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Title:  Fatty acid-acylated insulin analogs

United States Patent:  6,444,641

Issued:  September 3, 2002

Inventors:  Flora; David Benjamin (Greenfield, IN)

Assignee:  Eli Lilly Company (Indianapolis, IN)

Appl. No.:  530038

Filed:  July 7, 2000

PCT Filed:  October 22, 1998

PCT NO:  PCT/US98/22313

371 Date:  July 7, 2000

102(e) Date:  July 7, 2000

PCT PUB.NO.:  WO99/21573

PCT PUB. Date:  May 6, 1999

Abstract

Fatty acid-acylated insulin analogs are soluble at moderately acidic pH and provide long-acting basal control of glucose levels. In such a molecule, the insulin analog portion comprises an insulin A-chain, or an analog thereof, properly cross-linked to an analog of the insulin B-chain, wherein the .epsilon.-amino group of a Lys residue at either positions 28 or 29 of the B-chain analog is acylated with a fatty acid. The insulin analog portion of the molecules comprises an A-chain of insulin, or an analog thereof, with an optional Arg at position 0, properly cross-linked to a B-chain analog that includes Arg at positions 31 and 32.

SUMMARY OF THE INVENTION

The present invention provides a fatty acid-acylated insulin analog having an isoelectric point that is higher than the isoelectric point of insulin, comprising an insulin analog to which a fatty acyl chain is joined by an amide bond. None of the many publications mentioned above disclose acylated insulin analogs that have increased isoelectric points, and none has suggested their desirability. Acylation of insulin analogs that have increased isoelectric points, relative to insulin, is associated with excellent blood glucose control and provides basal insulin levels that are unexpectedly desirable therapeutically.

The invention further provides a soluble formulation comprising a fatty acid-acylated insulin analog of the present invention, together with one or more excipients selected from a preservative, a metal ion, an isotonicity agent, and a pharmaceutically-acceptable buffer. The invention also provides a method of treating hyperglycemia comprising administering to a patient in need thereof an effective dose of the fatty acid-acylated insulin analog of the present invention. The invention also provides a method of treating hyperglycemia comprising administering to a patient in need thereof an effective dose of the fatty acid-acylated insulin analog of the present invention.

The present invention includes a fatty acid-acylated insulin analog having an isoelectric point that is higher than the isoelectric point of insulin, comprising an insulin analog with a fatty acyl chain bonded to the insulin analog by an amide bond. The present invention also includes a fatty acid-acylated insulin analog having an isoelectric point that is higher than the isoelectric point of insulin, wherein the acylated insulin analog has at least one more net positive charge than insulin. The invention also includes a fatty acid-acylated insulin analog having an isoelectric point that is higher than the isoelectric point of insulin, wherein the acylated insulin analog has at least two more net positive charges than insulin.

The present invention encompasses a mono-acylated insulin analog having the formula below, comprising:

(a) a polypeptide of SEQ ID NO:1 properly crosslinked to a polypeptide of SEQ ID NO:2, or a pharmaceutically acceptable salt thereof, wherein the polypeptide of SEQ ID NO:1 has the sequence:

 

    0   1               5                  10                15
    Xaa Gly Ile VaI Glu Gln Cys Cys Thr Ser IIe Cys Ser Leu Tyr Gln Leu
       (SEQ ID NO:1)
                20
    Glu Asn Tyr Cys Xaa

wherein:

Xaa at position 0 is either Arg or absent; and

Xaa at position 21 is any naturally occurring amino acid except Cys and Lys; and

the polypeptide of SEQ ID NO:2 has the sequence:

 

    1               5                   10                  15
    Phe Val Xaa Gln His Leu Cys Gly Ser His Leu VaI Glu AIa Leu Tyr
     (SEQ ID NO:2)
                20                  25                  30
    Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Xaa Xaa Xaa Xaa Arg Arg

wherein:

Xaa at position 3 is any naturally occurring amino acid except Cys and Lys;

Xaa at position 27 is either Thr or absent;

Xaa at position 28 is selected from the group consisting of Pro, Leu, Val, Ala, Lys, and Asp;

Xaa at position 29 is selected from the group consisting of Pro and Lys;

Xaa at position 30 is absent or any naturally occurring amino acid except Cys or Lys;

further wherein position 28 or position 29 is Lys, and

if position 28 is Lys, position 29 is not Lys; and

(b) Lys at position 28 or position 29 of SEQ ID NO:2 is acylated.

The invention further comprises a the following mono-acylated insulin analogs of the formula above, wherein: Xaa at position 30 of the polypeptide of SEQ ID NO:2 is Thr; wherein Xaa at position 21 of the polypeptide of SEQ ID NO:1 is Asn; or wherein Xaa at position 3 of the polypeptide of SEQ ID NO:2 is Asn.

The invention further comprises a mono-acylated insulin analog of the formula above, wherein Xaa at position 21 of the polypeptide of SEQ ID NO:1 is Asn, and wherein Xaa at position 3 of the polypeptide of SEQ ID NO:2 is Asn. The invention further comprises a mono-acylated insulin analog of the formula above, wherein Xaa at position 21 of the polypeptide of SEQ ID NO:1 is Asn, and wherein Xaa at position 3 of the polypeptide of SEQ ID NO:2 is Asn, and further wherein Xaa at position 28 of the polypeptide of SEQ ID NO:2 is Pro, and Xaa at position 29 of the poiypeptide of SEQ ID NO:2 is Lys. Another monoacylated insulin analog of the invention has the formula above, wherein Xaa at position 21 of the polypeptide of SEQ ID NO:1 is Asn, and wherein Xaa at position 3 of the polypeptide of SEQ ID NO:2 is Asn, and further wherein Xaa at position 28 of the polypeptide of SEQ ID NO:2 is Lys, and Xaa at position 29 of the polypeptide of SEQ ID NO:2 is Pro.

The invention also includes a mono-acylated insulin analog of the formula above, wherein Xaa at position 21 of the polypeptide of SEQ ID NO:1 is Gly, and Xaa at position 3 of the polypeptide of SEQ ID NO:2 is Gin. Still another analog as the formula above, wherein Xaa at position 21 of the polypeptide of SEQ ID NO:1 is Gly, and Xaa at position 3 of the polypeptide of SEQ ID NO:2 is Asp.

The invention further comprises a mono-acylated insulin analog of the formula above, wherein the Lys at position 28 or position 29 of the polypeptide of SEQ ID NO:2 is acylated with a C4-C21fatty acid. The invention also includes mono-acylated insulin analog of the formula above, wherein the Lys at position 28 or position 29 of the polypeptide of SEQ ID NO:2 is acylated with a C10-C18fatty acid. The invention also includes a mono-acylated insulin analog of the formula above, wherein the Lys at position 28 or position 29 of the polypeptide of SEQ ID NO:2 is acylated with a fatty acid selected from the group consisting of palmitic and myristic acid.

The invention further comprises a mono-acylated insulin analog of the formula above, wherein the Lys at position 28 or position 29 of the polypeptide of SEQ ID NO:2 is acylated with a C4-C8fatty acid. The invention additionally comprises a mono-acylated insulin analog of the formula above, wherein the Lys at position 28 or position 29 of the polypeptide of SEQ ID NO:2 is acylated with a fatty acid selected from the group consisting of octanoic and hexanoic acid.

Yet another analog has the formula above, wherein Xaa at position 0 of the polypeptide of SEQ ID NO:1 is Arg, which is a mono-acylated tri-arginine insulin analog. The invention comprises modifications of this tri-arginine insulin analog, such as an analog wherein Xaa at position 21 of the polypeptide of SEQ ID NO:1 is selected from the group consisting of Gly, Asn, Ala, and Gln. Another tri-arginine insulin analog has Xaa at position 21 of the polypeptide of SEQ ID NO:1 is Gly. Yet another tri-arginine insulin analog has Xaa at position 21 of the polypeptide of SEQ ID NO:1 is Asn.

Another mono-acylated tri-arginine insulin analog of the invention has the formula above, wherein Xaa at position 21 of the polypeptide of SEQ ID NO:1 is Gly, Xaa at position 3 of SEQ ID NO:2 is Asn; Xaa at position 27 of SEQ ID NO:2 is Thr; Xaa at position 28 of SEQ ID NO:2 is Pro; and Xaa at position 29 of SEQ ID NO:2 is Lys. Yet another mono-acylated tri-arginine insulin analog of the invention has the formula above, wherein Xaa at position 21 of the polypeptide of SEQ ID NO:1 is Gly, Xaa at position 3 of SEQ ID NO:2 is Gln; Xaa at position 27 of SEQ ID NO:2 is Thr; Xaa at position 28 of SEQ ID NO:2 is Pro; and Xaa at position 29 of SEQ ID NO:2 is Lys.

Still another mono-acylated tri-arginine insulin analog of the invention has the formula above, wherein Xaa at position 21 of the polypeptide of SEQ ID NO:1 is Gly, Xaa at position 3 of SEQ ID NO:2 is Asp; Xaa at position 27 of SEQ ID NO:2 is Thr; Xaa at position 28 of SEQ ID NO:2 is Pro; and Xaa at position 29 of SEQ ID NO:2 is Lys. Yet another mono-acylated tri-arginine insulin analog of the invention has the formula above, wherein Xaa at position 21 of the polypeptide of SEQ ID NO:1 is Asn, Xaa at position 3 of SEQ ID NO:2 is Asn; Xaa at position 27 of SEQ ID NO:2 is Thr, and Xaa at position 28 of SEQ ID NO:2 is Pro, and Xaa at position 29 of SEQ ID NO:2 is Lys.

The invention also encompasses a mono-acylated tri-arginine insulin analog of the formula above, wherein Xaa at position 21 of the polypeptide of SEQ ID NO:1 is Gly, Xaa at position 3 of SEQ ID NO:2 is Asn; Xaa at position 27 of SEQ ID NO:2 is Thr; Xaa at position 28 of SEQ ID NO:2 is Lys; and Xaa at position 29 of SEQ ID NO:2 is Pro. Still another mono-acylated tri-arginine insulin analog of the invention has the formula above, wherein Xaa at position 21 of the polypeptide of SEQ ID NO:1 is Gly, Xaa at position 3 of SEQ ID NO:2 is Gln; Xaa at position 27 of SEQ ID NO:2 is Thr; Xaa at position 28 of SEQ ID NO:2 is Lys; and Xaa at position 29 of SEQ ID NO:2 is Pro.

The invention includes yet another mono-acylated tri-arginine insulin analog of the formula above, wherein Xaa at position 21 of the polypeptide of SEQ ID NO:1 is Gly, Xaa at position 3 of SEQ ID NO:2 is Asp; Xaa at position 27 of SEQ ID NO:2 is Thr; Xaa at position 28 of SEQ ID NO:2 is Lys; and Xaa at position 29 of SEQ ID NO:2 is Pro. The invention also comprises a mono-acylated tri-arginine insulin analog of the formula above, wherein Xaa at position 21 of the polypeptide of SEQ ID NO:1 is Asn, Xaa at position 3 of SEQ ID NO:2 is Asn; Xaa at position 27 of SEQ ID NO:2 is Thr; Xaa at position 28 of SEQ ID NO:2 is Lys; and Xaa at position 29 of SEQ ID NO:2 is Pro.

Yet another analog has the formula above, wherein Xaa at position 0 of the polypeptide of SEQ ID NO:1 is absent, which is a mono-acylated di-arginine insulin analog. An analog of the invention is a mono-acylated di-arginine insulin analog having the formula above, wherein Xaa at position 21 of the polypeptide of SEQ ID NO:1 is selected from the group consisting of Gly, Asn, Ala, and Gln.

The invention also includes a mono-acylated di-arginine insulin analog having the formula above, wherein Xaa at position 21 of the polypeptide of SEQ ID NO:1 is Gly. The invention also includes a mono-acylated di-arginine insulin analog having the formula above, wherein Xaa at position 21 of the polypeptide of SEQ ID NO:1 is Asn.

The invention also includes a mono-acylated di-arginine insulin analog having the formula above, wherein Xaa at position 21 of the polypeptide of SEQ ID NO:1 is Gly, Xaa at position 3 of SEQ ID NO:2 is Asn; Xaa at position 27 of SEQ ID NO:2 is Thr; Xaa at position 28 of SEQ ID NO:2 is Pro; and Xaa at position 29 of SEQ ID NO:2 is Lys. The invention also includes a mono-acylated di-arginine insulin analog having the formula above, wherein Xaa at position 21 of the polypeptide of SEQ ID NO:1 is Gly, Xaa at position 3 of SEQ ID NO:2 is Gln; Xaa at position 27 of SEQ ID NO:2 is Thr; Xaa at position 28 of SEQ ID NO:2 is Pro; and Xaa at position 29 of SEQ ID NO:2 is Lys.

The invention also includes a mono-acylated di-arginine insulin analog having the formula above, wherein Xaa at position 21 of the polypeptide of SEQ ID NO:1 is Asn, Xaa at position 3 of SEQ ID NO:2 is Asn; Xaa at position 27 of SEQ ID NO:2 is Thr; Xaa at position 28 of SEQ ID NO:2 is Pro; and Xaa at position 29 of SEQ ID NO:2 is Lys. The invention further includes a mono-acylated di-arginine insulin analog having the formula above, wherein Xaa at position 21 of the polypeptide of SEQ ID NO:1 is Gly, Xaa at position 3 of SEQ ID NO:2 is Asn; Xaa at position 27 of SEQ ID NO:2 is Thr; Xaa at position 28 of SEQ ID NO:2 is Lys; and Xaa at position 29 of SEQ ID NO:2 is Pro.

The invention also includes a mono-acylated di-arginine insulin analog having the formula above, wherein Xaa at position 21 of the polypeptide of SEQ ID NO:1 is Gly, Xaa at position 3 of SEQ ID NO:2 is Gln; Xaa at position 27 of SEQ ID NO:2 is Thr; Xaa at position 28 of SEQ ID NO:2 is Lys; and Xaa at position 29 of SEQ ID NO:2 is Pro. The invention also includes a mono-acylated di-arginine insulin analog having the formula above, wherein Xaa at position 21 of the polypeptide of SEQ ID NO:1 is Asn, Xaa at position 3 of SEQ ID NO:2 is Asn; Xaa at position 27 of SEQ ID NO:2 is Thr; Xaa at position 28 of SEQ ID NO:2 is Lys; and Xaa at position 29 of SEQ ID NO:2 is Pro.

The invention includes the following inventive monoacylated insulin analogs: B29-N.epsilon. -GlyA21 ArgB31 ArgB32 -myristoyl human insulin; B29-N.epsilon. -GlyA21 GlnB3 ArgB31 ArgB32 -myristoyl human insulin; B29-N.epsilon. -ArgA0 GlyA21 ArgB31 ArgB32 -myristoyl human insulin; B29-N.epsilon. -ArgA0 GlyA21 GlnB3 ArgB31 ArgB32 -myristoyl human insulin; B29-N.epsilon. -ArgA0 GlyA21 AspB3 ArgB31 ArgB32 -myristoyl human insulin; B29-N.epsilon. -ArgB31 ArgB32 -myristoyl human insulin; B29-N.epsilon. -ArgA0 ArgB31 ArgB32 -myristoyl human insulin analog; B29-N.epsilon. -GlyA21 ArgB31 ArgB32 -octanoyl human insulin; B29-N.epsilon. -GlyA21 GlnB3 ArgB31 ArgB32 -octanoyl human insulin; B29-N.epsilon.-ArgA0 GlyA21 ArgB31 ArgB32 -octanoyl human insulin; B29-N.epsilon. -ArgA0 GlyA21 GlnB3 ArgB31 ArgB32 -octanoyl human insulin;B29-N.epsilon.-ArgA0 GlyA21 AspB3 ArgB31 ArgB32 -octanoyl human insulin; B29-N.epsilon. -ArgB31 ArgB32 -octanoyl human insulin; B29-N.epsilon. -ArgA0 ArgB31 ArgB32 -octanoyl human insulin analog; -N.epsilon. -GlyA21 LysB28 ProB29 ArgB31 ArgB32 -myristoyl human insulin; B28-N.epsilon. -GlyA21 GlnB3 LysB28 ProB29 ArgB31 ArgB32 -myristoyl human insulin; B28-N.epsilon. -ArgA0 GlyA21 LysB28 ProB29 ArgB31 ArgB32 -myristoyl human insulin; B28-N.epsilon. -ArgA0 GlyA21 GlnB3 LysB28 ProB29 ArgB31 ArgB32 -myristoyl human insulin; B28-N.epsilon. -ArgA0 GlyA21 AspB3 LysB28 ProB29 ArgB31 ArgB32 -myristoyl human insulin; B28-N.epsilon. -LysB28 ProB29 ArgB31 ArgB32 -myristoyl human insulin; B28-N.epsilon. -ArgA0 LysB28 ProB29 ArgB31 ArgB32 -myristoyl human insulin; B28-N.epsilon. -GlyA21 LysB28 ProB29 ArgB31 ArgB32 -octanoyl human insulin; B28-N.epsilon. -GlyA21 GlnB3 LysB28 ProB29 ArgB31 ArgB32 -octanoyl human insulin; B28-N.epsilon. -ArgA0 GlyA21 LysB28 ProB29 ArgB31 ArgB32 -octanoyl human insulin; B28-N.epsilon. -ArgA0 GlyA21 GlnB3 LysB28 ProB29 ArgB31 ArgB32 -octanoyl human insulin; B28-N.epsilon. -ArgA0 GlyA21 AspB3 LysB28 ProB29 ArgB31 ArgB32 -octanoyl human insulin; B28-N.epsilon. -LysB28 ProB29 ArgB31 ArgB32 -octanoyl human insulin; B28-N.epsilon. -ArgA0 LysB28 ProB29 ArgB31 ArgB32 -octanoyl human insulin.

The invention also encompasses various formulations comprising any of the above-described mono-acylated insulin analogs. Such a formulation may comprise a preservative, such as m-cresol or phenol, an isotonicity agent, a pharmaceutically-acceptable buffer, and/or a metal in the +2 oxidation state, such as cobalt or zinc. The invention also includes a formulation comprising a mono-acylated insulin analog of the invention with a pH of between about 3.0 to about 3.8. The invention also includes a formulation comprising a mono-acylated insulin analog of the invention with a pH of about 3.5.

The invention further comprises a formulation comprising a mono-acylated insulin analog of the invention with a pH of between about 4.5 and 7.6. The invention additionally includes a formulation comprising a mono-acylated insulin analog of the invention, with a pH of between about 5.0 and about 7.0. The invention further includes a formulation comprising a mono-acylated insulin analog of the invention, with a pH of about 6.5.

The invention includes therapeutic methods, comprising administering to a patient suffering from diabetes, a formulation comprising a mono-acylated insulin analog of the invention. In particular, any of the above-recited mono-acylated insulin analogs of the invention are suitable for such therapeutic methods. The formulation administered in the inventive therapeutic method may have a pH of between about 3.0 to about 3.8 or a pH of about 3.5. The formulation administered in the therapeutic method may also have a pH of between about 4.5 and 7.6, or between about 5.0 and about 7.0, or the formulation may have a pH of about 6.5.

Claim 1 of 39 Claims

What is claimed is:

1. A fatty acid-acylated insulin analog comprising an insulin analog to which a fatty acyl chain is joined by an amide bond, wherein said fatty insulin analog has an isoelectric point that is higher than the isoelectric point of insulin.
 


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