U.S. patent application number 08/801393 was filed with the patent office on 2002-04-18 for peptide derivatives.
This patent application is currently assigned to Novo Nordisk of North America, Inc.. Invention is credited to BALSCHMIDT, PER, SCHAFFER, LAUGE.
Application Number | 20020045731 08/801393 |
Document ID | / |
Family ID | 8090772 |
Filed Date | 2002-04-18 |
United States Patent
Application |
20020045731 |
Kind Code |
A1 |
SCHAFFER, LAUGE ; et
al. |
April 18, 2002 |
PEPTIDE DERIVATIVES
Abstract
The present invention relates to derivatives of insulin and
insulin analogues wherein the N-terminal amino group of the B-chain
and/or the .epsilon.-amino group of Lys in position B28, B29 or B30
has a substituent of the formula --CO--W--COOH wherein W is a
divalent long chain hydrocarbon group having from 12 to 22 carbon
atoms and zinc complexes thereof are soluble at physiological pH
values and exhibit a long disappearance half-life from the
injection site after subcutaneous injection.
Inventors: |
SCHAFFER, LAUGE;
(COPENHAGEN, DK) ; BALSCHMIDT, PER; (ESPERGAERDE,
DK) |
Correspondence
Address: |
STEVE T ZELSON
NOVO NORDISK OF NORTH AMERICA INC
405 LEXINGTON AVENUE SUITE 6400
NEW YORK
NY
101746401
|
Assignee: |
Novo Nordisk of North America,
Inc.
|
Family ID: |
8090772 |
Appl. No.: |
08/801393 |
Filed: |
February 20, 1997 |
Current U.S.
Class: |
530/303 |
Current CPC
Class: |
C07K 14/62 20130101;
A61P 3/10 20180101; A61P 5/50 20180101 |
Class at
Publication: |
530/303 ;
514/3 |
International
Class: |
C07K 014/00; A61K
038/28; C07K 005/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 21, 1996 |
DK |
0188/96 |
Claims
What we claim is:
1. An insulin derivative having the sequence shown in Formula I:
2wherein Xaa at positions A21 and B3 are, independently, any amino
acid residue which can be coded for by the genetic code except Lys,
Arg and Cys; Xaa at position BI is (a) Phe, which is optionally
substituted in the amino group with a substituent of the formula
--CO--W--COOH wherein W is a divalent long chain hydrocarbon group
having from 12 to 22 carbon atoms; or (b) deleted, in which case
the amino group of Val at position B2 is either free or has a
substituent of the formula --CO--W--COOH as defined above; Xaa at
position B28 is (a) Pro, in which case Xaa at position B29 is Lys
which optionally, in its .epsilon.-amino group, has a substituent
of the formula --CO--W--COOH as defined above; (b) Asp or Ser, in
any of which cases Xaa at position B29 is Lys which optionally, in
its .epsilon.-amino group, has a substituent of the formula
--CO--W--COOH as defined above; or (c) Lys which optionally, in its
.epsilon.-amino group, has a substituent of the formula
--CO--W--COOH as defined above, in which case, whether the
.epsilon.-amino group of the Lys has the optional substituent or
not, Xaa at position B29 is Pro; Xaa at position B30 is (a) Thr;
(b) Ala; or (c) deleted; and any zinc complexes thereof, provided
that the insulin derivative of formula I has at least one
lipophilic substituent of the formula --CO--W--COOH.
2. The insulin derivative according to claim 1, wherein Xaa at
position A21 is an amino acid residue selected from the group
comprising Ala, Gln, Gly, Ser and Asn.
3. The insulin derivative according to claim 2, wherein Xaa at
position A21 is Asn.
4. The insulin derivative according to claim 1, wherein Xaa at
position B3 is an amino acid residue selected from the group
comprising Asp, Gln, Thr and Asn.
5. The insulin derivative according to claim 4, wherein Xaa at
position B3 is Asn.
6. The insulin derivative according to claim 1, wherein Xaa at
position B1 is Phe.
7. The insulin derivative according to claim 1, wherein the amino
acid at position B1 is deleted.
8. The insulin derivative according to claim 1, wherein Xaa at
position B28 is Pro and Xaa at position B29 is Lys.
9. The insulin derivative according to claim 1, wherein Xaa at
position B28 is Ser and Xaa at position B29 is Lys.
10. The insulin derivative according to claim 1, wherein Xaa at
position B28 is Lys and Xaa at position B29 is Pro.
11. The insulin derivative according to claim 1, wherein Xaa at
position B30 is Thr.
12. The insulin derivative according to claim 1, wherein Xaa at
position B30 is Ala.
13. The insulin derivative according to claim 1, wherein Xaa at
position B30 is deleted.
14. The insulin derivative according to claim 1, wherein Xaa at
position B1 is Phe and wherein solely the amino group of this Phe
has a substituent of the general formula --CO--W--COOH.
15. The insulin derivative according to claim 1, wherein Xaa at
position B1 is deleted and wherein solely the amino group of Val in
position B2 has a substituent of the general formula
--CO--W--COOH.
16. The insulin derivative according to claim 1, wherein solely the
.epsilon.-amino group of Lys at position B28 has a substituent of
the general formula --CO--W--COOH.
17. The insulin derivative according to claim 1, wherein solely the
.epsilon.-amino group of Lys at position B29 has a substituent of
the general formula --CO--W--COOH.
18. The insulin derivative according to claim 1, wherein Xaa at
position B1 is Phe having a substituent of the general formula
--CO--W--COOH in the amino group and Xaa at position B28 is Lys
having a substituent of the general formula --CO--W--COOH in the
.epsilon.-amino group.
19. The insulin derivative according to claim 1, wherein Xaa at
position B1 is Phe having a substituent of the general formula
--CO--W--COOH in the amino group and Xaa at position B29 is Lys
having a substituent of the general formula --CO--W--COOH in the
.epsilon.-amino group.
20. The insulin derivative according to claim 1, wherein the amino
acid at position B1 is deleted and Val at position B2 has a
substituent of the general formula --CO--W--COOH in the amino group
and Xaa at position B28 is Lys having a substituent of the general
formula --CO--W--COOH in the .epsilon.-amino group.
21. The insulin derivative according to claim 1, wherein the amino
acid at position B1 is deleted and Val at position B2 has a
substituent of the general formula --CO--W--COOH in the amino group
and Xaa at position B29 is Lys having a substituent of the general
formula --CO--W--COOH in the .epsilon.-amino group.
22. The insulin derivative according to claim 1, wherein W is
selected from the group comprising --(CH.sub.2).sub.12--,
--(CH.sub.2).sub.14--, --(CH.sub.2).sub.16--,
--(CH.sub.2).sub.18--, --(CH.sub.2).sub.20-- and
--(CH.sub.2).sub.22--.
23. A pharmaceutical composition for the treatment of diabetes in a
patient in need of such treatment, comprising a therapeutically
effective amount of an insulin derivative according to claim 1
together with a pharmaceutically acceptable carrier.
24. A pharmaceutical composition according to claim 23, further
comprising an insulin or an insulin analogue which has a rapid
onset of action.
25. A method of treating diabetes in a patient in need of such a
treatment, comprising administering to the patient a pharmaceutical
composition according to claim 23.
26. A method of treating diabetes in a patient in need of such a
treatment, comprising administering to the patient a pharmaceutical
composition according to claim 24.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority of Danish application
0188/96 filed Feb. 21, 1996, the contents of which is fully
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to novel derivatives of
naturally occurring insulins and analogues thereof which
derivatives are soluble and have a protracted profile of action, to
methods of providing such derivatives, to pharmaceutical
compositions containing them and to the use of such derivatives in
the treatment of diabetes.
BACKGROUND OF THE INVENTION
[0003] Many diabetic patients are treated with multiple daily
insulin injections in a regimen comprising one or two daily
injections of a protracted insulin to cover the basal requirement
supplemented by bolus injections of a rapid acting insulin to cover
the requirement related to meals.
[0004] Protracted insulin compositions are well known in the art.
Thus, one main type of protracted insulin compositions comprises
injectable aqueous suspensions of insulin crystals or amorphous
insulin. In these compositions, the insulin compounds utilized
typically are protamine insulin, zinc insulin or protamine zinc
insulin.
[0005] Certain drawbacks are associated with the use of insulin
suspensions. Thus, in order to secure an accurate dosing, the
insulin particles must be suspended homogeneously by gentle shaking
before a defined volume of the suspension is withdrawn from a vial
or expelled from a cartridge. Also, for the storage of insulin
suspensions, the temperature must be kept within more narrow limits
than for insulin solutions in order to avoid lump formation or
coagulation.
[0006] While it was earlier believed that protamines were
non-immunogenic, it has now turned out that protamines can be
immunogenic in man and that their use for medical purposes may lead
to formation of antibodies (Samuel et al., Studies on the
immunogenicity of protamines in humans and experimental animals by
means of a micro-complement fixation test, Clin. Exp. Immunol. 33,
pp. 252-260 (1978)).
[0007] Also, evidence has been found that the protamine-insulin
complex is itself immunogenic (Kurtz et al., Circulating IgG
antibody to protamine in patients treated with protamine-insulins.
Diabetologica 25, pp. 322-324 (1983)). Therefore, with some
patients the use of protracted insulin compositions containing
protamines must be avoided.
[0008] Another type of protracted insulin compositions are
solutions having a pH value below physiological pH from which the
insulin will precipitate because of the rise in the pH value when
the solution is injected. A drawback is that the solid particles of
the insulin act as a local irritant causing inflammation of the
tissue at the site of injection.
[0009] WO 91/12817 (Novo Nordisk A/S) discloses protracted, soluble
insulin compositions comprising insulin complexes of cobalt (III).
The protraction of these complexes is only intermediate and the
bioavailability is reduced.
[0010] Human insulin has three primary amino groups: the N-terminal
group of the A-chain and of the B-chain and the .epsilon.-amino
group of LysB.sup.29. Several insulin derivatives which are
substituted in one or more of these groups are known in the prior
art. Thus, U.S. Pat. No. 3,528,960 (Eli Lilly) relates to
N-carboxyaroyl insulins in which one, two or three primary amino
groups of the insulin molecule has a carboxyaroyl group. No
specifically N.sup..epsilon.B29-substituted insulins are
disclosed.
[0011] According to GB Patent No. 1,492,997 (Nat. Res. Dev. Corp.),
it has been found that insulin with a carbamyl substitution at
N.sup..epsilon.B29 has an improved profile of hypoglycaemic
effect.
[0012] JP laid-open patent application No. 1-254699 (Kodama Co.,
Ltd.) discloses insulin wherein an alkanoyl group is bound to the
amino group of Phe.sup.B1 or to the .epsilon.-amino group of
Lys.sup.B29 or to both of these. The stated purpose of the
derivatisation is to obtain a pharmacologically acceptable, stable
insulin preparation.
[0013] Insulin analogues, which in the B30 position have an amino
acid having at least five carbon atoms which cannot necessarily be
coded for by a triplet of nucleotides, are described in JP
laid-open patent application No. 57-067548 (Shionogi). The insulin
analogues are claimed to be useful in the treatment of diabetes
mellitus, particularly in patients who are insulin resistant due to
generation of bovine or swine insulin antibodies.
[0014] U.S. Pat. No. 5,359,030 (Ekwuribe, Protein Delivery, Inc.)
describes conjugation-stabilized polypeptide compositions for oral
or parenteral administration comprising a polypeptide covalently
coupled with a polymer including a linear polyalkylene moiety and a
lipophilic moiety, said moieties being arranged so relative to each
other that the polypeptide has an enhanced in vivo resistance to
enzymatic degradation.
[0015] EP 511600 A2 relates i.a. to protein derivatives of the
formula [protein] [Z].sub.n wherein [protein] represents a protein
having n amino residues each derivable from an amino group by
removal of one of its hydrogen atoms, instead of amino groups, [Z]
is a residue represented by the formula --CO--W--COOH wherein W is
a divalent long chain hydrocarbon group which may also contain
certain hetero atoms and n represents an average of the number of
amide bonds between [Z] and [protein]. It is mentioned that the
protein derivatives of the invention have an extremely prolonged
serum half-life as compared with the proteins from which they are
derived and that they exhibit no antigenicity. It is also
mentioned, that insulin is one of the proteins from which
derivatives according to the invention can be made, but no specific
insulin derivatives are disclosed in EP 511600 nor is there any
indication of a preferred [Z] or (a) preferred position(s) in which
[Z] should be introduced in order to obtain useful insulin
derivatives.
[0016] WO 95/07931 (Novo Nordisk A/S) discloses insulin derivatives
in which the amino acid at position B30 is (a) a non-codable
lipophilic amino acid having from 10 to 24 carbon atoms in which
case the .epsilon.-amino group of Lys.sup.b29 has a lower acyl
substituent or (b) any codable amino acid, in which case the
.epsilon.-amino group of LysB.sup.29 has a lipophilic substituent
or (c) deleted, in which case the .epsilon.-amino group of
Lys.sup.B29 has a lipophilic substituent. The insulin derivatives
are soluble at physiological pH values and have a protracted
profile of action.
[0017] By "insulin derivative" as used herein is meant a peptide
having a molecular structure similar to that of human insulin
including the disulphide bridges between Cys.sup.A7 and Cys.sup.B7
and between Cys.sup.A20 and Cys.sup.B19 and an internal disulphide
bridge between Cys.sup.A6 and Cys.sup.A11, and which has insulin
activity. When the amino acid at position B1 is deleted, the
position of the remaining amino acids of the B-chain are not
renumbered.
[0018] Despite the many improvements already made in the field
there still is a need for novel protracted injectable insulin
compositions which are solutions and contain insulins which stay in
solution after injection and possess minimal inflammatory and
immunogenic properties.
[0019] One object of the present invention is to provide insulin
derivatives soluble at physiological pH values and having a
protracted profile of action.
[0020] Another object of the present invention is to provide
insulin derivatives which have a long disappearance half-life from
the injection site after subcutaneous injection.
[0021] A further object of the present invention is to provide a
pharmaceutical composition comprising the insulin derivatives
according to the invention.
[0022] A still further object of the invention is to provide a
non-immunogenic insulin derivative.
[0023] A still further object of the invention is to provide a
method of making the insulin derivatives of the invention.
[0024] A still further object of the invention is to provide a
method of treating diabetes.
SUMMARY OF THE INVENTION
[0025] Surprisingly, it has turned out that certain derivatives of
naturally occurring insulins and insulin analogues wherein the
amino group of the N-terminal amino acid of the B-chain and/or the
.epsilon.-amino group of Lys.sup.B29 has a lipophilic substituent
of the formula --CO--W--COOH as defined below have a protracted
profile of action and are soluble at physiological pH values.
[0026] Accordingly, in its broadest aspect, the present invention
relates to an insulin derivative as shown in formula I: 1
[0027] wherein
[0028] Xaa at positions A21 and B3 are, independently, any amino
acid residue which can be coded for by the genetic code except Lys,
Arg and Cys;
[0029] Xaa at position B1 is (a) Phe, which is optionally
substituted in the amino group with a substituent of the formula
--CO--W--COOH wherein W is a divalent long chain hydrocarbon group
having from 12 to 22 carbon atoms; or (b) deleted, in which case
the amino group of Val at position B2 is either free or has a
substituent of the formula --CO--W--COOH as defined above;
[0030] Xaa at position B28 is (a) Pro, in which case Xaa at
position B29 is Lys which optionally, in its .epsilon.-amino group,
has a substituent of the formula --CO--W--COOH as defined above;
(b) Ser, in which case Xaa at position B29 is Lys which optionally,
in its .epsilon.-amino group, has a substituent of the formula
--CO--W--COOH as defined above; or (c) Lys which optionally, in its
.epsilon.-amino group, has a substituent of the formula
--CO--W--COOH as defined above, in which case, whether the
.epsilon.-amino group of the Lys has the optional substituent or
not, Xaa at position B29 is Pro;
[0031] Xaa at position B30 is (a) Thr; (b) Ala; or (c) deleted; and
any zinc complexes thereof, with the proviso that the insulin
derivative of formula I has at least one lipophilic substituent of
the formula --CO-W-COOH as defined above.
[0032] In another aspect, the invention relates to an insulin
derivative of the general formula I above wherein Xaa at position
A21, B1 and B3 are as defined above, while Xaa at position B28 is
Asp, Xaa at position B29 is Lys which, in its .epsilon.-amino
group, has a substituent of the formula --CO--W--COOH as defined
above and Xaa at position B30 is Thr.
[0033] In another aspect, the invention relates to an insulin
derivative of the general formula I above wherein Xaa at position
A21, B1 and B3 are as defined above, while Xaa at position B28 is
Pro, Xaa at position B29 is Thr and Xaa at position B30 is Lys
which, in its .epsilon.-amino group, has a substituent of the
formula --CO--W--COOH as defined above and.
[0034] In one preferred embodiment of the invention, the divalent
long chain hydrocarbon group, W, is --(CH.sub.2).sub.12--.
[0035] In another preferred embodiment of the invention, the
divalent long chain hydrocarbon group, W, is
--(CH.sub.2).sub.13--.
[0036] In another preferred embodiment of the invention, the
divalent long chain hydrocarbon group, W, is
--(CH.sub.2).sub.14--.
[0037] In another preferred embodiment of the invention, the
divalent long chain hydrocarbon group, W, is
--(CH.sub.2).sub.15--.
[0038] In another preferred embodiment of the invention, the
divalent long chain hydrocarbon group, W, is
--(CH.sub.2).sub.16--.
[0039] In another preferred embodiment of the invention, the
divalent long chain hydrocarbon group, W, is
--(CH.sub.2).sub.17--.
[0040] In another preferred embodiment of the invention, the
divalent long chain hydrocarbon group, W, is
--(CH.sub.2).sub.18--.
[0041] In another preferred embodiment of the invention, the
divalent long chain hydrocarbon group, W, is
--(CH.sub.2).sub.19--.
[0042] In another preferred embodiment of the invention, the
divalent long chain hydrocarbon group, W, is
--(CH.sub.2).sub.20--.
[0043] In another preferred embodiment of the invention, the
divalent long chain hydrocarbon group, W, is
---(CH.sub.2).sub.21--.
[0044] In another preferred embodiment of the invention, the
divalent long chain hydrocarbon group, W, is
--(CH.sub.2).sub.22--.
[0045] Further preferred features of the present invention will
appear from the appended claims.
[0046] Examples of preferred insulin derivatives according to the
present invention are the following:
[0047] N.sup..epsilon.B29-(CO--(CH.sub.2).sub.14--COOH) human
insulin and any zinc complexes thereof;
[0048] N.sup..epsilon.B29-(CO--(CH.sub.2).sub.16--COOH) human
insulin and any zinc complexes thereof;
[0049] N.sup..epsilon.B29-(CO--(CH.sub.2).sub.18--COOH) human
insulin and any zinc complexes thereof;
[0050] N.sup..epsilon.B29-(CO--(CH.sub.2).sub.20--COOH) human
insulin and any zinc complexes thereof;
[0051] N.sup..epsilon.B29-(CO--(CH.sub.2).sub.22--COOH) human
insulin and any zinc complexes thereof;
[0052] N.sup..epsilon.B29-(CO--(CH.sub.2).sub.14--COOH)
ASp.sup.B28-human insulin and any zinc complexes thereof;
[0053] N.sup..epsilon.B29-(Co--(CH.sub.2).sub.16--COOH)
ASp.sup.B28-human insulin and any zinc complexes thereof;
[0054] N.sup..epsilon.B29-(Co--(CH.sub.2).sub.18--COOH)
ASp.sup.B28-human insulin and any zinc complexes thereof;
[0055] N.sup..epsilon.B29-(CO--(CH.sub.2).sub.20.times.CooH)
Asp.sup.B28-human insulin and any zinc complexes thereof;
[0056] N.sup..epsilon.B29-(CO--(CH.sub.2).sub.22--COOH)
Asp.sup.B28-human insulin and any zinc complexes thereof;
[0057] N.sup..epsilon.B30-(CO--(CH.sub.2).sub.14--COOH)
Asp.sup.B28L -humaninsulin and any zinc complexes thereof;
[0058] N.sup..epsilon.B30-(Co--(CH.sub.2).sub.16--COOH)
Thr.sup.B29Lys.sup.B30-human insulin and any zinc complexes
thereof;
[0059] N.sup..epsilon.B.sup.30-(CO--(CH.sub.2).sub.18--COOH)
Thr.sup.B29Lys.sup.B30-human insulin and any zinc complexes
thereof;
[0060] N.sup..epsilon.B30--(CO--(CH.sub.2).sub.28--COOH)
Thr.sup.B29Lys.sup.B30-human insulin and any zinc complexes
thereof;
[0061] N.sup..epsilon.B30-(CO--(CH.sub.2).sub.22--COOH)
Thr.sup.B29Lys.sup.B30-human insulin and any zinc complexes
thereof;
[0062] N.sup..epsilon.B.sup.28-(CO--(CH.sub.2).sub.14--COOH)
Lys.sup.B28Pro.sup.B29-human insulin and any zinc complexes
thereof;
[0063] N.sup..epsilon.B28-(CO--(CH.sub.2).sub.16--COOH)
LyS.sup.B28Pro.sup.B29-human insulin and any zinc complexes
thereof;
[0064] N.sup..epsilon.B28-(Co--(CH.sub.2).sub.16--COOH)
LyS.sup.B28Pro.sup.B29-human insulin and any zinc complexes
thereof;
[0065] N.sup..epsilon.B28--(CO--(CH.sub.2).sub.28--COOH)
LyS.sup.B28Pro.sup.B29-human insulin and any zinc complexes
thereof;
[0066] N.sup..epsilon.B28-(CO--(CH.sub.2).sub.22--COOH)
Lys.sup.B28Pro.sup.B29-human insulin and any zinc complexes
thereof;
[0067]
N.sup..epsilon.B28--(CO--(CH.sub.2).sub.22--COOH)Lys.sup.B28Pro.sup-
.B29-human insulin and any zinc complexes thereof;
[0068] N.sup..epsilon.B29-(CO--(CH.sub.2).sub.14--COOH) desB30
human insulin and any zinc complexes thereof;
[0069] N.sup..epsilon.B29-(CO--(CH.sub.2).sub.16--COOH) desB30
human insulin and any zinc complexes thereof;
[0070] N.sup..epsilon.B29--(CO--(CH.sub.2).sub.18--COOH) desB30
human insulin and any zinc complexes thereof.
[0071] N.sup..epsilon.B29-(CO--(CH.sub.2).sub.20--COOH) desB30
human insulin and any zinc complexes thereof; and
[0072] N.sup..epsilon.B29--(CO--(CH.sub.2).sub.22--COOH) desB30
human insulin and any zinc complexes thereof.
DETAILED DESCRIPTION OF THE INVENTION
[0073] Terminology
[0074] The three letter codes for the amino acid residues used
herein are those stated in J. Biol. Chem. 243, p. 3558 (1968).
[0075] The expression "a codable amino acid" is intended to
indicate an amino acid which can be coded for by the genetic code,
i.e. a triplet ("codon") of nucleotides.
[0076] Preparation of the Compounds of the Invention
[0077] The compounds of the invention can be prepared by methods
known per se. Thus, the group --CO--W--COOH of formula I can be
introduced into an insulin moiety via an activated ester or an
activated amide, e.g. an azolide, of the diacid HOOC-W-COOH. The
preparation of activated esters is described i.a. in EP 0 511 600
A2 (Kuraray Co., Ltd.) and in WO 95/07931 (Novo Nordisk A/S). The
preparation of azolides is described i.a. in W. Foerst, ed. Neure
Methoden Der Prparativen Organischen Chemie, Band V, p. 53-93
(Verlag Chemie, Weinheim (1967)).
[0078] The group --CO--W--COOH can be introduced into an insulin
moiety in which the amino group of the N-terminal amino groups of
the A-chain and the B-chain is protected. This is in analogy with
the methods described in in WO 95/07931. In this case a
deprotection step follows the introduction of the group
--CO--W--COOH as illustrated in the appended examples 1 and 2.
[0079] Alternatively, by selecting suitable reaction conditions as
described e.g. in EP 0 712 862 A2, it is possible to introduce the
group --CO--W--COOH selectively into the .epsilon.-amino group of a
Lys residue without resorting to protection of the N-terminal amino
groups of the A-chain and the B-chain. This is illustrated in the
appended examples 3 and 4.
[0080] Experimental Results Achieved with the Compounds of the
Invention.
[0081] Certain experimental data on the compounds of the invention
are given in Table 1.
[0082] Lipophilicity
[0083] The lipophilicity of the insulin derivatives relative to
human insulin, k".sub.rel, was measured on a LiChrosorb RP18 (5
.mu.m, 250.times.4 mm) HPLC column by isocratic elution at
40.degree. C. using mixtures of A) 0.1 M sodium phosphate buffer,
pH 7.3, containing 10% acetonitrile, and B) 50% acetonitrile in
water as eluents. The elution was monitored by following the UV
absorption of the eluate at 214 nm. Void time, t.sub.0, was found
by injecting 0.1 mM sodium nitrate. Retention time for human
insulin, t.sub.human, was adjusted to at least 2t.sub.0 by varying
the ratio between the A and B solutions.
k'.sub.rel=(t.sub.derivative-t.sub.0)/(t.sub.human-t.sub.0).
[0084] Determination of Disappearance Half-life, T.sub.50%, from
the Injection Site after Subcutaneous Injection of an Insulin
Derivative in Pigs.
[0085] T.sub.50% is the time when 50% of the A14
Tyr(.sup.125I)-labeled analogue has disappeared from the site of
injection as measured with an external .gamma.-counter (Ribel, U et
al., The Pig as a Model for Subcutaneous Absorption in Man. In: M.
Serrano-Rios and P. J. Lefebre (Eds): Diabetes 1985; Proceedings of
the 12th Congress of the International Diabetes Federation, Madrid,
Spain, 1985 (Excerpta Medica, Amsterdam, (1986) 891-96).
[0086] For use in the determination of T.sub.50% as described
above, samples of the products to be studied were iodinated with
.sup.125I using the standard lactoperoxidase method and the
Tyr.sup.A14-labeled product was isolated by isocratic ethanol/tris
HPLC.
[0087] Binding to Porcine Albumin.
[0088] The binding to porcine albumin was determined in an in vitro
assay. The values given in Table 1 under the heading "Albumin
binding" are relative to the reference compound EXA.
1 TABLE 1 Albumin Compound*.sup.) Lipophilicity T.sub.50%, hours
binding EX 1 1.4 5 0.7 EX 2 2.3 10 5 EX 3 17 18 51 EX 4 7.2 17.1 36
EX A 113 14 1 EX B 346 12 0.9 *.sup.)The compounds EX 1, EX 2, EX 3
and EX 4 are the title compounds of Examples 1, 2, 3 and 4,
respectively. The reference compound EX A is N.sup..epsilon.B29
-tetradecanoyl desB30 insulin and the reference compound EX B is
N.sup..epsilon.B29 -hexadecanoyl desB30 insulin.
[0089] Pharmaceutical Compositions
[0090] Pharmaceutical compositions containing an insulin derivative
according to the present invention may be administered parenterally
to patients in need of such a treatment. Parenteral administration
may be performed by subcutaneous, intramuscular or intravenous
injection by means of a syringe, optionally a pen-like syringe.
Alternatively, parenteral administration can be performed by means
of an infusion pump. A further option is a composition which may be
a powder or a liquid for the administration of the insulin
derivative in the form of a nasal spray. As a still further option,
it may also be possible to administer the insulin derivative
transdermally.
[0091] Pharmaceutical compositions containing an insulin derivative
of the present invention may be prepared by conventional
techniques, e.g. as described in Remington's Pharmaceutical
Sciences, 1985.
[0092] Thus, the injectable compositions of the insulin derivatives
of the invention can be prepared using the conventional techniques
of the pharmaceutical industry which involves dissolving and mixing
the ingredients as appropriate to give the desired end product.
[0093] Thus, according to one procedure, the insulin derivative is
dissolved in an amount of water which is somewhat less than the
final volume of the composition to be prepared. An isotonic agent,
a preservative and a buffer is added as required and the pH value
of the solution is adjusted--if necessary--using an acid, e.g.
hydrochloric acid, or a base, e.g. aqueous sodium hydroxide as
needed. Finally, the volume of the solution is adjusted with water
to give the desired concentration of the ingredients.
[0094] Examples of isotonic agents are sodium chloride, mannitol
and glycerol.
[0095] Examples of preservatives are phenol, m-cresol, methyl
p-hydroxybenzoate and benzyl alcohol.
[0096] Examples of suitable buffers are sodium acetate and sodium
phosphate.
[0097] Preferred pharmaceutical compositions of the particular
insulin derivatives of the present invention are solutions of
hexameric complexes. Typically, the hexameric complexes are
stabilised by two or more zinc ions and three or more molecules of
a phenolic compound like phenol or meta. cresol or mixtures thereof
per hexamer.
[0098] In a particular embodiment, a composition is provided which
contains two different insulins, one having a protracted profile of
action and one having a rapid onset of action, in the form of
soluble hexameric complexes. Typically the hexameric complexes are
stabilized by two or more zinc ions and three or more molecules of
a phenolic compound like phenol or meta-cresol or mixtures thereof
per hexamer. The complexes are mixtures of hexamers of the
particular insulins and mixed hexamers in which the ratio between
the two different insulins is from 1:5 to 5:1.
[0099] A composition for nasal administration of an insulin
derivative may, for example, be prepared as described in European
Patent No. 272097 (to Novo Nordisk A/S).
[0100] The insulin derivatives of this invention can be used in the
treatment of diabetes. The particular insulin derivative to be used
and the optimal dose level for any patient will depend on a variety
of factors including the efficacy of the specific insulin
derivative employed, the age, body weight, physical activity, and
diet of the patient, on a possible combination with other drugs,
and on the severity of the case. It is recommended that the dosage
of the insulin derivative of this invention be determined for each
individual patient by those skilled in the art in a similar way as
for known insulins.
[0101] The present invention is further illustrated by the
following examples which, however, are not to be construed as
limiting the scope of protection. The features disclosed in the
foregoing description and in the following examples may, both
separately and in any combination thereof, be material for
realizing the invention in diverse forms thereof.
EXAMPLES
[0102] The following acronyms for chemicals are used:
2 DMF: N,N-dimethylformamide. DIC: N,N'-diisopropylcarbodiimide.
HOBT: 1-hydroxybenzotriazole. TFA: trifluoroacetic acid.
[0103] Analytical
[0104] Molecular masses of the products prepared were obtained by
plasma desorption mass spectrometry (PDMS) using Bio-Ion 20
instrument (Bio-Ion Nordic AB, Uppsala, Sweden).
Example 1
[0105] Synthesis of N.sup..epsilon.B29-(CO--(CH).sub.12--COOH)
des(B30) human insulin.
[0106] Tetradecanedioic acid (Sigma, 10 mg), HOBT (10 mg) and
ethyldiisopropylamine (10 .mu.l) was dissolved in DMF (400 .mu.l)
and DIC (6 .mu.l) was added. The mixture was left at 25.degree. C.
for one hour and then DMF (600 .mu.l) and
A1,B1-(Boc).sub.2-des(B30) human insulin was added. After one hour
at 25.degree. C. water (200 .mu.l) was added and after further 15
minutes precipitation of the intermediate was achieved by addition
of methanol (1 ml) and ether (5 ml). The precipitate was isolated
by centrifugation, washed (twice) with ether and dried. The dry
intermediate was dissolved in TFA (1 ml) and after 15 minutes at
25.degree. C. the product was precipitated by addition of ether (5
ml). The precipitate was isolated by centrifugation, washed with
ether (three times) and dried.
[0107] Purification was carried out in a two-step reversed phase
HPLC process on a C18 reversed phase HPLC column. First step was an
isocratic run in ethanolltris-buffer (40% ethanol). The desired
material, which constituted the largest peak in the chromatogram,
was collected, desalted on a Sep-Pak.RTM. column, and
re-chromatographed in an acetonitrile/TFA gradient (20-60%
acetonitrile) with the product eluting at 45.8% acetonitrile. The
purity was estimated to be >95%.
[0108] The identity of the product was confirmed by PDMS (native,
reduced and digested with V8-protease) which gave MW's of 5947,
3571 and 1255 corresponding to native analogue, B-chain and the
C-terminal fragment of the B-chain, respectively.
EXAMPLE 2
[0109] Synthesis of
N.sup..epsilon.B29-(CO--(CH.sub.2).sub.14--COOH) des(B30) Human
Insulin.
[0110] The title compound was synthesized by proceeding as
described in Example 1, except that hexadecanedioic acid was used
instead of the tetradecanedioic acid.
[0111] The purification was carried out essentially as described in
Example 1. In the isocratic run ethanol/tris-buffer containing
42.4% ethanol was used. The desired material, which constituted the
largest peak in the chromatogram, was collected, desalted on a
Sep-Pak.RTM. column, and re-chromatographed in an acetonitrile/TFA
gradient (20-60% acetonitrile) with the product eluting at 48.2%
acetonitrile. The purity was estimated to be >95%.
[0112] The identity of the product was confirmed by PDMS (native,
reduced and digested with V8-protease) which gave MW's of 5976,
3601 and 1285 corresponding to native analogue, B-chain and the
C-terminal fragment of the B-chain, respectively.
Example 3
[0113] ynthesis of N.sup..epsilon.B29-(CO--(CH.sub.2).sub.18--COOH)
des(B30) Human Insulin.
[0114] 10 mg of eicosadioic acid, 10 mg of hydroxybenzotriazole and
2.5 .mu.l of diisopropylcarbodiimide were dissolved in 300 .mu.l of
of N-methylpyrrolidone and left at 25.degree. C for one hour. Then,
a solution of 150 mg of des(B30) human insulin in a mixture of 2 ml
of water, 2.6 ml of N-methyl pyrrolidone and 200 .mu.l of
diisopropylethylamine was added and the reaction mixture was left
at room temperature for one hour. The mixture was then diluted with
water, applied to a C18 reversed phase HPLC column and eluted with
tris buffer containing 48% of ethanol. Further purification was
achieved by reversed phase HPLC on the same column by eluting with
an acetonitrile/TFA gradient where the title compound eluted at 55%
acetonitrile.
Example 4
[0115] Synthesis of
N.sup..epsilon.B29-(CO--(CH.sub.2).sub.16--COOH) des(B30) Human
Insulin.
[0116] Des(B30) human insulin (99 mg.about.17.34 .mu.mol) was
dissolved in 6 ml N-methylpyrrolidone/water (30/70 v/v) and 84
.mu.l diisopropylethyl amine. 28.5 mg.about.69.4 .mu.mol of
N-(17-carboxyheptadecanoyloxy) succinimide (Mw 411) dissolved in
360 .mu.l N-methylpyrrolidone was added. After 1 h at room
temperature the reaction mixture was diluted with 6.5 ml ethanol,
the pH adjusted from 11.4 to 7.3 using 1 N HCl, and the dilution
subjected to anion exchange chromatography using a 1.times.25 cm
column packed with Source.TM. Q15 (Pharmacia Biotech). The column
was eluted at a rate of 40 ml/h using a linear gradient of KCl,
from 30 mM tris pH 7.3 buffer in 50% ethanol to 200 mM KCl, 30 mM
tris pH 7.3 buffer in 50% ethanol, and using 300 ml of each
solvent. The title compound emerged from the column after about 200
ml of eluent, and was collected in a volume of 15 ml. The pool was
diluted with 22.5 ml of water and the pH was adjusted to 6.0 using
1 N HCl. After precipitation overnight at 4.degree. C. the product
was isolated by centrifugation.
[0117] The precipitate was dissolved in 3.3 ml 20% acetonitrile
(v/v) in water and the title compound was purified using 2 runs on
a 1.times.25 cm column of dimethylbutyldimethyl substituted 5.mu.
silica spheres, having a pore size of 100 .ANG.ngstrom. Elution was
performed over 40 min at a rate of 5 ml/min, using a linear
gradient from 98/2 (v/v) of solvent A: 18.75 mM
(NH.sub.4).sub.2SO.sub.4, 12.5 mM tris pH 7.0 in 20% acetonitrile
and solvent B: 80% acetonitrile, to a ratio of 40/60 (v/v) of the
same solvents. The title compound emerged from the column after
21-24 min. The acetonitrile was removed by evaporation in vacuo,
and the product was desalted by gel filtration using PD-10
Sephadex.RTM. G-25M in 10 mM ammoniumhydrogencarbonate/ammonia
buffer pH 8.8. Finally, the product was isolated in the dry state
by lyophilization. Yield 43 mg. Purity 99%.
[0118] Molecular mass of title compound found by MS: 6000.+-.6;
theory: 6003.
Example 5
[0119] Crystallization of
N.sup..epsilon.B29-(CO--(CH.sub.2).sub.16--COOH) des(B30) Insulin
in the Presence of Zinc.
[0120] Zinc containing crystals of the title compound was obtained
in a tris-citrate buffer using a variety of conditions:
[0121] Insulin analogue: 7.5 mg/ml, range 2.5 to 20 mg/ml.
[0122] Zinc acetate: 1 mM.
[0123] Tris: 0.5 M
[0124] Trisodium citrate: 0.1-0.4 M.
[0125] Phenol or m-cresol: 0.05% (w/v), range 0.02-0.15%
[0126] pH: 8.2.
[0127] The crystals appear as birefringent, elongated
rhombohedra.
Example 6
[0128] Crystallization of
N.sup..epsilon.B29-(CO--(CH.sub.2).sub.16--COOH) des(B30) Insulin
in the Absence of Zinc.
[0129] Zinc free crystals of the title compound were obtained in
sodium acetate using a variety of conditions:
[0130] Insulin analogue: 20 mg/ml, range 10 to 40 mg/ml.
[0131] Sodium acetate: 0.35-0.5 M.
[0132] Ethanol: 18%, rangel5-25%.
[0133] Buffer: ammonium acetate/ammonia, 0.02 M, pH 9.0.
[0134] Phenol: optional, range 0-0.05%.
[0135] pH: 9.0
[0136] The crystals appear as non-birefringent, cubes or
rhombedodecahedrons.
Example 7
[0137] Pharmaceutical Preparations.
[0138] A pharmaceutical solution suitable for s. c. or i. m.
injection therapy may, for example, be composed as follows:
[0139] 600 nmol/ml (.about.600 .mu.M) of insulin analogue, e.g. of
N.sup..epsilon.B29-(CO--(CH.sub.2).sub.16--COOH) des(B30) human
insulin.
[0140] 5 mM sodium phosphate buffer, pH 7.5
[0141] 10 mM sodium chloride
[0142] 16 mM phenol
[0143] 16 mM m-cresol
[0144] 200-300 .mu.M zinc
[0145] 1.6% (w/v) glycerol
Sequence CWU 1
1
* * * * *