, Jeffrey E. (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

Insulin; related peptides

C514S003100

Reexamination Certificate

active

06323311

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates to derivatives of insulin that show improved properties as compared to the natural protein. More particularly, the invention relates to an improved method for the synthesis of such insulin derivatives and certain derivatives prepared by this and other methods.
It is well known that insulin consists of two polypeptide chains, termed the A chain and the B chain, linked together by disulfide bonds formed between cysteine residues. The N-terminal group of the A chain is a glycine residue (GlyA1), and the N-terminal group of the B chain is a phenylalanine residue (PheB1). Both N-terminal positions contain reactive free &agr;-amino groups. Adjacent to the C-terminal group of the B chain is a lysine residue (LysB29), which has a free &egr;-amino group. It is believed that these free amino groups contribute to the problem of aggregation of insulin molecules, with their eventual precipitation, as will be discussed in more detail below.
The self-association of insulin into dimers, hexamers, high molecular weight aggregates, and insoluble fibrils (at therapeutic concentrations) has been recognized as a problem in the treatment of diabetes mellitus, J. Brange, Galenics of Insulin (1987), especially in formulations intended for implantable insulin pumps. It seems that this process requires the presence of monomeric insulin with a concomitant change of its conformation, J. Brange et al., Studies of the Insulin Fibrillation Process, in Advanced Models for the Therapy of Insulin-Dependent Diabetes 85-90 (P. Brunetti & W. Waldhausl eds. 1987); V. Sluzky et al., 88 Proc. Nat'l Acad. Sci. USA 9377-9381 (1991), and is promoted by contacts with hydrophobic surfaces, W. D. Loughseed et al., 32 Diabetes 424-432 (1983), often present in insulin pumps. Attempts to stabilize insulin solutions have included the use of additives such as phenol derivatives, U. Derewenta et al., 338 Nature 594-596 (1989), nonionic and ionic surfactants, propylene glycol, glycerol, carbohydrates, and even calcium ions, J. Brange, Galenics of Insulin (1987). Sugar-based nonionic detergents have proven to be very efficient stabilizers. L. Hovgaard et al., 19 J. Controlled Release 135-138 (1992); V. Sluzky et al., 40 Biotechnol. Bioeng. 895-903 (1992).
The chemical modification of insulin through glycosylation at the three available amino groups (GlyA1, PheB1, and LysB29) has been described. S. Y. Jeong et al., 1 J. Controlled Release 57-66 (1984); U.S. Pat. Nos. 4,444,683; 4,478,746; 4,478,830; 4,483,792; 4,489,063; 4,489,064; 4,536,572. This modified insulin was evaluated extensively as a component for a glucose-responsive artificial pancreas using concanavalin A. S. Y. Jeong et al., 2 J. Controlled Release 143-152 (1985). It was hypothesized that glycosylation of amino groups, especially at the PheB1 and LysB29 sites, should lead to insulin derivatives with a suppressed tendency to self-associate. This is due to the fact that LysB29 is located on the edge of the dimerization surface of the monomer, and PheB1 is involved in the assembly of three dimers into insulin hexamers. T. L. Blundell et al., 26 Adv. Protein Chem. 279-402 (1972). It has been shown that monomeric or dimeric insulin derivatives, S. E. Shoelson et al., 31 Biochemistry 1757-1767 (1992); J. Brange et al., 333 Nature 679-682 (1988), when administered parenterally exhibit faster absorption profiles from the injection site and the intravenous profile is more attuned to endogenous insulin release. J. Brange et al., 13 Diabetes Care 923-94 (1990). Moreover, by incorporating the covalently bound carbohydrate onto the insulin molecule, the nonspecific hydrophobic aggregation process may be reduced by hydrophilization of its surface, while maintaining the bioactivity of the insulin derivative.
The physical stabilization of insulin by covalent attachment of p-succinylamidophenyl glucopyranoside (SAPG) groups to insulin resulted in seven glycosylated insulin derivatives: three monosubstituted, three disubstituted, and one trisubstituted. M. Baudy{haeck over (s)} et al., Physical Stabilization of Insulin by Glycosylation, 84 J. Pharm. Sci. 28-33 (1995). Nearly all of these derivatives retained in vivo biological activity comparable to insulin, the sole exception being the GlyA1-LysB29-disubstituted derivative, which yielded about 65% of the activity of native insulin. Protein self-association was most suppressed and physical stability was most improved with the disubstituted derivatives, especially those substituted at PheB1, and the trisubstituted derivative.
In view of the foregoing, it will be appreciated that an improved method for synthesis of insulin derivatives and certain insulin derivatives prepared by this or other methods would be a significant advancement in the art.
BRIEF SUMMARY OF THE INVENTION
It is an object of the present invention to provide an improved method for the synthesis of insulin derivatives wherein the process can be carried out without the need to isolate and purify intermediates, i.e. a “one-pot” method.
It is also an object of the invention to provide insulin derivatives wherein only the &agr;-amino group of the PheB1 residue is linked to a polyethylene glycol moiety or derivative thereof.
It is another object of the invention to provide insulin derivatives wherein only the &agr;-amino group of the PheB1 residue is linked to a glycoside.
These and other objects can be achieved by providing a method for the synthesis of an insulin derivative having a hydrophilic compound coupled to the PheB1 amino group comprising:
(a) coupling an acyl protective group to the GlyA1 and LysB29 amino groups of insulin comprising reacting insulin with a cyclic anhydride of a dicarboxylic acid in the presence of a tertiary amine thereby obtaining Gly-N
&agr;A1
, LysN
&egr;B29
-disubstituted insulin;
(b) reacting the Gly-N
&agr;A1
, LysN
&egr;B29
-disubstituted insulin with an activated hydrophilic compound thereby covalently bonding the hydrophilic compound to the PheB1 amino group;
(c) quantitatively hydrolyzing the acyl protective group from the GlyA1 and LysB29 residues, thereby obtaining the insulin derivative having the hydrophilic compound coupled to the PheB1 amino group.
In a preferred embodiment of the invention the cyclic anhydride of a dicarboxylic acid is a member selected from the group consisting of maleic anhydride, citraconic anhydride, phthalic anhydride, exo-cis-3,6-endoxo-&Dgr;
4
-tetrahydrophthalic anhydride, and mixtures thereof. In another preferred embodiment of the invention, the tertiary amine is a member selected from the group consisting of triethylamine and N-methylmorpholine. The activated hydrophilic compound is preferably a derivative of polyethylene glycol represented by the formula:
(R—(O—CH
2
CH
2
)
n
)
m
—X
wherein R is hydrogen or lower alkyl having from about 1 to 6 carbon atoms; n is an integer from about 3 to about 400; m is an integer of 1 or 2; and X is a connecting spacer with a reactive end group having the formula:
—O—(CH
2
)
r
—COCl
where r is an integer from 1 to about 6; or
—O—(CH
2
)
r
—SO
2
Cl
where r is an integer from 1 to 6; or
—O—CH
2
CH
2
—O—SO
2
CH
2
CF
3
or
wherein r is an integer from 1 to about 6 and p is an integer from 1 to about 8; or
wherein r is an integer from 1 to about 6 and p is an integer from 1 to about 8.
The step of quantitatively hydrolyzing the acyl protective group from the GlyA1 and LysB29 residues preferably comprises mild acid treatment, more preferably treatment with dilute acetic acid, and most preferably treatment with a medium comprising 1 M acetic acid and 7 M urea.
Another preferred embodiment of the invention comprises an insulin derivative wherein a hydrophilic compound is coupled to the PheB1 amino group wherein the insulin derivative is prepared according to the method described above.
Still another preferred embodiment of the invention comprises an insulin derivative represented by the formula:
Insulin-X-(PEG)
m
wherein X is an organic spacer and PEG is a polyethylene glycol or alkoxy derivative thereof or a branch

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