U.S. patent application number 12/781134 was filed with the patent office on 2010-09-09 for insulin derivatives.
This patent application is currently assigned to Novo Nordisk A/S. Invention is credited to Patrick William Garibay, Svend Havelund, Thomas Hoeg-Jensen, Palle Jakobsen, Ib Jonassen, Peter Madsen.
Application Number | 20100227796 12/781134 |
Document ID | / |
Family ID | 36095520 |
Filed Date | 2010-09-09 |
United States Patent
Application |
20100227796 |
Kind Code |
A1 |
Garibay; Patrick William ;
et al. |
September 9, 2010 |
Insulin Derivatives
Abstract
The present invention is related to insulin derivatives having a
side chain attached either to the .alpha.-amino group of the
N-terminal amino acid residue of B chain or to an .epsilon.-amino
group of a Lys residue present in the B chain of the parent insulin
molecule via an amide bond which side chain comprises one or more
residues of ethylenglycol, propyleneglycol and/or butyleneglycol
containing independently at each termini a group selected from
--NH.sub.2 and --COOH; a fatty diacid moiety with 4 to 22 carbon
atoms, at least one free carboxylic acid group or a group which is
negatively charged at neutral pH; and possible linkers which link
the individual components in the side chain together via amide or
ether bonds, said linkers optionally comprising a free carboxylic
acid group.
Inventors: |
Garibay; Patrick William;
(Holte, DK) ; Hoeg-Jensen; Thomas; (Klampenborg,
DK) ; Jonassen; Ib; (Valby, DK) ; Havelund;
Svend; (Bagsvaerd, DK) ; Madsen; Peter;
(Bagsvaerd, DK) ; Jakobsen; Palle; (Vaerlose,
DK) |
Correspondence
Address: |
NOVO NORDISK, INC.;INTELLECTUAL PROPERTY DEPARTMENT
100 COLLEGE ROAD WEST
PRINCETON
NJ
08540
US
|
Assignee: |
Novo Nordisk A/S
Bagsvaerd
DK
|
Family ID: |
36095520 |
Appl. No.: |
12/781134 |
Filed: |
May 17, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11814374 |
Jan 29, 2008 |
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PCT/EP06/50594 |
Feb 1, 2006 |
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12781134 |
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60651271 |
Feb 8, 2005 |
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Current U.S.
Class: |
514/6.3 ;
530/303 |
Current CPC
Class: |
A61P 3/10 20180101; C07K
14/62 20130101; A61K 38/00 20130101; A61K 47/60 20170801 |
Class at
Publication: |
514/3 ;
530/303 |
International
Class: |
A61K 38/28 20060101
A61K038/28; C07K 14/62 20060101 C07K014/62; A61P 3/10 20060101
A61P003/10 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 2, 2005 |
DK |
PA 2005 00156 |
Claims
1. An insulin derivatives having a side chain attached either to
the .alpha.-amino group of the N-terminal amino acid residue of B
chain or to an .epsilon.-amino group of a Lys residue present in
the B chain of the parent insulin molecule via an amide bond
wherein said side chain comprises one or more residues of
ethylenglycol, propyleneglycol and/or butyleneglycol containing
independently at each termini a group selected from --NH.sub.2 and
--COOH; a fatty diacid moiety with from 4 to 22 carbon atoms, at
least one free carboxylic acid group or a group which is negatively
charged at neutral pH; and possible linkers which link the
individual components in the side chain together via amide or ether
bonds, said linkers optionally comprising a free carboxylic acid
group.
2. The insulin derivative according to claim 1, wherein PEG or PPG
or PBG group has from 2 to 20; from 2 to 10 or from 2 to 5 residues
of ethyleneglycol, propyleneglycol or butyleneglycol.
3. The insulin derivative according to claim 1, wherein the
sidechain comprises a single residue of ethyleneglycol.
4. The insulin derivative according to claim 1, wherein the
sidechain comprises single residues of ethylenglycol,
propyleneglycol and butyleneglycol alone or in combination.
5. The insulin derivative according to claim 4, wherein the
sidechain comprises one residue of propyleneglycol and one residue
of butyleneglycol.
6. The insulin derivative according to claim 1, wherein the fatty
diacid comprises from 4 to 22 carbon atoms in the carbon chain.
7. The insulin derivative according to claim 6, wherein the fatty
diacid comprises from 6 to 22, from 8 to 20, from 8 to 18, from 4
to 18, from 6 to 18, from 8 to 16, from 8 to 22, from 8 to 17 or
from 8 to 15 carbon atoms in the carbon chain.
8. The insulin derivative according to claim 1, wherein the linker
is an amino acid residue, a peptide chain of 2-4 amino acid
residues or has the motif .alpha.-Asp, .beta.-Asp, .alpha.-Glu,
.gamma.-Glu, .alpha.-hGlu, .delta.-gGlu,
--N(CH.sub.2COOH)CH.sub.2CO--,
--N(CH.sub.2CH.sub.2COOH)CH.sub.2CH.sub.2CO--,
--N(CH.sub.2COOH)CH.sub.2CH.sub.2CO-- or
--N(CH.sub.2CH.sub.2COOH)CH.sub.2CO--
9. The insulin derivative according to claim 1, wherein the Lys
residue in the B chain of the parent insulin in either position B3,
B29 or in one of positions B23-30.
10. The insulin derivative according to claim 1 having the formula
##STR00071## wherein Ins is the parent insulin moiety which via the
.alpha.-amino group of the N-terminal amino acid residue of the B
chain or an .epsilon.-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.sub.1, Q.sub.2, Q.sub.3, and Q.sub.4 independently of each
other can be selected from: (CH.sub.2CH.sub.2O).sub.s--;
(CH.sub.2CH.sub.2CH.sub.2O).sub.s--;
(CH.sub.2CH.sub.2CH.sub.2CH.sub.2O).sub.s--;
(CH.sub.2CH.sub.2OCH.sub.2CH.sub.2CH.sub.2CH.sub.2O).sub.s-- or
(CH.sub.2CH.sub.2CH.sub.2OCH.sub.2CH.sub.2CH.sub.2CH.sub.2O).sub.s--
where s is 1-20; --(CH.sub.2).sub.r-- where r is an integer from 4
to 22; or a divalent hydrocarbon chain comprising 1, 2 or 3
--CH.dbd.CH-- groups and a number of --CH.sub.2-- groups sufficient
to give a total number of carbon atoms in the chain in the range of
4 to 22; --(CH.sub.2).sub.t-- or --(CH.sub.2OCH.sub.2).sub.t--,
where t is an integer from 1 to 6; --(CR.sub.1R.sub.2).sub.q--,
where R.sub.1 and R.sub.2 independently of each other can be H,
--COOH, (CH.sub.2).sub.1-6COOH and R.sub.1 and R.sub.2 can be
different at each carbon, and q is 1-6;
--((CR.sub.3R.sub.4).sub.q1).sub.1--(NHCO--(CR.sub.3R.sub.4).sub.q1--NHCO-
).sub.1-2--((CR.sub.3R.sub.4).sub.q1).sub.1 or
--((CR.sub.3R.sub.4).sub.q1).sub.1--(CONH--(CR.sub.3R.sub.4).sub.q1--CONH-
).sub.1-2--((CR.sub.3R.sub.4).sub.q1--)--,
--((CR.sub.3R.sub.4).sub.q1).sub.1--(NHCO--(CR.sub.3R.sub.4).sub.q1--CONH-
).sub.1-2--((CR.sub.3R.sub.4).sub.q1).sub.1 or
--((CR.sub.3R.sub.4).sub.q1).sub.1--(CONH--(CR.sub.3R.sub.4).sub.q1--NHCO-
).sub.1-2--((CR.sub.3R.sub.4).sub.q1).sub.1 where R.sub.3 and
R.sub.4 independently of each other can be H, --COOH, and R.sub.3
and R.sub.4 can be different at each carbon, and q.sub.1 is 1-6-;
and a bond; with the proviso that Q.sub.1-Q.sub.4 are different;
X.sub.1, X.sub.2 and X.sub.3 are independently selected from: O; a
bond; ##STR00072## where R is hydrogen or --(CH.sub.2).sub.p--COOH,
--(CH.sub.2).sub.p--SO.sub.3H, --(CH.sub.2).sub.p--PO.sub.3H.sub.2,
--(CH.sub.2).sub.p--O--SO.sub.3H;
--(CH.sub.2).sub.p--O--PO.sub.3H.sub.2; or
--(CH.sub.2).sub.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 selected
from: --COOH; --CO-Asp; --CO-Glu; --CO-Gly; --CO-Sar;
--CH(COOH).sub.2; --N(CH.sub.2COOH).sub.2; --SO.sub.3H;
--OSO.sub.3H; --OPO3H.sub.2, --PO.sub.3H.sub.2 and -tetrazol-5-yl
and any Zn.sup.2+ complex thereof.
11. The insulin derivative according to claim 10, wherein s is in
the range of 2-12, 2-4 or 2-3
12. The insulin derivative according to claim 10, wherein s is
preferably 1.
13. The insulin derivative according to claim 10, wherein Z is
--COOH.
14. The insulin derivative according to claim 1, wherein the parent
insulin is a desB30 human insulin analogue.
15. The insulin derivative according to claim 1, wherein the parent
insulin is selected from the group consisting of human insulin;
desB1 human insulin; desB30 human insulin; GlyA21 human insulin;
GlyA21 desB30 human insulin; AspB28 human insulin; porcine insulin;
LysB28 ProB29 human insulin; GlyA21 ArgB31 ArgB32 human insulin;
and LysB3 GIuB29 human insulin or AspB28 desB30 human insulin.
16. The insulin derivative according to claim 1 selected from the
group consisting of
N.sup..epsilon.B29-(3-[2-{2-(2-[.omega.-carboxy-pentadecanoyl-.gamma.-glu-
tamyl-(2-amino-ethoxy)]-ethoxy)-ethoxy}-ethoxy]-propinoyl) desB30
human insulin,
N.sup..epsilon.B29-(3-[2-{2-(2-[.omega.-carboxy-heptadecanoyl-.g-
amma.-glutamyl-(2-amino-ethoxy)]-ethoxy)-ethoxy}-ethoxy]-propinoyl)
desB30 human insulin,
N.sup..epsilon.B29-{3-[2-(2-{2-[2-(.omega.-carboxy-pentadecanoylamino)-et-
hoxy]-ethoxy}-ethoxy)-ethoxy]-propionyl-.gamma.-glutamyl desB30
human insulin,
N.sup..epsilon.B29-(.omega.-[2-(2-{2-[2-(2-carboxy-ethoxy)-ethox-
y]-ethoxy}-ethoxy)-ethylcarbamoyl]-heptadecanoyl-.alpha.-glutamyl)
desB30 human insulin,
N.sup..epsilon.B29-(.omega.-[2-(2-{2-[2-(2-carboxy-ethoxy)-ethoxy]-ethoxy-
}-ethoxy)-ethylcarbamoyl]-heptadecanoyl-.gamma.-glutamyl) desB30
human insulin,
N.sup..epsilon.B29-3-[2-(2-{2-[2-(.omega.-carboxy-heptadecanoyla-
mino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionyl-.gamma.-glutamyl
desB30 human insulin,
N.sup..epsilon.B29-(3-(3-{2-[2-(3-[7-carboxyheptanoylamino]propoxy)ethoxy-
]-ethoxy}propylcarbamoyl)propionyl) desB30 human insulin,
N.sup..epsilon.B29-(3-(3-{4-[3-(7-Carboxyheptanoylamino)propoxy]butoxy}pr-
opylcarbamoyl)-propionyl-.gamma.-glutamyl) desB30 human insulin,
N.sup..epsilon.B29-(3-(3-{2-[2-(3-[9-Carboxynonanoylamino]propoxy)ethoxy]-
ethoxy}-propylcarbamoyl)propionyl) desB30 human insulin,
N.sup..epsilon.B29-(3-(2-{2-[2-(9-carboxynonanoylamino)ethoxy]ethoxy}ethy-
lcarbamoyl)propionyl-.gamma.-glutamyl) desB30 human insulin,
N.sup..epsilon.B29-(3-(3-{4-[3-(9-Carboxynonanoylamino)propoxy]butoxy}-pr-
opylcarbamoyl)propionyl-.gamma.-glutamyl) desB30 human insulin,
N.sup..epsilon.B29-(2-[3-(2-(2-{2-(7-carboxyheptanoylamino)ethoxy}ethoxy)-
-ethylcarbamoyl]propionyl-.gamma.-glutamyl) desB30 human insulin,
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxypentadecanoylamino)etho-
xy]ethoxy}ethoxy)ethoxy]propionyl)) desB30 human insulin,
N.sup..epsilon.B29-(3-(2-{2-[2-(2-{2-[2-(2-{2-[2-(2-{2-[2-(.omega.-carbox-
y-tridecanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy-
]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionoyl-.gamma.-glutamyl)
desB30 human insulin,
N.sup..epsilon.B29'-(3-[2-(2-{2-[2-(.omega.-Carboxy-tridecanoylamino)-eth-
oxy]-ethoxy}-ethoxy)-ethoxy]-propionoyl-.gamma.-glutamyl) desB30
human insulin,
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(2-{2-[2-(.omega.-carboxy-trid-
ecanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-etho-
xy}-propionoyl-.gamma.-glutamyl) desB30 human insulin,
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-Carboxy-pentadecanoylamino)-ethox-
y]-ethoxy}-ethylcarbamoyl)-propionyl-.gamma.-glutamyl) desB30 human
insulin,
N.sup..epsilon.B29-(3-(3-{2-[2-(3-[.omega.-Carboxypentadecanoyla-
mino]propoxy)ethoxy]-ethoxy}propylcarbamoyl)propionyl-.gamma.-glutamyl)
desB30 human insulin,
N.sup..epsilon.B29-(3-(3-{4-[3-(.omega.-Carboxyundecanoylamino)propoxy]bu-
toxypropylcarbamoyl)propionyl-.gamma.-glutamyl) desB30 human
insulin,
N.sup..epsilon.B29-(3-(3-{4-[3-(.omega.-carboxytridecanoylamino)propoxy]b-
utoxypropyl-carbamoyl)propionyl-.gamma.-glutamyl) desB30 human
insulin,
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-Carboxyundecanoylamino)ethoxy]eth-
oxy}ethylcarbamoyl)propionyl-.gamma.-glutamyl) desB30 human
insulin,
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxytridecanoylamino)ethoxy]et-
hoxy}ethylcarbamoyl)-propionyl-.gamma.-glutamyl) desB30 human
insulin,
N.sup..epsilon.B29-{3-[2-(2-{2-[2-(.omega.-carboxy-pentadecanoylamino)eth-
oxy]ethoxy}ethoxy)ethoxy]propionyl-gamma-.gamma.-D-glutamyl) desB30
human insulin,
N.sup..epsilon.B28-{3-[2-(2-{2-[2-(7-carboxyheptanoylamino)ethox-
y]ethoxy}ethoxy)ethoxy]-propionyl-.gamma.-glutamyl} desB30 human
insulin,
N.sup..epsilon.B29-{3-[2-(2-{2-[2-(9-carboxynonanoylamino)ethoxy]ethoxy}e-
thoxy)ethoxy]propioniyl-.gamma.-glutamyll desB30 human insulin,
N.sup..epsilon.B29-{3-[2-(2-{2-[2-(.omega.-carboxyundecanoylamino)ethoxy]-
ethoxy}ethoxy)ethoxy]-propionyl-.gamma.-glutamyl} desB30 human
insulin,
N.sup..epsilon.B29-{3-[2-(2-{2-[2-(.omega.-carboxytridecanoylamino)ethoxy-
]ethoxy}ethoxy)ethoxy]propionyl-.gamma.-glutamyl} desB30 human
insulin.
17. A method of treating diabetes in a patient in need of such a
treatment, comprising administering to the patient a
therapeutically effective amount of an insulin derivative, said
insulin derivative having a side chain attached either to the
.alpha.-amino group of the N-terminal amino acid residue of B chain
or to an s-amino group of a Lys residue present in the B chain of
the parent insulin molecule via an amide bond which side chain
comprises one or more residues of ethylenglycol, propyleneglycol
and/or butyleneglycol containing independently at each termini a
group selected from --NH.sub.2 and --COOH; a fatty diacid moiety
with from 4 to 22 carbon atoms, at least one free carboxylic acid
group or a group which is negatively charged at neutral pH; and
possible linkers which link the individual components in the side
chain together via amide or ether bonds, said linkers optionally
comprising a free carboxylic acid group together with a
pharmaceutically acceptable carrier.
18. A method of treating diabetes in a patient in need of such a
treatment, comprising administering to the patient a
therapeutically effective amount of an insulin derivative, said
insulin derivative having a side chain attached either to the
.alpha.-amino group of the N-terminal amino acid residue of B chain
or to an s-amino group of a Lys residue present in the B chain of
the parent insulin molecule via an amide bond which side chain
comprises one or more residues of ethylenglycol, propyleneglycol
and/or butyleneglycol containing independently at each termini a
group selected from --NH.sub.2 and --COOH; a fatty diacid moiety
with from 4 to 22 carbon atoms, at least one free carboxylic acid
group or a group which is negatively charged at neutral pH; and
possible linkers which link the individual components in the side
chain together via amide or ether bonds, said linkers optionally
comprising a free carboxylic acid group in mixture with an insulin
or an insulin analogue which has a rapid onset of action, together
with a pharmaceutically acceptable carrier.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/814,374, filed Jul. 20, 2007, which is a 35 U.S.C. .sctn.371
national stage application of International Patent Application
PCT/EP2006/050594 (published as WO 2006/082205), filed Feb. 1,
2006, which claimed priority of Danish Patent Application PA 2005
00156, filed Feb. 2, 2005; this application further claims priority
under 35 U.S.C. .sctn.119 of U.S. Provisional Application
60/651,271, filed Feb. 8, 2005.
FIELD OF THE INVENTION
[0002] The present invention relates to novel human insulin
derivatives which are soluble at physiological pH values and have a
prolonged profile of action. The invention also relates to methods
of providing such derivatives, to pharmaceutical compositions
containing them, to a method of treating diabetes and
hyperglycaemia using the insulin derivatives of the invention and
to the use of such insulin derivatives in the treatment of diabetes
and hyperglycaemia.
BACKGROUND OF THE INVENTION
[0003] Currently, the treatment of diabetes, both type 1 diabetes
and type 2 diabetes, relies to an increasing extent on the
so-called intensive insulin treatment. According to this regimen,
the patients are treated with multiple daily insulin injections
comprising one or two daily injections of long acting insulin to
cover the basal insulin requirement supplemented by bolus
injections of a rapid acting insulin to cover the insulin
requirement related to meals.
[0004] Long acting insulin compositions are well known in the art.
Thus, one main type of long acting 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] Another type of long acting 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 with these solutions is that
the particle size distribution of the precipitate formed in the
tissue on injection, and thus the release profile of the
medication, depends on the blood flow at the injection site and
other parameters in a somewhat unpredictable manner. A further
drawback is that the solid particles of the insulin may act as a
local irritant causing inflammation of the tissue at the site of
injection.
[0007] 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 LysB29. 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.
[0008] 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.
[0009] JP laid-open patent application No. 1-254699 (Kodama Co.,
Ltd.) discloses insulin wherein a fatty acid is bound to the amino
group of PheB1 or to the .epsilon.-amino group of LysB29 or to both
of these. The stated purpose of the derivatisation is to obtain a
pharmacologically acceptable, stable insulin preparation.
[0010] Insulins, 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 porcine insulin antibodies.
[0011] WO 95/07931 (Novo Nordisk A/S) discloses human insulin
derivatives wherein the .epsilon.-amino group of LysB29 has a
lipophilic substituent. These insulin derivatives have a prolonged
profile of action and are soluble at physiological pH values.
[0012] EP 894095 discloses insulin derivatives wherein the
N-terminal 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 where W can be a long chain hydrocarbon group. These
insulin derivatives have a prolonged profile of action and are
soluble at physiological pH values.
[0013] Unfortunately, many diabetics are unwilling to undertake
intensive therapy due to the discomfort associated with the many
injections required to maintain close control of glucose levels.
This type of therapy can be both psychologically and physically
painful. Upon oral administration, insulin is rapidly degraded in
the gastro intestinal tract and is not absorbed into the blood
stream. Therefore, many investigators have studied alternate routes
for administering insulin, such as oral, rectal, transdermal, and
nasal routes. Thus far, however, these routes of administration
have not resulted in effective insulin absorption.
[0014] Efficient pulmonary delivery of a protein is dependent on
the ability to deliver the protein to the deep lung alveolar
epithelium. Proteins that are deposited in the upper airway
epithelium are not absorbed to a significant extent. This is due to
the overlying mucus which is approximately 30-40 .mu.m thick and
acts as a barrier to absorption. In addition, proteins deposited on
this epithelium are cleared by mucociliary transport up the airways
and then eliminated via the gastrointestinal tract. This mechanism
also contributes substantially to the low absorption of some
protein particles. The extent to which proteins are not absorbed
and instead eliminated by these routes depends on their solubility,
their size, as well as other less understood characteristics.
[0015] It is however well recognised that the properties of
peptides can be enhanced by grafting organic chain-like molecules
onto them. Such grafting can improve pharmaceutical properties such
as half life in serum, stability against proteolytical degradation,
and reduced immunogenicity.
[0016] The organic chain-like molecules often used to enhance
properties are polyethylene glycol-based or polyethylene based
chains, i.e., chains that are based on the repeating unit
--CH.sub.2CH.sub.2O--. Hereinafter, the abbreviation "PEG" is used
for polyethyleneglycol.
[0017] Classical PEG technology takes advantage of providing
polypeptides with increased size (Stoke radius) by attaching a
soluble organic molecule to the polypeptide (Kochendoerfer, G., et
al., Science (299) 884-, 2003). This technology leads to reduced
clearance in man and animals of a hormone polypeptide compared to
the native polypeptide. However this technique is often hampered by
reduced potency of the hormone polypeptides subjected to this
technique (Hinds, K., et al., Bioconjugate Chem. (11), 195-, 2000).
WO 02/20033 discloses a general method for the synthesis of well
defined polymer modified peptides.
[0018] However, there is still a need for insulins having a more
prolonged profile of action than the insulin derivatives known up
till now and which at the same time are soluble at physiological pH
values and have a potency which is comparable to that of human
insulin. Furthermore, there is need for further insulin
formulations which are well suited for pulmonary application.
SUMMARY OF THE INVENTION
[0019] The present invention is based on the recognition that
acylation of insulin with one or more residues of ethylenglycol,
propyleneglycol and/or butyleneglycol in combination with fatty
diacid residues has surprisingly shown a good bioavailability.
[0020] Organic chain-like molecules, which can be used to enhance
properties, are poly-ethyleneglycol based, polypropyleneglycol
based or polybutyleneglycol based chains, i.e., chains that are
based on the repeating unit CH.sub.2CH.sub.2O--,
CH.sub.2CH.sub.2CH.sub.2O-- or CH.sub.2CH.sub.2CH.sub.2CH.sub.2O--.
Hereinafter, the abbreviation "PEG" is used for polyethyleneglycol,
"PPG" is used for polypropyleneglycol and "PBG" is used for
polybutyleneglycol.
[0021] In one aspect the present invention is related to insulin
derivatives having a side chain attached either to the
.alpha.-amino group of the N-terminal amino acid residue of the B
chain or to an .epsilon.-amino group of a Lys residue present in
the B chain of the parent insulin molecule via an amide bond which
side chain comprises one or more residues of ethylenglycol,
propyleneglycol and/or butyleneglycol containing independently at
each termini a group selected from --NH.sub.2 and --COOH; a fatty
diacid moiety with 4 to 22 carbon atoms; at least one free
carboxylic acid group or a group which is negatively charged at
neutral pH; and possible linkers which link the individual
components in the side chain together via amide, ether or amine
bonds, said linkers optionally comprising a free carboxylic acid
group.
[0022] In one aspect the insulin derivatives contain a difunctional
PEG, PPG or PBG group that has from 2 to 20; from 2 to 10 or from 2
to 5 residues of ethyleneglycol, propyleneglycol or butyleneglycol,
respectively.
[0023] In one aspect the side chain of the insulin derivative
comprise one single residue of ethyleneglycol.
[0024] In one aspect the side chain of the insulin derivative
comprise one single residue of propyleneglycol.
[0025] In one aspect the side chain of the insulin derivative
comprise one single residue of butyleneglycol.
[0026] In one aspect the side chain of the insulin derivative has
single residues of ethylenglycol, propyleneglycol or butyleneglycol
alone or in combination.
[0027] In one aspect the side chain of the insulin derivative has
one residue of propyleneglycol and one residue of
butyleneglycol.
[0028] In one aspect the fatty diacid comprises from 4 to 22 carbon
atoms in the carbon chain.
[0029] In one aspect the fatty diacid comprises from 6 to 22, from
8 to 20, from 8 to 18, from 4 to 18, from 6 to 18, from 8 to 16,
from 8 to 22, from 8 to 17 or from 8 to 15 carbon atoms in the
carbon chain.
[0030] In one aspect the linker is an amino acid residue, a peptide
chain of 2-4 amino acid residues or has the motif is .alpha.-Asp;
.beta.-Asp; .alpha.-Glu; .delta.-Glu; .alpha.-hGlu; .delta.-hGlu;
--N(CH.sub.2COOH)CH.sub.2CO--;
--N(CH.sub.2CH.sub.2COOH)CH.sub.2CH.sub.2CO--;
--N(CH.sub.2COOH)CH.sub.2CH.sub.2CO-- or
--N(CH.sub.2CH.sub.2COOH)CH.sub.2CO--.
[0031] In one aspect the Lys residue in the B chain will be
position B3, B29 or in one of positions B23-B30.
[0032] In another aspect the invention is related to an insulin
derivative having the formula
##STR00001##
wherein Ins is the parent insulin moiety which via the
.alpha.-amino group of the N-terminal amino acid residue of the B
chain or an .epsilon.-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; [0033] Q.sub.1, Q.sub.2, Q.sub.3, and Q.sub.4 independently
of each other can be [0034] (CH.sub.2CH.sub.2O).sub.s--;
(CH.sub.2CH.sub.2CH.sub.2O).sub.s--;
(CH.sub.2CH.sub.2CH.sub.2CH.sub.2O).sub.s--;
(CH.sub.2CH.sub.2OCH.sub.2CH.sub.2CH.sub.2CH.sub.2O).sub.s-- or
(CH.sub.2CH.sub.2CH.sub.2OCH.sub.2CH.sub.2CH.sub.2CH.sub.2O).sub.s--
where s is 1-20 [0035] --(CH.sub.2).sub.r-- where r is an integer
from 4 to 22; or a divalent hydrocarbon chain comprising 1, 2 or 3
--CH.dbd.CH-- groups and a number of --CH.sub.2-- groups sufficient
to give a total number of carbon atoms in the chain in the range of
4 to 22; [0036] --(CH.sub.2).sub.t-- or
--(CH.sub.2OCH.sub.2).sub.t--, where t is an integer from 1 to 6;
[0037] --(CR.sub.1R.sub.2).sub.q--, where R.sub.1 and R.sub.2
independently of each other can be H, --COOH,
(CH.sub.2).sub.1-6COOH and R.sub.1 and R.sub.2 can be different at
each carbon, and q is 1-6, [0038]
--((CR.sub.3R.sub.4).sub.q1).sub.1--(NHCO--(CR.sub.3R.sub.4).sub.q1--NHCO-
).sub.1-2--((CR.sub.3R.sub.4).sub.q1).sub.1 or
--((CR.sub.3R.sub.4).sub.q1).sub.1--(CONH--(CR.sub.3R.sub.4).sub.q1--CONH-
).sub.1-2--((CR.sub.3R.sub.4).sub.q1--)--,
--((CR.sub.3R.sub.4).sub.q1).sub.1--(NHCO--(CR.sub.3R.sub.4).sub.q1--CONH-
).sub.1-2--((CR.sub.3R.sub.4).sub.q1).sub.1 or
--((CR.sub.3R.sub.4).sub.q1).sub.1--(CONH--(CR.sub.3R.sub.4).sub.q1--NHCO-
).sub.1-2--((CR.sub.3R.sub.4).sub.q1).sub.1 where R.sub.3 and
R.sub.4 independently of each other can be H, --COOH, and R.sub.3
and R.sub.4 can be different at each carbon, and q.sub.1 is 1-6-,
or [0039] a bond;
[0040] with the proviso that Q.sub.1-Q.sub.4 are different;
[0041] X.sub.1, X.sub.2 and X.sub.3 are independently [0042] O;
[0043] a bond; or
##STR00002##
[0044] where R is hydrogen or --(CH.sub.2).sub.p--COOH,
--(CH.sub.2).sub.p--SO.sub.3H, --(CH.sub.2).sub.p--PO.sub.3H.sub.2,
--(CH.sub.2).sub.p--O--SO.sub.3H;
--(CH.sub.2).sub.p--O--PO.sub.3H.sub.2; or
--(CH.sub.2).sub.p-tetrazol-5-yl, where each p independently of the
other p's is an integer in the range of 1 to 6; and
[0045] Z is:
[0046] --COOH;
[0047] --CO-Asp;
[0048] --CO-Glu;
[0049] --CO-Gly;
[0050] --CO-Sar;
[0051] --CH(COOH).sub.2,
[0052] --N(CH.sub.2COOH).sub.2;
[0053] --SO.sub.3H
[0054] --OSO.sub.3H
[0055] --OPO3H.sub.2
[0056] --PO.sub.3H.sub.2 or
[0057] -tetrazol-5-yl
[0058] and any Zn.sup.2+ complex thereof.
[0059] Where mentioned that R.sub.1, R.sub.2, R.sub.3 and R.sub.4
can be different at each carbon is meant that R.sub.1, R.sub.2,
R.sub.3 and R.sub.4 can be different for each value of q or
q.sub.1.
[0060] In one aspect r is from 6 to 22, from 8 to 20, from 8 to 18,
from 4 to 18, from 6 to 18 from 8 to 16 from 8 to 22 from 8 to 17
from 8 to 15.
[0061] In another aspect s is in the range of 2-12, 2-4 or 2-3.
[0062] In another aspect s is 1.
[0063] In one aspect n is from 1-6, from 2-6, from 2-5, from 2-4,
from 0-2 or from 2-3.
[0064] In one aspect q is from 1-5, from1-4, from 1-3 or from
1-2.
[0065] In one aspect q.sub.1 is from 1-5, from 1-4, from 1-3 or
from 1-2.
[0066] In one aspect t is from 1-6, from 1-5, from 1-4, from 1-3 or
from 1-2.
[0067] In one aspect Z is --COOH.
[0068] In one aspect Z is --CO-Asp.
[0069] In another aspect Z is --CO-Glu.
[0070] In another aspect Z is --CO-Gly.
[0071] In another aspect Z is --CO-Sar.
[0072] In another aspect Z is --CH(COOH).sub.2.
[0073] In another aspect Z is --N(CH.sub.2COOH).sub.2.
[0074] In another aspect Z is --SO.sub.3H.
[0075] In another aspect Z is --PO.sub.3H.
[0076] In another aspect Z is O--SO.sub.3H;
[0077] In another aspect Z is O--PO.sub.3H.sub.2;
[0078] In another aspect Z is tetrazol-5-yl.
[0079] In a further aspect the parent insulin is a desB30 human
insulin analogue.
[0080] Non limiting examples of parent insulins are human insulin;
desB1 human insulin; desB30 human insulin; GlyA21 human insulin;
GlyA21 desB30 human insulin; AspB28 human insulin; porcine insulin;
LysB28 ProB29 human insulin; GlyA21 ArgB31 ArgB32 human insulin;
LysB3 GIuB29 human insulin or AspB28 desB30 human insulin.
[0081] In a still further aspect the insulin derivative are
selected from the group consisting of
N.sup..epsilon.B29-(3-[2-{2-(2-[.omega.-carboxy-pentadecanoyl-.gamma.-glu-
tamyl-(2-amino-ethoxy)]-ethoxy)-ethoxy}-ethoxy]-propinoyl) desB30
human insulin,
N.sup..epsilon.B29-(3-[2-{2-(2-[.omega.-carboxy-heptadecanoyl-.d-
elta.-glutamyl-(2-amino-ethoxy)]-ethoxy)-ethoxyl-ethoxy]-propinoyl)
desB30 human insulin,
N.sup..epsilon.629-{3-[2-(2-{2-[2-(.omega.-carboxy-pentadecanoylamino)-et-
hoxy]-ethoxy}-ethoxy)-ethoxy]-propionyl-.gamma.-glutamyl desB30
human insulin,
N.sup..epsilon.B29-(.omega.-[2-(2-{2-[2-(2-carboxy-ethoxy)-ethox-
y]-ethoxy}-ethoxy)-ethylcarbamoyl]-heptadecanoyl-.alpha.-glutamyl)
desB30 human insulin,
N.sup..epsilon.B29-(.omega.-[2-(2-{2-[2-(2-carboxy-ethoxy)-ethoxy]-ethoxy-
l-ethoxy)-ethylcarbamoyl]-heptadecanoyl-.gamma.-glutamyl) desB30
human insulin,
N.sup..epsilon.B29-3-[2-(2-{2-[2-(.omega.-carboxy-heptadecanoyla-
mino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionyl-.gamma.-glutamyl
desB30 human insulin,
N.sup..epsilon.B29-(3-(3-{2-[2-(3-[7-carboxyheptanoylamino]propoxy)ethoxy-
]-ethoxy}propylcarbamoyl)propionyl) desB30 human insulin,
N.sup..epsilon.B29-(3-(3-{4-[3-(7-Carboxyheptanoylamino)propoxy]butoxy}pr-
opylcarbamoyl)-propionyl-.gamma.-glutamyl) desB30 human insulin,
N.sup..epsilon.B29-(3-(3-{2-[2-(3-[9-Carboxynonanoylamino]propoxy)ethoxy]-
ethoxy}-propylcarbamoyl)propionyl) desB30 human insulin,
N.sup..epsilon.B29-(3-(2-{2-[2-(9-carboxynonanoylamino)ethoxy]ethoxy}ethy-
lcarbamoyl)propionyl-.gamma.-glutamyl) desB30 human insulin,
N.sup..epsilon.B29-(3-(3-{4-[3-(9-Carboxynonanoylamino)propoxy]butoxy}-pr-
opylcarbamoyl)propionyl-.gamma.-glutamyl) desB30 human insulin,
N.sup..epsilon.B29-(2-[3-(2-(2-{2-(7-carboxyheptanoylamino)ethoxy}ethoxy)-
-ethylcarbamoyl]propionyl-.gamma.-glutamyl) desB30 human insulin,
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxypentadecanoylamino)etho-
xy]ethoxy}ethoxy)ethoxy]propionyl)) desB30 human insulin,
N.sup..epsilon.B29-(3-(2-{2-[2-(2-{2-[2-(2-{2-[2-(2-{2-[2-(.omega.-carbox-
y-tridecanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy-
]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionoyl-.gamma.-glutamyl)
desB30 human insulin,
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-Carboxy-tridecanoylamino)-etho-
xy]-ethoxy}-ethoxy)-ethoxy]-propionoyl-.gamma.-glutamyl) desB30
human insulin,
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(2-{2-[2-(.omega.-carboxy-trid-
ecanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-etho-
xy}-propionoyl-.gamma.-glutamyl) desB30 human insulin,
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-Carboxy-pentadecanoylamino)-ethox-
y]-ethoxy}-ethylcarbamoyl)-propionyl-.gamma.-glutamyl) desB30 human
insulin,
N.sup..epsilon.B29-(3-(3-{2-[2-(3-[.omega.-Carboxypentadecanoyla-
mino]propoxy)ethoxy]-ethoxy}propylcarbamoyl)propionyl-.gamma.-glutamyl)
desB30 human insulin,
N.sup..epsilon.B29-(3-(3-{4-[3-(.omega.-Carboxyundecanoylamino)propoxy]bu-
toxypropylcarbamoyl)propionyl-.gamma.-glutamyl) desB30 human
insulin,
N.sup..epsilon.B29-(3-(3-{4-[3-(.omega.-carboxytridecanoylamino)propoxy]b-
utoxypropyl-carbamoyl)propionyl-.gamma.-glutamyl) desB30 human
insulin,
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-Carboxyundecanoylamino)ethoxy]eth-
oxy}ethylcarbamoyl)propionyl-.gamma.-glutamyl) desB30 human
insulin,
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxytridecanoylamino)ethoxy]et-
hoxy}ethylcarbamoyl)-propionyl-.gamma.-glutamyl) desB30 human
insulin,
N.sup..epsilon.B29-{3-[2-(2-{2-[2-(.omega.-carboxy-pentadecanoylamino)eth-
oxy]ethoxy}ethoxy)ethoxy]propionyl-gamma-.gamma.-D-glutamyl) desB30
human insulin,
N.sup..epsilon.B29-{3-[2-(2-{2-[2-(7-carboxyheptanoylamino)ethox-
y]ethoxy}ethoxy)ethoxy]-propionyl-.gamma.-glutamyl} desB30 human
insulin,
N.sup..epsilon.B29-{3-[2-(2-{2-[2-(9-carboxynonanoylamino)ethoxy]ethoxy}e-
thoxy)ethoxy]propionyl-.gamma.-glutamyl} desB30 human insulin,
N.sup..epsilon.B29-{3-[2-(2-{2-[2-(.omega.-carboxyundecanoylamino)ethoxy]-
ethoxy}ethoxy)ethoxy]-propionyl-.gamma.-glutamyl} desB30 human
insulin,
N.sup..epsilon.B29-{3-[2-(2-{2-[2-(.omega.-carboxytridecanoylamino)ethoxy-
]ethoxy}ethoxy)ethoxy]propionyl-.gamma.-glutamyl} desB30 human
insulin.
[0082] Insulin derivatives according to the invention may be
provided in the form of essentially zinc free compounds or in the
form of zinc complexes. When zinc complexes of an insulin
derivative according to the invention are provided, two Zn.sup.2+
ions, three Zn.sup.2+ ions or four Zn.sup.2+ ions can be bound to
each insulin hexamer. Solutions of zinc complexes of the insulin
derivatives will contain mixtures of such species.
[0083] In a further aspect the invention is related to a
pharmaceutical composition comprising a therapeutically effective
amount of an insulin derivative according to the invention together
with a pharmaceutically acceptable carrier can be provided for the
treatment of type 1 diabetes, type 2 diabetes and other states that
cause hyperglycaemia in patients in need of such a treatment. An
insulin derivative according to the invention can be used for the
manufacture of a pharmaceutical composition for use in the
treatment of type 1 diabetes, type 2 diabetes and other states that
cause hyperglycaemia.
[0084] In a further aspect of the invention, there is provided a
pharmaceutical composition for treating type 1 diabetes, type 2
diabetes and other states that cause hyperglycaemia in a patient in
need of such a treatment, comprising a therapeutically effective
amount of an insulin derivative according to the invention in
mixture with an insulin or an insulin analogue which has a rapid
onset of action, together with pharmaceutically acceptable carriers
and additives.
[0085] In a further aspect the invention is related to a pulmonary
application for treating type 1 diabetes, type 2 diabetes and other
states that cause hyperglycaemia in a patient in need of such a
treatment, comprising a therapeutically effective amount of an
insulin derivative according to the invention optionally in mixture
with an insulin or an insulin analogue which has a rapid onset of
action, together with pharmaceutically acceptable carriers and
additives.
[0086] In one aspect the invention provides a pharmaceutical
composition being a mixture of an insulin derivative according to
the invention and a rapid acting insulin analogue selected group
consisting of AspB28 human insulin; LysB28 ProB29 human insulin and
LysB3 GluB29 human insulin.
[0087] The insulin derivative according to the invention and the
rapid acting insulin analogue can be mixed in a ratio from about
90/10%; about 70/30% or about 50/50%.
[0088] In a further aspect of the invention, there is provided a
method of treating type 1 diabetes, type 2 diabetes and other
states that cause hyperglycaemia in a patient in need of such a
treatment, comprising administering to the patient a
therapeutically effective amount of an insulin derivative according
to the invention together with a pharmaceutically acceptable
carrier and pharmaceutical acceptable additives.
[0089] In a further aspect of the invention, there is provided a
method of treating type 1 diabetes, type 2 diabetes and other
states that cause hyperglycaemia in a patient in need of such a
treatment, comprising administering to the patient a
therapeutically effective amount of an insulin derivative according
to the invention in mixture with an insulin or an insulin analogue
which has a rapid onset of action, together with a pharmaceutically
acceptable carrier and pharmaceutical acceptable additives.
[0090] In another aspect of the invention the insulin derivatives
has a side chain attached either to the .alpha.-amino group of the
N-terminal amino acid residue of B chain or to an .epsilon.-amino
group of a Lys residue present in the B chain of the parent insulin
molecule via an amide bond which side chain comprises a
monodisperse, diffunctionel PEG group containing independently at
each termini a group selected from --OH; --NH.sub.2 and --COOH; a
fatty diacid moiety with 4 to 22 carbon atoms, at least one free
carboxylic acid group or a group which is negatively charged at
neutral pH; and possible linkers which link the individual
components in the side chain together via amide, ether or amine
bonds, said linkers optionally comprising a free carboxylic acid
group.
[0091] In another aspect of the invention the PEG group of the
insulin derivative has from 1 to 20; from 1 to 10 or from 1 to 5
ethylene residues.
[0092] In another aspect of the invention the insulin derivatives
has a side chain attached either to the .alpha.-amino group of the
N-terminal amino acid residue of B chain or to an .epsilon.-amino
group of a Lys residue present in the B chain of the parent insulin
molecule via an amide bond which side chain comprises a
monodisperse, diffunctionel PEG group containing independently at
each termini a group selected from --OH; --NH.sub.2 and --COOH; a
fatty diacid moiety with 4 to 22 carbon atoms, at least one free
carboxylic acid group or a group which is negatively charged at
neutral pH; and possible linkers which link the individual
components in the side chain together via amide, ether or amine
bonds, said linkers optionally comprising a free carboxylic acid
group.
[0093] In a further aspect of the invention the insulin derivatives
comprises a difunctionel PEG group which has from 1 to 20; from 1
to 10 or from 1 to 5 ethylene units.
[0094] In a further aspect of the invention the insulin derivatives
comprises a fatty diacid which comprises from 4 to 22 carbon atoms
in the carbon chain.
[0095] In a further aspect of the invention the insulin derivatives
comprises a fatty acid, wherein the fatty diacid comprises from 6
to 22, from 8 to 20, from 8 to 18, from 4 to 18, from 6 to 18, from
8 to 16, from 8 to 22, from 8 to 17 or from 8 to 15 carbon atoms in
the carbon chain.
[0096] In a further aspect of the invention the insulin derivatives
comprises a linker wherein the linker is an amino acid residue, a
peptide chain of 2-4 amino acid residues or has the motif
.alpha.-Asp, .beta.-Asp, .alpha.-Glu, .gamma.-Glu, .alpha.-hGlu,
.delta.-hGlu, --N(CH.sub.2COOH)CH.sub.2CO--,
--N(CH.sub.2CH.sub.2COOH)CH.sub.2CH.sub.2CO--,
--N(CH.sub.2COOH)CH.sub.2CH.sub.2CO-- or
--N(CH.sub.2CH.sub.2COOH)CH.sub.2CO--
[0097] In a further aspect of the invention the insulin derivatives
comprises a Lys residue wherein the Lys residue in the B chain of
the parent insulin in in either position B3 or in one of positions
B23-30.
[0098] In a further aspect of the invention the insulin derivatives
has the formula
##STR00003##
wherein lns is the parent insulin moiety which via the
.alpha.-amino group of the N-terminal amino acid residue of the B
chain or an .epsilon.-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; [0099] each n is independently 0, 1, 2, 3,
4, 5 or 6; [0100] Q.sub.1, Q.sub.2, Q.sub.3, and Q.sub.4
independently of each other can be [0101]
(CH.sub.2CH.sub.2O).sub.s-- where s is 1-20, [0102]
--(CH.sub.2).sub.r-- where r is an integer from 4 to 22; or a
divalent hydrocarbon chain comprising 1, 2 or 3 --CH.dbd.CH--
groups and a number of --CH.sub.2-- groups sufficient to give a
total number of carbon atoms in the chain in the range of 4 to 22;
[0103] --(CH.sub.2).sub.t-- or --(CH.sub.2OCH.sub.2).sub.t--, where
t is an integer from 1 to 6; [0104] --(CR.sub.1R.sub.2).sub.q--,
where R.sub.1 and R.sub.2 independently of each other can be H,
--COOH, and R.sub.1 and R.sub.2 can be different at each carbon,
and q is 1-6, [0105]
--((CR.sub.3R.sub.4).sub.q1).sub.1--(NHCO--(CR.sub.3R.sub.4).sub.q1--NHCO-
).sub.1-2--((CR.sub.3R.sub.4).sub.q1).sub.1 or
--((CR.sub.3R.sub.4).sub.q1).sub.1--(CONH--(CR.sub.3R.sub.4).sub.q1--CONH-
).sub.1-2--((CR.sub.3R.sub.4).sub.q1--, where R.sub.3 and R.sub.4
independently of each other can be H, --COOH, and R.sub.3 and
R.sub.4 can be different at each carbon, and q.sub.1 is 1-6-, or
[0106] a bond;
[0107] with the proviso that Q.sub.1-Q.sub.4 are different;
[0108] X and V and G are independently [0109] O; [0110] a bond;
or
##STR00004##
[0111] where R is hydrogen or --(CH.sub.2).sub.p--COOH,
--(CH.sub.2).sub.p--SO.sub.3H, --(CH.sub.2).sub.p--PO.sub.3H.sub.2,
--(CH.sub.2).sub.p--O--SO.sub.3H;
--(CH.sub.2).sub.p--O--PO.sub.3H.sub.2; or
--(CH.sub.2).sub.p-tetrazolyl, where each p independently of the
other p's is an integer in the range of 1 to 6; and
[0112] Z is:
[0113] --COOH;
[0114] --CO-Asp;
[0115] --CO-Glu;
[0116] --CO-Gly;
[0117] --CO-Sar;
[0118] --CH(COOH).sub.2,
[0119] --N(CH.sub.2COOH).sub.2;
[0120] --SO.sub.3H
[0121] --OSO.sub.3H
[0122] --OPO3H.sub.2
[0123] --PO.sub.3H.sub.2 or
[0124] -tetrazolyl.
[0125] In a further aspect of the invention the insulin derivatives
according to the formula, s is from 6 to 22, from 8 to 20, from 8
to 18, from 4 to 18, from 6 to 18, from 8 to 16, from 8 to 22, from
8 to 17 or from 8 to 15.
[0126] In a further aspect of the invention the insulin derivatives
according to the formula s is from 1-20, from 1-10 or from 1-5.
[0127] In a further aspect of the invention the insulin derivative
according to the formula, Z is --COOH.
[0128] In a further aspect of the invention the insulin derivative
according to the invention, the parent insulin is a desB30 human
insulin analogue.
[0129] In a further aspect of the invention the insulin derivative
according to the invention, the parent insulin is selected from the
group consisting of human insulin; desB1 human insulin; desB30
human insulin; GlyA21 human insulin; GlyA21 desB30human insulin;
AspB28 human insulin; porcine insulin; LysB28ProB29 human insulin;
GlyA21ArgB31ArgB32 human insulin; and LysB3GluB29 human
insulin.
[0130] In a further aspect of the invention the insulin derivative
according the invention is selected from the group consisting of
N.sup..epsilon.B29--(N.sup..alpha.--(HOOC(CH.sub.2).sub.14CO)-.gamma.-L-G-
lu-HN(CH.sub.2CH.sub.2O).sub.4CH.sub.2CH.sub.2CO) desB30 human
insulin;
N.sup..epsilon.B29--(N.sup..alpha.--(HOOC(CH.sub.2).sub.16CO)-.gamma.-L-G-
lu-HN(CH.sub.2CH.sub.2O).sub.4CH.sub.2CH.sub.2CO) des(B30) human
insulin;
N.sup..epsilon.B29--(N.sup..alpha.--(HOOC(CH.sub.2CH.sub.2O).sub.4CH.sub.-
2CH.sub.2NH--OC(CH.sub.2).sub.16CO)-.alpha.-L-Glu-) des(B30) human
insulin;
N.sup..epsilon.B29-{3-[2-(2-{2-[2-(15-Carboxy-pentadecanoylamino-
)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionyl-gamma-Glu desB30
insulin; and
N.sup..epsilon.B29-3-[2-(2-{2-[2-(17-Carboxy-heptadecanoylamino)-ethoxy]--
ethoxy}-ethoxy)-ethoxy]-propionyl .gamma.-Glu desB30 insulin
[0131] In a further aspect of the invention there is provided 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 the
invention together with a pharmaceutically acceptable carrier.
[0132] In a further aspect of the invention there is provided 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 the
invention in mixture with an insulin or an insulin analogue which
has a rapid onset of action, together with a pharmaceutically
acceptable carrier.
[0133] In a further aspect of the invention there is provided a
pharmaceutical composition according to the invention intended for
pulmonal administration.
[0134] In a further aspect of the invention there is provided a
method of treating diabetes in a patient in need of such a
treatment, comprising administering to the patient a
therapeutically effective amount of an insulin derivative according
to claim 1 together with a pharmaceutically acceptable carrier.
[0135] In a further aspect of the invention there is provided a
method of treating diabetes in a patient in need of such a
treatment, comprising administering to the patient a
therapeutically effective amount of an insulin derivative according
to claim 1 in mixture with an insulin or an insulin analogue which
has a rapid onset of action, together with a pharmaceutically
acceptable carrier.
DETAILED DESCRIPTION OF THE INVENTION
[0136] The present insulin derivatives are characterized by having
a side chain attached to a Lys group in the B chain or to the
N-terminal amino group in the B-chain of the parent insulin
molecule which side chain comprises one or more residues of
ethylenglycol, propylene-glycol and/or butyleneglycol and a fatty
diacid moiety.
[0137] The insulin derivative according to the invention is
furthermore characterized in having at least one free carboxylic
acid group in the side chain and may comprise up to 2, 3 or 4 free
carboxylic acid groups or a group which is negatively charged at
neutral pH.
[0138] The insulin derivatives will only contain one lysine
residue. This lysine residue may either be in position B29 as in
human insulin or in one of position B3, B30 or B23 to B28.
[0139] The residues of ethylenglycol, propyleneglycol and/or
butyleneglycol will have any combination of the three groups --OH;
--NH.sub.2 and --COOH at each end. The residues of ethylenglycol,
propyleneglycol and/or butyleneglycol will typically be in the form
of an ethyleneglycol residue followed by a butyleneglycol residue
or have a chain length of 2 to 20 PEG, PPG or PBG residues
corresponding to a molecular weight of about 200 to 800.
[0140] The residues of ethylenglycol, propyleneglycol and/or
butyleneglycol will typically be in the form of an ethyleneglycol
residue followed by a butylen residue
--(CH.sub.2CH.sub.2OCH.sub.2CH.sub.2CH.sub.2CH.sub.2O).sub.m where
m is 1 to 20.
[0141] The difunctional PEG or PPG or PBG will have any combination
of the three groups --OH; --NH.sub.2, and --COOH at each end and
will typically have a chain length of 1 to 20 PEG residues
corresponding to a molecular weight of about 200 to 1000.
[0142] Non limiting examples of amino PEG moieties are
H.sub.2N--(CH.sub.2).sub.u--(OCH.sub.2CH.sub.2).sub.m--O(CH.sub.2).sub.u--
-COOH and
H.sub.2N--(CH.sub.2).sub.v--NH--CO--(CH.sub.2).sub.u--(OCH.sub.2-
CH.sub.2).sub.m--(CH.sub.2).sub.u--COOH, where u are independently
1 to 6, m is 2 to 20 and v is 1 to 6.
[0143] Non limiting examples of amino PPG moieties are
H.sub.2N--(CH.sub.2).sub.u--(OCH.sub.2CH.sub.2CH.sub.2).sub.m--O(CH.sub.2-
).sub.u--COOH and
H.sub.2N--(CH.sub.2).sub.v--NH--CO--(CH.sub.2).sub.u--(OCH.sub.2CH.sub.2C-
H.sub.2).sub.m--(CH.sub.2).sub.u--COOH, where u are independently 1
to 6, m is 2 to 20 and v is 1 to 6.
[0144] Non limiting examples of amino PBG moieties are
H.sub.2N--(CH.sub.2).sub.u--(OCH.sub.2CH.sub.2CH.sub.2CH.sub.2).sub.m--O(-
CH.sub.2).sub.u--COOH and
H.sub.2N--(CH.sub.2).sub.v--NH--CO--(CH.sub.2).sub.u--(OCH.sub.2CH.sub.2C-
H.sub.2CH.sub.2).sub.m--(CH.sub.2).sub.u--COOH, where u are
independently 1 to 6, m is 2 to 20 and v is 1 to 6.
[0145] The fatty diacid will typically comprise from 4 to 22, from
6 to 22, from 8 to 20, from 8 to 18, from 4 to 18, from 6 to 18,
from 8 to 16, from 8 to 22, from 8 to 12, from 8 to 10, from 8 to
17 or from 8 to 15 carbon atoms in the carbon chain.
[0146] Non limiting examples of the fatty diacid moiety are diacids
with the formula HOOC--(CH.sub.2).sub.r1--COOH, where r1 is 4 to
22. Examples of fatty diacids are succinic acid, hexanedioic acid,
octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid or octadecandedioic
acid.
[0147] The insulin moiety--in the present text also referred to as
the parent insulin--or insulin derivative according to the
invention can be a naturally occurring insulin such as human
insulin or porcine insulin. Alternatively, the parent insulin can
be an insulin analogue.
[0148] In one group of parent insulin analogues, the amino acid
residue at position A21 is Asn.
[0149] In another group of parent insulin analogues, the amino acid
residue at position B1 has been deleted. A specific example from
this group of parent insulin analogues is desB1 human insulin.
[0150] In another group of parent insulin analogues, the amino acid
residue at position B30 has been deleted. A specific example from
this group of parent insulin analogues is desB30 human insulin.
[0151] In another group of parent insulin analogues, the amino acid
residue at position B28 is Asp. A specific example from this group
of parent insulin analogues is AspB28 human insulin.
[0152] In another group of parent insulin analogues, the amino acid
residue at position B28 is Lys and the amino acid residue at
position B29 is Pro. A specific example from this group of parent
insulin analogues is LysB28 ProB29 human insulin.
[0153] In another group of parent insulin analogues the amino acid
residue in position B30 is Lys and the amino acid residue in
position B29 is any codable amino acid except Cys, Arg and Lys. An
example is an insulin analogue where the amino acid residue at
position B29 is Thr and the amino acid residue at position B30 is
Lys. A specific example from this group of parent insulin analogues
is ThrB29 LysB30 human insulin.
[0154] In another group of parent insulin analogues, the amino acid
residue at position B3 is Lys and the amino acid residue at
position B29 is Glu. A specific example from this group of parent
insulin analogues is LysB3 GluB29 human insulin.
[0155] The linkers will typically be an amino acid residue or a
chain of amino acid residue comprising up to four amino acids.
Thus, the linker may be selected from the group consisting of
.alpha.-Asp; .beta.-Asp; .alpha.-Glu; .gamma.-Glu; .alpha.-hGlu;
.delta.-hGlu; --N(CH.sub.2COOH)CH.sub.2CO--,
--N(CH.sub.2CH.sub.2COOH)CH.sub.2CH.sub.2CO--;
--N(CH.sub.2COOH)CH.sub.2CH.sub.2CO-- or
--N(CH.sub.2CH.sub.2COOH)CH.sub.2CO--
[0156] In a further aspect the linker can be a chain composed of
two amino acid residues of which one has from 4 to 10 carbon atoms
and a carboxylic acid group in the side chain while the other has
from 2 to 11 carbon atoms but no free carboxylic acid group. The
amino acid residue with no free carboxylic acid group can be a
neutral, codable .alpha.-amino acid residue. Examples of such
linkers are are: .alpha.-Asp-Gly; Gly-.alpha.-Asp; .beta.-Asp-Gly;
Gly-.beta.-Asp; .alpha.-Glu-Gly; Gly-.alpha.-Glu; .gamma.-Glu-Gly;
Gly-.gamma.-Glu; .alpha.-hGlu-Gly; Gly-.alpha.-hGlu;
.delta.-hGlu-Gly; and Gly-.delta.-hGlu.
[0157] In a further aspect the linker is a chain composed of two
amino acid residues, independently having from 4 to 10 carbon
atoms, and both having a carboxylic acid group in the side chain.
Examples of such linkers are: .alpha.-Asp-.alpha.-Asp;
.alpha.-Asp-.alpha.-Glu; .alpha.-Asp-.alpha.-hGlu;
.alpha.-Asp-.beta.-Asp; .alpha.-Asp-.gamma.-Glu;
.alpha.-Asp-.delta.-hGlu; .beta.-Asp-.alpha.-Asp;
.beta.-Asp-.alpha.-Glu; .beta.-Asp-.alpha.-hGlu;
.beta.-Asp-.beta.-Asp; .beta.-Asp-.gamma.-Glu;
.beta.-Asp-.delta.-hGlu; .alpha.-Glu-.alpha.-Asp;
.alpha.-Glu-.alpha.-Glu; .alpha.-Glu-.alpha.-hGlu;
.alpha.-Glu-.beta.-Asp; .alpha.-Glu-.delta.-Glu;
.alpha.-Glu-.delta.-hGlu; .gamma.-Glu-.alpha.-Asp;
.gamma.-Glu-.alpha.-Glu; .gamma.-Glu-.alpha.-hGlu;
.gamma.-Glu-.beta.-Asp; .gamma.-Glu-.gamma.-Glu;
.gamma.-Glu-.delta.-hGlu; .alpha.-hGlu-.alpha.-Asp;
.alpha.-hGlu-.alpha.-Glu; .alpha.-hGlu-.alpha.-hGlu;
.alpha.-hGlu-.beta.-Asp; .alpha.-hGlu-.gamma.-Glu;
.alpha.-hGlu-.delta.-hGlu; .delta.-hGlu-.alpha.-Asp;
.delta.-hGlu-.alpha.-Glu; .delta.-hGlu-.alpha.-hGlu;
.delta.-hGlu-.beta.-Asp; .delta.-hGlu-.gamma.-Glu; and
.delta.-hGlu-.delta.-hGlu.
[0158] In a further aspect the linker is a chain composed of three
amino acid residues, independently having from 4 to 10 carbon
atoms, the amino acid residues of the chain being selected from the
group of residues having a neutral side chain and residues having a
carboxylic acid group in the side chain so that the chain has at
least one residue which has a carboxylic acid group in the side
chain. In one aspect, the amino acid residues are codable
residues.
[0159] In a further aspect, the linker is a chain composed of four
amino acid residues, independently having from 4 to 10 carbon
atoms, the amino acid residues of the chain being selected from the
group having a neutral side chain and residues having a carboxylic
acid group in the side chain so that the chain has at least one
residue which has a carboxylic acid group in the side chain. In one
aspect, the amino acid residues are codable residues.
[0160] Examples of insulin derivatives according to the invention
are the following compounds:
[0161]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxyheptadecanoylamin-
o)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-.gamma.-glutamyl) desB30
human insulin;
[0162]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxyhexadecanoylamino-
)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-.gamma.-glutamyl) desB30
human insulin;
[0163]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxytridecanoylamino)-
ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-.gamma.-glutamyl) desB30
human insulin;
[0164]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxyundecanoylamino)e-
thoxy]ethoxy}ethoxy)-ethoxy]propionyl-.gamma.-glutamyl) desB30
human insulin;
[0165]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxynonanoylamino)eth-
oxy]ethoxy}ethoxy)-ethoxy]propionyl-.gamma.-glutamyl) desB30 human
insulin;
[0166]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxyheptanoylamino)et-
hoxy]ethoxy}ethoxy)-ethoxy]propionyl-.gamma.-glutamyl) desB30 human
insulin;
[0167]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxyheptadecanoylamin-
o)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl) desB30 human insulin;
[0168]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxyhexadecanoylamino-
)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl) desB30 human insulin;
[0169]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxytridecanoylamino)-
ethoxy]ethoxy}ethoxy)-ethoxy]propionyl) desB30 human insulin;
[0170]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxyundecanoylamino)e-
thoxy]ethoxy}ethoxy)-ethoxy]propionyl) desB30 human insulin;
[0171]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxynonanoylamino)eth-
oxy]ethoxy}ethoxy)-ethoxy]propionyl) desB30 human insulin;
[0172]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxyheptanoylamino)et-
hoxy]ethoxy}ethoxy)-ethoxy]propionyl) desB30 human insulin;
[0173]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyheptadecanoylamino)p-
ropoxy]ethoxy}-propylcarbamoyl)propionyl) desB30 human insulin;
[0174]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyhexadecanoylamino)pr-
opoxy]ethoxy}-propylcarbamoyl)propionyl) desB30 human insulin;
[0175]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxypentadecanoylamino)p-
ropoxy]ethoxy}-propylcarbamoyl)propionyl) desB30 human insulin;
[0176]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxytridecanoylamino)pro-
poxy]ethoxy}-propylcarbamoyl)propionyl) desB30 human insulin;
[0177]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyundecanoylamino)prop-
oxy]ethoxy}-propylcarbamoyl)propionyl) desB30 human insulin;
[0178]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxynonanoylamino)propox-
y]ethoxy}-propylcarbamoyl)propionyl) desB30 human insulin;
[0179]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyheptanoylamino)propo-
xy]ethoxy}-propylcarbamoyl)propionyl) desB30 human insulin;
[0180]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyheptadecanoylamino)p-
ropoxy]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) desB30
human insulin;
[0181]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyhexadecanoylamino)pr-
opoxy]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) desB30
human insulin;
[0182]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxypentadecanoylamino)p-
ropoxy]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) desB30
human insulin;
[0183]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxytridecanoylamino)pro-
poxy]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) desB30
human insulin;
[0184]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyundecanoylamino)prop-
oxy]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) desB30
human insulin;
[0185]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxynonanoylamino)propox-
y]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) desB30 human
insulin;
[0186]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyheptanoylamino)propo-
xy]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) desB30 human
insulin;
[0187]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxypentadecanoylamino)e-
thoxy]ethoxy}-ethylcarbamoyl)propionyl) desB30 human insulin;
[0188]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyhexadecanoylamino)et-
hoxy]ethoxy}-ethylcarbamoyl)propionyl) desB30 human insulin;
[0189]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxypentadecanoylamino)e-
thoxy]ethoxy}-ethylcarbamoyl)propionyl) desB30 human insulin;
[0190]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxytridecanoylamino)eth-
oxy]ethoxy}-ethylcarbamoyl)propionyl) desB30 human insulin;
[0191]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyundecanoylamino)etho-
xy]ethoxy}-ethylcarbamoyl)propionyl) desB30 human insulin;
[0192]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxynonanoylamino)ethoxy-
]ethoxy}-ethylcarbamoyl)propionyl) desB30 human insulin;
[0193]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyheptanoylamino)ethox-
y]ethoxy}-ethylcarbamoyl)propionyl) desB30 human insulin;
[0194]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyheptadecanoylamino)e-
thoxy]ethoxy}-ethylcarbamoyl)propionyl-.gamma.-glutamyl) desB30
human insulin;
[0195]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyhexadecanoylamino)et-
hoxy]ethoxy}-ethylcarbamoyl)propionyl-.gamma.-glutamyl) desB30
human insulin;
[0196]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxypentadecanoylamino)e-
thoxy]ethoxy}-ethylcarbamoyl)propionyl-.gamma.-glutamyl) desB30
human insulin;
[0197]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxytridecanoylamino)eth-
oxy]ethoxy}-ethylcarbamoyl)propionyl-.gamma.-glutamyl) desB30 human
insulin;
[0198]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyundecanoylamino)etho-
xy]ethoxy}-ethylcarbamoyl)propionyl-.gamma.-glutamyl) desB30 human
insulin;
[0199]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxynonanoylamino)ethoxy-
]ethoxy}-ethylcarbamoyl)propionyl-.gamma.-glutamyl) desB30 human
insulin
[0200]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyheptanoylamino)ethox-
y]ethoxy}-ethylcarbamoyl)propionyl-.gamma.-glutamyl) desB30 human
insulin;
[0201]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyheptadecanoylamin-
o)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
desB30 human insulin;
[0202]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxypentadecanoylamin-
o)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
desB30 human insulin;
[0203]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxypentadecanoylamin-
o)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
desB30 human insulin;
[0204]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxytridecanoylamino)-
propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
desB30 human insulin;
[0205]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyundecanoylamino)p-
ropoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
desB30 human insulin;
[0206]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxynonanoylamino)pro-
poxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
desB30 human insulin;
[0207]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyheptanoylamino)pr-
opoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
desB30 human insulin;
[0208]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyheptadecanoylamin-
o)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) desB30 human
insulin;
[0209]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyhexadecanoylamino-
)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) desB30 human
insulin;
[0210]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxypentadecanoylamin-
o)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) desB30 human
insulin;
[0211]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxytridecanoylamino)-
propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) desB30 human
insulin;
[0212]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyundecanoylamino)p-
ropoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) desB30 human
insulin;
[0213]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxynonanoylamino)pro-
poxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) desB30 human
insulin;
[0214]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyheptanoylamino)pr-
opoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) desB30 human
insulin;
[0215]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxyheptadecanoylamin-
o)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-.gamma.-glutamyl) human
insulin;
[0216]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxyhexadecanoylamino-
)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-.gamma.-glutamyl) human
insulin;
[0217]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxytridecanoylamino)-
ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-.gamma.-glutamyl) human
insulin;
[0218]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxyundecanoylamino)e-
thoxy]ethoxy}ethoxy)-ethoxy]propionyl-.gamma.-glutamyl) human
insulin;
[0219]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxynonanoylamino)eth-
oxy]ethoxy}ethoxy)-ethoxy]propionyl-.gamma.-glutamyl) human
insulin;
[0220]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxyheptanoylamino)et-
hoxy]ethoxy}ethoxy)-ethoxy]propionyl-.gamma.-glutamyl) human
insulin;
[0221]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxyheptadecanoylamin-
o)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl) human insulin;
[0222]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxyhexadecanoylamino-
)ethoxy]ethoxy}ethoxy)ethoxy]propionyl) human insulin
[0223]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxytridecanoylamino)-
ethoxy]ethoxy}ethoxy)ethoxy]propionyl) human insulin
[0224]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxyundecanoylamino)e-
thoxy]ethoxy}ethoxy)-ethoxy]propionyl) human insulin;
[0225]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxynonanoylamino)eth-
oxy]ethoxy}ethoxy)-ethoxy]propionyl) human insulin;
[0226]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxyheptanoylamino)et-
hoxy]ethoxy}ethoxy)-ethoxy]propionyl) human insulin;
[0227]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyheptadecanoylamino)p-
ropoxy]ethoxy}-propylcarbamoyl)propionyl) human insulin;
[0228]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyhexadecanoylamino)pr-
opoxy]ethoxy}-propylcarbamoyl)propionyl) human insulin;
[0229]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxypentadecanoylamino)p-
ropoxy]ethoxy}-propylcarbamoyl)propionyl) human insulin;
[0230]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxytridecanoylamino)pro-
poxy]ethoxy}-propylcarbamoyl)propionyl) human insulin;
[0231]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyundecanoylamino)prop-
oxy]ethoxy}-propylcarbamoyl)propionyl) human insulin;
[0232]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxynonanoylamino)propox-
y]ethoxy}-propylcarbamoyl)propionyl) human insulin;
[0233]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyheptanoylamino)propo-
xy]ethoxy}-propylcarbamoyl)propionyl) human insulin;
[0234]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyheptadecanoylamino)p-
ropoxy]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) human
insulin;
[0235]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyhexadecanoylamino)pr-
opoxy]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) human
insulin;
[0236]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxypentadecanoylamino)p-
ropoxy]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) human
insulin;
[0237]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxytridecanoylamino)pro-
poxy]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) human
insulin;
[0238]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyundecanoylamino)prop-
oxy]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) human
insulin;
[0239]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxynonanoylamino)propox-
y]ethoxy}propylcarbamoyl)propionyl-.gamma.-glutamyl) human
insulin
[0240]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyheptanoylamino)propo-
xy]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) human
insulin;
[0241]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxypentadecanoylamino)e-
thoxy]ethoxy}-ethylcarbamoyl)propionyl) human insulin;
[0242]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyhexadecanoylamino)et-
hoxy]ethoxy}-ethylcarbamoyl)propionyl) human insulin;
[0243]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxypentadecanoylamino)e-
thoxy]ethoxy}-ethylcarbamoyl)propionyl) human insulin;
[0244]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxytridecanoylamino)eth-
oxy]ethoxy}-ethylcarbamoyl)propionyl) human insulin;
[0245]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyundecanoylamino)etho-
xy]ethoxy}-ethylcarbamoyl)propionyl) human insulin;
[0246]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxynonanoylamino)ethoxy-
]ethoxy}-ethylcarbamoyl)propionyl) human insulin;
[0247]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyheptanoylamino)ethox-
y]ethoxy}-ethylcarbamoyl)propionyl) human insulin;
[0248]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyheptadecanoylamino)e-
thoxy]ethoxy}-ethylcarbamoyl)propionyl-.gamma.-glutamyl) human
insulin;
[0249]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyhexadecanoylamino)et-
hoxy]ethoxy}-ethylcarbamoyl)propionyl-.gamma.-glutamyl) human
insulin;
[0250]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxypentadecanoylamino)e-
thoxy]ethoxy}-ethylcarbamoyl)propionyl-.gamma.-glutamyl) human
insulin;
[0251]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxytridecanoylamino)eth-
oxy]ethoxy}ethylcarbamoyl)-propionyl-.gamma.-glutamyl) human
insulin;
[0252]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyundecanoylamino)etho-
xy]ethoxy}ethylcarbamoyl)propionyl-.gamma.-glutamyl) human
insulin
[0253]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxynonanoylamino)ethoxy-
]ethoxy}ethylcarbamoyl)-propionyl-.gamma.-glutamyl) human
insulin;
[0254]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyheptanoylamino)ethox-
y]ethoxy}ethylcarbamoyl)-propionyl-.gamma.-glutamyl) human
insulin;
[0255]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyheptadecanoylamin-
o)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
human insulin;
[0256]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxypentadecanoylamin-
o)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
human insulin;
[0257]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxypentadecanoylamin-
o)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
human insulin;
[0258]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxytridecanoylamino)-
propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
human insulin;
[0259]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyundecanoylamino)p-
ropoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
human insulin;
[0260]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxynonanoylamino)pro-
poxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
human insulin;
[0261]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyheptanoylamino)pr-
opoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
human insulin;
[0262]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyheptadecanoylamin-
o)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) human
insulin;
[0263]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyhexadecanoylamino-
)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) human
insulin;
[0264]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxypentadecanoylamin-
o)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) human
insulin;
[0265]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxytridecanoylamino)-
propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) human
insulin;
[0266]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyundecanoylamino)p-
ropoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) human insulin;
[0267]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxynonanoylamino)pro-
poxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) human insulin;
[0268]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyheptanoylamino)pr-
opoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) human insulin:
[0269]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxyheptadecanoylamin-
o)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-.gamma.-glutamyl) B28D
human insulin;
[0270]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxyhexadecanoylamino-
)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-.gamma.-glutamyl) B28D
human insulin;
[0271]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxytridecanoylamino)-
ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-.gamma.-glutamyl) B28D human
insulin;
[0272]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxyundecanoylamino)e-
thoxy]ethoxy}ethoxy)-ethoxy]propionyl-.gamma.-glutamyl) B28D human
insulin;
[0273]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxynonanoylamino)eth-
oxy]ethoxy}ethoxy)-ethoxy]propionyl-.gamma.-glutamyl) B28D human
insulin;
[0274]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxyheptanoylamino)et-
hoxy]ethoxy}ethoxy)-ethoxy]propionyl-.gamma.-glutamyl) B28D human
insulin;
[0275]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxyheptadecanoylamin-
o)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl) B28D human insulin;
[0276]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxyhexadecanoylamino-
)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl) B28D human insulin;
[0277]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxytridecanoylamino)-
ethoxy]ethoxy}ethoxy)-ethoxy]propionyl) B28D human insulin;
[0278]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxyundecanoylamino)e-
thoxy]ethoxy}ethoxy)-ethoxy]propionyl) B28D human insulin;
[0279]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxynonanoylamino)eth-
oxy]ethoxy}ethoxy)-ethoxy]propionyl) B28D human insulin;
[0280]
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(.omega.-carboxyheptanoylamino)et-
hoxy]ethoxy}ethoxy)-ethoxy]propionyl) B28D human insulin;
[0281]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyheptadecanoylamino)p-
ropoxy]ethoxy}-propylcarbamoyl)propionyl) B28D human insulin;
[0282]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyhexadecanoylamino)pr-
opoxy]ethoxy}-propylcarbamoyl)propionyl) B28D human insulin;
[0283]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxypentadecanoylamino)p-
ropoxy]ethoxy}-propylcarbamoyl)propionyl) B28D human insulin;
[0284]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxytridecanoylamino)pro-
poxy]ethoxy}-propylcarbamoyl)propionyl) B28D human insulin;
[0285]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyundecanoylamino)prop-
oxy]ethoxy}-propylcarbamoyl)propionyl) B28D human insulin;
[0286]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxynonanoylamino)propox-
y]ethoxy}-propylcarbamoyl)propionyl) B28D human insulin;
[0287]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyheptanoylamino)propo-
xy]ethoxy}-propylcarbamoyl)propionyl) B28D human insulin;
[0288]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyheptadecanoylamino)p-
ropoxy]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) B28D
human insulin;
[0289]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyhexadecanoylamino)pr-
opoxy]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) B28D
human insulin;
[0290]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxypentadecanoylamino)p-
ropoxy]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) B28D
human insulin;
[0291]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxytridecanoylamino)pro-
poxy]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) B28D human
insulin;
[0292]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyundecanoylamino)prop-
oxy]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) B28D human
insulin;
[0293]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxynonanoylamino)propox-
y]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) B28D human
insulin;
[0294]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyheptanoylamino)propo-
xy]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) B28D human
insulin;
[0295]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxypentadecanoylamino)e-
thoxy]ethoxy}-ethylcarbamoyl)propionyl) B28D human insulin;
[0296]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyhexadecanoylamino)et-
hoxy]ethoxy}-ethylcarbamoyl)propionyl) B28D human insulin;
[0297]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxypentadecanoylamino)e-
thoxy]ethoxy}-ethylcarbamoyl)propionyl) B28D human insulin;
[0298]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxytridecanoylamino)eth-
oxy]ethoxy}ethylcarbamoyl)-propionyl) B28D human insulin;
[0299]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyundecanoylamino)etho-
xy]ethoxy}-ethylcarbamoyl)propionyl) B28D human insulin;
[0300]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxynonanoylamino)ethoxy-
]ethoxy}ethylcarbamoyl)-propionyl) B28D human insulin;
[0301]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyheptanoylamino)ethox-
y]ethoxy}ethylcarbamoyl)-propionyl) B28D human insulin;
[0302]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyheptadecanoylamino)e-
thoxy]ethoxy}-ethylcarbamoyl)propionyl-.gamma.-glutamyl) B28D human
insulin;
[0303]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyhexadecanoylamino)et-
hoxy]ethoxy}-ethylcarbamoyl)propionyl-.gamma.-glutamyl) B28D human
insulin;
[0304]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxypentadecanoylamino)e-
thoxy]ethoxy}-ethylcarbamoyl)propionyl-.gamma.-glutamyl) B28D human
insulin;
[0305]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxytridecanoylamino)eth-
oxy]ethoxy}ethylcarbamoyl)-propionyl-.gamma.-glutamyl) B28D human
insulin;
[0306]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyundecanoylamino)etho-
xy]ethoxy}-ethylcarbamoyl)propionyl-.gamma.-glutamyl) B28D human
insulin;
[0307]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxynonanoylamino)ethoxy-
]ethoxy}ethylcarbamoyl)-propionyl-.gamma.-glutamyl) B28D human
insulin;
[0308]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyheptanoylamino)ethox-
y]ethoxy}ethylcarbamoyl)-propionyl-.gamma.-glutamyl) B28D human
insulin;
[0309]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyheptadecanoylamin-
o)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
B28D human insulin;
[0310]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxypentadecanoylamin-
o)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
B28D human insulin;
[0311]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxypentadecanoylamin-
o)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
B28D human insulin;
[0312]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxytridecanoylamino)-
propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
B28D human insulin;
[0313]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyundecanoylamino)p-
ropoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
B28D human insulin;
[0314]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxynonanoylamino)pro-
poxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
B28D human insulin;
[0315]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyheptanoylamino)pr-
opoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
B28D human insulin;
[0316]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyheptadecanoylamin-
o)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) B28D human
insulin;
[0317]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyhexadecanoylamino-
)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) B28D human
insulin;
[0318]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxypentadecanoylamin-
o)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) B28D human
insulin;
[0319]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxytridecanoylamino)-
propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) B28D human
insulin;
[0320]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyundecanoylamino)p-
ropoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) B28D human
insulin;
[0321]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxynonanoylamino)pro-
poxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) B28D human
insulin;
[0322]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyheptanoylamino)pr-
opoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) B28D human
insulin;
[0323]
N.sup..epsilon.B29-(3-[3-(2-{2-[2-(.omega.-carboxyheptadecanoylamin-
o)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-.gamma.-glutamyl) B28D,
desB30 human insulin;
[0324]
N.sup..epsilon.B29-(3-[3-(2-{2-[2-(.omega.-carboxyhexadecanoylamino-
)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-.gamma.-glutamyl) B28D,
desB30 human insulin;
[0325]
N.sup..epsilon.B29-(3-[3-(2-{2-[2-(.omega.-carboxytridecanoylamino)-
ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-.gamma.-glutamyl) B28D,
desB30 human insulin;
[0326]
N.sup..epsilon.B29-(3-[3-(2-{2-[2-(.omega.-carboxyundecanoylamino)e-
thoxy]ethoxy}ethoxy)-ethoxy]propionyl-.gamma.-glutamyl) B28D,
desB30 human insulin;
[0327]
N.sup..epsilon.B29-(3-[3-(2-{2-[2-(.omega.-carboxynonanoylamino)eth-
oxy]ethoxy}ethoxy)-ethoxy]propionyl-.gamma.-glutamyl) B28D, desB30
human insulin;
[0328]
N.sup..epsilon.B29-(3-[3-(2-{2-[2-(.omega.-carboxyheptanoylamino)et-
hoxy]ethoxy}ethoxy)ethoxy]-propionyl-.gamma.-glutamyl) B28D, desB30
human insulin;
[0329]
N.sup..epsilon.B29-(3-[3-(2-{2-[2-(.omega.-carboxyheptadecanoylamin-
o)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl) B28D, desB30 human
insulin;
[0330]
N.sup..epsilon.B29-(3-[3-(2-{2-[2-(.omega.-carboxyhexadecanoylamino-
)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl) B28D, desB30 human
insulin;
[0331]
N.sup..epsilon.B29-(3-[3-(2-{2-[2-(.omega.-carboxytridecanoylamino)-
ethoxy]ethoxy}ethoxy)-ethoxy]propionyl) B28D, desB30 human
insulin;
[0332]
N.sup..epsilon.B29-(3-[3-(2-{2-[2-(.omega.-carboxyundecanoylamino)e-
thoxy]ethoxy}ethoxy)-ethoxy]propionyl) B28D, desB30 human
insulin;
[0333]
N.sup..epsilon.B29-(3-[3-(2-{2-[2-(.omega.-carboxynonanoylamino)eth-
oxy]ethoxy}ethoxy)-ethoxy]propionyl) B28D, desB30 human
insulin;
[0334]
N.sup..epsilon.B29-(3-[3-(2-{2-[2-(.omega.-carboxyheptanoylamino)et-
hoxy]ethoxy}ethoxy)-ethoxy]propionyl) B28D, desB30 human
insulin;
[0335]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyheptadecanoylamino)p-
ropoxy]ethoxy}-propylcarbamoyl)propionyl) B28D, desB30 human
insulin;
[0336]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyhexadecanoylamino)pr-
opoxy]ethoxy}-propylcarbamoyl)propionyl) B28D, desB30 human
insulin;
[0337]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxypentadecanoylamino)p-
ropoxy]ethoxy}-propylcarbamoyl)propionyl) B28D, desB30 human
insulin;
[0338]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxytridecanoylamino)pro-
poxy]ethoxy}-propylcarbamoyl)propionyl) B28D, desB30 human
insulin;
[0339]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyundecanoylamino)prop-
oxy]ethoxy}-propylcarbamoyl)propionyl) B28D, desB30 human
insulin;
[0340]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxynonanoylamino)propox-
y]ethoxy}-propylcarbamoyl)propionyl) B28D, desB30 human
insulin;
[0341]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyheptanoylamino)propo-
xy]ethoxy}-propylcarbamoyl)propionyl) B28D, desB30 human
insulin;
[0342]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyheptadecanoylamino)p-
ropoxy]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) B28D,
desB30 human insulin;
[0343]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyhexadecanoylamino)pr-
opoxy]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) B28D,
desB30 human insulin;
[0344]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxypentadecanoylamino)p-
ropoxy]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) B28D,
desB30 human insulin;
[0345]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxytridecanoylamino)pro-
poxy]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) B28D,
desB30 human insulin;
[0346]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyundecanoylamino)prop-
oxy]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) B28D,
desB30 human insulin;
[0347]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxynonanoylamino)propox-
y]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) B28D, desB30
human insulin;
[0348]
N.sup..epsilon.B29-(3-(3-{2-[3-(.omega.-carboxyheptanoylamino)propo-
xy]ethoxy}-propylcarbamoyl)propionyl-.gamma.-glutamyl) B28D, desB30
human insulin;
[0349]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxypentadecanoylamino)e-
thoxy]ethoxy}-ethylcarbamoyl)propionyl) B28D, desB30 human
insulin;
[0350]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyhexadecanoylamino)et-
hoxy]ethoxy}-ethylcarbamoyl)propionyl) B28D, desB30 human
insulin;
[0351]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxypentadecanoylamino)e-
thoxy]ethoxy}-ethylcarbamoyl)propionyl) B28D, desB30 human
insulin;
[0352]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxytridecanoylamino)eth-
oxy]ethoxy}-ethylcarbamoyl)propionyl) B28D, desB30 human
insulin;
[0353]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyundecanoylamino)etho-
xy]ethoxy}-ethylcarbamoyl)propionyl) B28D, desB30 human
insulin;
[0354]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxynonanoylamino)ethoxy-
]ethoxy}-ethylcarbamoyl)propionyl) B28D, desB30 human insulin;
[0355]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyheptanoylamino)ethox-
y]ethoxy}-ethylcarbamoyl)propionyl) B28D, desB30 human insulin;
[0356]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyheptadecanoylamino)e-
thoxy]ethoxy}-ethylcarbamoyl)propionyl-.gamma.-glutamyl) B28D,
desB30 human insulin;
[0357]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyhexadecanoylamino)et-
hoxy]ethoxy}-ethylcarbamoyl)propionyl-.gamma.-glutamyl) B28D,
desB30 human insulin;
[0358]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxypentadecanoylamino)e-
thoxy]ethoxy}-ethylcarbamoyl)propionyl-.gamma.-glutamyl) B28D,
desB30 human insulin;
[0359]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxytridecanoylamino)eth-
oxy]ethoxy}-ethylcarbamoyl)propionyl-.gamma.-glutamyl) B28D, desB30
human insulin;
[0360]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyundecanoylamino)etho-
xy]ethoxy}-ethylcarbamoyl)propionyl-.gamma.-glutamyl) B28D, desB30
human insulin;
[0361]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxynonanoylamino)ethoxy-
]ethoxy}-ethylcarbamoyl)propionyl-.gamma.-glutamyl) B28D, desB30
human insulin;
[0362]
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxyheptanoylamino)ethox-
y]ethoxy}-ethylcarbamoyl)propionyl-.gamma.-glutamyl) B28D, desB30
human insulin;
[0363]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyheptadecanoylamin-
o)propoxy]ethoxy}-ethoxy)propylcarbamoyl]propionyl-.gamma.-glutamyl)
B28D, desB30 human insulin
[0364]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxypentadecanoylamin-
o)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
B28D, desB30 human insulin;
[0365]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxypentadecanoylamin-
o)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
B28D, desB30 human insulin;
[0366]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxytridecanoylamino)-
propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
B28D, desB30 human insulin;
[0367]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyundecanoylamino)p-
ropoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
B28D, desB30 human insulin;
[0368]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxynonanoylamino)pro-
poxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
B28D, desB30 human insulin;
[0369]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyheptanoylamino)pr-
opoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-.gamma.-glutamyl)
B28D, desB30 human insulin;
[0370]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyheptadecanoylamin-
o)propoxy]ethoxy}-ethoxy)propylcarbamoyl]propionyl) B28D, desB30
human insulin;
[0371]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyhexadecanoylamino-
)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) B28D, desB30
human insulin;
[0372]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxypentadecanoylamin-
o)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) B28D, desB30
human insulin;
[0373]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxytridecanoylamino)-
propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) B28D, desB30
human insulin;
[0374]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyundecanoylamino)p-
ropoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) B28D, desB30 human
insulin;
[0375]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxynonanoylamino)pro-
poxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) B28D, desB30 human
insulin; and
[0376]
N.sup..epsilon.B29-(3-[3-(2-{2-[3-(.omega.-carboxyheptanoylamino)pr-
opoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl) B28D, desB30 human
insulin
[0377] Representative formulas are:
##STR00005## ##STR00006## ##STR00007## ##STR00008## ##STR00009##
##STR00010## ##STR00011## ##STR00012##
[0378] In a further aspect, the present invention relates to
insulin derivatives which have an overall hydrophobicity which is
essentially similar to that of human insulin.
[0379] In a further aspect, the insulin derivatives of the present
invention have a hydrophobic index, k'rel, which is in the range
from about 0.02 to about 10, from about 0.1 to about 5; from about
0.5 to about 5; from about 0.2 to about 2; from about 0.2 to about
1; from about 0.1 to about 2; or from about 0.5 to about 2.
[0380] According to one aspect of the present invention, the
insulin derivatives will comprise a side chain of general formula
(I) as defined above which have at least one free carboxylic acid
group and according to a further aspect, the side chain will
optionally hold one or more free carboxylic acid groups.
[0381] The hydrophobicity (hydrophobic index) of the insulin
derivatives of the invention 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).
[0382] In another aspect, the invention relates to a pharmaceutical
composition comprising an insulin derivative according to the
invention which is soluble at physiological pH values.
[0383] In another aspect, the invention relates to a pharmaceutical
composition comprising an insulin derivative according to the
invention which is soluble at pH values in the interval from about
6.5 to about 8.5.
[0384] In another aspect, the invention relates to a pharmaceutical
composition with a prolonged profile of action which comprises an
insulin derivative according to the invention.
[0385] In another aspect, the invention relates to a pharmaceutical
composition which is a solution containing from about 120 nmol/ml
to about 2400 nmol/ml, from about 400 nmol/ml to about 2400
nmol/ml, from about 400 nmol/ml to about 1200 nmol/ml, from about
600 nmol/ml to about 2400 nmol/ml, or from about 600 nmol/ml to
about 1200 nmol/ml of an insulin derivative according to the
invention or of a mixture of the insulin derivative according to
the invention with a rapid acting insulin analogue.
[0386] The starting product for the acylation, the parent insulin
or insulin analogue or a precursor thereof can be produced by
either well-know peptide synthesis or by well known recombinant
production in suitable transformed microorganisms. Thus the insulin
starting product can be produced by a method which comprises
culturing a host cell containing a DNA sequence encoding the
polypeptide and capable of expressing the polypeptide in a suitable
nutrient medium under conditions permitting the expression of the
peptide, after which the resulting peptide is recovered from the
culture.
[0387] As an example desB30 human insulin can be produced from a
human insulin precursor B(1-29)-Ala-Ala-Lys-A(1-21) which is
produced in yeast as disclosed in U.S. Pat. No. 4,916,212. This
insulin precursor can then be converted into desB30 human insulin
by ALP cleavage of the Ala-Ala-Lys peptide chain to give desB30
human insulin which can then be acylated to give the present
insulintives.
[0388] The medium used to culture the cells may be any conventional
medium suitable for growing the host cells, such as minimal or
complex media containing appropriate supplements. Suitable media
are available from commercial suppliers or may be prepared
according to published recipes (e.g. in catalogues of the American
Type Culture Collection). The peptide produced by the cells may
then be recovered from the culture medium by conventional
procedures including separating the host cells from the medium by
centrifugation or filtration, precipitating the proteinaceous
components of the supernatant or filtrate by means of a salt, e.g.
ammonium sulphate, purification by a variety of chromatographic
procedures, e.g. ion exchange chromatography, gel filtration
chromatography, affinity chromatography, or the like, dependent on
the type of peptide in question.
[0389] The DNA sequence encoding the parent insulin may suitably be
of genomic or cDNA origin, for instance obtained by preparing a
genomic or cDNA library and screening for DNA sequences coding for
all or part of the polypeptide by hybridisation using synthetic
oligonucleotide probes in accordance with standard techniques (see,
for example, Sambrook, J, Fritsch, E F and Maniatis, T, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
New York, 1989). The DNA sequence encoding the parent insulin may
also be prepared synthetically by established standard methods,
e.g. the phosphoamidite method described by Beaucage and Caruthers,
Tetrahedron Letters 22 (1981), 1859-1869, or the method described
by Matthes et al., EMBO Journal 3 (1984), 801-805. The DNA sequence
may also be prepared by polymerase chain reaction using specific
primers, for instance as described in U.S. Pat. No. 4,683,202 or
Saiki et al., Science 239 (1988), 487-491.
[0390] The DNA sequence may be inserted into any vector which may
conveniently be subjected to recombinant DNA procedures, and the
choice of vector will often depend on the host cell into which it
is to be introduced. Thus, the vector may be an autonomously
replicating vector, i.e. a vector which exists as an
extrachromosomal entity, the replication of which is independent of
chromosomal replication, e.g. a plasmid. Alternatively, the vector
may be one which, when introduced into a host cell, is integrated
into the host cell genome and replicated together with the
chromosome(s) into which it has been integrated.
[0391] The vector is preferably an expression vector in which the
DNA sequence encoding the parent insulin is operably linked to
additional segments required for transcription of the DNA, such as
a promoter. The promoter may be any DNA sequence which shows
transcriptional activity in the host cell of choice and may be
derived from genes encoding proteins either homologous or
heterologous to the host cell. Examples of suitable promoters for
directing the transcription of the DNA encoding the parent insulin
in a variety of host cells are well known in the art, cf. for
instance Sambrook et al., supra.
[0392] The DNA sequence encoding the parent insulin may also, if
necessary, be operably connected to a suitable terminator,
polyadenylation signals, transcriptional enhancer sequences, and
translational enhancer sequences. The recombinant vector of the
invention may further comprise a DNA sequence enabling the vector
to replicate in the host cell in question.
[0393] The vector may also comprise a selectable marker, e.g. a
gene the product of which complements a defect in the host cell or
one which confers resistance to a drug, e.g. ampicillin, kanamycin,
tetracyclin, chloramphenicol, neomycin, hygromycin or
methotrexate.
[0394] To direct a peptide of the present invention into the
secretory pathway of the host cells, a secretory signal sequence
(also known as a leader sequence, prepro sequence or pre sequence)
may be provided in the recombinant vector. The secretory signal
sequence is joined to the DNA sequence encoding the peptide in the
correct reading frame. Secretory signal sequences are commonly
positioned 5' to the DNA sequence encoding the peptide. The
secretory signal sequence may be that normally associated with the
peptide or may be from a gene encoding another secreted
protein.
[0395] The procedures used to ligate the DNA sequences coding for
the parent insulin, the promoter and optionally the terminator
and/or secretory signal sequence, respectively, and to insert them
into suitable vectors containing the information necessary for
replication, are well known to persons skilled in the art (cf., for
instance, Sambrook et al., supra).
[0396] The host cell into which the DNA sequence or the recombinant
vector is introduced may be any cell which is capable of producing
the present peptide and includes bacteria, yeast, fungi and higher
eukaryotic cells. Examples of suitable host cells well known and
used in the art are, without limitation, E. coli, Saccharomyces
cerevisiae, or mammalian BHK or CHO cell lines.
[0397] The parent insulin molecule is then converted into the
insulin derivatives of the invention by introducing of the relevant
side chain in either the B1 position or in the chosen Lys position
in the B-chain. The side chain can be introduced by any convenient
method and many methods are disclosed in the prior art for
acylation of an amino group. More details will appear from the
following examples.
Pharmaceutical Compositions
[0398] The insulin derivatives of this invention of the claimed
formula can, for example, be administered subcutaneously, orally,
or pulmonary.
[0399] For subcutaneous administration, the compounds of the
formula are formulated analogously with the formulation of known
insulins. Furthermore, for subcutaneous administration, the
compounds of the formula are administered analogously with the
administration of known insulins and, generally, the physicians are
familiar with this procedure.
[0400] The insulin derivatives of this invention may be
administered by inhalation in a dose effective manner to increase
circulating insulin levels and/or to lower circulating glucose
levels. Such administration can be effective for treating disorders
such as diabetes or hyperglycemia. Achieving effective doses of
insulin requires administration of an inhaled dose of insulin
derivative of this invention of more than about 0.5 .mu.g/kg to
about 50 .mu.g/kg. A therapeutically effective amount can be
determined by a knowledgeable practitioner, who will take into
account factors including insulin level, blood glucose levels, the
physical condition of the patient, the patient's pulmonary status,
or the like.
[0401] According to the invention, insulin derivative of this
invention may be delivered by inhalation to achieve prolonged
duration of action. Administration by inhalation can result in
pharmacokinetics comparable to subcutaneous administration of
insulins. Different inhalation devices typically provide similar
pharmacokinetics when similar particle sizes and similar levels of
lung deposition are compared.
[0402] According to the invention, insulin derivative of this
invention may be delivered by any of a variety of inhalation
devices known in the art for administration of a therapeutic agent
by inhalation. These devices include metered dose inhalers,
nebulizers, dry powder generators, sprayers, and the like.
Preferably, insulin derivative of this invention is delivered by a
dry powder inhaler or a sprayer. There are a several desirable
features of an inhalation device for administering insulin
derivative of this invention. For example, delivery by the
inhalation device is advantageously reliable, reproducible, and
accurate. The inhalation device should deliver small particles, for
example, less than about 10 .mu.m, for example about 1-5 .mu.m, for
good respirability. Some specific examples of commercially
available inhalation devices suitable for the practice of this
invention are Turbohaler.TM. (Astra), Rotahaler.RTM. (Glaxo),
Diskus.RTM. (Glaxo), Spiros.TM. inhaler (Dura), devices marketed by
Inhale Therapeutics, AERx.TM. (Aradigm), the Ultravent.RTM.
nebulizer (Mallinckrodt), the Acorn II.RTM. nebulizer (Marquest
Medical Products), the Ventolin.RTM. metered dose inhaler (Glaxo),
the Spinhaler.RTM. powder inhaler (Fisons), or the like.
[0403] As those skilled in the art will recognize, the formulation
of insulin derivative of this invention, the quantity of the
formulation delivered, and the duration of administration of a
single dose depend on the type of inhalation device employed. For
some aerosol delivery systems, such as nebulizers, the frequency of
administration and length of time for which the system is activated
will depend mainly on the concentration of insulin conjugate in the
aerosol. For example, shorter periods of administration can be used
at higher concentrations of insulin conjugate in the nebulizer
solution. Devices such as metered dose inhalers can produce higher
aerosol concentrations, and can be operated for shorter periods to
deliver the desired amount of insulin conjugate. Devices such as
powder inhalers deliver active agent until a given charge of agent
is expelled from the device. In this type of inhaler, the amount of
insulin derivative of this invention in a given quantity of the
powder determines the dose delivered in a single
administration.
[0404] The particle size of insulin derivative of this invention in
the formulation delivered by the inhalation device is critical with
respect to the ability of insulin to make it into the lungs, and
preferably into the lower airways or alveoli. Preferably, the
insulin derivative of this invention is formulated so that at least
about 10% of the insulin conjugate delivered is deposited in the
lung, preferably about 10 to about 20%, or more. It is known that
the maximum efficiency of pulmonary deposition for mouth breathing
humans is obtained with particle sizes of about 2 .mu.m to about 3
.mu.m. When particle sizes are above about 5 .mu.m pulmonary
deposition decreases substantially. Particle sizes below about 1
.mu.m cause pulmonary deposition to decrease, and it becomes
difficult to deliver particles with sufficient mass to be
therapeutically effective. Thus, particles of the insulin
derivative delivered by inhalation have a particle size preferably
less than about 10 .mu.m, more preferably in the range of about 1
.mu.m to about 5 .mu.m. The formulation of the insulin derivative
is selected to yield the desired particle size in the chosen
inhalation device.
[0405] Advantageously for administration as a dry powder, an
insulin derivative of this invention is prepared in a particulate
form with a particle size of less than about 10 .mu.m, preferably
about 1 to about 5 .mu.m. The preferred particle size is effective
for delivery to the alveoli of the patient's lung. Preferably, the
dry powder is largely composed of particles produced so that a
majority of the particles have a size in the desired range.
Advantageously, at least about 50% of the dry powder is made of
particles having a diameter less than about 10 .mu.m. Such
formulations can be achieved by spray drying, milling, or critical
point condensation of a solution containing insulin conjugate and
other desired ingredients. Other methods also suitable for
generating particles useful in the current invention are known in
the art.
[0406] The particles are usually separated from a dry powder
formulation in a container and then transported into the lung of a
patient via a carrier air stream. Typically, in current dry powder
inhalers, the force for breaking up the solid is provided solely by
the patient's inhalation. In another type of inhaler, air flow
generated by the patient's inhalation activates an impeller motor
which deagglomerates the particles.
[0407] Formulations of insulin derivatives of this invention for
administration from a dry powder inhaler typically include a finely
divided dry powder containing the derivative, but the powder can
also include a bulking agent, carrier, excipient, another additive,
or the like. Additives can be included in a dry powder formulation
of insulin conjugate, for example, to dilute the powder as required
for delivery from the particular powder inhaler, to facilitate
processing of the formulation, to provide advantageous powder
properties to the formulation, to facilitate dispersion of the
powder from the inhalation device, to stabilize the formulation
(for example, antioxidants or buffers), to provide taste to the
formulation, or the like. Advantageously, the additive does not
adversely affect the patient's airways. The insulin derivative can
be mixed with an additive at a molecular level or the solid
formulation can include particles of the insulin conjugate mixed
with or coated on particles of the additive. Typical additives
include mono-, di-, and polysaccharides; sugar alcohols and other
polyols, such as, for example, lactose, glucose, raffinose,
melezitose, lactitol, maltitol, trehalose, sucrose, mannitol,
starch, or combinations thereof; surfactants, such as sorbitols,
diphosphatidyl choline, or lecithin; or the like. Typically an
additive, such as a bulking agent, is present in an amount
effective for a purpose described above, often at about 50% to
about 90% by weight of the formulation. Additional agents known in
the art for formulation of a protein such as insulin analogue
protein can also be included in the formulation.
[0408] A spray including the insulin derivatives of this invention
can be produced by forcing a suspension or solution of insulin
conjugate through a nozzle under pressure. The nozzle size and
configuration, the applied pressure, and the liquid feed rate can
be chosen to achieve the desired output and particle size. An
electrospray can be produced, for example, by an electric field in
connection with a capillary or nozzle feed. Advantageously,
particles of insulin conjugate delivered by a sprayer have a
particle size less than about 10 .mu.m, preferably in the range of
about 1 .mu.m to about 5 .mu.m.
[0409] Formulations of insulin derivatives of this invention
suitable for use with a sprayer will typically include the insulin
derivative in an aqueous solution at a concentration of about 1 mg
to about 20 mg of insulin conjugate per ml of solution. The
formulation can include agents such as an excipient, a buffer, an
isotonicity agent, a preservative, a surfactant, and, preferably,
zinc. The formulation can also include an excipient or agent for
stabilization of the insulin derivative, such as a buffer, a
reducing agent, a bulk protein, or a carbohydrate. Bulk proteins
useful in formulating insulin conjugates include albumin,
protamine, or the like. Typical carbohydrates useful in formulating
insulin conjugates include sucrose, mannitol, lactose, trehalose,
glucose, or the like. The insulin derivative formulation can also
include a surfactant, which can reduce or prevent surface-induced
aggregation of the insulin conjugate caused by atomization of the
solution in forming an aerosol. Various conventional surfactants
can be employed, such as polyoxyethylene fatty acid esters and
alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts
will generally range between about 0.001 and about 4% by weight of
the formulation.
[0410] Pharmaceutical compositions containing an insulin derivative
according to the present invention may also 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. Further options are to administer the
insulin nasally or pulmonally, preferably in compositions, powders
or liquids, specifically designed for the purpose.
[0411] Injectable compositions of the insulin derivatives of the
invention can be prepared using the conventional techniques of the
pharmaceutical industry which involve dissolving and mixing the
ingredients as appropriate to give the desired end product. Thus,
according to one procedure, an insulin derivative according to the
invention 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.
[0412] In a further aspect of the invention the buffer is selected
from the group consisting of sodium acetate, sodium carbonate,
citrate, glycylglycine, histidine, glycine, lysine, arginine,
sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium
phosphate, and tris(hydroxymethyl)-aminomethan, bicine, tricine,
malic acid, succinate, maleic acid, fumaric acid, tartaric acid,
aspartic acid or mixtures thereof. Each one of these specific
buffers constitutes an alternative aspect of the invention.
[0413] In a further aspect of the invention the formulation further
comprises a pharmaceutically acceptable preservative which may be
selected from the group consisting of phenol, o-cresol, m-cresol,
p-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate,
2-phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethanol, benzyl
alcohol, chlorobutanol, and thiomerosal, bronopol, benzoic acid,
imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol,
ethyl p-hydroxybenzoate, benzethonium chloride, chlorphenesine
(3p-chlorphenoxypropane-1,2-diol) or mixtures thereof. In a further
aspect of the invention the preservative is present in a
concentration from 0.1 mg/ml to 20 mg/ml. In a further aspect of
the invention the preservative is present in a concentration from
0.1 mg/ml to 5 mg/ml. In a further aspect of the invention the
preservative is present in a concentration from 5 mg/ml to 10
mg/ml. In a further aspect of the invention the preservative is
present in a concentration from 10 mg/ml to 20 mg/ml. Each one of
these specific preservatives constitutes an alternative aspect of
the invention. The use of a preservative in pharmaceutical
compositions is well-known to the skilled person. For convenience
reference is made to Remington: The Science and Practice of
Pharmacy, 19.sup.th edition, 1995.
[0414] In a further aspect of the invention the formulation further
comprises an isotonic agent which may be selected from the group
consisting of a salt (e.g. sodium chloride), a sugar or sugar
alcohol, an amino acid (e.g. L-glycine, L-histidine, arginine,
lysine, isoleucine, aspartic acid, tryptophan, threonine), an
alditol (e.g. glycerol (glycerine), 1,2-propanediol
(propyleneglycol), 1,3-propanediol, 1,3-butanediol)
polyethyleneglycol (e.g. PEG400), or mixtures thereof. Any sugar
such as mono-, di-, or polysaccharides, or water-soluble glucans,
including for example fructose, glucose, mannose, sorbose, xylose,
maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin,
cyclodextrin, soluble starch, hydroxyethyl starch and
carboxymethylcellulose-Na may be used. In one aspect the sugar
additive is sucrose. Sugar alcohol is defined as a C4-C8
hydrocarbon having at least one --OH group and includes, for
example, mannitol, sorbitol, inositol, galactitol, dulcitol,
xylitol, and arabitol. In one aspect the sugar alcohol additive is
mannitol. The sugars or sugar alcohols mentioned above may be used
individually or in combination. There is no fixed limit to the
amount used, as long as the sugar or sugar alcohol is soluble in
the liquid preparation and does not adversely effect the
stabilizing effects achieved using the methods of the invention. In
one aspect, the sugar or sugar alcohol concentration is between
about 1 mg/ml and about 150 mg/ml. In a further aspect of the
invention the isotonic agent is present in a concentration from 1
mg/ml to 50 mg/ml. In a further aspect of the invention the
isotonic agent is present in a concentration from 1 mg/ml to 7
mg/ml. In a further aspect of the invention the isotonic agent is
present in a concentration from 8 mg/ml to 24 mg/ml. In a further
aspect of the invention the isotonic agent is present in a
concentration from 25 mg/ml to 50 mg/ml. Each one of these specific
isotonic agents constitutes an alternative aspect of the invention.
The use of an isotonic agent in pharmaceutical compositions is
well-known to the skilled person. For convenience reference is made
to Remington: The Science and Practice of Pharmacy, 19.sup.th
edition, 1995.
[0415] Typical isotonic agents are sodium chloride, mannitol,
dimethyl sulfone and glycerol and typical preservatives are phenol,
m-cresol, methyl p-hydroxybenzoate and benzyl alcohol.
[0416] Examples of suitable buffers are sodium acetate,
glycylglycine, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid) and sodium phosphate.
[0417] A composition for nasal administration of an insulin
derivative according to the present invention may, for example, be
prepared as described in European Patent No. 272097 (to Novo
Nordisk A/S).
[0418] Compositions containing insulin derivatives of this
invention can be used in the treatment of states which are
sensitive to insulin. Thus, they can be used in the treatment of
type 1 diabetes, type 2 diabetes and hyperglycaemia for example as
sometimes seen in seriously injured persons and persons who have
undergone major surgery. 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 state to
be treated. It is recommended that the daily 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
insulin compositions.
[0419] Where expedient, the insulin derivatives of this invention
may be used in mixture with other types of insulin, e.g. insulin
analogues with a more rapid onset of action. Examples of such
insulin analogues are described e.g. in the European patent
applications having the publication Nos. EP 214826 (Novo Nordisk
A/S), EP 375437 (Novo Nordisk A/S) and EP 383472 (Eli Lilly &
Co.).
[0420] In a further aspect of the present invention the present
compounds are administered in combination with one or more further
active substances in any suitable ratios. Such further active
agents may be selected from antidiabetic agents, antihyperlipidemic
agents, antiobesity agents, antihypertensive agents and agents for
the treatment of complications resulting from or associated with
diabetes.
[0421] Suitable antidiabetic agents include insulin, GLP-1
(glucagon like peptide-1) derivatives such as those disclosed in WO
98/08871 (Novo Nordisk A/S), which is incorporated herein by
reference, as well as orally active hypoglycemic agents.
[0422] Suitable orally active hypoglycemic agents preferably
include imidazolines, sulfonylureas, biguanides, meglitinides,
oxadiazolidinediones, thiazolidinediones, insulin sensitizers,
.alpha.-glucosidase inhibitors, agents acting on the ATP-dependent
potassium channel of the pancreatic .beta.-cells eg potassium
channel openers such as those disclosed in WO 97/26265, WO 99/03861
and WO 00/37474 (Novo Nordisk A/S) which are incorporated herein by
reference, potassium channel openers, such as ormitiglinide,
potassium channel blockers such as nateglinide or BTS-67582,
glucagon antagonists such as those disclosed in WO 99/01423 and WO
00/39088 (Novo Nordisk A/S and Agouron Pharmaceuticals, Inc.), all
of which are incorporated herein by reference, GLP-1 agonists such
as those disclosed in WO 00/42026 (Novo Nordisk A/S and Agouron
Pharmaceuticals, Inc.), which are incorporated herein by reference,
DPP-IV (dipeptidyl peptidase-IV) inhibitors, PTPase (protein
tyrosine phosphatase) inhibitors, inhibitors of hepatic enzymes
involved in stimulation of gluconeogenesis and/or glycogenolysis,
glucose uptake modulators, GSK-3 (glycogen synthase kinase-3)
inhibitors, compounds modifying the lipid metabolism such as
antihyperlipidemic agents and antilipidemic agents, compounds
lowering food intake, and PPAR (peroxisome proliferator-activated
receptor) and RXR (retinoid X receptor) agonists such as ALRT-268,
LG-1268 or LG-1069.
Definitions
[0423] With "desB30 insulin", "desB30 human insulin" is meant a
natural insulin or an analogue thereof lacking the B30 amino acid
residue. Similarly, "desB29desB30 insulin or "desB29desB30 human
insulin" means a natural insulin or an analogue thereof lacking the
B29 and B30 amino acid residues.
[0424] With "B(1-29)" is meant a natural insulin B chain or an
analogue thereof lacking the B30 amino acid residue. "A(1-21)"
means the natural insulin A chain or an analogue thereof.
[0425] With "B1", "A1" etc. is meant the amino acid residue in
position 1 in the B chain of insulin (counted from the N-terminal
end) and the amino acid residue in position 1 in the A chain of
insulin (counted from the N-terminal end), respectively. The amino
acid residue in a specific position may also be denoted as e.g.
Phe.sup.B1 which means that the amino acid residue in position B1
is a phenylalanine residue.
[0426] With "Insulin" as used herein is meant human insulin with
disulfide bridges between Cys.sup.A7 and Cys.sup.B7 and between
Cys.sup.A20 and Cys.sup.B19 and an internal disulfide bridge
between Cys.sup.A6 and Cys.sup.A11, porcine insulin and bovine
insulin.
[0427] By "insulin analogue" as used herein is meant a polypeptide
which has a molecular structure which formally can be derived from
the structure of a naturally occurring insulin, for example that of
human insulin, by deleting and/or substituting at least one amino
acid residue occurring in the natural insulin and/or by adding at
least one amino acid residue. The added and/or substituted amino
acid residues can either be codable amino acid residues or other
naturally occurring amino acid residues or purely synthetic amino
acid residues.
[0428] The insulin analogues may be such wherein position 28 of the
B chain may be modified from the natural Pro residue to one of Asp,
Lys, or Ile. In another aspect Lys at position B29 is modified to
Pro. In one aspect B30 may be Lys and then B29 can be any codable
amino acid except Cys, Met, Arg and Lys. Also, Asn at position A21
may be modified to Ala, Gln, Glu, Gly, His, Ile, Leu, Met, Ser,
Thr, Trp, Tyr or Val, in particular to Gly, Ala, Ser, or Thr and
preferably to Gly. Furthermore, Asn at position B3 may be modified
to Lys or Asp. Further examples of insulin analogues are desB30
human insulin, desB30 human insulin analogues; insulin analogues
wherein one or both of B1 and B2 have been deleted; insulin
analogues wherein the A-chain and/or the B-chain have an N-terminal
extension and insulin analogues wherein the A-chain and/or the
B-chain have a C-terminal extension. Further insulin analogues are
such wherein. Thus one or two Arg may be added to position B1. Also
one or more of B26-B30 may have been deleted
[0429] By "insulin derivative" as used herein is meant a naturally
occurring insulin or an insulin analogue which has been chemically
modified, e.g. by introducing a side chain in one or more positions
of the insulin backbone or by oxidizing or reducing groups of the
amino acid residues in the insulin or by converting a free
carboxylic group to an ester group or acylating a free amino group
or a hydroxy group.
[0430] The expression "a codable amino acid" or "a codable amino
acid residue" is used to indicate an amino acid or amino acid
residue which can be coded for by a triplet ("codon") of
nucleotides.
[0431] .alpha.-Asp is the L-form of --HNCH(CO--)CH.sub.2COOH.
[0432] .beta.-Asp is the L-form of --HNCH(COOH)CH.sub.2CO--.
[0433] .alpha.-Glu is the L-form of
--HNCH(CO--)CH.sub.2CH.sub.2COOH.
[0434] .gamma.-Glu is the L-form of
--HNCH(COOH)CH.sub.2CH.sub.2CO--.
[0435] The expression "an amino acid residue having a carboxylic
acid group in the side chain" designates amino acid residues like
Asp, Glu and hGlu. The amino acids can be in either the L- or
D-configuration. If nothing is specified it is understood that the
amino acid residue is in the L configuration.
[0436] The expression "an amino acid residue having a neutral side
chain" designates amino acid residues like Gly, Ala, Val, Leu, Ile,
Phe, Pro, Ser, Thr, Cys, Met, Tyr, Asn and Gln.
[0437] When an insulin derivative according to the invention is
stated to be "soluble at physiological pH values" it means that the
insulin derivative can be used for preparing insulin compositions
that are fully dissolved at physiological pH values. Such
favourable solubility may either be due to the inherent properties
of the insulin derivative alone or a result of a favourable
interaction between the insulin derivative and one or more
ingredients contained in the vehicle.
[0438] The following abbreviations have been used in the
specification and examples:
[0439] Aad: Alpha-amino-adipic acid (homoglutamic acid)
[0440] Bzl=Bn: benzyl
[0441] CN: Alpha-cyano-4-hydroxycinnamic acid
[0442] DIEA: N, N-diisopropylethylamine
[0443] DMF: N,N-dimethylformamide
[0444] IDA: Iminodiacetic acid
[0445] Sar: Sarcosine (N-methyl-glycine)
[0446] tBu: tert-butyl
[0447] TSTU: O-(N-succinimidyI)-1,1,3,3-tetramethyluronium
tetrafluoroborate
[0448] THF: Tetrahydrofuran
[0449] EtOAc: Ethyl acetate
[0450] DIPEA: N, N-Diisopropylethylamine
[0451] HOAt: 1-Hydroxy-7-azabenzotriazole
[0452] TEA: Triethyl amine
[0453] SA: Sinapic acid
[0454] Su: succinimidyl=2,5-dioxo-pyrrolidin-1-yl
[0455] TFA: Trifluoracetic acid
[0456] DCM: Dichloromethane
[0457] DMSO: Dimethyl sulphoxide
[0458] PEG: Polyethyleneglycol
[0459] PBG: Poly-1,4-butyleneglycol
[0460] PPG: Poly-1,3-propyleneglycol
[0461] TLC: Thin Layer Chromatography
[0462] RT: Room temperature
[0463] With "fatty diacid" is meant a linear or branched
dicarboxylic acids having at least 2 carbon atoms and being
saturated or unsaturated. Non limiting examples of fatty diacids
are succinic acid, hexanedioic acid, octanedioic acid, decanedioic
acid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic
acid and octadecanedioic acid.
[0464] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference in
their entirety and to the same extent as if each reference were
individually and specifically indicated to be incorporated by
reference and were set forth in its entirety herein (to the maximum
extent permitted by law).
[0465] All headings and sub-headings are used herein for
convenience only and should not be construed as limiting the
invention in any way.
[0466] The use of any and all examples, or exemplary language
(e.g., "such as") provided herein, is intended merely to better
illuminate the invention and does not pose a limitation on the
scope of the invention unless otherwise claimed. No language in the
specification should be construed as indicating any non-claimed
element as essential to the practice of the invention.
[0467] The citation and incorporation of patent documents herein is
done for convenience only and does not reflect any view of the
validity, patentability, and/or enforceability of such patent
documents.
[0468] This invention includes all modifications and equivalents of
the subject matter recited in the claims appended hereto as
permitted by applicable law.
Examples
[0469] The following examples and general procedures refer to
intermediate compounds and final products identified in the
specification and in the synthesis schemes. The preparation of the
compounds of the present invention is described in detail using the
following examples, but the chemical reactions described are
disclosed in terms of their general applicability to the
preparation of compounds of the invention. Occasionally, the
reaction may not be applicable as described to each compound
included within the disclosed scope of the invention. The compounds
for which this occurs will be readily recognised by those skilled
in the art. In these cases the reactions can be successfully
performed by conventional modifications known to those skilled in
the art, that is, by appropriate protection of interfering groups,
by changing to other conventional reagents, or by routine
modification of reaction conditions. Alternatively, other reactions
disclosed herein or otherwise conventional will be applicable to
the preparation of the corresponding compounds of the invention. In
all preparative methods, all starting materials are known or may
easily be prepared from known starting materials. All temperatures
are set forth in degrees Celsius and unless otherwise indicated,
all parts and percentages are by weight when referring to yields
and all parts are by volume when referring to solvents and
eluents.
[0470] The compounds of the invention can be purified by employing
one or more of the following procedures which are typical within
the art. These procedures can--if needed--be modified with regard
to gradients, pH, salts, concentrations, flow, columns and so
forth. Depending on factors such as impurity profile, solubility of
the insulins in question etcetera, these modifications can readily
be recognised and made by a person skilled in the art.
[0471] After acidic HPLC or desalting, the compounds are isolated
by lyophilisation of the pure fractions.
[0472] After neutral HPLC or anion exchange chromatography, the
compounds are desalted, precipitated at isoelectrical pH, or
purified by acidic HPLC.
[0473] Typical Purification Procedures:
[0474] The HPLC system is a Gilson system consisting of the
following: Model 215 Liquid handler, Model 322-H2 Pump and a Model
155 UV Dector. Detection is typically at 210 nm and 280 nm.
[0475] The Akta Purifier FPLC system (Amersham Biosciences)
consists of the following: Model P-900 Pump, Model UV-900 UV
detector, Model pH/C-900 pH and conductivity detector, Model
Frac-950 Frction collector. UV detection is typically at 214 nm,
254 nm and 276 nm.
[0476] Acidic HPLC:
TABLE-US-00001 Column: Macherey-Nagel SP 250/21 Nucleusil 300-7 C4
Flow: 8 ml/min Buffer A: 0.1% TFA in acetonitrile Buffer B: 0.1%
TFA in water. Gradient: 0.0-5.0 min: 10% A 5.00-30.0 min: 10% A to
90% A 30.0-35.0 min: 90% A 35.0-40.0 min: 100% A
[0477] Neutral HPLC:
TABLE-US-00002 Column: Phenomenex, Jupiter, C4 5 .mu.m 250 .times.
10.00 mm, 300 .ANG. Flow: 6 ml/min Buffer A: 5 mM TRIS, 7.5 mM
(NH.sub.4).sub.2SO.sub.4, pH = 7.3, 20% CH.sub.3CN Buffer B: 60%
CH3CN, 40% water Gradient: 0-5 min: 10% B 5-35 min: 10-60% B 35-39
min: 60% B 39-40 min: 70% B 40-43.5 min: 70% B
[0478] Anion Exchange Chromatography:
TABLE-US-00003 00Column: RessourceQ, 1 ml Flow: 6 ml/min Buffer A:
0.09% NH.sub.4HCO.sub.3, 0.25% NH.sub.4OAc, 42.5% ethanol pH 8.4
Buffer B: 0.09% NH.sub.4HCO.sub.3, 2.5% NH.sub.4OAc, 42.5% ethanol
pH 8.4 Gradient: 100% A to 100% B during 30 column volumes
[0479] Desalting:
TABLE-US-00004 Column: HiPrep 26/10 Flow: 10 ml/min, 6 column
volumes Buffer: 10 mM NH.sub.4HCO.sub.3
[0480] Analytical Procedures:
[0481] Method 1:
TABLE-US-00005 Two Waters 510 HPLC pumps Waters 2487 Dual .lamda.
Absorbance detector Buffer A: 0.1% TFA in acetonitrile. Buffer B:
0.1% TFA in water. Flow: 1.5 ml/min. Gradient: 1-17 min: 25% B to
85% B, 17-22 min: 85% B, 22-23 min: 85% B to 25% B, 23-30 min 25%
B, 30-31 min 25% B flow: 0.15 ml/min. Column: C4 5.mu. 150 .times.
4.60 mm Phenomenex (Jupiter). Detection: UV 214 nm.
[0482] Method 2:
TABLE-US-00006 Two Waters 510 HPLC pumps Waters 2487 Dual .lamda.
Absorbance detector Buffer A: 0.1% TFA, 10% CH.sub.3CN, 89.9%
water. Buffer B: 0.1% TFA, 80% CH.sub.3CN, 19.9% water. Flow: 1.5
ml/min. Gradient: 0-17 min: 20%-90% B, 17-21 min 90% B. Column: C4
5.mu. 150 .times. 4.60 mm Phenomenex (Jupiter), kept at 40.degree.
C. Detection: UV 214 nm.
[0483] Method 3:Two Waters 510 HPLC Pumps
TABLE-US-00007 Waters 486 Tunable Absorbance Detector Waters 717
Autosampler Column: C4 5.mu. 150 .times. 4_60 mm Phenomenex
(Jupiter). Injection: 20 .mu.l. Buffer A: 80% 0.0125 M Tris, 0.0187
M (NH.sub.4).sub.2SO.sub.4 pH = 7, 20% CH.sub.3CN. Buffer B: 80%
CH.sub.3CN, 20% water. Flow: 1.5 ml/min. Gradient: 0 min 5% B ->
20 min 55% B -> 22 min 80% B -> 24 min 80% B -> 25 min 5%
B 32 min 5% B. Detection: UV 214 nm.
[0484] Method 4:
TABLE-US-00008 Two Waters 510 HPLC pumps Waters 2487 Dual .lamda.
Absorbance detector Column: C4 5.mu. 150 .times. 4.60 mm Phenomenex
(Jupiter). Injection: 20 .mu.l Buffer A: 80% 0.0125 M Tris, 0.0187
M (NH.sub.4).sub.2SO.sub.4 pH = 7, 20% CH.sub.3CN Buffer B: 80%
CH.sub.3CN, 20% water Flow: 1.5 ml/min Gradient: 0 min 10% B ->
20 min 50% B -> 22 min 60% B -> 23 min 10% B -> 30 min 10%
B -> 31 min 10% B flow 0.15 min Detection: 214 nm
[0485] Method 5:
TABLE-US-00009 Waters 2695 separations module Waters 996 Photodiode
Array Detector Column: C4 5.mu. 150 .times. 4.60 mm Phenomenex
(Jupiter). Injection: 25 .mu.l Buffer A: 80% 0.01 M Tris, 0.015 M
(NH.sub.4).sub.2SO.sub.4 pH = 7.3; 20% CH.sub.3CN Buffer B: 20%
water; 80% CH.sub.3CN Flow: 1.5 ml/min Gradient: 1-20 min: 5-50% B,
20-22 min: 50-60% B, 22-23 min: 60-5% B, 23-30 min 0% B 30-31 min
0-5% B, flow: 0.15 ml/min. Detection: 214 nm
[0486] Method 6:
[0487] Waters 2795 Separations Module
[0488] Waters 2996 Photodiode Array Detector
[0489] Waters Micromass ZQ 4000 Electrospray Mass Spectrometer
[0490] LC-Method:
TABLE-US-00010 Column: Phenomenex, Jupiter 5.mu. C4 300 .ANG. 50
.times. 4.60 mm Buffer A: 0.1% TFA in water Buffer B: CH.sub.3CN
Flow: 1 ml/min Gradient: 0-7.5 min: 10-90% B 7.5-8.5 min: 90-10% B
8.5-9.5 min 10% B 9.5-10.00 min 10% B, flow: 0.1 ml/min
[0491] MS method: Mw: 500-2000 ES+ [0492] Cone Voltage 60V [0493]
Scantime 1 [0494] Interscan delay: 0.1
[0495] Method 7:
TABLE-US-00011 Agilent 1100 series Column: GraceVydac Protein C4,
5um 4.6 .times. 250 mm (Cat# 214TP54) Buffer A: 10 mM Tris, 15 mM
(NH.sub.4).sub.2SO.sub.4, 20% CH3CN in water pH 7.3 Buffer B: 20%
water in CH3CN Flow: 1.5 ml/min Gradient: 1-20 min: 10% B to 50% B,
20-22 min: 50% B to 60% B, 22-23 min: 60% B to 10% B, 23-30 min 10%
B 30-31 min 10% B, flow 0.15 ml/min. Detection: 214 nm
[0496] Method 8: HPLC-MS
[0497] The following instrumentation is used: [0498] Hewlett
Packard series 1100 G1312A Bin Pump [0499] Hewlett Packard series
1100 G13 15A DAD diode array detector [0500] Sciex3000
triplequadropole mass spectrometer [0501] Gilson 215 micro injector
[0502] Sedex55 evaporative light scattering detector
[0503] Pumps and detectors are controlled by MassChrom 1.1.1
software running on a Macintosh G3 computer. Gilson Unipoint
Version 1.90 controls the auto-injector.
[0504] The HPLC pump is connected to two eluent reservoirs
containing:
[0505] A: 0.01% TFA in water
[0506] B: 0.01% TFA in acetonitrile
[0507] The analysis is performed at room temperature by injecting
an appropriate volume of the sample (preferably 10 .mu.l) onto the
column, which is eluted, with a gradient of acetonitrile. The
eluate from the column passed through the UV detector to meet a
flow splitter, which passed approximately 30 .mu.l/min (1/50)
through to the API Turbo ion-spray interface of API 3000
spectrometer. The remaining 1.48 ml/min (49/50) is passed through
to the ELS detector.
[0508] The HPLC conditions, detector settings and mass spectrometer
settings used are giving in the following table.
TABLE-US-00012 Column Waters X-Terra C18, 5.mu., 50 mm .times. 3 mm
id Gradient 5%-90% acetonitrile linearly during 7.5 min at 1.5
ml/min Detection 210 nm (analogue output from DAD) MS ionisation
mode API Turbo ion-spray ELS Gain 8 and 40.degree. C.
[0509] Method 9: HPLC-MS [0510] Hewlett Packard series 1100 G1312A
Bin Pump [0511] Hewlett Packard series 1100 Column compartment
[0512] Hewlett Packard series 1100 G1315A DAD diode array detector
[0513] Hewlett Packard series 1100 MSD [0514] Sedere 75 Evaporative
Light Scattering detector
[0515] The instrument was controlled by HP Chemstation
software.
[0516] The HPLC pump was connected to two eluent reservoirs
containing:
[0517] A: 0.01% TFA in water
[0518] B: 0.01% TFA in acetonitrile
[0519] The analysis is performed at 40.degree. C. by injecting an
appropriate volume of the sample (preferably 1 .mu.l) onto the
column which is eluted with a gradient of acetonitrile.
[0520] The HPLC conditions, detector settings and mass spectrometer
settings used are giving in the following:
TABLE-US-00013 Column: Waters Xterra MS C-18 .times. 3 mm id 5
.mu.m Gradient: 5%-100% acetonitrile linear during 7.5 min at 1.5
ml/min Detection: 210 nm (analogue output from DAD) ELS (analogue
output from ELS)
[0521] After the DAD the flow was divided yielding approx 1 ml/min
to the ELS and 0.5 ml/min to the MS.
[0522] MALDI-TOF-MS spectra were recorded on a Bruker Autoflex II
TOF/TOF operating in linear mode using a matrix of sinnapinic acid,
a nitrogen laser and positive ion detection. Accelerating voltage:
20 kV.
Example 1
Synthesis of
N.sup..epsilon.B29-(3-[2-{2-(2-[.omega.-carboxy-pentadecanoyl-.gamma.-dlu-
tamyl-(2-amino-ethoxy)]-ethoxy)-ethoxy}-ethoxy]-propinoyl) DesB30
Human Insulin
##STR00013##
[0524] DesB30 human insulin (400 mg, 0.070 mmol) was dissolved in
100 mM Na.sub.2CO.sub.3 (5 ml, pH 10.2) at room temperature.
Succinimidyl
3-[2-{2-(2-[.omega.-tert-butyl-carboxy-pentadecanoyl-.gamma.-glutamyl-.al-
pha.-tert-butyl-(2-amino-ethoxy)]-ethoxy)-ethoxy}-ethoxy]-propinoyl
(72 mg, 0.084 mmol, prepared as described below), was dissolved in
acetonitrile (5 ml) and subsequently added to the insulin solution.
After 30 mins, 0.2 M methylamine (0.5 ml) was added. pH was
adjusted by HCl to 5.5, and the isoelectric precipitate was
collected by centrifugation and dried in vacuo to give 345 mg. The
coupling yield was 64% (RP-HPLC, C4 column; Buffer A: 10% MeCN in
0.1% TFA-water, Buffer B: 80% MeCN in 0.1% TFA-water; gradient 20%
to 90% B in 16 minutes). The protected product was dissolved in TFA
(10 ml), left 30 mins, and evaporated in vacuo. The crude product
was dissolved in water and lyophilized.
[0525]
N.sup..epsilon.B29-(3-[2-{2-(2-[.omega.-carboxy-pentadecanoyl-.gamm-
a.-glutamyl-(2-amino-ethoxy)]-ethoxy)-ethoxy}-ethoxy]-propinoyl)
desB30 human was purified by RP-HPLC on C4-column, buffer A: 20%
EtOH+0.1% TFA, buffer B: 80% EtOH+0.1% TFA; gradient 15-60% B,
followed by HPLC on C4-column, buffer A: 10 mM Tris+15 mM ammonium
sulphate in 20% EtOH, pH 7.3, buffer B: 80% EtOH, gradient 15-60%
B. The collected fractions were desalted on Sep-Pak with 70%
acetonitrile+0.1% TFA, neutralized by addition of ammonia and
freeze-dried. The unoptimized yield was 60 mg, 13%. The purity as
evaluated by HPLC was >98%. MALDI-TOF-MS 6349,
C.sub.285H.sub.432N.sub.66O.sub.86S.sub.6 requires 6351.
[0526] Preparation of succinmidyl
3-[2-{2-(2-[.omega.-tert-butyl-carboxy-pentadecanoyl-.gamma.-glytamyl-.al-
pha.-tert-butyl-(2-amino-ethoxy)]-ethoxy)-ethoxy}-ethoxy]-propinoyl).
[0527] Hexadecadioic acid (40.0 g, 140 mmol) was suspended in
toluene (250 ml) and the mixture was heated to reflux.
N,N-dimethylformamide di-tert-butyl acetal (76.3 g, 375 mmol) was
added drop-wise over 4 hours. The mixture was refluxed overnight.
The solvent was removed in vacuo at 50.degree. C., and the crude
material was suspended in DCM/AcOEt (500 ml, 1:1) and stirred for
15 mins. The solids were collected by filtration and triturated
with DCM (200 ml). The filtrated were evaporated in vacuo to give
crude mono-tert-butyl hexadecandioate, 30 grams. This material was
suspended in DCM (50 ml), cooled with ice for 10 mins, and
filtered. The solvent was removed in vacuo to leave 25 gram crude
mono-tert-butyl hexadecandioate, which was recrystallized from
heptane (200 ml) to give mono-tert-butyl hexadecandioate, 15.9 g
(33%). Alternatively to recrystallization, the mono-ester can be
purified by silica chromatography in AcOEt/heptane.
[0528] .sup.1H-NMR (CDCl.sub.3) .delta.: 2.35 (t, 2H), 2.20 (t,
2H), 1.65-1.55 (m, 4H), 1.44 (s, 9H), 1.34-1.20 (m, 20H).
[0529] The mono tert-butyl ester (2 g, 5.8 mmol) was dissolved in
THF (20 ml) and treated with TSTU (2.1 g, 7.0 mmol) and DIEA (1.2
ml, 7.0 mmol) and stirred overnight. The mixture was filtered, and
the filtrate was evaporated in vacuo. The residue was dissolved in
AcOEt and washed twice with cold 0.1 M HCl and water. Drying over
MgSO4 and evaporation in vacuo gave succinimidyl tert-butyl
hexadecandioate, 2.02 g (79%).
[0530] 1H-NMR (CDCl3) .delta.: 2.84 (s, 4H), 2.60 (t, 2H), 2.20 (t,
2H), 1.74 (p, 2H), 1.56 (m, 2H), 1.44 (s, 9H), 1.40 (m, 2H),
1.30-1.20 (m, 18H).
[0531] Succinimidyl tert-butyl hexadecandioate (1 g, 2.27 mmol) was
dissolved in DMF (15 ml) and treated with L-Glu-OtBu (0.51 g, 2.5
mmol) and DIEA (0.58 ml, 3.41 mmol) and the mixture was stirred
overnight. The solvent was evaporated in vacuo, and the crude
product was dissolved in AcOEt, and washed twice with 0.2M HCl,
with water and brine. Drying over MgSO.sub.4 and evaporation in
vacuo gave w-tert-butyl
carboxy-pentadecanoyl-L-glutamyl-.alpha.-tert-butyl ester, 1.2 g
(100%).
[0532] 1H-NMR (CDCl3) .delta.: 6.25 (d, 1H), 4.53 (m, 1H), 2.42 (m,
2H), 2.21 (m, 4H), 1.92 (m, 1H), 1.58 (m, 4H), 1.47 (s, 9H), 1.43
(s, 9H), 1.43-1.22 (m, 18H).
[0533]
15-tert-butyl-carboxy-pentadecanyl-L-glutamyl-.alpha.-tert-butyl
ester (1.2 g, 2.27 mmol) was dissolved in THF (15 ml) and treated
with TSTU (0.82 g, 2.72 mmol) and DIEA (0.47 ml, 2.72 mmol) and
stirred overnight. The mixture was filtered, and the filtrate was
evaporated in vacuo. The residue was dissolved in AcOEt and washed
twice with cold 0.1 M HCl and water. Drying over MgSO.sub.4 and
evaporation in vacuo gave succinimidyl
w-tert-butyl-carboxy-pentadecanyl-L-glumtayl-.alpha.-tert-butyl
ester, 1.30 g (92%).
[0534] .sup.1H-NMR (CDCl.sub.3) .delta.: 6.17 (d, 1H), 4.60 (m,
1H), 2.84 (s, 4H), 2.72 (m, 1H), 2.64 (m, 1H), 2.32 (m, 1H), 2.20
(m, 4H), 2.08 (m, 1H), 1.6 (m, 4H), 1.47 (s, 9H), 1.43 (s, 9H),
1.33-1.21 (m, 20H).
[0535] Succinimidyl
15-tert-butyl-carboxy-pentadecanyl-L-glumtayl-.alpha.-tert-butyl
ester (109 mg, 0.17 mmol) was dissolved in DCM (2 ml) and treated
with 3-(2-{2-[2-(2-amino-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionic
acid (51 mg, 0.19 mmol, Quanta Biodesign, OH, USA) and DIEA (45
.mu.L, 0.26 mmol). The mixture was stirred overnight and evaporated
in vacuo. The residue was dissolved in AcOEt and washed twice with
cold 0.2 M HCl, water and brine. Drying over MgSO.sub.4 and
evaporation in vacuo gave
3-[2-{2-(2-[.omega.-tert-butyl-carboxy-pentadecanoyl-.gamma.-glumtayl-.al-
pha.-tert-butyl-(2-amino-ethoxy)]-ethoxy)-ethoxy}-ethoxy]-propionic
acid), 119 mg (88%).
[0536] .sup.1H-NMR (CDCl.sub.3) .delta.: 7.01 (t, 1H), 6.58 (d,
1H), 4.42 (m, 1H), 3.76 (d, 2H), 3.62 (m, 16H), 3.55 (t, 2H), 3.42
(m, 1H), 2.58 (t, 2H), 2.28 (m, 2H), 2.17 (m, 2H), 2.11 (m, 1H),
1.94 (m, 1H), 1.57 (m, 4H), 1.43 (s, 9H), 1.42 (s, 9H), 1.22 (m,
20H).
[0537]
3-[2-{2-(2-[.omega.-tert-Butyl-carboxy-pentadecanoyl-.gamma.-glumta-
yl-.alpha.-tert-butyl-(2-amino-ethoxy)]-ethoxy)-ethoxy}-ethoxy]-propionic
acid) (119 mg, 0.15 mmol) was dissolved in THF (2 ml) and treated
with TSTU (55 mg, 018 mmol) and DIEA (31 .mu.L, 0.18 mmol) and
stirred overnight. The mixture was filtered, and the filtrate was
evaporated in vacuo. The residue was dissolved in AcOEt and washed
twice with cold 0.1 M HCl and water. Drying over MgSO.sub.4 and
evaporation in vacuo gave succinimidyl
3-[2-{2-(2-[.omega.-tert-butyl-carboxy-pentadecanoyl-.gamma.-glutamyl-.al-
pha.-tert-butyl-(2-amino-ethoxy)]-ethoxy)-ethoxy}-ethoxy]-propinoyl),
123 mg (92%).
[0538] .sup.1H-NMR (CDCl.sub.3) .delta.: 6.64 (t, 1H), 6.54 (d,
1H), 4.35 (m, 1H), 3.80 (d, 2H), 3.59 (m, 16H), 3.51 (t, 2H), 3.39
(m, 1H), 2.85 (t, 2H), 2.79 (s, 4H), 2.22 (m, 2H), 2.15 (m, 2H),
2.08 (m, 1H), 1.90 (m, 1H), 1.55 (m, 4H), 1.41 (s, 9H), 1.39 (s,
9H), 1.20 (m, 20H).
Example 2
Synthesis of
N.sup..epsilon.B29-(3-[2-{2-(2-[.omega.-carboxy-hebtadecanoyl-.gamma.-glu-
tamyl-(2-amino-ethoxy)]-ethoxy)-ethoxy}-ethoxy]-propinoyl) DesB30
Human Insulin
##STR00014##
[0540] This compound was prepared in analogy with example 1 via
reaction of L-GluOtBu with tert-butyl succinimidyl octadecandioate
followed by activation with TSTU, activation with TSTU, reaction
with 3-(2-{2-[2-(2-Amino-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionic
acid activation with TSTU, coupling with DesB30 human insulin and
deprotection by TFA.
[0541] MALDI-TOF-MS 6380, calculated 6379.
Example 3
Synthesis of
N.sup..epsilon.B29-{3-[2-(2-{2-[2-(15-carboxy-pentadecanoylamino)-ethoxy]-
-ethoxy}-ethoxy)-ethoxy]-propionyl-.gamma.-glutamyl DesB30 Human
Insulin
##STR00015##
[0542] Step 1:
.omega.-[2-(2-{2-[2-(2-Carboxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethylcarba-
moyl]-pentadecanoic acid tert-butyl ester
##STR00016##
[0544] Hexadecanedioic acid tert-butyl ester 2,5-dioxo
pyrrolidin-1-yl ester (0.12 g, 0.283 mmol) was dissolved in DMF
(2.5 ml),
3-(2-{2-[2-(2-amino-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionic acid
(75 mg, 0.283 mmol) was added and the mixture was stirred at rt for
16 h. The reaction mixture was combined with another reaction
mixture performed on a 0.038 mmol scale. AcOEt (25 ml) was added an
the solution was washed with acidified water (15 ml+300 .mu.l of
0.1 N HCl) and water (3.times.15 ml), dried over MgSO.sub.4 and
concentrated under vacuum, adding some DCM and concentrating again
twice, thus yielding a white greasy residue (0.15 g, 79%)
[0545] HPLC-MS m/z: 590 (M+1), Rt=5.24 min.
[0546] 1H-NMR (CDCl.sub.3, 400 MHz) .delta. 6.48 (br, 1H), 3.79 (t,
2H), 3.6-3.7 (m, 14H), 3.47 (m, 2H), 2.60 (t, 2H), 2.17-2.22 (m,
4H), 1.57-1.64 (m, 4H), 1.44 (s, 9H), 1.2-1.3 (m, 20H).
Step 2:
2-{3-[2-(2-{2-[2-(.omega.-tert-Butoxycarbonylpentadecanoylamino)et-
hoxy]-ethoxy}ethoxy)ethoxy]propionylamino}pentanedioic acid
5-benzyl ester 1-tert-butyl ester
##STR00017##
[0548] Hexadecanoic acid tert-butyl ester 0.15 g, 0.254 mmol) was
dissolved in DMF (2.5 ml) and HOBt (48 mg, 0.356 mmol) and EDAC (63
mg, 0.331 mmol) were added. The solution was stirred at it for 30
min and H-Glu-(OBzl)-OtBu (117 mg, 0.356 mmol) was added. The
reaction was stirred at it for 16 h, and AcOEt (25 ml) was added.
The solution was washed with water (10 ml), 0.2 N HCl (3.times.10
ml), 1:1 Sat. NaCl/water (3.times.10 ml), dried over MgSO.sub.4 and
concentrated to yield an oil (0.24 g). The product was purified by
flash chrometography (silica, 95:5 DCM/methanol) to yield an oil
0.2g.
[0549] HPLC-MS (method 9): m/z: 866 (M+1), R.sub.t=6.99-7.09
min
[0550] 1H-NMR (CDCl.sub.3, 400 MHz) .delta. 7.34-7.38 (m, 5H), 6.83
(d, 1H), 6.10 (br, 1H), 5.11 (s, 2H), 4.50-4.55 (m, 1H), 3.71-3.75
(m, 2H), 3.60-3.65 (m, 12H), 3.55 (t, 2H), 3.36-3.42 (m, 2H),
2.36-2.51 (m, 4H), 2.14-2.24 (m, 5H), 1.93-2.00 (m, 1H), 1.57-1.63
(m, 4H), 1.46 (s, 9H), 1.44 (s, 9H), 1.2-1.3 (m, 20H).
Step 3:
2-{3-[2-(2-{2-[2-(.omega.-tert-butoxycarbonyl-pentadecanoylamino)e-
thoxy]ethoxy}ethoxy)ethoxy]-propionylamino}pentanedioic acid
1-tert-butyl ester
##STR00018##
[0552]
2-{3-[2-(2-{2-[2-(.omega.-tert-Butoxycarbonylpentadecanoylamino)eth-
oxy]-ethoxy}ethoxy)ethoxy]propionylamino}pentanedioic acid 5-benzyl
ester 1-tert-butyl ester (0.2 g, 0.23 mmol) was dissolved in THF.
The flask was filled with N.sub.2, and palladium (0.3 g, 10% on
carbon, 50% water) was added, and the flask was equipped with a
balloon filled with H.sub.2. The mixture was stirred for 16 h at
rt, and filtered through celite, washing with THF. The filtrate was
concentrated to yield an oil (0.16 g, 89%).
[0553] HPLC-MS (method 9m/z: 775 (M+1), Rt=5.46 min.
Step 4:
2-{3-[2-(2-{2-[2-(.omega.-tert-Butoxycarbonyl-pentadecanoylamino)e-
thoxy]-ethoxy}ethoxy)ethoxy]-propionylamino}pentanedioic acid
5-tert-butyl ester 1-(2,5-dioxopyrrolidin-1-yl)ester
##STR00019##
[0555]
2-{3-[2-(2-{2-[2-(.omega.-tert-Butoxycarbonyl-pentadecanoylamino)et-
hoxy]-ethoxy}ethoxy)ethoxy]-propionylamino}pentanedioic acid
1-tert-butyl ester (0.16 g, 0.21 mmol) was dissolved in DMF (2 ml)
and THF (4 ml) and DIEA (42 pl, 0.25 mmol) was added. The solution
was cooled to 0.degree. C., and TSTU (74 mg, 0.21 mmol) was added.
The reaction was stirred over night at rt. the solvent was removed
under vacuum and AcOEt (25 ml) was added. The mixture was washed
with 0.2 N HCl (3.times.10 ml), sat NaHCO.sub.3 (3.times.10 ml),
dried over MgSO.sub.4 and concentrated under vacuum to yield an oil
(0.16 g). The product was purified by flash chromatography (silica,
95:5 DCM/methanol) to yield an oil (0.11 g, 61%).
[0556] HPLC-MS (method 9) m/z: 872 (M+1), Rt=5.67 min.
Step 5:
N.sup..epsilon.B29-{3-[2-(2-{2-[2-(.omega.-carboxy-pentadecanoylam-
ino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionyl-.delta.-glutamyl
DesB30 Human Insulin
##STR00020##
[0558]
2-{3-[2-(2-{2-[2-(.omega.-tert-Butoxycarbonyl-pentadecanoylamino)et-
hoxy]-ethoxy}ethoxy)ethoxy]-propionylamino}pentanedioic acid
.alpha.-tert-butyl ester 1-(2,5-dioxopyrrolidin-1-yl)ester was
coupled to desB30 insulin in similar fashion as described in
Example 1 The intermediate product was purified by preparative HPLC
(C.sub.18-5 cm dia.) before treating with TFA. The final product
was purified by preparative HPLC (C.sub.4, 2 cm dia.) then
(C.sub.4, 1 cm dia.) (20-60% acetonitrile).
[0559] MALDI-TOF-MS: 6355, Calculated: 6351
Example 4
Synthesis of
N.sup..epsilon.B29-(.omega.-[2-(2-{2-[2-(2-carboxy-ethoxy)-ethoxy]-ethoxy-
}-ethoxy)-ethylcarbamoyl]-hebtadecanoyl-.alpha.-glutamyl) DesB30
Human Insulin
##STR00021##
[0561] This compound was prepared in analogy with example 1 via
reaction of H.sub.2N(CH.sub.2CH.sub.2O).sub.4CH.sub.2CH.sub.2COOtBu
(Quanta Biodesign, OH, USA) with mono-succinimidyl oc-tadecandioate
followed by activation with TSTU, reaction with L-Glu(OtBu),
activation with TSTU, coupling with DesB30 human insulin and
deprotection by TFA. LCMS 6380, method 6, calculated 6379.
Example 5
Synthesis of
N.sup..epsilon.B29-(.omega.-[2-(2-{2-[2-(2-carboxy-ethoxy)-ethoxy]-ethoxy-
}-ethoxy)-ethylcarbamoyl]-heptadecanoyl-.gamma.-glutamyl) DesB30
Human Insulin
##STR00022##
[0563] This compound was prepared in analogy with example 1 via
reaction of H.sub.2N(CH.sub.2CH.sub.2O).sub.4CH.sub.2CH.sub.2COOtBu
(Quanta Biodesign, OH, USA) with mono-succinimidyl oc-tadecandioate
followed by activation with TSTU, reaction with L-Glu-OtBu,
activation with TSTU, coupling with DesB30 human insulin and
deprotection by TFA.
[0564] LCMS 6378.4, method 6, calculated 6379.4.
Example 6
Synthesis of
N.sup..epsilon.B29-3-[2-(2-{2-[2-(.omega.-carboxy-heptadecanoylamino)-eth-
oxy]-ethoxy}-ethoxy)-ethoxy]-propionyl-.gamma.-glutamyl DesB30
Human Insulin
##STR00023##
[0566] The compound was prepared in the same manner as with
N.sup..epsilon.B29-3-[2-(2-{2-[2-(.omega.-carboxy-pentadecanoylamino)-eth-
oxy]-ethoxy}-ethoxy)-ethoxy]-propionyl-.gamma.-glutamyl desB30
insulin using octadecanedioic acid tert-butyl ester
2,5-dioxo-pyrrolidin-1-yl ester as the starting material.
Step 1:
.omega.-[2-(2-{2-[2-(2-carboxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-eth-
ylcarbamoyl]-heptadecanoic acid tert-butyl ester
##STR00024##
[0568] HPLC-MS (method 9) m/z: 618 (M+1), Rt=5.92 min.
[0569] 1H-NMR (CDCl.sub.3, 300 MHz) .delta. 6.46 (br, 1H), 3.79 (t,
2H), 3.61-3.69 (m, 14H), 3.44-3.49 (m, 2H), 2.60 (t, 2H), 2.16-2.22
(m, 4H), 1.51-1.68 (m, 4H), 1.44 (s, 9H), 1.19-1.36 (m, 24H).
Step 2:
2-{3-[2-(2-{2-[2-(17-tert-Butoxycarbonylheptadecanoylamino)ethoxy]-
-ethoxy}ethoxy)ethoxy]-propionylamino}pentanedioic acid 5-benzyl
ester 1-tert-butyl ester
##STR00025##
[0571] HPLC-MS (method 9) m/z: 894 (M+1), Rt=7.82-7.89 min. 1H-NMR
(CDCl.sub.3, 300 MHz) 8 7.29-7.42 (m, 5H), 6.83 (d, 1H), 6.13 (br,
1H), 5.11 (s, 2H), 4.46-4.59 (m, 1H), 3.68-3.81 (m, 2H), 3.57-3.68
(m, 12H), 3.55 (t, 2H), 3.39-3.49 (m, 2H), 2.32-2.55 (m, 4H),
2.12-2.28 (m, 5H), 1.86-2.07 (m, 1H), 1.51-1.68 (m, 4H), 1.46 (s,
9H), 1.44 (s, 9H), 1.17-1.36 (m, 24H).
Step 3:
2-{3-[2-(2-{2-[2-(17-tert-Butoxycarbonylheptadecanoylamino)ethoxy]-
-ethoxy}ethoxy)ethoxy]propionylamino}pentanedioic acid 1-tert-butyl
ester
##STR00026##
[0573] HPLC-MS (method 9) m/z: 804 (M+1), Rt=5.81 min.
Step 4:
2-{3-[2-(2-{2-[2-(.omega.-tert-Butoxycarbonyl-heptadecanoylamino)e-
thoxy]-ethoxy}ethoxy)ethoxy]propionylamino}pentanedioic acid
5-tert-butyl ester 1-(2,5-dioxo-pyrrolidin-1-yl)ester
##STR00027##
[0575] HPLC-MS (method 9) m/z: 901 (M+1), Rt=6.00 min.
[0576] 1H-NMR (CDCl.sub.3, 300 MHz) .delta. 6.94 (d, 1H), 6.15 (br,
1H), 4.55-4.62 (m, 1H), 3.71-3.79 (m, 2H), 3.59-3.71 (m, 12H), 3.55
(t, 2H), 3.42-3.47 (m, 2H), 2.84 (s, 4H), 2.58-2.79 (m, 2H), 2.52
(t, 2H), 2.24-2.41 (m, 1H), 2.13-2.24 (m, 4H), 2.04-2.10 (m, 1H),
1.51-1.70 (m, 4H), 1.48 (s, 9H), 1.44 (s, 9H) 1.19-1.37 (m,
24H).
Step 5:
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(17-carboxy-heptadecanoylamino)--
ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionyl gamma-glutamyl DesB30
Human Insulin
[0577] The final product was purified by HPLC (C.sub.18-5cm
dia.).
[0578] HPLC-MS (method 9) m/z: 1596.4 (M+4/4), Calculated 6379,
Rt=4.05 min
Example 7
Synthesis of
N.sup..epsilon.B29-(3-(3-{2-[2-(3-17-carboxyheptanoylamino]propoxy)ethoxy-
]-ethoxy}propylcarbamoyl)propionyl) DesB30 Human Insulin
##STR00028##
[0580] This compound was prepared using the same synthesis steps as
reported for the synthesis of example 1.
Step 1.
N-(3-{2-[2-(3-tert-Butoxycarbonylaminopropoxy)ethoxy]ethoxy}propyl-
)succinamic acid
##STR00029##
[0581] preparation from
1-(tert-butoxycarbonylamino)-4,7,10-trioxa-13-tridecanamine (5g)
and succinic anhydride (1.98) gave 7 g crude product. LCMS (Method
6): Rt 3.34 min; m/z (M+1) 421 Calcd: 421
Step 2.
7-[3-(2-{2-[3-(3-Carboxypropionylamino)-propoxy]-ethoxy}-ethoxy)-p-
ropylcarbamoyl]-heptanoic acid tert-butyl ester
##STR00030##
[0583] This compound was prepared by deprotection of
N-(3-{2-[2-(3-tert-butoxycarbonylaminopropoxy)-ethoxy]ethoxy}propyl)succi-
namic acid (1.56 mmol) by means of TFA, followed by reaction with
octanedioic acid tert-butyl ester 2,5-dioxo-pyrrolidin-1-yl ester
(1.56 mmol). as described in example 8 step3.
[0584] The crude product was purified on Gilson using acidic HPLC
on a C18 column (Jones, Kromasil RP18 5 .mu.m 15.times.225 mm).
[0585] Gradient: 0.0-4.0 min 20% A; 4.0-11.0 min 20-90% A; 11-16
min 90% A.
[0586] The product was collected in fractions from 15.0-17.0 min.
The combined fractions were evaporatied yielding the wanted product
(0.78 g)
[0587] LCMS (Method 9): Rt 4.03 min; m/z (M+1) 533, Calcd.:
533.
Step 3.
7-{3-[2-(2-{3-[3-(2,5-Dioxopyrrolidin-1-yloxycarbonyl)-propionylam-
ino]-propoxy}-ethoxy)-ethoxy]-propylcarbamoyl}-heptanoic acid
tert-butyl ester
##STR00031##
[0589]
7-[3-(2-{2-[3-(3-Carboxypropionylamino)-propoxy]-ethoxy}-ethoxy)-pr-
opylcarbamoyl]-heptanoic acid tert-butyl ester (0.78g,1.46 mmol)
was activated by means of TSTU as described in example 8 step 4.
Crude yield 360 mg, LCMS Method 6: Rt 4.40 min; m/z (M+1) 630;
Calcd.: 630. The compound was used without further
purification.
Step 4.
N.sup..epsilon.B29-(3-(3-{2-[2-(3-[7-carboxyheptanoylamino]propoxy-
)ethoxy]-ethoxy}propylcarbamoyl)propionyl-.gamma.-glutamyl) DesB30
Human Insulin
##STR00032##
[0591] Preparation following step 6 in example 8 resulted in 0.78 g
of the target product after purification on Gilson using acidic
HPLC on a C18 column (Jones, Kromasil RP18 5 .mu.m 15.times.225
mm). Gradient: 0.0-1.0 min: 30% CH3CN, 1.00-15.0 min: 30-50% CH3CN,
15.0-20.0 min: 50% CH3CN Flow: 10 ml/min. Rt=14.5-16.0 min.
[0592] MALDI-TOF-MS (matrix SA): m/z 6167; calc. 6165.
[0593] HPLC (method 5); Rt 3.973 min.
Example 8
Synthesis of
N.sup..epsilon.B29-(3-(3-{4-[3-(7-Carboxyheptanoylamino)propoxy]butoxy}pr-
opylcarbamoyl)-propionyl-.gamma.-glutamyl) DesB30 Human Insulin
##STR00033##
[0594] Step 1:
N-{3-[4-(3-tert-Butoxycarbonylaminopropoxy)-butoxy]-propyl}succinamic
acid
##STR00034##
[0596] 1-(tert-Butoxycarbonylamino)-4,9-dioxa-12-dodecanamine (5.0
g, 16.45 mmol) was dissolved in THF (30 mL), succinic anhydride
(1.81 g, 18.1 mmol) in acetonitrile (10 mL) was added and the
mixture was heated to 60 C for 4 h, and subsequently stirred at RT
over-night.
[0597] The mixture was evaporated to dryness and EtAc (50 mL) was
added.
[0598] The EtAc phase was washed with HCl (0.1 M) 3 times, dried
with MgSO.sub.4 and subsequently the organic phase was evaporated
to dryness which gave 5.86 g (88%) of a thick oil.
[0599] LCMS (Method 6): Rt 2.86 min; m/z (M+1) 405. Calcd: 405.
[0600] This product was used without further purification.
Step 2. Octanedioic acid tert-butyl ester 2,5-dioxo-pyrrolidin-1-yl
ester
##STR00035##
[0602] Octanedioic acid mono-tert-butyl ester (3.14 g, 13.63 mmol)
was dissolved in THF (100 mL). TSTU (4.9 g, 16.3 mmol) was added
and pH was adjusted to 8.5 with DIPEA (2.85 mL).
[0603] The mixture was stirred under nitrogen overnight, evaporated
to dryness, dissolved in EtAc (50 mL) which subsequently was
extracted 2 times with HCL (0.1 M). The organic phase was dried
with MgSO.sub.4, filtered and evaporated resulting in an slightly
yellow oil (5 g, containing small amounts of solvent)
[0604] LCMS (Method 6): Rt 6.56 min; m/z (M+1) 328. Calcd: 328.
Step 3:
7-(3-{4-[3-(3-Carboxypropionylamino)propoxy]butoxy}propylcarbamoyl-
)heptanoic acid tert-butyl ester
##STR00036##
[0606]
N-{3-[4-(3-tert-butoxycarbonylaminopropoxy)-butoxy]-propyl}succinam-
ic acid (4.60 g, 11.37 mmol) was stirred with TFA (20 mL) at RT for
60 min, after evaporation the residue was stripped with DCM (30
mL.times.2) and evaporated to dryness.
[0607] The resulting oil was dissolved in acetonitrile (30 mL) and
octanedioic acid tert-butyl ester 2,5-dioxo-pyrrolidin-1-yl ester
(4.46 g, 13.6 mmol) in DMF (20 mL) was added.
[0608] pH was adjusted to 8.5 with DIPEA and the mixture was
stirred overnight under nitrogen. The mixture was subsequently
evaporated to dryness and redissolved in EtAc (50 mL). The EtAc
phase was extracted .times.3 with HCl (0.1 M), the organic layer
dried over magnesium sulphate, filtered and evaporated resulting in
a slightly yellow crystalline oil (6.5 g, content of solvent
residues)
[0609] LCMS (Method 6): Rt 4.31 min; m/z (M+1) 517. Calcd: 517.
[0610] The crude product was used for further reaction without
further purification.
Step 4.
2-[3-(3-{4-[3-(7-tert-butoxycarbonylheptanoylamino)propoxy]butoxy}-
-propylcarbamoyl)-propionylamino]pentanedioic acid 1-tert-butyl
ester
##STR00037##
[0612]
7-(3-{4-[3-(3-Carboxypropionylamino)propoxy]butoxy}propylcarbamoyl)-
heptanoic acid tert-butyl ester (5.9 g), the crude product from
above, was dissolved in THF (20 mL), TSTU (4.13 g, 13.7 mmol) was
added together with DMF (6 mL), pH was adjusted to 8.2 with DIPEA
(2.6 mL). The mixture was stirred overnight under nitrogen.
[0613] The mixture was evaporated and the residue dissolved in EtAc
which was extracted with HCl (0.1 M) 3 times.
[0614] The organic layer was dried with magnesium sulphate,
filtered and the filtrate evaporated to give an oil.
[0615] LCMS (Method 6): Rt 4.57 min; m/z 614 corresponding to the
activated acid.
[0616] This was dissolved in THF (30 mL), pH was adjusted to 8.2
with DIPEA (0.4 mL) and H-glu-OtBu (1.7 g, 4.9 mmol) was added
together with DMF (10 mL). The mixture was stirred at RT for 3 h,
filtration followed by evaporation afforded a thick yellow oil.
[0617] This was extracted between EtAc and HCl (0.1 M) as reported
above, and the resulting dried EtAc layer gave 3.5 g crude product
on evaporation. LCMS (Method 6): Rt 4.77 min; m/z (M+1) 702.
[0618] The crude product was purified on Gilson using acidic HPLC
on a C18 column (Jones, Kromasil RP18 5 .mu.m 15.times.225 mm).
[0619] Gradient: 0.0-10.0 min 35% A; 10.0-25.0 min 35-80% A; 25-30
min 90% A; 30-35 min 100% A.
[0620] The product was collected in fractions from 21-22.5 min. The
combined fractions were evaporated yielding the wanted product (1.8
g)
[0621] LCMS (Method 6): Rt 4.77 min; m/z (M+1) 702, Calcd. 702.
Step 5.
2-[3-(3-{4-[3-(7-tert-butoxycarbonylheptanoylamino)propoxy]butoxy}-
propylcarbamoyl)-propionylamino]pentanedioic acid 5-tert-butyl
ester 1-(2,5-dioxopyrrolidin-1-yl)ester
##STR00038##
[0623]
2-[3-(3-{4-[3-(7-tert-butoxycarbonylheptanoylamino)propoxy]butoxy}--
propylcarbamoyl)-propionylamino]pentanedioic acid 1-tert-butyl
ester (1.5 g, 2.14 mmol) was dissolved in THF (20 mL), pH was
adjusted to 8.5 with DIPEA (0.9 mL), TSTU (0.83 g, 2.77 mmol) was
added in DMF (5 mL). The mixture was stirred under nitrogen
overnight , subsequent evaporation and extraction between EtAc and
HCl as described above resulted in 1.75 g crude product.
[0624] LCMS (Method 6): Rt 5.10 min; m/z (M+1) 800, Calcd.:
800.
Step 6.
N.sup..epsilon.B29-(3-(3-{4-[3-(7-carboxyheptanoylamino)propoxy]bu-
toxy}propylcarbamoyl)-propionyl-.delta.-glytamyl) DesB30 Human
Insulin
##STR00039##
[0625]
2-[3-(3-{4-[3-(7-tert-butoxycarbonylheptanoylamino)propoxy]butoxy}p-
ropylcarbamoyl)-propionylamino]pentanedioic acid 5-tert-butyl ester
1-(2,5-dioxopyrrolidin-1-yl)ester (0.255 g, 0.319 mmol) was
dissolved in acetonitrile (10 mL) and added to a solution of desB30
human insulin (1.82 g) dissolved in Na.sub.2CO.sub.3 solution (10
mL, pH 10.3), pH was adjusted to 10.1 with NaOH (0.1 M). The
mixture was stirred at RT for 2 h, then pH was adjusted to 5.5 by
means of HCl (2M, 3 mL) resulting in the precipitation of an oily
crystalline mass.
[0626] This was isolated and dissolved in water acetic acid (1M)
and freeze dried.
[0627] The resulting product was dissolved in water and purified on
Gilson using acidic HPLC on a C18 column (Jones, Kromasil RP18 5
.mu.m 15.times.225 mm).
[0628] Gradient: 0.0-5.0 min 35% A; 5.0-25.0 min 35-80% A; 25-30
min 90% A; 30-35 min 100% A. Fractions around Rt 15 min were
collected, mixed and evaporated.
[0629] The product was treated with TFA/DCM 1/1 (20 mL) by stirring
at RT for 1 h, subsequent evaporation to dryness and stripping with
DCM 40 mL.times.2 resulted in the deprotected product which was
dissolved in water and freeze dried giving 540 mg of the wanted
product.
[0630] MALDI.TOF-MS: m/z 6276.66; calc. 6276.
[0631] HPLC (method 5); Rt 10.19 min.
Example 9
Synthesis of
N.sup..epsilon.B29-(3-(3-{2-[2-(3-[9-Carboxynonanoylamino]propoxy)ethoxy]-
ethoxy}-propylcarbamoyl)propionyl) DesB30 Human Insulin
##STR00040##
[0633] Following the procedure from example 7, but exchanging the
diacid part gave the product.
[0634] Preparation following step 6 in example 8 using 0.114 mmol
of desB30 insulin resulted in 0.96 g of the protected compound.
[0635] Gilson purification using acidic HPLC on a C18 column
(Jones, Kromasil RP18 5 .mu.m 15.times.225 mm). Gradient: 0.0-1.0
min: 35% CH3CN, 1.00-15.0 min: 35-55% CH3CN, 15.0-20.0 min: 55%
CH3CN Flow: 10 ml/min. Rt=12.5-14.0 min.
[0636] Deprotection my means of TFA gave 0.141 g colourless
compound after freeze drying.
[0637] MALDI-TOF-MS (matrix SA): m/z 6195; calc. 6186
[0638] HPLC (method 5): Rt; 4.094 min.
Decanedioic acid tert-butyl ester 2,5-dioxo-pyrrolidin-1-yl
ester
##STR00041##
[0640] Preparation as described in step 2 example 8 gave 5.57 g
crude product which was used without further purification. LCMS
(Method 6): Rt 5.82 min; m/z (M+1) 356, Calcd.: 355.
Example 10
Synthesis of
N.sup..epsilon.B29-(3-(2-{2-[2-(9-carboxynonanoylamino)ethoxy]ethoxy}ethy-
lcarbamoyl)propionyl-.gamma.-olutamyl) DesB30 Human Insulin
##STR00042##
[0642] The preparation was performed using the methodology
described in example 8
[0643]
(S)-2-[3-(2-{2-[2-(9-tert-Butoxycarbonylnonanoylamino)ethoxy]ethoxy-
}-ethylcarbamoyl)propionylamino]pentanedioic acid 1-tert-butyl
ester (0.59 g, 0.876 mmol) was activated with TSTU, 0.132 g (0.171
mmol) the crude reaction product was reacted with desB30 insulin
(0.154 mmol) as described in example 8
[0644] This resulted in 740 mg of oily precipitate which was
freeze-dried and purified on
[0645] Gilson using acidic HPLC on a C18 column (Jones, Kromasil
RP18 5 .mu.m 15.times.225 mm).
[0646] Gradient: 0.0-5.0 min 30% A; 5.0-20.0 min 35-50% A.
[0647] 105 mg of target compound was isolated. MALDI.TOF-MS (matrix
Cyano): m/z 6245.9; calc. 6243.
[0648] HPLC (method 5); Rt 8.759 min.
N-{2-[2-(2-tert-butoxycarbonylamino-ethoxy)-ethoxy]-ethyl}-succinamic
acid
##STR00043##
[0650] Preparation from
1-(t-butyloxycarbonylamino)-3,6-dioxa-8-octaneamine) (5 g, 20.16
mmol) and succinic anhydride (2.218 g, 22,18 mmol) gave a thick
yellow oil which crystallised on standing (6.5 g, yield 98%). LCMS
(Method 6): Rt 2.99 min; m/z (M+1) 349; Calcd.: 349.
9-(2-{2-[2-(3-carboxypropionylamino)ethoxy]ethoxy}ethylcarbamoyl)nonanoic
acid tert-butyl ester
##STR00044##
[0652] Preparation from decanedioic acid tert-butyl ester
2,5-dioxo-pyrrolidin-1-yl ester (1.13 g, 3.45 mmol) and
N-{2-[2-(2-tert-butoxycarbonylamino-ethoxy)-ethoxy]-ethyl}-succinamic
acid (1 g, 2.84 mmol) as described in step 3 example 8 gave 1.68 g
crude product which was used without further purification. LCMS
(Method 6): Rt 3.86 min; m/z (M+1) 489; Calcd.: 489.
(S)-2-[3-(2-{2-[2-(9-tert-Butoxycarbonylnonanoylamino)ethoxy]ethoxy}-ethyl-
carbamoyl)propionylamino]pentanedioic acid 1-tert-butyl ester
##STR00045##
[0654] Preparation from
9-(2-{2-[2-(3-carboxypropionylamino)ethoxy]ethoxy}-ethylcarbamoyl)nonanoi-
c acid tert-butyl ester (1.4 g, 2.86 mmol) and glu-OtBu (0.87 g,
4.29 mmol) folllowing the method described step4 example 8 gave 1.8
g crude product. LCMS (Method 6): Rt 5.1 min; m/z (M+1) 674;
Calcd.: 674.
[0655] Gilson purification using acidic HPLC on a C18 column
(Jones, Kromasil RP18 5 .mu.m 15.times.225 mm), Gradient: 0.0-10.0
min: 35% CH.sub.3CN, 10.00-25.0 min: 35-90% CH3CN, Flow: 10 ml/min.
Fractions at Rt=20.0-25.0 min were collected and evaporated to
dryness giving 0.590 g of a yellow oil. LCMS (Method 6): Rt 5.1
min; m/z (M+1) 674; Calcd.: 674.
Example 11
Synthesis of
N.sup..epsilon.B29-(3-(3-{4-[3-(9-Carboxynonanoylamino)propoxy]butoxy}-pr-
opylcarbamoyl)propionyl-.gamma.-glutamyl) DesB30 Human Insulin
##STR00046##
[0657]
(S)-2-[3-(3-{4-[3-(9-tert-Butoxycarbonylnonanoylamino)propoxy]butox-
y}-propylcarbamoyl)propionylamino]pentanedioic acid 5-tert-butyl
ester 1-(2,5-dioxopyrrolidin-1-yl)ester (0.06 g, 0.073 mmol) and
desB30 insulin (0.065 mmol) were reacted as described in example
8.The TFA treated product was purified on Gilson using acidic HPLC
on a C18 column (Jones, Kromasil RP18 5 .mu.m 15.times.225 mm).
[0658] Gradient: 0.0-5.0 min 30% A; 5.0-20.0 min 35-50% A.
fractions at Rt 16.0 min-17.5 min were collected evaporated and
subsequently freeze-dried. Yield 34 mg.
[0659] MALDI.TOF-MS: m/z 6305.69; calc. 6299.
[0660] HPLC method 5; Rt 8.850 min.
9-(3-{4-[3-(3-Carboxypropionylamino)propoxy]butoxy}propylcarbamoyl)nonanoi-
c acid tert-butyl ester
##STR00047##
[0662] Preparation from decanedioic acid tert-butyl ester
2,5-dioxo-pyrrolidin-1-yl ester (0.88 g,
[0663] 2.47 mmol) and
N-{3-[4-(3-tert-butoxycarbonylaminopropoxy)-butoxy]-propyl}succinamic
acid
[0664] (1 g, 2.47 mmol) as described in example 8 afforded 150 mg
compound after purification.
[0665] LCMS (Method 6): Rt 4.31 min; m/z (M+1) 545; Calcd.:
545.
(S)-2-[3-(3-{4-[3-(9-tert-butoxycarbonylnonanoylamino)propoxy]butoxy}propy-
lcarbamoyl)propionylamino]pentanedioic acid 1-tert-butyl ester
##STR00048##
[0666]
9-(3-{4-[3-(3-Carboxypropionylamino)propoxy]butoxy}propylcarbamoyl)-
nonanoic acid tert-butyl ester (0.15 g, 0.276 mmol) was activated
with TSTU , the resulting OSu-derivative was reacted with
H-Glu-OtBu (0.076 g, 0.37 mmol) as described previously. After work
up the resulting oil was purified on Gilson using acidic HPLC on a
C18 column (Jones, Kromasil RP18 5 .mu.m 15.times.225 mm).
[0667] Gradient: 0.0-5.0 min 20% A; 5.0-20.0 min 20-90% A.
fractions at Rt 24.5 min-25.5 min were collected evaporated and
subsequently freeze-dried. Yield 50 mg. LCMS Method 6: Rt 5.43 min;
m/z (M+1) 730; Calcd.: 730.
[0668] This compound was activated with TSTU resulting in 60 mg
crude
(S)-2-[3-(3-{4-[3-(9-tert-Butoxycarbonylnonanoylamino)propoxy]butoxy}prop-
ylcarbamoyl)propionylamino]-pentanedioic acid 5-tert-butyl ester
1-(2,5-dioxopyrrolidin-1-yl)ester
##STR00049##
[0669] LCMS Method 6: Rt 5.79 min; m/z (M+Na) 850; Calcd.: 850.
[0670] The crude product was used without further purification.
Example 12
Synthesis of
N.sup..epsilon.B29-(2-[3-(2-(2-{2-(7-carboxyheptanoylamino)ethoxy}ethoxy)-
-ethylcarbamoyl]propionyl-.gamma.-glutamyl) DesB30 Human
Insulin
##STR00050##
[0671]
(S)-2-[3-(2-{2-[2-(7-tert-Butoxycarbonylheptanoylamino)ethoxy]ethox-
y}ethylcarbamoyl)-propionylamino]pentanedioic acid 5-tert-butyl
ester 1-(2,5-dioxopyrrolidin-1-yl)ester (0.126 g, 0.17 mmol) was
reacted with desB30 insulin (0.153 mmol) as described above. The
crude product after TFA treatment (0.750 mg) was purified two times
on Gilson using acidic HPLC on a C18 column (Jones, Kromasil RP18 5
.mu.m 15.times.225 mm).
[0672] Gradient: 0.0-5.0 min 25% A; 5.0-20.0 min 20-50%. Fractions
from Rt 21.0-22.0 min collected and evaporated resulting in 13 mg
compound.
[0673] MALDI.TOF-MS (matrix SA): m/z 6221.15; calc. 6215.
[0674] LCMS (Method 6): Rt 3.53 min; m/z (M+414) 1556; Calcd.:
1554
7-(2-{2-[2-(3-Carboxy-propionylamino)-ethoxy]-ethoxy}-ethylcarbamoyl)-hept-
anoic acid tert-butyl ester
##STR00051##
[0676] Octanedioic acid tert-butyl ester 2,5-dioxo-pyrrolidin-1-yl
ester (1.13 g, 3.45 mmol) and
N-{2-[2-(2-tert-butoxycarbonylamino-ethoxy)-ethoxy]-ethyl}-succinamic
acid (1 g, 2.874 mmol) were reacted as described above. 1.75 g
crude product was isolated and used without further purification.
LCMS (Method 6): Rt 3.86 min; m/z (M+1) 461; Calcd.: 461.
(S)-2-[3-(2-{2-[2-(7-tert-Butoxycarbonylheptanoylamino)ethoxy]ethoxy}-ethy-
lcarbamoyl)propionylamino]pentanedioic acid 1-tert-butyl ester
##STR00052##
[0678]
7-(2-{2-[2-(3-Carboxy-propionylamino)-ethoxy]-ethoxy}-ethylcarbamoy-
l)-heptanoic acid tert-butyl ester (1.3 g, 2.83 mmol) was activated
with TSTU and subsequently the crude product was reacted with
H-glu-OtBu (0.86 g, 4.2 mmol). After work up using the method
described in example 8, the product was further purified on Gilson
using acidic HPLC on a C18 column (Jones, Kromasil RP18 5 .mu.m
15.times.225 mm).
[0679] Gradient: 0.0-10.0 min 30% A; 10.0-25.0 min 30-90% A,
fractions at Rt 20-25 min were collected and evaporated resulting
in 600 mg product which was used for TSTU activation described
below. LCMS (Method 6): Rt 4.51 min; m/z (M+1) 646; Calcd.:
646.
(S)-2-[3-(2-{2-[2-(7-tert-Butoxycarbonylheptanoylamino)ethoxy]ethoxy}-ethy-
lcarbamoyl)propionylamino]pentanedioic acid 5-tert-butyl ester
1-(2,5-dioxopyrrolidin-1-yl)ester
##STR00053##
[0681]
(S)-2-[3-(2-{2-[2-(7-tert-Butoxycarbonylheptanoylamino)ethoxy]ethox-
y}-ethylcarbamoyl)propionylamino]pentanedioic acid 1-tert-butyl
ester (0.6 g, 0.93 mmol) was activated with TSTU using the
procedure described above.
[0682] This resulted in 0.75 g crude compound which was used
without further purification.
[0683] LCMS (Method 6): Rt 4.81 min; m/z (M+1) 743; Calcd.: 743
Example 13
Synthesis of
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(15-carboxypentadecanoylamino)ethoxy]et-
hoxy}ethoxy)-ethoxy]propionyl)) DesB30 Human Insulin
##STR00054##
[0685] This compound was prepared similarly as described in example
4. The intermediate
15-[2-(2-{2-[2-(2-Carboxyethoxy)ethoxy]ethoxy}ethoxy)ethylcarbamoyl]penta-
decanoic acid tert-butyl ester was activated to the OSu-ester using
TSTU and coupled to desB30 human insulin. Deprotection using TFA
afforded the title compound.
[0686] MALDI-TOF MS: m/z=6222. Calculated: 6222
[0687] HPLC (Method 1): R.sub.t=11.12 min.
[0688] HPLC (Method 5): R.sub.t=12.03 min.
Example 14
Synthesis of
N.sup..epsilon.B29-(3-(2-{2-[2-(2-{2-[2-(2-{2-[2-(2-{2-[2-(13-carboxy-tri-
decanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-eth-
oxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionoyl-.gamma.-glutamyl)
DesB30 Human Insulin
##STR00055##
[0690] This compound was prepared in analogy with example 1 via
reaction of H.sub.2N(CH.sub.2CH.sub.2O).sub.12CH.sub.2CH.sub.2COOH
(Quanta Biodesign, OH, USA) with tert-butyl O-succinimidyl
tetradecandioate followed by activation with TSTU, reaction with
L-Glu-OtBu, activation with TSTU, coupling with DesB30 human
insulin and deprotection by TFA.
[0691] LCMS 6676.0, method 6, calculated 6675.8.
Example 15
Synthesis of
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(13-Carboxy-tridecanoylamino)-ethoxy]-e-
thoxy}-ethoxy)-ethoxy]-propionoy-.gamma.-glutamyl) DesB30 Human
Insulin
##STR00056##
[0693] This compound was prepared in analogy with example 1 via
reaction of H.sub.2N(CH.sub.2CH.sub.2O).sub.4CH.sub.2CH.sub.2COOH
(Quanta Biodesign, OH, USA) with tert-butyl O-succinimidyl
tetradecandioate followed by activation with TSTU, reaction with
L-Glu-OtBu, activation with TSTU, coupling with DesB30 human
insulin and deprotection by TFA.
[0694] LCMS 6323.2, (method 6) calculated 6323.3.
Example 16
Synthesis of
N.sup..epsilon.B29-(3-[2-(2-{2-[2-(2-{2-[2-(13-carboxy-tridecanoylamino)--
ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-propionoyl-
-.gamma.-glutamyl) DesB30 Human Insulin
##STR00057##
[0696] This compound was prepared in analogy with example 1 via
reaction of H.sub.2N(CH.sub.2CH.sub.2O).sub.8CH.sub.2CH.sub.2COOH
(Quanta Biodesign, OH, USA) with tert-butyl O-succinimidyl
tetradecandioate followed by activation with TSTU, reaction with
L-Glu-OtBu, activation with TSTU, coupling with Des(B30) human
insulin and deprotection by TFA.
[0697] LCMS 6498.8, method 6, calculated 6499.6.
Example 17
Synthesis of
N.sup..epsilon.B29-(3-(2-{2-[2-(15-Carboxy-pentadecanoylamino)-ethoxy]-et-
hoxy}-ethylcarbamoyl)-propionyl-.gamma.-glutamyl) DesB30 Human
Insulin
##STR00058##
[0699] MALDI-TOF MS (matrix: SA): m/z=6336. Calculated: 6334
[0700] HPLC (Method 1): R.sub.t=11.71 min.
[0701] HPLC (Method 5): R.sub.t=9.37 min.
Example 18
Synthesis of
N.sup..epsilon.B29-(3-(3-{2-[2-(3-[15-Carboxypentadecanoylamino]propoxy)e-
thoxy]-ethoxy}propylcarbamoyl)propionyl-.gamma.-glutamyl) DesB30
Human Insulin
##STR00059##
[0702]
15-{3-[2-(2-{3-[3-(2,5-Dioxopyrrolidin-1-yloxycarbonyl)propionylami-
no]propoxy}ethoxy)ethoxy]-propylcarbamoyl}pentadecanoic acid
tert-butyl ester (crude product 0.196 g, 0.264 mmol) reacted with
desB30 insulin (0.132mmol) as described above resulting in 400 mg
precipitate which was Gilson purified, gradient: 0.0-5.0 min 40% A;
5.0-15.0 min 40-80% A, fractions at Rt 15.5-16.0 min were collected
and evaporated to dryness.
[0703] The resulting mass was subsequently treated with TFA/DCM 1/1
(100mL) in order to deprotect the carboxy groups. After evaporation
the resulting product was purified 3 times on
[0704] Gilson HPLC on a C18 column (Jones, Kromasil RP18 5 .mu.m
15.times.225 mm).
[0705] Gradient: 0.0-5.0 min 35% A; 5.0-20.0 min 20-90%. Fractions
from Rt 15.0-16.0 min collected and evaporated resulting in 23 mg
compound.
[0706] MALDI-TOF-MS: m/z 6277.15; calc. 6270.
[0707] HPLC (method 5): Rt 9.50 min.
.omega.-{3-[2-(2-{3-[3-(2,5-Dioxopyrrolidin-1-yloxycarbonyl)propionylamino-
]propoxy}ethoxy)-ethoxy]propylcarbamoyl}pentadecanoic acid
tert-butyl ester
##STR00060##
[0709] Preparation from
.omega.-[3-(2-{2-[3-(3-carboxypropionylamino)propoxy]ethoxy}ethoxy)propyl-
carbamoyl-]pentadecanoic acid tert-butyl ester (0.17 g, 0.264 mmol)
and TSTU gave 196 mg crude product which was used without further
purification. LCMS Method 6: Rt 7.36 min; m/z (M+1) 742; Calcd.:
742.
.omega.-[3-(2-{2-[3-(3-carboxypropionylamino)propoxy]ethoxy}ethoxy)-propyl-
carbamoyl]pentadecanoic acid tert-butyl ester
##STR00061##
[0711] Hexadecanedioic acid tert-butyl ester
2,5-dioxo-pyrrolidin-1-yl ester (0.5 g, 1.13 mmol) and
N-(3-{2-[2-(3-aminopropoxy)ethoxy]ethoxy}propyl)succinamic acid
(0.36 g, 1.13 mmol) were reacted as described above.
[0712] Purification of the crude product on Gilson HPLC on a C18
column (Jones, Kromasil RP18 5 .mu.m 15.times.225 mm). Gradient:
0.0-1.0 min 50% A; 1.0-30.0 min 50-90%. Fractions with Rt 24.0-26.0
min collecte4d and evaporated resulting in 170 mg of the target
product.
[0713] LCMS (Method 6): Rt 7.06 min; m/z (M+1) 645; Calcd.:
645.
Example 19
Synthesis of
N.sup..epsilon.B29-(3-(3-{4-[3-(.omega.-Carboxyundecanoylamino)propoxy]bu-
toxypropylcarbamoyl)-propionyl-.gamma.-glutamyl) DesB30 Human
Insulin
##STR00062##
[0715] This compound was prepared similarly as described in example
8 using dodecanoic acid mono tert-butyl ester.
[0716] Data for the title compound:
[0717] MALDI-TOF-MS: m/z=6332. Calculated: 6334
[0718] HPLC (Method 1): R.sub.t=9.57 min.
[0719] HPLC (Method 5): R.sub.t=7.50 min.
[0720] HPLC (Method 6): R.sub.t=4.11 min; m/z: 1584 (M+4)/4. Calcd:
1584.
Example 20
N.sup..epsilon.B29-(3-(3-{4-[3-(.omega.-Carboxytridecanoylamino)propoxy]bu-
toxypropylcarbamoyl)-propionyl-.gamma.-glutamyl) DesB30 Human
Insulin
##STR00063##
[0721] Example 21
N.sup..epsilon.B29-(3-(2-{2-[2-(11-Carboxyundecanoylamino)ethoxy]ethoxy}et-
hylcarbamoyl)propionyl-.gamma.-glutamyl) DesB30 Human Insulin
##STR00064##
[0722] Example 22
N.sup..epsilon.B29-(3-(2-{2-[2-(.omega.-carboxytridecanoylamino)ethoxy]eth-
oxy}ethylcarbamoyl)propionyl-.gamma.-glutamyl) DesB30 Human
Insulin
##STR00065##
[0723] Example 23
N.sup..epsilon.B29-{3-[2-(2-{2-[2-(.omega.-Carboxy-pentadecanoylamino)etho-
xy]ethoxy}ethoxy)ethoxy]propionyl-gamma-.gamma.-D-glutamyl) DesB30
Human Insulin
##STR00066##
[0724] Example 24
N.sup..epsilon.B29-{3-[2-(2-{2-[2-(7-carboxyheptanoylamino)ethoxy]ethoxy}e-
thoxy)ethoxy]propionyl-.gamma.-glutamyl} DesB30 Human Insulin
##STR00067##
[0725] Example 25
N.sup..epsilon.B29-{3-[2-(2-{2-[2-(9-carboxynonanoylamino)ethoxy]ethoxy}et-
hoxy)ethoxy]propioniyl-.gamma.-glutamyl} DesB30 Human Insulin
##STR00068##
[0726] Example 26
N.sup..epsilon.B29-{3-[2-(2-{2-[2-(11-carboxyundecanoylamino)ethoxy]ethoxy-
}ethoxy)ethoxy]propionyl-.gamma.-glutamyl} DesB30 Human Insulin
##STR00069##
[0727] Example 27
N.sup..epsilon.B29-{3-[2-(2-{2-[2-(13-carboxytridecanoylamino)ethoxy}ethox-
y]ethoxy)ethoxy]propionyl-.gamma.-glutamyl} DesB30 Human
Insulin
##STR00070##
[0728] Example 28
Insulin Receptor Binding of the Insulin Derivatives of the
Invention
[0729] The affinity of the insulin analogues of the invention for
the human insulin receptor was determined by a SPA assay
(Scintillation Proximity Assay) microtiterplate antibody capture
assay. SPA-PVT antibody-binding beads, anti-mouse reagent (Amersham
Biosciences, Cat No. PRNQ0017) were mixed with 25 ml of binding
buffer (100 mM HEPES pH 7.8; 100 mM sodium chloride, 10 mM
MgSO.sub.4, 0.025% Tween-20). Reagent mix for a single Packard
Optiplate (Packard No. 6005190) is composed of 2.4 .mu.l of a
1:5000 diluted purified recombinant human insulin receptor-exon 11,
an amount of a stock solution of A14 Tyr[.sup.125I]-human insulin
corresponding to 5000 cpm per 100 .mu.l of reagent mix, 12 .mu.l of
a 1:1000 dilution of F12 antibody, 3 ml of SPA-beads and binding
buffer to a total of 12 ml. A total of 100 .mu.l was then added and
a dilution series is made from appropriate samples. To the dilution
series was then added 100 .mu.l of reagent mix and the samples were
incubated for 16 hours while gently shaken. The phases were the
then separated by centrifugation for 1 min and the plates counted
in a Topcounter. The binding data were fitted using the nonlinear
regression algorithm in the GraphPad Prism 2.01 (GraphPad Software,
San Diego, Calif.).
Human Serum Albumin Affinity Assay
[0730] Relative binding constant of 125I-TyrA14-analogue to human
serum albumin immobilised on Minileak particles and measured at
23.degree. C. (detemir=1 in saline buffer)
TABLE-US-00014 Insulin receptor affinity in Albumin affinity in
relation to Compound relation to human insulin insulin Determir
example 1 11 2.381 example 2 12 10.649 example 3 14 4.273 example 4
9.2 1.201 example 5 5.6 0.48 example 6 8.3 3.59 example 7 35
example 8 34 example 9 34 example 10 27 example 11 16 example 12 38
example 13 17 example 14 15 example 15 35 example 16 31 example 17
17
Example 29
Pulmonary Delivery of Insulin Derivatives to Rats
[0731] The test substance will be dosed pulmonary by the drop
instillation method. In brief, male Wistar rats (app.250 g) are
anaesthesized in app. 60 ml fentanyl/dehydrodenzperidol/dormicum
given as a 6.6 ml/kg sc primingdose and followed by 3 maintainance
doses of 3.3 ml/kg sc with an interval of 30 min. Ten minutes after
the induction of anaesthesia, basal samples are obtained from the
tail vein (t=-20 min) followed by a basal sample immediately prior
to the dosing of test substance (t=0). At t=0, the test substance
is dosed intra tracheally into one lung. A special cannula with
rounded ending is mounted on a syringe containing the 200 ul air
and test substance (1 ml/kg). Via the orifice, the cannula is
introduced into the trachea and is forwarded into one of the main
bronchi--just passing the bifurcature. During the insertion, the
neck is palpated from the exterior to assure intratracheal
positioning. The content of the syringe is injected followed by 2
sec pause. Thereafter, the cannula is slowly drawn back. The rats
are kept anaesthesized during the test (blood samples for up to 4
hrs) and are euthanized after the experiment.
* * * * *