U.S. patent application number 17/089993 was filed with the patent office on 2021-04-01 for novel acylated insulin analogues and uses thereof.
The applicant listed for this patent is Novo Nordisk A/S. Invention is credited to Jakob Brandt, Bo Falck Hansen, Grith Skytte Olsen, Ingrid Pettersson, Lauge Schaeffer, Rita Slaaby.
Application Number | 20210094999 17/089993 |
Document ID | / |
Family ID | 1000005264031 |
Filed Date | 2021-04-01 |
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United States Patent
Application |
20210094999 |
Kind Code |
A1 |
Olsen; Grith Skytte ; et
al. |
April 1, 2021 |
Novel Acylated Insulin Analogues and Uses Thereof
Abstract
The present invention relates to novel insulin analogues and
derivatives thereof, such as acylated insulin analogues, and their
pharmaceutical use, in particular in the treatment or prevention of
medical conditions relating to diabetes, obesity and cardiovascular
diseases.
Inventors: |
Olsen; Grith Skytte;
(Vaerloese, DK) ; Hansen; Bo Falck; (Virum,
DK) ; Schaeffer; Lauge; (Lyngby, DK) ;
Pettersson; Ingrid; (Frederiksberg, DK) ; Slaaby;
Rita; (Lyngby, DK) ; Brandt; Jakob;
(Broenshoej, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novo Nordisk A/S |
Bagsvaerd |
|
DK |
|
|
Family ID: |
1000005264031 |
Appl. No.: |
17/089993 |
Filed: |
November 5, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
15998755 |
Aug 16, 2018 |
10919949 |
|
|
17089993 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 3/04 20180101; A61P
35/00 20180101; G01N 33/50 20130101; C07K 14/62 20130101; C07K
2319/90 20130101; G01N 2440/14 20130101; A61P 9/00 20180101; A61P
3/10 20180101; A61K 38/00 20130101 |
International
Class: |
C07K 14/62 20060101
C07K014/62; A61P 9/00 20060101 A61P009/00; A61P 3/04 20060101
A61P003/04; A61P 3/10 20060101 A61P003/10; A61P 35/00 20060101
A61P035/00; G01N 33/50 20060101 G01N033/50 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 17, 2017 |
EP |
17186612.2 |
Dec 1, 2017 |
EP |
17204872.0 |
Claims
1. An insulin derivative, comprising an insulin analogue comprising
B5Y or B5F and a substituent comprising an acyl group, or a
pharmaceutically acceptable salt, amide or ester thereof.
2. The insulin derivative according to claim 1, wherein said
insulin analogue further comprises B26G or B26A.
3. The insulin derivative according to claim 1, wherein said
insulin analogue further comprises B28K, B26K or B29 K and said
acyl group is attached to B28K, B26K or B29K.
4. The insulin derivative according to claim 2, wherein said
insulin analogue further comprises B28K, B26K or B29 K and said
acyl group is attached to B28K, B26K or B29K.
5. The insulin derivative according to claim 1, wherein said
substituent has the following formula (I): Acy-L1-L2-L3 wherein:
Acy is an acyl group and is represented by lithocholic acid or
comprises at least one functional group of formulae:
--CO--(CH.sub.2).sub.x--COOH; or Chem. 1:
--CO--(CH.sub.2).sub.x-tetrazolyl; Chem. 2: wherein x represents an
integer in the range of from 12 to 20; and the tetrazolyl group is
1H-tetrazol-5-yl. or is a fatty acid of formula:
--CO--(CH.sub.2).sub.x--CH.sub.3 Chem. 3: wherein x represents an
integer in the range from 8 to 16, L1 is absent and represents a
covalent bond or represents OEG, gGlu, DgGlu or sulfonimide C-4 L2
is absent and represents a covalent bond or represents OEG, gGlu,
DgGlu or sulfonimide C-4 L3 is absent and represents a covalent
bond or represents OEG, gGlu, DgGlu or sulfonimide C-4 wherein gGlu
represents a gamma glutamic acid residue and OEG represents
[2-(2-aminoethoxy)ethoxy]acetyl.
6. The insulin derivative according to claim 1, wherein said
insulin analogue further comprises A14E and/or desB30 and/or
desB29-30 and/or desB27-30.
7. The insulin derivative according to claim 1, wherein said
insulin analogue comprises i. A14E, B5Y, B26A, B28K, desB29-30; ii.
A14E, B5Y, B26G, B28K, desB29-30; iii. B5Y, B26A, B28K, desB29-30;
iv. B5Y, B26G, B28K, desB29-30; v. B5Y, B28K, desB29-30; vi. A14E,
B5F, B26G, B28K, desB29-30; vii. B5F, B28K, desB29-30; viii. B5Y,
B26K, desB27-desB30; ix. B5Y, desB30; x. B5Y, B26G, desB30; xi.
B5Y, B26A, desB30; xii. B5F, B26A, B28K, desB29-30; xiii. B5Y,
B26A, B28K, desB29-30; xiv. B5Y, B26G, B28K, desB29-30; xv. B5F,
B26G, B28K, desB29-30; xvi. A14E, B5F, B26A, B28K, desB29-30; xvii.
A14E, B5Y, B26A, B28K, desB29-30; xviii. A14E, B5Y, B26G, B28K,
desB29-30; xix. A14E, B5Y, B28K, desB29-30; xx. A14E, B5F, B28K,
desB29-30; xxi. A14E, B5Y, B26K, desB27-desB30; xxii. A14E, B5Y,
desB30; xxiii. A14E, B5Y, B26G, desB30; or xxiv. A14E, B5Y, B26A,
desB30.
8. The insulin derivative according to claim 5, wherein said
insulin analogue comprises i. A14E, B5Y, B26A, B28K, desB29-30; ii.
A14E, B5Y, B26G, B28K, desB29-30; iii. B5Y, B26A, B28K, desB29-30;
iv. B5Y, B26G, B28K, desB29-30; v. B5Y, B28K, desB29-30; vi. A14E,
B5F, B26G, B28K, desB29-30; vii. B5F, B28K, desB29-30; viii. B5Y,
B26K, desB27-desB30; ix. B5Y, desB30; x. B5Y, B26G, desB30; xi.
B5Y, B26A, desB30; xii. B5F, B26A, B28K, desB29-30; xiii. B5Y,
B26A, B28K, desB29-30; xiv. B5Y, B26G, B28K, desB29-30; xv. B5F,
B26G, B28K, desB29-30; xvi. A14E, B5F, B26A, B28K, desB29-30; xvii.
A14E, B5Y, B26A, B28K, desB29-30; xviii. A14E, B5Y, B26G, B28K,
desB29-30; xix. A14E, B5Y, B28K, desB29-30; xx. A14E, B5F, B28K,
desB29-30; xxi. A14E, B5Y, B26K, desB27-desB30; xxii. A14E, B5Y,
desB30; xxiii. A14E, B5Y, B26G, desB30; or xxiv. A14E, B5Y, B26A,
desB30.
9. The insulin derivative according to claim 7, wherein Acy is
selected from the group consisting of: lithocholic acid,
1,16-hexadecanedioic acid, 1,18-octadecanedioic acid,
1,20-eicosanedioic acid, tetrazole-C16, tetrazole-C17, tetrazole
C18 and tetradecanoic acid.
10. The insulin derivative according to claim 8, wherein -L1-L2-L3
represents a divalent linking group selected from group consisting
of DgGlu, gGlu, gGlu-gGlu, gGlu-OEG, gGlu-OEG-OEG, OEG,
sulfonimide-C4, and sulfonimide-C4-sulfonimide-C4.
11. The insulin derivative according to claim 1, wherein said
insulin derivative is
N{Epsilon-B28}-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)-
butanoyl]amino]ethoxy]ethoxy]acetyl]-[GluA14,TyrB5,GlyB26,LysB28],des-(B29-
-B30)-lnsulin ##STR00080##
12. An insulin analogue comprising i. A14E, B5Y, B26A, B28K,
desB29-30; ii. A14E, B5Y, B26G, B28K, desB29-30; iii. B5Y, B26A,
B28K, desB29-30; iv. B5Y, B26G, B28K, desB29-30; v. B5Y, B28K,
desB29-30; vi. A14E, B5F, B26G, B28K, desB29-30; vii. B5F, B28K,
desB29-30; viii. B5Y, B26K, desB27-desB30; ix. B5Y, desB30; x. B5Y,
B26G, desB30; xi. B5Y, B26A, desB30; xii. B5F, B26A, B28K,
desB29-30; xiii. B5Y, B26A, B28K, desB29-30; xiv. B5Y, B26G, B28K,
desB29-30; xv. B5F, B26G, B28K, desB29-30; xvi. A14E, B5F, B26A,
B28K, desB29-30; xvii. A14E, B5Y, B26A, B28K, desB29-30; xviii.
A14E, B5Y, B26G, B28K, desB29-30; xix. A14E, B5Y, B28K, desB29-30;
xx. A14E, B5F, B28K, desB29-30; xxi. A14E, B5Y, B26K,
desB27-desB30; xxii. A14E, B5Y, desB30; xxiii. A14E, B5Y, B26G,
desB30; or xxiv. A14E, B5Y, B26A, desB30. or a pharmaceutically
acceptable salt, amide or ester thereof.
13. An insulin derivative according to claim 1, wherein said
insulin analogue comprising an amino acid sequence selected from
the group consisting of SEQ ID NO: 1, and SEQ ID NOs: 3-15.
14. An insulin derivative according to claim 5, wherein said
insulin analogue comprising an amino acid sequence selected from
the group consisting of SEQ ID NO: 1, and SEQ ID NOs: 3-15.
15. A method of treating diabetes, cardiovascular disease,
atherosclerosis, or endothelial dysfunction, or reducing liver
triglyceride content or reducing body weight gain, comprising
administering to a subject in need thereof a therapeutically
effective amount of an insulin derivative according to claim 1.
16. A method of treating diabetes, cardiovascular disease,
atherosclerosis, or endothelial dysfunction, or reducing liver
triglyceride content or reducing body weight gain, comprising
administering to a subject in need thereof a therapeutically
effective amount of an insulin derivative according to claim 8.
17. A method for treating diabetes, diabetes of Type 1, diabetes of
Type 2, impaired glucose tolerance, hyperglycemia, dyslipidemia,
obesity, metabolic syndrome X, insulin resistance syndrome,
hypertension, cognitive disorders, atherosclerosis, myocardial
infarction, stroke, cardiovascular disorders, coronary heart
disease, stroke, inflammatory bowel syndrome, dyspepsia,
hypotension or gastric ulcers, comprising administrating to a
subject in need thereof a therapeutically effective amount of an
insulin derivative according to claim 1.
18. A method for treating diabetes, diabetes of Type 1, diabetes of
Type 2, impaired glucose tolerance, hyperglycemia, dyslipidemia,
obesity, metabolic syndrome X, insulin resistance syndrome,
hypertension, cognitive disorders, atherosclerosis, myocardial
infarction, stroke, cardiovascular disorders, coronary heart
disease, stroke, inflammatory bowel syndrome, dyspepsia,
hypotension or gastric ulcers, comprising administrating to a
subject in need thereof a therapeutically effective amount of an
insulin derivative according to claim 8.
19. A method for treating diabetes, diabetes of Type 1, diabetes of
Type 2, impaired glucose tolerance, hyperglycemia, dyslipidemia,
obesity, metabolic syndrome X, insulin resistance syndrome,
hypertension, cognitive disorders, atherosclerosis, myocardial
infarction, stroke, cardiovascular disorders, coronary heart
disease, stroke, inflammatory bowel syndrome, dyspepsia,
hypotension or gastric ulcers, comprising administrating to a
subject in need thereof a therapeutically effective amount of an
insulin analogue according to claim 11.
20. A method for determining selectivity of an insulin compound
comprising the following steps: measuring the maximal AKT
phosphorylation induced by said insulin compound relative to human
insulin measuring the maximal ERK activation induced by said
insulin compound relative to human insulin, wherein the ERK/AKT
ratio is less than 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. application Ser.
No. 15/998,755, filed Aug. 16, 2018, which claims priority to
European Patent Application No. 17186612.2, filed Aug. 17, 2017 and
European Patent Application No. 17204872.0, filed Dec. 1, 2017; the
contents of which are incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to novel insulin analogues and
derivatives thereof, such as acylated insulin analogues, and their
pharmaceutical use, in particular in the treatment or prevention of
medical conditions relating to diabetes, obesity and cardiovascular
diseases.
INCORPORATION-BY-REFERENCE OF THE SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted in ASCII format and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Oct. 29,
2020, is named 170002US01_SeqList.txt and is 6 kilobytes in
size.
BACKGROUND
[0004] Diabetes mellitus is a metabolic disorder, in which the
ability to utilise glucose is partly or completely lost. More than
5% of the global population live with diabetes, with millions more
at risk of developing the disease.
[0005] With current therapies, still about 50% of people with
diabetes die of cardiovascular disease and because people with
diabetes also are at risk of developing microvascular complications
(like nephropathy, retinopathy and neuropathy), there is a
continuous need for developing new drugs for improved treatment.
Especially in the case of Type 2 diabetes mellitus, since these
patients, in addition to hyperglycemia, often suffer from various
metabolic dysfunctions, such as e.g. dyslipidemia, obesity and
cardiovascular complications for which current insulin therapy only
have limited beneficial effect.
[0006] In the liver, insulin suppresses gluconeogenesis and
glycogenolysis, and increases glycogen synthesis, resulting in
decreased glucose output from the liver. These processes are
markedly impaired by hepatocyte insulin resistance, and this is the
major cause of fasting hyperglycemia in the metabolic syndrome. In
addition to the effects on glucose metabolism, insulin also
increases the synthesis of fatty acids and triglycerides in the
liver through activation of the transcription factor SREBP-1c,
which in turn increases transcription of genes for lipogenic
enzymes, including acetyl-coenzyme A carboxylase and fatty acid
synthase (FAS). However, in contrast to the effects on glycogen
synthesis and glucose production, insulin-induced increase in lipid
synthesis is not affected at all, or at least less impaired in
insulin resistant rodent models of type II diabetes as well as in
humans suffering from diabetes caused by lipodystrophy or mutations
affecting specific nodes in the intracellular insulin signalling
pathways. Accordingly, the hyperinsulinemia associated with
metabolic syndrome can directly cause increases in hepatic lipid
synthesis and thereby exacerbate the increases in circulating
levels of triglycerides. This is a very likely reason why several
studies indicate that hepatic insulin resistance contributes to
human dyslipidaemia, hepatic steatosis, cardiovascular vascular
dysfunction, and even reduced kidney function observed in people
with Type 2 diabetes. In line with this, treatment of insulin
resistant subjects with a high concentration of insulin in order to
lower blood glucose levels may result in over-stimulation of the
non-resistant pathways involved in e.g. de novo lipogenesis. In
order to lower blood glucose levels without over-stimulating
non-resistant pathways (e.g. the lipogenic pathways), it would be
desirable to have access to insulin analogues, which selectively
activate the glucose lowering pathways. Such functionally selective
insulin analogues would, beyond lowering blood glucose levels,
exhibit improved effects on dyslipidaemia, hepatic steatosis,
atherosclerosis and cardiovascular diseases (CVD).
[0007] WO 2005 054291 allegedly describes single chain insulins
with B28K acylation. WO 2009 112583 allegedly describes protease
stabilised insulin analogues comprising B28K. WO90/07511,
WO96/15804, WO200043034, US2012241356, WO2012015692 and WO9731022
allegedly disclose insulin analogues comprising B28K, some of which
allegedly also discloses acylation at the same position.
[0008] A great variety of insulin analogues and derivatives have
been reported. However they all activate the insulin receptor in
quite the same manner, i.e. the downstream effects of the insulin
receptor activation are rather similar, regardless of whether the
activation results from binding a high-affinity analogue or a
low-affinity analogue--only the potency differs.
[0009] Thus, there is still a need for insulin analogues and
derivatives having functionally selective properties on the
resistant and non-resistant pathways, e.g. gluconeogenesis and
lipid metabolism pathways.
SUMMARY
[0010] In a first aspect, the present invention relates to an
insulin derivative, wherein said insulin derivative comprises B5Y
and a substituent comprising an acyl group.
[0011] In a second aspect, the present invention relates to an
insulin derivative, wherein said insulin derivative comprises B5F
and a substituent comprising an acyl group.
[0012] In another aspect, the present invention relates to an
insulin derivative, wherein said insulin derivative comprises B5Y
and B26G or B26A and a substituent comprising an acyl group
attached to B28K, B26K or B29K.
[0013] In another aspect, the present invention relates to an
insulin derivative, wherein said insulin derivative comprises B5F
and B26G or B26A and a substituent comprising an acyl group
attached to B28K, B26K or B29K.
[0014] In another aspect, the invention provides pharmaceutical
compositions comprising the insulin derivative of the invention,
and one or more pharmaceutically acceptable carriers or
diluents.
[0015] In further aspects, the invention relates to the use of the
insulin derivatives according to the invention for the manufacture
of a medicament for the treatment or prevention of diabetes, Type 1
diabetes, Type 2 diabetes, impaired glucose tolerance,
hyperglycemia, dyslipidemia, obesity, metabolic syndrome (metabolic
syndrome X, insulin resistance syndrome), hypertension, cognitive
disorders, atherosclerosis, myocardial infarction, stroke,
cardiovascular disorders, coronary heart disease, stroke,
inflammatory bowel syndrome, dyspepsia, hypotension or gastric
ulcers.
[0016] Also, or alternatively in a second aspect, the invention
provides insulin derivatives with improved effects on processes
related to obesity, dyslipidemia or cardiovascular complications,
such as a lower weight gain, lower increase in body fat mass, lower
increase in liver triglycerides or improved endothelial
function.
[0017] In one aspect, the insulin derivatives of the present
invention are capable of lowering blood glucose without negatively
impacting lipid metabolism.
[0018] In another aspect, the insulin derivatives of the present
invention induce a submaximal insulin receptor phosphorylation, and
induce selective signalling, and thus selective cellular response,
i.e. give a lower maximal response on lipid metabolism pathways
than on glucose lowering pathway, when compared to human
insulin.
[0019] In yet another aspect, the insulin derivatives of the
present invention, besides the glucose lowering, also have lower
weight gain, in particular by a lower increase in fat mass.
[0020] In one aspect the invention provides insulin derivatives
with improved effects on processes related to hepatic
dyslipidaemia, hepatic steatosis or non-alcoholic fatty liver
disease (NAFLD), in particular by increased lowering of liver
triglycerides compared to human insulin.
[0021] In another aspect, the invention provides insulin
derivatives with improved effects on processes related to
cardiovascular disorders such as improved endothelial function
compared to human insulin.
[0022] In yet another aspect, the insulin derivatives of the
present invention provide lower incidence of adverse cardiovascular
events in patients with diabetes.
[0023] In one aspect the invention provides insulin derivatives
with improved stability in formulation.
[0024] The invention may also solve further problems that will be
apparent from the disclosure of the exemplary embodiments.
BRIEF DESCRIPTION OF DRAWINGS
[0025] FIG. 1 shows representative receptor binding curves for
human insulin (.circle-solid.) and compound of example 15
(.tangle-solidup.) from a competition binding assay with
solubilised IR-A (CPM=counts per minute);
[0026] FIG. 2 shows representative dose-response curves for human
insulin (.circle-solid.) and compound of example 15
(.tangle-solidup.) from an IRpY1158 phosphorylation assay in CHO-hl
R cells overexpressing the IR-A;
[0027] FIG. 3 shows representative dose-response curves for human
insulin (.circle-solid.) and compound of example 15
(.tangle-solidup.) from an AKT phosphorylation assay
(phosphorylation of serine residue number 473) in CHO-hIR cells
overexpressing the IR-A;
[0028] FIG. 4 shows representative dose-response curves for human
insulin (.circle-solid.) and compound of example 15
(.tangle-solidup.) from an ERK phosphorylation assay in CHO-hIR
cells overexpressing the IR-A;
[0029] FIG. 5 shows representative dose-response curves for human
insulin (.circle-solid.) and compound of example 15
(.tangle-solidup.) from a lipogenesis assay in primary rat
adipocytes (DPM=disintegrations per minute);
[0030] FIG. 6 shows representative dose-response curves for human
insulin (.circle-solid.) and compound of example 15
(.tangle-solidup.) from a glycogen synthesis assay in primary rat
hepatocytes;
[0031] FIG. 7 shows representative dose-response curves for human
insulin (.circle-solid.) and compound of example 15
(.tangle-solidup.) from a quantitative real-time polymerase chain
reaction (RT-PCR) assay for fasn performed on cDNA isolated from
primary rat hepatocytes;
[0032] FIG. 8 shows representative dose-response curves for human
insulin (.circle-solid.) and compound of example 15
(.tangle-solidup.) from a quantitative real-time polymerase chain
reaction (RT-PCR) assay for g6pc performed on cDNA isolated from
primary rat hepatocytes;
[0033] FIG. 9 shows representative dose-response curves for human
insulin (.circle-solid.) and compound of example 15
(.tangle-solidup.) from a de novo lipogenesis assay in primary rat
hepatocytes (CPM=counts per minute);
[0034] FIG. 10 shows representative curves for HbA1c levels from a
sub-chronic in vivo study in diabetic STZ-DIO mice dosed
subcutaneously twice daily for six weeks with vehicle, the PK
Comparator (comparator no. 5) and compound of example 15.
[0035] FIG. 11 shows representative curves for body weight from a
sub-chronic in vivo study in diabetic STZ-DIO mice dosed
subcutaneously twice daily for six weeks with vehicle, the PK
Comparator (comparator no. 5) and compound of example 15.
[0036] FIG. 12 shows representative curves for body fat mass from a
sub-chronic in vivo study in diabetic STZ-DIO mice dosed
subcutaneously twice daily for six weeks with vehicle, the PK
Comparator (comparator no. 5) and compound of example 15.
[0037] FIG. 13 shows representative curves for liver TG from a
sub-chronic in vivo study in diabetic STZ-DIO mice dosed
subcutaneously twice daily for six weeks with vehicle, the PK
Comparator (comparator no. 5) and compound of example 15.
[0038] FIG. 14 shows representative curves for ACh-stimulated
vasorelaxation of mesenteric arteries from a sub-chronic in vivo
study in diabetic STZ-DIO mice dosed subcutaneously twice daily for
six weeks with vehicle, the PK Comparator (comparator no. 5) and
compound of example 15.
[0039] FIG. 15 shows representative SEC data for a formulation of
0.6 mM of a comparator compound i.e. comparator 9 (black line)
including 3 Zn/6ins, 30 mM phenol, 1.6% glycerol and 7 mM
tris(hydroxymethyl)aminomethane at pH 7.6. A SEC reference mixture
of human albumin, Co(III) insulin hexamer and a monomer insulin
analogue (B9Asp, B27Glu) is included (grey line).
[0040] FIG. 16 shows representative SEC data of a similar
formulation of compound of example 3 (black line).
[0041] FIG. 17 shows representative SEC data of a similar
formulation of compound of example 11 (black line).
[0042] FIG. 18 shows representative SEC data of a similar
formulation of compound of example 12 (black line)
[0043] FIG. 19 shows representative SEC data of a similar
formulation of compound of example 15 (black line).
DESCRIPTION
[0044] The present invention provides novel analogues of human
insulin, which are acylated and show functionally selective
properties on the pathways involved in gluconeogenesis and lipid
metabolism.
[0045] The present invention relates broadly to insulin derivatives
comprising B5Y or B5F and a substituent comprising an acyl
group.
[0046] In one embodiment, the insulin derivatives of the present
invention induce a submaximal phosphorylation of insulin receptor
of about or below 65%, when compared to human insulin.
[0047] In one embodiment, the insulin derivative comprises a
substituent comprising an acyl group.
[0048] In one embodiment, the substituent is attached to B28K, B26K
or B29K.
[0049] In one embodiment, the substituent is attached to B28K.
[0050] In one embodiment, the substituent is attached to B26K.
[0051] In one embodiment, the substituent is attached to B29K.
[0052] In another embodiment, the insulin derivative comprises B26G
or B26A.
[0053] In another embodiment, the insulin derivative comprises
A14E.
[0054] In another embodiment, the insulin derivative comprises
desB30, desB29-30 or desB27-30.
[0055] In another embodiment, the insulin derivative comprises
desB30.
[0056] In another embodiment, the insulin derivative comprises
desB29-30.
[0057] In another embodiment, the insulin derivative comprises
desB27-30.
[0058] In another embodiment the insulin derivative of the
invention is selected from the group consisting of the compounds of
examples 1-46:
TABLE-US-00001 Acyl Ex Name Mutations Linker group Acylation site 1
N{Epsilon-B28}-15- B5Y, B28K, none C16 B28K carboxypentadecanoyl-
desB29-B30 diacid [TyrB5, LysB28], des-(B29-B30)-Insulin 2
N{Epsilon-B28}-[(4S)-4-carboxy-4- B5Y, B28K, gGlu C16 B28K (15-
desB29-B30 diacid carboxypentadecanoylami- no)butanoyl]-[TyrB5,
LysB28], des-(B29-B30)-Insulin 3 N{Epsilon-B28}-17- B5Y, B28K, none
C18 B28K carboxyheptadecanoyl- desB29-B30 diacid [TyrB5, LysB28],
des-(B29-B30)-Insulin 4 N{Epsilon-B28}-[(4S)-4-carboxy-4- B5Y,
B28K, gGlu C18 B28K (17- desB29-B30 diacid carboxyheptadecanoylami-
no)butanoyl]-[TyrB5, LysB28], des-(B29-B30)-Insulin 5
N{Epsilon-B28}-[2-[2-[2-(17- B5Y, B28K, OEG C18 B28K
carboxyheptadecanoylami- desB29-B30 diacid
no)ethoxy]ethoxy]acetyl]- [TyrB5, LysB28], des-(B29-B30)-Insulin 6
N{Epsilon-B28}-[(4S)-4-carboxy-4- B5Y, B28K, 2xgGlu C18 B28K
[[(4S)-4-carboxy-4-(17- desB29-B30 diacid carboxyheptadecanoylami-
no)butanoyl]amino]butanoyl]- [TyrB5, LysB28], des-(B29-B30)-Insulin
7 N{Epsilon-B28}-[2-[2-[2-[[(4S)-4- B5Y, B28K, gGlu- C18 B28K
carboxy-4-(17- desB29-B30 OEG diacid carboxyheptadecanoylami-
no)butanoyl]amino]ethoxy]ethoxy]acetyl]- [TyrB5, LysB28],
des-(B29-B30)-Insulin 8 N{Epsilon-B28}-[(4S)-4-carboxy-4- B5Y,
B28K, gGlu Litocholic acid B28K [[(4R)-4-[(3R,10S,13R,17R)-3-
desB29-B30 hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17- tetradecahydro-1H-
cyclopenta[a]phenanthren-17- yl]pentanoyl]amino]butanoyl]- [TyrB5,
LysB28], des-(B29-B30)-Insulin 9 N{Epsilon-B28}-[(4R)-4- B5Y, B28K,
none Litocholic acid B28K [(3R,10S,13R,17R)-3-hydroxy- desB29-B30
10,13-dimethyl- 2,3,4,5,6,7,8,9,11,12,14,15,16,17-
tetradecahydro-1H- cyclopenta[a]phenanthren-17-
yl]pentanoyl]-[TyrB5, LysB28], des-(B29-B30)-Insulin 10
N{Epsilon-B28}-15- B5Y, B26G, none C16 B28K carboxypentadecanoyl-
B28K, diacid [TyrB5, GlyB26, LysB28], desB29-B30
des-(B29-B30)-Insulin 11 N{Epsilon-B28}-17- B5Y, B26G, none C18
B28K carboxyheptadecanoyl- B28K, diacid [TyrB5, GlyB26, LysB28],
desB29-B30 des-(B29-B30)-Insulin 12 N{Epsilon-B28}-17- A14E, B5Y,
none C18 B28K carboxyheptadecanoyl- B26G, B28K, diacid [GluA14,
TyrB5, GlyB26, LysB28], desB29-B30 des-(B29-B30)-Insulin 13
N{Epsilon-B28}-[(4S)-4-carboxy-4- A14E, B5Y, gGlu C18 B28K (17-
B26G, B28K, diacid carboxyheptadecanoylamino)butanoyl]- desB29-B30
[GluA14, TyrB5, GlyB26, LysB28], des-(B29-B30)-Insulin 14
N{Epsilon-B28}-[(4S)-4-carboxy-4- A14E, B5Y, 2xgGlu C18 B28K
[[(4S)-4-carboxy-4-(17- B26G, B28K, diacid carboxyheptadecanoylami-
desB29-B30 no)butanoyl]amino]butanoyl]- [GluA14, TyrB5, GlyB26,
LysB28], des-(B29-B30)-Insulin 15 N{Epsilon-B28}-[2-[2-[2-[[(4S)-4-
A14E, B5Y, gGlu- C18 B28K carboxy-4-(17- B26G, B28K, OEG diacid
carboxyheptadecanoylamino)butanoyl]ami- desB29-B30
no]ethoxy]ethoxy]acetyl]- [GluA14, TyrB5, GlyB26, LysB28],
des-(B29-B30)-Insulin 16 N{Epsilon-B28}-[2-[2-[2-[[(4S)-4- A14E,
B5Y, gGlu- C20 B28K carboxy-4-(19- B26G, B28K, OEG diacid
carboxynonadecanoylamino)butanoyl]ami- desB29-B30
no]ethoxy]ethoxy]acetyl]- [GluA14, TyrB5, GlyB26, LysB28],
des-(B29-B30)-Insulin 17 N{Epsilon-B28}-[2-[2-[2-[[2-[2-[2- A14E,
B5Y, gGlu- C20 B28K [[(4S)-4-carboxy-4-(19- B26G, B28K, 2xOEG
diacid carboxynonadecanoylami- desB29-B30
no)butanoyl]amino]ethoxy]ethoxy]acetyl]ami-
no]ethoxy]ethoxy]acetyl]- [GluA14, TyrB5, GlyB26, LysB28],
des-(B29-B30)-Insulin 18 N{Epsilon-B28}-[(4S)-4-carboxy-4- A14E,
B5Y, gGlu C20 B28K (19- B26G, B28K, diacid
carboxynonadecanoylamino)butanoyl]- desB29-B30 [GluA14, TyrB5,
GlyB26, LysB28], des-(B29-B30)-Insulin 19 N{Epsilon-B28}-19- A14E,
B5Y, none C20 B28K carboxynonadecanoyl- B26G, B28K, diacid [GluA14,
TyrB5, GlyB26, LysB28], desB29-B30 des-(B29-B30)-Insulin 20
N{Epsilon-B28}-15-(1H-tetrazol-5- A14E, B5Y, none Tetrazole-C16
B28K yl)pentadecanoyl- B26G, B28K, [GluA14, TyrB5, GlyB26, LysB28],
desB29-B30 des-(B29-B30)-Insulin 21
N{Epsilon-B28}-17-(1H-tetrazol-5- A14E, B5Y, none Tetrazole-C18
B28K yl)heptadecanoyl- B26G, B28K, [GluA14, TyrB5, GlyB26, LysB28],
desB29-B30 des-(B29-B30)-Insulin 22
N{Epsilon-B28}-16-(1H-tetrazol-5- A14E, B5Y, none Tetrazole-C17
B28K yl)hexadecanoyl- B26G, B28K, [GluA14, TyrB5, GlyB26, LysB28],
desB29-B30 des-(B29-B30)-Insulin 23
N{Epsilon-B28}-4-[16-(1H-tetrazol- A14E, B5Y, sulfonimide-
Tetrazole-C17 B28K 5- B26G, B28K, C4 yl)hexadecanoylsulfamo-
desB29-B30 yl]butanoyl-[GluA14, TyrB5, GlyB26, LysB28],
des-(B29-B30)-Insulin 24 N{Epsilon-B28}-4-[4-[15-(1H- A14E, B5Y,
2xsulfonimide-C4 Tetrazole-C16 B28K tetrazol-5- B26G, B28K,
yl)pentadecanoylsulfamoyl]butanoylsulfamo- desB29-B30 yl]butanoyl-
[GluA14, TyrB5, GlyB26, LysB28], des-(B29-B30)-Insulin 25
N{Epsilon-B28}-4-[17-(1H-tetrazol- A14E, B5Y, sulfonimide-
Tetrazole-C18 B28K 5- B26G, B28K, C4 yl)heptadecanoylsulfamo-
desB29-B30 yl]butanoyl- [GluA14, TyrB5, GlyB26, LysB28],
des-(B29-B30)-Insulin 26 N{Epsilon-B28}-[2-[2-[2-[[2-[2-[2- A14E,
B5Y, gGlu- Tetrazole-C16 B28K [[(4S)-4-carboxy-4-[15-(1H- B26G,
B28K, 2xOEG tetrazol-5- desB29-B30
yl)pentadecanoylamino]butanoyl]ami-
no]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]- [GluA14,
TyrB5, GlyB26, LysB28], des-(B29-B30)-Insulin 27
N{Epsilon-B28}-4-[4-[17-(1H- A14E, B5Y, 2xsulfonimide-
Tetrazole-C18 B28K tetrazol-5- B26G, B28K, C4
yl)heptadecanoylsulfamoyl]butanoylsulfamoyl]butanoyl- desB29-B30
[GluA14, TyrB5, GlyB26, LysB28], des-(B29-B30)-Insulin 28
N{Epsilon-B28}-4-(17- A14E, B5Y, sulfonimide- C18 B28K
carboxyheptadecanoylsulfamoyl)butanoyl- B26G, B28K, C4 diacid
[GluA14, TyrB5, GlyB26, LysB28], desB29-B30 des-(B29-B30)-Insulin
29 N{Epsilon-B28}-[2-[2-[2-[[(4S)-4- A14E, B5Y, gGlu- Tetrazole-C16
B28K carboxy-4-[15-(1H-tetrazol-5- B26G, B28K, OEG
yl)pentadecanoylamino]butanoyl]ami- desB29-B30
no]ethoxy]ethoxy]acetyl]- [GluA14, TyrB5, GlyB26, LysB28],
des-(B29-B30)-Insulin 30 N{Epsilon-B28}-[(4R)-4-carboxy-4- A14E,
B5Y, DgGlu C18 B28K (17- B26G, B28K, diacid
carboxyheptadecanoylamino)butanoyl]- desB29-B30 [GluA14, TyrB5,
GlyB26, LysB28], des-(B29-B30)-Insulin 31
N{Epsilon-B28}-[2-[2-[2-[[(4S)-4- B5Y, B26G, gGlu- C18 B28K
carboxy-4-(17- B28K, OEG diacid
carboxyheptadecanoylamino)butanoyl]ami- desB29-B30
no]ethoxy]ethoxy]acetyl]- [TyrB5, GlyB26, LysB28],
des-(B29-B30)-Insulin 32 N{Epsilon-B28}-[2-[2-[2-[[(4S)-4- A14E,
B5Y, gGlu- C18 B28K carboxy-4-(17- B26A, B28K, OEG diacid
carboxyheptadecanoylamino)butanoyl]ami- desB29-B30
no]ethoxy]ethoxy]acetyl]- [GluA14, TyrB5, AlaB26, LysB28],
des-(B29-B30)-Insulin 33 N{Epsilon-B28}-[2-[2-[2-[[(4S)-4- B5Y,
B26A, gGlu- C18 B28K carboxy-4-(17- B28K, OEG diacid
carboxyheptadecanoylamino)butanoyl]ami- desB29-B30
no]ethoxy]ethoxy]acetyl]- [TyrB5, AlaB26, LysB28],
des-(B29-B30)-Insulin 34 N{Epsilon-B28}-[(4S)-4-carboxy-4- A14E,
B5Y, gGlu C18 B28K (17- B26A, B28K, diacid
carboxyheptadecanoylamino)butanoyl]- desB29-B30 [GluA14, TyrB5,
AlaB26, LysB28], des-(B29-B30)-Insulin 35 N{Epsilon-B28}-17- B5F,
B28K, none C18 B28K carboxyheptadecanoyl- desB29-B30 diacid [PheB5,
LysB28], des-(B29-B30)-Insulin 36 N{Epsilon-B28}-17- A14E, B5F,
none C18 B28K carboxyheptadecanoyl- B26G, B28K, diacid [GluA14,
PheB5, GlyB26, LysB28], desB29-B30 des-(B29-B30)-Insulin 37
N{Epsilon-B26}-17- B5Y, B26K, none C18 B26K carboxyheptadecanoyl-
desB27-B30 diacid [TyrB5,LysB26], des-(B27-B30)-Insulin 38
N{Epsilon-B26}-[(4S)-4-carboxy-4- B5Y, B26K, gGlu C18 B26K (17-
desB27-B30 diacid carboxyheptadecanoylamino)butanoyl]- [TyrB5,
LysB26], des-(B27-B30)-Insulin 39
N{Epsilon-B26}-[2-[2-[2-[[2-[2-[2- B5Y, B26K, gGlu- C18 B26K
[[(4S)-4-carboxy-4-(17- desB27-B30 2xOEG diacid
carboxyheptadecanoylamino)butanoyl]ami-
no]ethoxy]ethoxy]acetyl]ami- no]ethoxy]ethoxy]acetyl]- [TyrB5,
LysB26], des-(B27-B30)-Insulin 40 N{Epsilon-B29}-[(4S)-4-carboxy-4-
B5Y, desB30 gGlu C16 B29K (15- diacid
carboxypentadecanoylamino)butanoyl]-[TyrB5], des-ThrB30-Insulin 41
N{Epsilon-B29}-tetradecanoyl- B5Y, desB30 none C14 B29K [TyrB5],
des-ThrB30-Insulin 42 N{Epsilon-B29}-[(4S)-4-carboxy-4- B5Y, B26G,
gGlu C16 B29K (15- desB30 diacid carboxypentadecanoylamino)buta
noyl]-[TyrB5, GlyB26], des-ThrB30-Insulin 43
N{Epsilon-B29}-[(4S)-4-carboxy-4- B5Y, B26A, gGlu C16 B29K (15-
desB30 diacid carboxypentadecanoylamino)butanoyl]- [TyrB5, AlaB26],
des-ThrB30-Insulin 44 N{Epsilon-B29}-[(4S)-4-carboxy-4- B5Y, B26G,
gGlu C18 B29K (17- desB30 diacid
carboxyheptadecanoylamino)butanoyl]- [TyrB5, GlyB26],
des-ThrB30-Insulin 45 N{Epsilon-B29}-[(4S)-4-carboxy-4- B5Y, B26A,
gGlu C18 B29K (17- desB30 diacid
carboxyheptadecanoylamino)butanoyl]- [TyrB5, AlaB26],
des-ThrB30-Insulin 46 N{Epsilon-B26}-[2-[2-[2-[[(4S)-4- B5Y, B26K,
gGlu- C18 B26K carboxy-4-(17- desB27-B30 OEG diacid
carboxyheptadecanoylamino)butanoyl]ami- no]ethoxy]ethoxy]acetyl]-
[TyrB5, LysB26], des-(B27-B30)-Insulin Ex: example nr.
[0059] The present invention relates to the use of the insulin
derivatives according to the invention for the manufacture of a
medicament for the treatment or prevention of diabetes.
[0060] In animal models it has surprisingly been found, that
insulinderivatives of the present invention are capable of lowering
blood glucose with less negative impact on lipid metabolism or
endothelial dysfunction. This is believed to lead to a lower
incidence of adverse cardiovascular events, and these animal
experiments also show that the glucose lowering is accompanied by a
lower weight gain, and in particular by a lower increase in fat
mass, when compared to conventional insulin treatment.
[0061] This is likely to lead to beneficial effects on liver
disease development or progression as the animal experiments also
show that the glucose lowering is accompanied by an increased
lowering in liver triglycerides when compared to conventional
insulin treatment.
[0062] Furthermore, when examining the down-stream signalling in
vitro, the insulin derivatives of the present invention have shown
to induce a submaximal insulin receptor phosphorylation, and to
induce selective signalling, and thus selective cellular response,
i.e. to give a lower maximal response on lipid metabolism pathways
than on glucose lowering pathway, when compared to human
insulin.
[0063] Finally, the insulin derivatives of the present invention
also have a desired selective signalling and improved stability in
formulation.
[0064] Insulin
[0065] The term "human insulin" as used herein means the human
insulin hormone whose structure and properties are well-known.
Human insulin has two polypeptide chains, named the A-chain and the
B-chain. The A-chain is a 21 amino acid peptide and the B-chain is
a 30 amino acid peptide, the two chains being connected by
disulphide bridges: a first bridge between the cysteine in position
7 of the A-chain and the cysteine in position 7 of the B-chain, and
a second bridge between the cysteine in position 20 of the A-chain
and the cysteine in position 19 of the B-chain. A third bridge is
present between the cysteines in position 6 and 11 of the
A-chain.
[0066] The human insulin A-chain has the following sequence:
TABLE-US-00002 (SEQ ID NO: 1) GIVEQCCTSICSLYQLENYCN,
while the B-chain has the following sequence:
TABLE-US-00003 (SEQ ID NO: 2) FVNQHLOGSHLVEALYLVOGERGFFYTPKT.
[0067] In the human body, the hormone is synthesized as a
single-chain precursor proinsulin (preproinsulin) consisting of a
prepeptide of 24 amino acids followed by proinsulin containing 86
amino acids in the configuration: prepeptide-B-Arg Arg-C-Lys Arg-A,
in which C is a connecting peptide of 31 amino acids. Arg-Arg and
Lys-Arg are cleavage sites for cleavage of the connecting peptide
from the A and B chains.
[0068] "An insulin" according to the invention is herein to be
understood as human insulin or an insulin from another species,
such as porcine or bovine insulin.
[0069] The term "insulin peptide" as used herein means a peptide
which is either human insulin or an analogue or a derivative
thereof with insulin activity.
[0070] Insulin Analogue
[0071] The term "insulin analogue" as used herein means a single
modified human insulin molecule wherein one or more amino acid
residues of the insulin have been substituted by other amino acid
residues and/or wherein one or more amino acid residues have been
deleted from the insulin and/or wherein one or more amino acid
residues have been added and/or inserted to the insulin.
[0072] In one embodiment an insulin analogue comprises less than 10
amino acid modifications (substitutions, deletions, additions
(including insertions) and any combination thereof) relative to
human insulin, alternatively less than 9, 8, 7, 6, 5, 4, 3, 2 or 1
modification relative to human insulin.
[0073] Modifications in the insulin molecule are denoted stating
the chain (A or B), the position, and the one or three letter code
for the amino acid residue substituting the native amino acid
residue.
[0074] By "connecting peptide" or "C-peptide" is meant a connection
moiety "C" of the B-C-A polypeptide sequence of a single chain
proinsulin-molecule. In the human insulin chain, the C-peptide
connects position 30 of the B chain and position 1 of the A chain
and is 35 amino acid residue long. The connecting peptide includes
two terminal dibasic amino acid sequence, e.g., Arg-Arg and Lys-Arg
which serve as cleavage sites for cleavage off of the connecting
peptide from the A and B chains to form the two-chain insulin
molecule.
[0075] By "desB30" or "B(1-29)" is meant a natural insulin B chain
or an analogue thereof lacking the B30 amino acid and "A(1-21)"
means the natural insulin A chain. Thus, e.g., B5Y, B28K,
desB29-desB30 human insulin is an analogue of human insulin where
the amino acid in position 5 in the B chain is substituted with
tyrosine (Tyr or Y), the amino acid in position 28 in the B chain
is substituted with lysine (Lys or K), and the amino acids in
positions 29 and 30 in the B chain are deleted.
[0076] Herein terms like "A1", "A2" and "A3" etc. indicates the
amino acid in position 1, 2 and 3 etc., respectively, in the A
chain of insulin (counted from the N-terminal end). Similarly,
terms like B1, B2 and B3 etc. indicates the amino acid in position
1, 2 and 3 etc., respectively, in the B chain of insulin (counted
from the N-terminal end).
[0077] Herein, the term "amino acid residue" is an amino acid from
which, formally, a hydroxy group has been removed from a carboxy
group and/or from which, formally, a hydrogen atom has been removed
from an amino group.
[0078] In one embodiment, the insulin analogue of the present
invention is an analogue of human insulin comprising 2-10
mutations.
[0079] In one embodiment, the insulin analogue of the present
invention is an analogue of human insulin comprising 2-6
mutations.
[0080] In one embodiment, the insulin analogue of the present
invention is an analogue of human insulin comprising 2-3
mutations.
[0081] In one embodiment, the insulin analogue of the present
invention is an analogue of human insulin comprising two
mutations.
[0082] In one embodiment, the insulin analogue of the present
invention is an analogue of human insulin comprising three
mutations.
[0083] In one embodiment, the insulin analogue of the present
invention is an analogue of human insulin comprising four
mutations.
[0084] In one embodiment, the insulin analogue of the present
invention is an analogue of human insulin comprising five
mutations.
[0085] In one embodiment, the insulin analogue of the present
invention is an analogue of human insulin comprising six
mutations.
[0086] In one embodiment, the insulin analogue of the present
invention is an analogue of human insulin comprising seven
mutations.
[0087] In one embodiment, the insulin analogue of the present
invention is an analogue of human insulin comprising eight
mutations.
[0088] In one embodiment, the insulin analogue of the present
invention is an analogue of human insulin comprising nine
mutations.
[0089] In one embodiment, the insulin analogue of the present
invention is an analogue of human insulin comprising 10
mutations.
[0090] In one embodiment, the insulin analogue of the present
invention is an analogue of human insulin comprising less than 10
mutations.
[0091] In one embodiment, the insulin analogue of the present
invention is an analogue of human insulin comprising less than 7
mutations.
[0092] In one embodiment, the insulin analogue of the present
invention is an analogue of human insulin comprising less than 5
mutations.
[0093] In one embodiment, the insulin analogue of the present
invention is an analogue of human insulin comprising less than 4
mutations.
[0094] In one embodiment, the insulin analogue of the present
invention is an analogue of human insulin comprising less than 3
mutations.
[0095] In one embodiment the insulin analogue comprises B5Y.
[0096] In one embodiment the insulin analogue comprises B5F.
[0097] In one embodiment the insulin analogue comprises B26A or
B26G.
[0098] In one embodiment, the insulin analogue comprises B26A.
[0099] In one embodiment, the insulin analogue comprises B26G.
[0100] In another embodiment, the insulin analogue comprises
A14E.
[0101] In one embodiment, the insulin analogue comprises B5Y and
B26A.
[0102] In one embodiment, the insulin analogue comprises B5Y and
B26G.
[0103] In one embodiment, the insulin analogue comprises B5F and
B26A.
[0104] In one embodiment, the insulin analogue comprises B5F and
B26G.
[0105] In one embodiment, the insulin analogue comprises B28K, B26K
or B29K. In one embodiment, the insulin analogue comprises
B28K.
[0106] In one embodiment, the insulin analogue comprises B26K.
[0107] In one embodiment, the insulin analogue comprises B29K.
[0108] In another embodiment, the insulin analogue comprises B26G
or B26A.
[0109] In another embodiment, the insulin analogue comprises
desB30, desB29-30 or desB27-30.
[0110] In another embodiment, the insulin analogue comprises
desB30.
[0111] In another embodiment, the insulin analogue comprises
desB29-30.
[0112] In another embodiment, the insulin analogue comprises
desB27-30.
[0113] In one embodiment, the insulin analogue may further comprise
up to 10 substitutions in addition to B5Y.
[0114] In one embodiment, the insulin analogue further comprises up
to 5 substitutions in addition to B5Y.
[0115] In one embodiment, the insulin analogue further comprises
two substitutions in addition to B5Y.
[0116] In one embodiment, the insulin analogue further comprises
three substitutions in addition to B5Y.
[0117] In one embodiment, the insulin analogue further comprises
four substitutions in addition to B5Y.
[0118] In one embodiment, the insulin analogue further comprises
five substitutions in addition to B5Y.
[0119] In one embodiment, the insulin analogue further comprises
six substitutions in addition to B5Y.
[0120] In one embodiment, the insulin analogue further comprises
seven substitutions in addition to B5Y.
[0121] In one embodiment, the insulin analogue further comprises
eight substitutions in addition to B5Y.
[0122] In one embodiment, the insulin analogue further comprises
nine substitutions in addition to B5Y.
[0123] In one embodiment, the insulin analogue may further comprise
up to 10 substitutions in addition to B5F.
[0124] In one embodiment, the insulin analogue further comprises up
to 5 substitutions in addition to B5F.
[0125] In one embodiment, the insulin analogue further comprises
two substitutions in addition to B5F.
[0126] In one embodiment, the insulin analogue further comprises
three substitutions in addition to B5F.
[0127] In one embodiment, the insulin analogue further comprises
four substitutions in addition to B5F.
[0128] In one embodiment, the insulin analogue further comprises
five substitutions in addition to B5F.
[0129] In one embodiment, the insulin analogue further comprises
six substitutions in addition to B5F.
[0130] In one embodiment, the insulin analogue further comprises
seven substitutions in addition to B5F.
[0131] In one embodiment, the insulin analogue further comprises
eight substitutions in addition to B5F.
[0132] In one embodiment, the insulin analogue further comprises
nine substitutions in addition to B5F.
[0133] Non-limiting examples of insulin analogues of the present
invention include: [0134] A14E, B5Y, B26A, B28K, desB29-30 (SEQ ID
NO: 3 and 4) [0135] A14E, B5Y, B26G, B28K, desB29-30 (SEQ ID NO: 3
and 5) [0136] B5Y, B26A, B28K, desB29-30 (SEQ ID NO: 1 and 7)
[0137] B5Y, B26G, B28K, desB29-30 (SEQ ID NO: 1 and 8) [0138] B5Y,
B28K, desB29-30 (SEQ ID NO: 1 and 9) [0139] A14E, B5F, B26G, B28K,
desB29-30 (SEQ ID NO: 3 and 10) [0140] B5F, B28K, desB29-30 (SEQ ID
NO: 1 and 11) [0141] B5Y, B26K, desB27-desB30 (SEQ ID 1 and 12)
[0142] B5Y, desB30 (SEQ ID 1 and 13) [0143] B5Y, B26G, desB30 (SEQ
ID 1 and 14) [0144] B5Y, B26A, desB30 (SEQ ID 1 and 15)
[0145] In one embodiment, the insulin analogues of the present
invention comprise: [0146] A14E, B5Y, B26A, B28K, desB29-30 (SEQ ID
NO: 3 and 4) [0147] A14E, B5Y, B26G, B28K, desB29-30 (SEQ ID NO: 3
and 5) [0148] B5Y, B26A, B28K, desB29-30 (SEQ ID NO: 1 and 7)
[0149] B5Y, B26G, B28K, desB29-30 (SEQ ID NO: 1 and 8) [0150] B5Y,
B28K, desB29-30 (SEQ ID NO: 1 and 9) [0151] A14E, B5F, B26G, B28K,
desB29-30 (SEQ ID NO: 3 and 10) [0152] B5F, B28K, desB29-30 (SEQ ID
NO: 1 and 11) [0153] B5Y, B26K, desB27-desB30 (SEQ ID 1 and 12)
[0154] B5Y, desB30 (SEQ ID 1 and 13) [0155] B5Y, B26G, desB30 (SEQ
ID 1 and 14) [0156] B5Y, B26A, desB30 (SEQ ID 1 and 15) [0157] B5F,
B26A, B28K, desB29-30 (SEQ ID 1 and 6) [0158] B5Y, B26A, B28K,
desB29-30 (SEQ ID NO: 1 and 4) [0159] B5Y, B26G, B28K, desB29-30
(SEQ ID NO: 1 and 5) [0160] B5F, B26G, B28K, desB29-30 (SEQ ID NO:
1 and 10) [0161] A14E, B5F, B26A, B28K, desB29-30 (SEQ ID 3 and 6)
[0162] A14E, B5Y, B26A, B28K, desB29-30 (SEQ ID NO: 3 and 7) [0163]
A14E, B5Y, B26G, B28K, desB29-30 (SEQ ID NO: 3 and 8) [0164] A14E,
B5Y, B28K, desB29-30 (SEQ ID NO: 3 and 9) [0165] A14E, B5F, B28K,
desB29-30 (SEQ ID NO: 3 and 11) [0166] A14E, B5Y, B26K,
desB27-desB30 (SEQ ID 3 and 12) [0167] A14E, B5Y, desB30 (SEQ ID 3
and 13) [0168] A14E, B5Y, B26G, desB30 (SEQ ID 3 and 14) and [0169]
A14E, B5Y, B26A, desB30 (SEQ ID 3 and 15).
[0170] Insulin Derivative
[0171] The term "insulin derivative" as used herein means a
chemically modified parent insulin or analogue thereof, in which
one or more side chains have been covalently attached to the
peptide. The term "side chain" as used herein may also be referred
to as a "substituent" or "albumin binding moiety". Non-limiting
examples of side chains are amides, carbohydrates, alkyl groups,
acyl groups, esters, PEGylations, and the like, which may further
comprise a linker.
[0172] The term "albumin binding moiety" as used herein refers to
any chemical group capable of non-covalent binding to albumin, i.e.
has albumin binding affinity. In some embodiments the albumin
binding moiety comprises an acyl group.
[0173] In another particular embodiment the side chain comprises a
portion which is particularly relevant for the albumin binding and
thereby the protraction, which portion may accordingly be referred
to as a "protracting moiety" or "protractor" or "acyl group". The
protracting moiety may be near, and preferably at the terminal (or
distal, or free) end of the albumin binding moiety, relative to its
point of attachment to the peptide.
[0174] The "substituent", "side chain" or "albumin binding moiety"
according to the present invention has the following formula
(I):
Acy-L1-L2-L3
wherein: [0175] Acy is an acyl group and is represented by
lithocholic acid, by a functional group of the formulae:
[0175] --CO--(CH.sub.2).sub.x--COOH; or Chem. 1:
--CO--(CH.sub.2).sub.x-tetrazolyl; Chem. 2:
wherein x represents an integer in the range of from 12 to 20; and
the tetrazolyl group is 1H-tetrazol-5-yl
[0176] or by a fatty acid of formula:
--CO--(CH.sub.2).sub.x--CH.sub.3 Chem. 3:
wherein x represents an integer in the range from 8 to 16 [0177] L1
is absent or represents OEG, gGlu, DgGlu or sulfonimide C-4 [0178]
L2 is absent or represents OEG, gGlu, DgGlu or sulfonimide C-4
[0179] L3 is absent or represents OEG, gGlu, DgGlu or sulfonimide
C-4 wherein: [0180] OEG represents [2-(2-aminoethoxy)ethoxy]acetyl
or amino acid residue 8-amino-3,6-dioxaoctanoic acid
--NH(CH.sub.2).sub.2O(CH.sub.2).sub.2OCH.sub.2CO-- and is
represented by the following structure:
[0180] ##STR00001## [0181] gGlu represents a gamma glutamic acid
residue represented by the following structure:
##STR00002##
[0181] wherein the carboxyl group on the right of the structure
drawing is the gamma-carboxy group which forms the bond to the
neighbouring amino group [0182] DgGlu represents a gamma glutamic
acid residue represented by the following structure:
##STR00003##
[0182] wherein the carboxyl group on the right of the structure
drawing is the gamma-carboxy group which forms the bond to the
neighbouring amino group [0183] and sulfonimide C-4 is represented
by the following structure:
##STR00004##
[0184] In one embodiment, L1-L2-L3 of formula (I) is represented
independently by: [0185] none [0186] gGlu [0187] OEG [0188]
2.times.gGlu [0189] gGlu-OEG [0190] gGlu-2.times.OEG [0191]
sulfonimide-C4 [0192] 2.times.sulfonimide-C4 [0193] DgGlu
[0194] In one embodiment, the substituent has formula (I)
Acy-L1-L2-L3 and is represented independently by: [0195]
Lithocholic acid [0196] Lithocholic acid-gGlu [0197] C14 [0198] C16
diacid [0199] C16 diacid-gGlu [0200] C18 diacid [0201] C18
diacid-gGlu [0202] C18 diacid-2.times.gGlu [0203] C18 diacid-DgGlu
[0204] C18 diacid-gGlu-OEG [0205] C18 diacid-gGlu-2.times.OEG
[0206] C18 diacid-OEG [0207] C18 diacid-sulfonimide-C4 [0208] C20
diacid [0209] C20 diacid-gGlu [0210] C20 diacid-gGlu-OEG [0211] C20
diacid-gGlu-2.times.OEG [0212] Tetrazole-C16 [0213]
Tetrazole-C16-gGlu-OEG [0214] Tetrazole-C16-gGlu-2.times.OEG [0215]
Tetrazole-C16-2.times.sulfonimide-C4 [0216] Tetrazole-C17 [0217]
Tetrazole-C17-sulfonimide-C4 [0218] Tetrazole-C18; [0219]
Tetrazole-C18-sulfonimide-C4 [0220]
Tetrazole-C18-2.times.sulfonimide-C4
[0221] In one embodiment, the substituent is the substituent of
compound of example 15, wherein Acy-L1-L2-L3 is represented by
C18diacid-gGlu-OEG and by the structure:
##STR00005##
[0222] In one embodiment, the insulin derivatives of the invention
are selected from the group consisting of the compounds of Examples
1-46.
[0223] In yet another embodiment, the insulin derivatives of the
invention are selected from the group consisting of the compounds
of Examples 1-36.
[0224] In yet another embodiment the insulin derivative of the
invention is selected from the group consisting of the compounds of
Examples 10-34.
[0225] In yet another embodiment the insulin derivative of the
invention is selected from the group consisting of the compounds of
Examples 3, 4, 12-16, 18-20, 22, 23, 25, 26, 28-30.
[0226] In yet another embodiment the insulin derivative of the
invention is selected from the group consisting of the compounds of
Examples 14-16, 18, 20 and 26.
[0227] In yet another embodiment, the insulin derivatives of the
invention are selected from the group consisting of the compounds
of Examples 37-39 or 46.
[0228] In yet another embodiment, the insulin derivatives of the
invention are selected from the group consisting of the compounds
of Examples 40-45.
[0229] In one embodiment the insulin derivative of the invention is
the compound of Example 15:
N{Epsilon-B28}-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)-
butanoyl]amino]ethoxy]ethoxy]acetyl]-[GluA14,TyrB5,GlyB26,LysB28],des-(B29-
-B30)-Insulin
##STR00006##
[0230] Acyl Group In one embodiment, the insulin derivatives of the
invention comprise an acyl group. The insulin derivatives
comprising an acyl group can therefore be referred to as "acylated
insulin analogues".
[0231] In a preferred embodiment, the insulin derivatives of the
invention comprise an acyl group (Acy) wherein the acyl group
represents lithocholic acid, or a functional group of formulae:
--CO--(CH.sub.2).sub.x--COOH; or Chem. 1:
--CO--(CH.sub.2).sub.x-tetrazolyl- Chem. 2:
wherein x represents an integer in the range of from 12 to 20; and
the tetrazolyl group is 1H-tetrazol-5-yl or a fatty acid of
formula:
--CO--(CH.sub.2).sub.x--CH.sub.3 Chem. 3:
wherein x represents an integer in the range from 8 to 16.
[0232] In one embodiment, Acy is selected from the group consisting
of: lithocholic acid, 1,16-hexadecanedioic acid,
1,18-octadecanedioic acid, 1,20-eicosanedioic acid, tetrazole-C16,
tetrazole-C17, tetrazole C18 and tetradecanoic acid.
[0233] In one embodiment, the insulin derivative comprises an acyl
group, which comprises a dicarboxylic acid.
[0234] In one embodiment, the insulin derivative comprises an acyl
group of the formula of Chem 1, wherein x represents an integer in
the range of from 12 to 20.
[0235] In one embodiment, the insulin derivative comprises an acyl
group of the formula of the formula of Chem 1, wherein x represents
an integer in the range of from 12 to 18.
[0236] In one embodiment, the insulin derivative comprises an acyl
group of the formula of the formula of Chem 1, wherein x represents
an integer in the range of from 12 to 16.
[0237] In one embodiment, the insulin derivative comprises an acyl
group of the formula of the formula of Chem 1, wherein x represents
an integer in the range of from 12 to 14.
[0238] In one embodiment, the insulin derivative comprises an acyl
group of the formula of the formula of Chem 1; wherein x represents
integer 14, 16 or 18, i.e., the fatty diacid group
1,16-hexadecanedioic acid, 1,18-octadecanedioic acid, and
1,20-eicosanedioic acid, respectively.
[0239] In one embodiment, the insulin derivative comprises an acyl
group of the formula --CO--(CH.sub.2).sub.12--COOH and is
represented by the following structure:
##STR00007##
[0240] In one embodiment, the insulin derivative comprises an acyl
group of the formula --CO--(CH.sub.2).sub.14--COOH also named
1,16-hexadecanedioic acid and is represented by the following
structure:
##STR00008##
[0241] In one embodiment, the insulin derivative comprises an acyl
group of the formula --CO--(CH.sub.2).sub.16--COOH, also referred
to as 1,18-octadecanedioic acid and is represented by the following
structure.
##STR00009##
[0242] In one embodiment, the insulin derivative comprises an acyl
group of the formula --CO--(CH.sub.2).sub.18--COOH also referred to
as 1,20-eicosanedioic acid and is represented by the following
structure.
##STR00010##
[0243] In one embodiment, the insulin derivative comprises an acyl
group of the formula --CO--(CH.sub.2).sub.20--COOH and is
represented by the following structure:
##STR00011##
[0244] In one embodiment, the insulin derivative comprises an acyl
group comprising lithocholic acid and is represented by the
following structure:
##STR00012##
[0245] In one embodiment, the insulin derivative comprises an acyl
group comprising a fatty acid of the formula
--CO--(CH.sub.2).sub.12--CH.sub.3 and is represented by the
following structure:
##STR00013##
[0246] In another embodiment, the insulin derivative comprises an
acyl group comprising at least one functional group.
[0247] In one embodiment, the insulin derivative comprises an acyl
group comprising a functional group selected from the group
consisting of a carboxylic acid and a tetrazole moiety.
[0248] In one embodiment, the insulin derivative comprises an acyl
group comprising a functional group selected from formulae:
--CO--(CH.sub.2).sub.x--COOH; or Chem. 1:
--CO--(CH.sub.2).sub.x-tetrazolyl- Chem. 2:
[0249] wherein x represents an integer in the range of from 12 to
20; and the tetrazolyl group is 1H-tetrazol-5-yl.
[0250] In one embodiment, the insulin derivative comprises a
functional group comprising a tetrazole moiety which is selected
from the group consisting of tetrazole-C16, tetrazole-C17 and
tetrazole C18.
[0251] In one embodiment, the insulin derivative comprises a
functional group which is tetrazole-C16 represented by the
following structure:
##STR00014##
[0252] In one embodiment, the insulin derivative comprises a
functional group which is tetrazole-C17 represented by the
following structure:
##STR00015##
[0253] In one embodiment, the insulin derivative comprises a
functional group which is tetrazole-C18 represented by the
following structure:
##STR00016##
[0254] In one embodiment, the insulin derivative comprises a fatty
acid.
[0255] In one embodiment, the insulin derivative comprises a fatty
acid of formula:
--CO--(CH.sub.2).sub.x--CH.sub.3 Chem. 3:
wherein x represents an integer in the range from 8 to 16.
[0256] Linker
[0257] The term "linker" as used herein includes suitable side
chains that can join a moiety, such as a acyl group, to the insulin
or insulin analogue. Thus, the linker and the acyl group become a
side chain together. The moiety joined to the linker may be any
suitable moiety. Examples include an albumin binding moiety.
[0258] The linker can contribute to and/or enhance the binding
effect of the moiety (for example the albumin binding moiety), e.g.
a linker comprising gGlu can enhance the albumin binding effect of
insulin or the insulin analogue.
[0259] In one embodiment the side chain comprises a portion between
the acyl group and the point of attachment to the insulin or
insulin analogue, which portion may be referred to as a "linker",
"linker moiety", "linker group", "linking group", "spacer", or the
like. The linker may be optional, and hence in that case the side
chain may be identical to the acyl group.
[0260] The acyl group or the linker may be covalently attached to a
lysine residue of the insulin peptide by acylation, i.e. via an
amide bond formed between a carboxylic acid group thereof (of the
acyl group, the albumin binding moiety, the protracting moiety, or
the linker) and an amino group of the lysine residue or amino acid
residue in the N-terminal. Additional or alternative conjugation
chemistry include alkylation, ester formation, amide formation, or
coupling to a cysteine residue, such as by maleimide or
haloacetamide (such as bromo-/chloro-/iodo-) coupling.
[0261] In a preferred embodiment, an active ester of the acyl
group, preferably comprising a protracting moiety and a linker, is
covalently linked to an amino group of a lysine residue, preferably
the epsilon amino group thereof, under formation of an amide bond,
as explained above.
[0262] In another embodiment, the linker group is absent and
represents a covalent bond.
[0263] In one embodiment, the insulin derivatives of the invention
comprise a linker group of formula -L1-L2-L3, wherein: [0264] L1 is
absent or represents OEG, gGlu, DgGlu or sulfonimide C-4 [0265] L2
is absent or represents OEG, gGlu, DgGlu or sulfonimide C-4 [0266]
L3 is absent or represents OEG, gGlu, DgGlu or sulfonimide C-4
wherein [0267] gGlu represents a gamma glutamic acid residue
represented by the following structure:
##STR00017##
[0267] wherein the carboxyl group on the right of the structure
drawing is the gamma-carboxy group which forms the bond to the
neighbouring amino group [0268] DgGlu represents a gamma glutamic
acid residue represented by the following structure:
##STR00018##
[0268] wherein the carboxyl group on the right of the structure
drawing is the gamma-carboxy group which forms the bond to the
neighbouring amino group [0269] OEG represents
[2-(2-aminoethoxy)ethoxy]acetyl and is represented by the following
structure:
[0269] ##STR00019## [0270] sulfonimide C-4 is represented by the
following structure:
##STR00020##
[0270] and [0271] 2.times.sulfonimide-C4 or
sulfonimide-C4-sulfonimide-C4, is represented by the following
structure:
##STR00021##
[0272] In one embodiment, the insulin peptides of the invention
comprise a linker group which represents a divalent linking group
selected from DgGlu, gGlu, gGlu-gGlu, gGlu-OEG, gGlu-OEG-OEG, OEG,
sulfonimide-C4, and 2.times. sulfonimide-C4, wherein [0273] gGlu
represents a gamma glutamic acid residue; and [0274] OEG represents
the amino acid with the formula
NH.sub.2--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--CH.sub.2--COO-
H, corresponding to the group or residue
--NH--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--CH.sub.2--CO--,
also designated [2-(2-aminoethoxy)ethoxy]acetyl. [0275]
sulfonimide-C4, which is represented by the following
structure:
[0275] ##STR00022## [0276] 2.times.sulfonimide-C4 or
sulfonimide-C4-sulfonimide-C4, which is represented by the
following structure:
##STR00023##
[0277] Non-limiting examples of linkers are selected from the list
consisting of: DgGlu, gGlu, gGlu-gGlu, gGlu-OEG, gGlu-OEG-OEG, OEG,
sulfonimide-C4, 2.times. sulfonimide-C4.
[0278] In one embodiment, the linking group is absent.
[0279] In one embodiment, the linking group is DgGlu.
[0280] In one embodiment, the linking group is gGlu.
[0281] In one embodiment, the linking group is gGlu-gGlu.
[0282] In one embodiment, the linking group is gGlu-OEG.
[0283] In one embodiment, the linking group is gGlu-OEG-OEG.
[0284] In one embodiment, the linking group is OEG.
[0285] In one embodiment, the linking group is sulfonimide-C4.
[0286] In one embodiment, the linking group is
sulfonimide-C4-sulfonimide-C4 or 2.times.sulfonimide C-4.
[0287] Intermediate Products
[0288] The invention furthermore relates to an intermediate product
in the form of a novel backbone, which when attached to the
substituents of the invention, leads to the insulin derivative
peptides of the invention.
[0289] The invention also relates to an intermediate product in the
form of the novel backbone of the insulin peptides of the
invention, selected from the group consisting of: [0290] i. A14E,
B5Y, B26A, B28K, desB29-30 (SEQ ID NO: 3 and 4) [0291] ii. A14E,
B5Y, B26G, B28K, desB29-30 (SEQ ID NO: 3 and 5) [0292] iii. B5Y,
B26A, B28K, desB29-30 (SEQ ID NO: 1 and 7) [0293] iv. B5Y, B26G,
B28K, desB29-30 (SEQ ID NO: 1 and 8) [0294] v. B5Y, B28K, desB29-30
(SEQ ID NO: 1 and 9) [0295] vi. A14E, B5F, B26G, B28K, desB29-30
(SEQ ID NO: 3 and 10) [0296] vii. B5F, B28K, desB29-30 (SEQ ID NO:
1 and 11) [0297] viii. B5Y, B26K, desB27-desB30 (SEQ ID 1 and 12)
[0298] ix. B5Y, desB30 (SEQ ID 1 and 13) [0299] x. B5Y, B26G,
desB30 (SEQ ID 1 and 14) [0300] xi. B5Y, B26A, desB30 (SEQ ID 1 and
15) [0301] xii. B5F, B26A, B28K, desB29-30 (SEQ ID 1 and 6) [0302]
xiii. B5Y, B26A, B28K, desB29-30 (SEQ ID NO: 1 and 4) [0303] xiv.
B5Y, B26G, B28K, desB29-30 (SEQ ID NO: 1 and 5) [0304] xv. B5F,
B26G, B28K, desB29-30 (SEQ ID NO: 1 and 10) [0305] xvi. A14E, B5F,
B26A, B28K, desB29-30 (SEQ ID 3 and 6) [0306] xvii. A14E, B5Y,
B26A, B28K, desB29-30 (SEQ ID NO: 3 and 7) [0307] xviii. A14E, B5Y,
B26G, B28K, desB29-30 (SEQ ID NO: 3 and 8) [0308] xix. A14E, B5Y,
B28K, desB29-30 (SEQ ID NO: 3 and 9) [0309] xx. A14E, B5F, B28K,
desB29-30 (SEQ ID NO: 3 and 11) [0310] xxi. A14E, B5Y, B26K,
desB27-desB30 (SEQ ID 3 and 12) [0311] xxii. A14E, B5Y, desB30 (SEQ
ID 3 and 13) [0312] xxiii. A14E, B5Y, B26G, desB30 (SEQ ID 3 and
14) [0313] xxiv. A14E, B5Y, B26A, desB30 (SEQ ID 3 and 15) or a
pharmaceutically acceptable salt, amide or ester thereof.
[0314] Pharmaceutically Acceptable Salt, Amide, or Ester
[0315] The intermediate products, analogues and derivatives of the
invention may be in the form of a pharmaceutically acceptable salt,
amide, or ester.
[0316] Salts are e.g. formed by a chemical reaction between a base
and an acid, e.g.:
2NH.sub.3+H.sub.2SO.sub.4.fwdarw.(NH.sub.4).sub.2SO.sub.4.
[0317] The salt may be a basic salt, an acid salt, or it may be
neither nor (i.e. a neutral salt). Basic salts produce hydroxide
ions and acid salts hydronium ions in water.
[0318] The salts of the derivatives of the invention may be formed
with added cations or anions that react with anionic or cationic
groups, respectively. These groups may be situated in the peptide
moiety, and/or in the side chain of the derivatives of the
invention.
[0319] Non-limiting examples of anionic groups of the derivatives
of the invention include free carboxylic groups in the side chain,
if any, as well as in the peptide moiety. The peptide moiety often
includes a free carboxylic acid group at the C-terminus, and it may
also include free carboxylic groups at internal acid amino acid
residues such as Asp and Glu.
[0320] Non-limiting examples of cationic groups in the peptide
moiety include the free amino group at the N-terminus, if present,
as well as any free amino group of internal basic amino acid
residues such as His, Arg, and Lys.
[0321] The ester of the derivatives of the invention may, e.g., be
formed by the reaction of a free carboxylic acid group with an
alcohol or a phenol, which leads to replacement of at least one
hydroxyl group by an alkoxy or aryloxy group.
[0322] The ester formation may involve the free carboxylic group at
the C-terminus of the peptide, and/or any free carboxylic group in
the side chain.
[0323] The amide of the derivatives of the invention may, e.g., be
formed by the reaction of an activated form of a free carboxylic
acid group with an amine or a substituted amine, or by reaction of
a free or substituted amino group with an activated form of a
carboxylic acid.
[0324] The amide formation may involve the free carboxylic group at
the C-terminus of the peptide, any free carboxylic group in the
side chain, the free amino group at the N-terminus of the peptide,
and/or any free or substituted amino group of the peptide in the
peptide and/or the side chain.
[0325] In a particular embodiment, the peptide or derivative is in
the form of a pharmaceutically acceptable salt. In another
particular embodiment, the derivative is in the form of a
pharmaceutically acceptable amide, preferably with an amide group
at the C-terminus of the peptide. In a still further particular
embodiment, the peptide or derivative is in the form a
pharmaceutically acceptable ester.
[0326] Stability
[0327] Stability of an insulin derivative is defined as the ability
to maintain a three dimensional structure (physical stability) as
well as the ability to withstand covalent changes in the structure
(chemical stability). A favourable stability may be due to inherent
properties of the insulin derivative alone or a result of
favourable interactions between the insulin derivative and one or
more ingredients contained in the vehicle.
[0328] Satisfactory stability of an insulin derivative is defined
as stability comparable to or better than insulin aspart.
[0329] In one aspect the invention provides insulin derivatives
with satisfactory stability in formulation.
[0330] In one aspect the invention provides insulin derivatives
with improved stability in formulation.
[0331] The inventors have found that the insulin derivatives
according to the invention have satisfactory stability in
formulation. The inventors have surprisingly found that the human
insulin derivatives according to the invention have both
satisfactory stability and retain the partial activation of the
insulin receptor.
[0332] Stability may be determined by conventional methods and
various standard methods known to the person skilled in the
art.
[0333] Insulin derivatives can for example be screened for
stability in formulations including zinc and phenol. In one
embodiment the aim is to obtain a formulation including a single
insulin self-association state (e.g. a hexameric state) which is
not changed during storage or at increased temperature.
[0334] An example of a method to measure stability is Differential
Scanning calorimetry (DSC), which is a common method to evaluate
protein stability, typically by evaluation of onset temperature
(T.sub.onset) of unfolding or, more frequently, by the midpoint of
the thermal unfolding (T.sub.m) at insulin derivative
self-association. It has been described in the literature (Huus et
al, Biochemistry (2005) 44, 11171-11177) how the thermal unfolding
(by DSC) of insulin correlates to the stability of the zinc-hexamer
and with that also with the formulation stability of insulin (Huus
et al, Pharm Res (2006) 23(11), 2611-20). A low onset temperature
might be seen as insulin dissociation whereas a higher onset
temperature indicates a stable insulin derivative complex until
unfolding temperature. A satisfactory stability is obtained at
T.sub.onset and T.sub.m about or above T.sub.onset and T.sub.m for
insulin aspart (B28D human insulin) at conditions resembling a
pharmaceutical formulation.
[0335] Another example of such a method is evaluation of insulin
self-association by size exclusion chromatography (SEC) using an
eluent resembling formulation condition adding phenol and keeping
ion strength low. Broad and tailing peaks are sign of several
self-association states changing during the chromatography whereas
a single sharp peak indicates a stable self-association state.
[0336] In one embodiment, an insulin derivative according to the
invention has satisfactory chemical and/or physical stability
relative to insulin aspart.
[0337] In another embodiment, an insulin derivative according to
the invention has satisfactory chemical and/or physical stability
relative to the corresponding insulin derivative without the B5Y or
B5F mutation.
[0338] Unless otherwise indicated in the specification, terms
presented in singular form also include the plural situation.
[0339] In Vitro Biology
[0340] Insulin binding and receptor activation/phosphorylation
[0341] Activation of the insulin receptor by insulin leads to
activation of the receptor, i.e. phosphorylation of several
residues on the receptor, which activation results in a cascade of
cellular responses, including cellular processes that contribute to
lowering the plasma glucose concentration, cellular processes that
regulate lipid metabolism, and cellular processes that promote cell
growth and proliferation.
[0342] Although the insulin derivatives of the invention are fully
capable of displacing human insulin from the insulin receptor, they
in fact induce submaximal phosphorylation of the insulin receptor.
The insulin derivatives of the present invention therefore may be
considered partial agonists with respect to insulin receptor
phosphorylation, and they may be referred to as "partial insulin
derivatives".
[0343] In the context of this invention "partial insulin
derivatives" are defined as insulin analogues, which induce insulin
receptor phosphorylation, but the maximum response obtained, i.e.
the maximum insulin receptor phosphorylation level, is less than
the maximum response induced by human insulin, and i.e. is less
than 65% of the maximum response induced by human insulin.
[0344] A submaximal response or submaximal effect may be defined as
a maximum response induced by a partial insulin derivative of the
invention that is lower than the maximum response induced by human
insulin. To determine if an insulin derivative has a submaximal
effect, the maximal response of said derivative has to be
determined in an assay and compared to the maximal response of
human insulin. The maximum response of an insulin derivative may be
determined by measuring the response in the presence of increasing
amounts of the insulin derivative, in order to obtain a
dose-response curve. The maximum response (Top) can be calculated
from the dose-response curve and compared with the maximum response
induced by human insulin.
[0345] Insulin binding and receptor phosphorylation may be
determined by conventional methods, and various standard assays are
known to the person skilled in the art. Such standard assays for in
vitro determination include i.a. insulin radio-receptor assays, in
which the relative affinity of an insulin derivative is defined as
the ratio of insulin to insulin derivative required to displace 50%
of labelled .sup.125I-insulin specifically bound to insulin
receptors, e.g. on whole cells, on cell membrane fractions, or on
purified receptors, as well as insulin receptor phosphorylation
assays, in which the ability of the insulin derivative to activate
the insulin receptor is determined, e.g. by measuring the
phosphorylation of tyrosine residues on the insulin receptor, using
e.g. enzyme-linked immunosorbent assay (ELISA) or Western blot
techniques.
[0346] In one embodiment, the insulin derivatives of the present
invention induce a submaximal phosphorylation of insulin receptor
of about or below 65%, when compared to human insulin.
[0347] In one embodiment, the insulin derivatives of the present
invention induce a submaximal phosphorylation of insulin receptor
of about or below 60%, when compared to human insulin.
[0348] In one embodiment, the insulin derivatives of the present
invention induce a submaximal phosphorylation of insulin receptor
of about or below 50%, when compared to human insulin.
[0349] In one embodiment, the insulin derivatives of the present
invention induce a submaximal phosphorylation of insulin receptor
of about or below 40%, when compared to human insulin.
[0350] In one embodiment, the insulin derivatives of the present
invention induce a submaximal phosphorylation of insulin receptor
of about or below 30%, when compared to human insulin.
[0351] In one embodiment, the insulin derivatives of the present
invention induce a submaximal phosphorylation of insulin receptor
of about or below 20%, when compared to human insulin.
[0352] In one embodiment, the insulin derivatives of the present
invention induce a submaximal phosphorylation of insulin receptor
of about or below 15%, when compared to human insulin.
[0353] In one embodiment, the insulin derivatives of the present
invention induce a submaximal phosphorylation of insulin receptor
of about or below 12%, when compared to human insulin.
[0354] In one embodiment, the insulin derivatives of the present
invention induce a phosphorylation of the insulin receptor of about
or above 1%, when compared to human insulin.
[0355] In one embodiment, the insulin derivatives of the present
invention are capable of inducing phosphorylation of the insulin
receptor.
[0356] Insulin Receptor Signalling
[0357] Activation of the insulin receptor initiates a cascade of
intracellular responses, such as phosphorylation of residues on,
and activation of, various intracellular proteins, including the
Extracellular regulated kinase (ERK), also known as MAP kinases
(MAPK), and AKT, also known as Protein Kinase B (PKB). Activation
of AKT is important for inducing the cellular processes that
contributes to lowering the blood glucose concentration, whereas
activation of ERK is important for inducing the cellular processes
that contributes to promoting cell growth and proliferation in
growing cells.
[0358] The AKT signalling pathway is an example of a signal
transduction pathway that is important for the glucose lowering
pathways, wherein AKT is a key protein. In people with Type 2
diabetes, activation of the AKT signalling pathway is greatly
impaired, which leads to e.g. a decrease in glucose uptake in to
skeletal muscle and adipose tissue. However, the ERK signalling
pathway remains responsive to insulin and is an example of a
non-resistant pathway that can be over-stimulated during treatment.
The activation of the ERK pathway by insulin also encourages the
migration and proliferation of VSMCs and collagen synthesis, which
are critical steps for the progression of atherosclerotic lesions.
In a preferred embodiment the insulin derivatives of the invention
induce a submaximal effect on the ERK signalling pathway.
[0359] Assays for measuring insulin activated signalling, e.g. the
ability to phosphorylate signalling molecules such as ERK and AKT
in vitro, are known to the person skilled in the art, and include
i.a. Western blot techniques, Surefire and/or ELISA techniques.
[0360] The insulin derivatives of the present invention have a
lower submaximal effect on ERK activation (phosphorylation) than on
AKT activation (phosphorylation), when compared to the effect of
human insulin.
[0361] Surprisingly we have found that there is a correlation
between the maximum insulin receptor phosphorylation (i.e. degree
of insulin receptor partiality) that a test compound is able to
induce and its ability to activate the glucose lowering pathways
without over-stimulating non-resistant pathways. For example, the
compounds of the present invention have a lower submaximal effect
on ERK activation (phosphorylation) than on AKT activation
(phosphorylation), when compared to human insulin and they are
capable of reducing blood glucose levels, but have improved effects
on processes related to e.g. lipid metabolism, such as less weight
gain, less increase in body fat mass and increased lowering of
liver TG, and/or improved endothelial function.
[0362] Partiality is defined as having the maximal insulin receptor
phosphorylation of test compound/maximal insulin receptor
phosphorylation of human insulin ratio less than 1.
[0363] In one embodiment, the present invention relates to a method
for measuring insulin receptor partiality, comprising the following
steps:
a) measuring the maximal insulin receptor phosphorylation induced
by a test compound b) measuring the maximal insulin receptor
phosphorylation induced by human insulin wherein the ratio of a)/b)
is less than 1.
[0364] In one embodiment said insulin receptor partiality is less
than 0.65.
[0365] In one embodiment said insulin receptor partiality is less
than 0.6.
[0366] In one embodiment said insulin receptor partiality is less
than 0.5.
[0367] In one embodiment said insulin receptor partiality is less
than 0.4.
[0368] In one embodiment said insulin receptor partiality is less
than 0.3.
[0369] In one embodiment said insulin receptor partiality is less
than 0.2.
[0370] In one embodiment said insulin receptor partiality is less
than 0.15.
[0371] In one embodiment said insulin receptor partiality is less
than 0.12.
[0372] Selectivity is defined as the ability of a test compound to
activate the glucose lowering pathways without over-stimulating
non-resistant pathways, wherein the ratio is less than 1.
[0373] In one embodiment, the present invention relates to a method
for measuring selectivity, comprising measuring independently the
maximal phosphorylation of glucose lowering pathways and
non-resistant pathways compared to human insulin and determining if
the ratio between glucose lowering pathways/non-resistant pathways
is less than 1.
[0374] In one embodiment, the present invention relates to a method
for measuring selectivity, comprising measuring independently the
maximal phosphorylation of ERK and AKT compared to human insulin
and determining if the ratio between ERK/AKT is less than 1.
[0375] In one embodiment said ratio is less than 0.8.
[0376] In one embodiment said ratio is less than 0.7.
[0377] In one embodiment said ratio is less than 0.6.
[0378] In one embodiment said ratio is less than 0.5.
[0379] In one embodiment said ratio is less than 0.4.
[0380] Lipid Metabolism/Non-Resistant Pathways
[0381] The insulin derivatives of the present invention, when
compared to human insulin are able to lower blood glucose levels
without over-stimulating non-resistant pathways, e.g. exhibit
submaximal effects on lipid metabolism pathways.
[0382] As described above, the signalling cascades initiated by
insulin through the insulin receptor lead to a wide range of
cellular effects, including effects on lipid metabolism pathways.
As defined herein the term "lipid metabolism pathways" covers
biological actions induced or inhibited by insulin, which actions
affect e.g. the synthesis of triglycerides in the liver. An example
of a non-resistant pathway is the ERK signalling pathway.
[0383] In a preferred embodiment the insulin derivatives of the
invention induce a submaximal effect on metabolic pathways related
to de novo lipid synthesis (DNL) in primary hepatocytes, as
compared to the effect of human insulin. Standard assays for
measuring the effect on fatty acid synthesis in liver are known to
the skilled person and include i.a. determining the effect on the
de novo lipogenesis by measuring the conversion of
.sup.14C-labelled acetate into organic-extractable material (i.e.
lipids) in primary hepatocytes.
[0384] The signalling cascades initiated by insulin through the
insulin receptor leads to induction of the enzymes necessary for
regulating lipid metabolism. Example of such enzyme is Fatty Acid
Synthase (FAS). FAS is a key enzyme in the cellular processes that
serve to regulate lipid synthesis.
[0385] The insulin derivatives of the invention also have a
submaximal effect on mRNA encoding factors involved in lipid
synthesis, e.g. FAS that are only submaximally induced by the
insulin derivatives of the present invention in primary
hepatocytes, when compared to human insulin.
[0386] Standard assays for measuring the ability of insulin and its
analogues and derivatives to induce or inhibit mRNA expression of
genes involved in lipid metabolism pathways, are known to the
person skilled in the art, and include i.a measuring mRNA
expression using quantitative real-time polymerase chain reaction
(RT-PCR).
[0387] Glucose Lowering Pathways
[0388] While the insulin derivatives of the invention exhibit a
lower submaximal effect on the pathways relating to non-resistant
pathways such as lipid metabolism, they are capable of inducing the
same maximal response on the glucose lowering pathways as human
insulin.
[0389] In the context of this invention the term "glucose lowering
pathways" cover biological actions induced by insulin that cause
lowering of the plasma glucose concentration, such as promoting the
storage of glucose in the liver, where insulin activates enzymes
that promote glycolysis and glycogenesis, and suppresses those
involved in gluconeogenesis, such as promoting incorporation of
glucose into lipids in adipocytes, and increasing the uptake of
glucose in e.g. muscle.
[0390] As described above, the signalling cascades initiated by
insulin through the insulin receptor leads to a wide range of
cellular effects, including effects on glucose lowering pathways.
Examples of insulin stimulated cellular processes that serve to
lower plasma glucose concentration are facilitating entry of
glucose into muscle, adipose and several other tissues, and
stimulating the liver to store glucose in the form of glycogen. The
AKT signalling pathway is an example of a signal transduction
pathway that is important for the glucose lowering pathways,
wherein AKT is a key protein.
[0391] In vitro assays for measuring the effect of human insulin,
or analogues and derivatives hereof, on glucose lowering pathways
are known to the skilled person, and include i.a. assays with
isolated rat hepatocytes, where glycogen synthesis can be
determined by measuring glycogen accumulation, and lipogenesis
assays, performed, e.g., with rat adipocytes wherein the amount of
[3-.sup.3H] glucose converted into organic-extractable material
(i.e. lipids) is measured.
[0392] The signalling cascades initiated by insulin through the
insulin receptor also lead to activation and/or induction of
enzymes and transcription factors necessary for regulating the
glucose lowering pathways. An example of such an enzyme is
Glucose-6-phosphatase (G6Pc). G6Pc is a rate limiting enzyme in
gluconeogenesis that results in the generation of glucose from
non-carbohydrate carbon substrates, such as pyruvate, lactate,
glycerol, glucogenic amino acids, and odd-chain fatty acids.
[0393] The insulin derivatives of the invention exhibit the same
maximal effect on stimulating storage of glucose in the form of
glycogen in hepatocytes and/or muscles.
[0394] The insulin derivatives of the invention have the same
maximal effect as human insulin on mRNA encoding factors involved
in gluconeogenesis in primary rat hepatocytes.
[0395] Standard assays for measuring the ability of human insulin
or its analogues and derivatives to induce or inhibit mRNA
expression of genes involved in glucose lowering pathways assays
are also known to the person skilled in the art, and include i.e.
measuring mRNA expression using quantitative RT-PCR.
[0396] The insulin derivatives of the invention exhibit full
(maximal) effect on the glucose lowering pathways, such as
expression of mRNA encoding enzymes involved in gluconeogenesis,
stimulation of glycogen synthesis, incorporation of glucose into
lipids, in primary cells, e.g. muscle, hepatocytes and/or
adipocytes, when comparing to the effects of human insulin.
[0397] The insulin derivatives exhibit the same maximal response on
stimulating incorporation of glucose into lipid in primary rat
adipocytes as human insulin.
[0398] In Vivo Biology
[0399] In another particular embodiment the insulin derivatives of
the invention are capable of reducing blood glucose levels in vivo,
and have improved effects on processes related to lipid metabolism,
such as less weight gain, less increase in body fat mass, increased
lowering of liver triglycerides, or improved endothelial function
compared to human insulin, which may be determined as is known in
the art in any suitable animal model, as well as in clinical
trials.
[0400] The C57BL/6J-Diet-Induced Obese (DIO) mouse is one example
of a suitable animal model, and the blood glucose and/or effects on
processes related to lipid metabolism, such as less weight gain,
less increase in body fat mass or increased or lowering of liver
triglycerides, may be determined in such mice, e.g. as described in
Assay (II).
[0401] The structural and functional integrity of the endothelium
is crucial to maintain vascular homeostasis and prevent
atherosclerosis. Endothelial dysfunction comprises a number of
functional alterations in the vascular endothelium and impairment
of arterial endothelial function is an early event in
atherosclerosis and correlates with the major risk factors for
cardiovascular disease. Thus, it would be a major advantage to have
insulin derivatives which prevents or reduce endothelial
dysfunction as these would have a beneficial effect on
atherosclerosis and/or CVD.
[0402] In one embodiment, the present invention provides insulin
derivatives, which are cable of reducing blood glucose levels and
have improved effects on endothelial function compared to
conventional insulin therapy e.g. prevent or reduce endothelial
dysfunction.
[0403] In another embodiment the present invention provides insulin
derivatives that prevent or reduce a cardiovascular disease in a
diabetic subject and/or prevent or reduce development of
atherosclerosis.
[0404] Arterial endothelial function can be measured by methods
known to a person skilled in the art such as measurement of the
effect on acetyl-choline induced vasorelaxation in mesenteric
arteries e.g. as described in Assay (II).
[0405] Hepatic liver metabolism is severely dysregulated in
diabetes. Normally, less than 5% of the liver volume is fat, but in
patients with non-alcoholic steatohepatitis (NASH) or non alcoholic
fatty liver diseases (NAFLD), up to 50%-80% of liver weight may be
made up of fat, mostly in the form of triglycerides, see e.g.:
Sanyal, A. J. in "AGA technical review on non-alcoholic fatty liver
disease", Gastroenterology 2002; 123, 1705-1725.
[0406] In a preferred embodiment, the present invention provides
insulin derivatives, which are cable of reducing blood glucose
levels, but results in lower liver triglyceride content compared to
conventional insulin therapy.
[0407] Liver triglyceride content can be measured by methods known
to a person skilled in the art e.g. as described in (Assay II).
[0408] Production of Insulin Peptides
[0409] The production of polypeptides, e.g., insulins, is well
known in the art. The insulin may for instance be produced by
classical peptide synthesis, e.g., solid phase peptide synthesis
using t-Boc or Fmoc chemistry or other well established techniques,
see, e.g., Greene and Wuts, "Protective Groups in Organic
Synthesis", John Wley & Sons, 1999. The insulin may also be
produced by a method which comprises culturing a host cell
containing a DNA sequence encoding the analogue and capable of
expressing the insulin analogue in a suitable nutrient medium under
conditions permitting the expression of the insulin peptide.
Several recombinant methods may be used in the production of human
insulin and human insulin analogues. Examples of methods which may
be used in the production of insulin in microorganisms such as,
e.g., Escherichia coli and Saccharomyces cerevisiae are, e.g.,
disclosed in WO2008034881.
[0410] Typically, the insulin analogue is produced by expressing a
DNA sequence encoding the insulin analogue in question or a
precursor thereof in a suitable host cell by well-known technique
as disclosed in e.g. EP1246845 or WO2008034881.
[0411] The insulin analogue may be expressed with an N-terminal
extension as disclosed in EP 1,246,845. After secretion to the
culture medium and recovery, the insulin precursor will be
subjected to various in vitro procedures to remove the possible
N-terminal extension sequence and connecting peptide to give the
insulin analogue. Such methods include enzymatic conversion by
means of trypsin or an Achromobacter lyticus protease in the
presence of an L-threonine ester followed by conversion of the
threonine ester of the insulin analogue into the insulin analogue
by basic or acid hydrolysis as described in U.S. Pat. No. 4,343,898
or 4,916,212
[0412] Examples of N-terminal extensions of the type suitable in
the present invention are disclosed in U.S. Pat. No. 5,395,922 and
EP765395.
[0413] For insulin analogues comprising non-natural amino acid
residues, the recombinant cell should be modified such that the
non-natural amino acids are incorporated into the analogue, for
instance by use of tRNA mutants. Hence, briefly, the insulin
peptides according to the invention are prepared analogously to the
preparation of known insulin analogues.
[0414] Protein Purification
[0415] The insulin analogues used for making the insulin
derivatives of the invention are recovered from the cell culture
medium. The insulin analogue used for making insulin derivatives of
the present invention may be purified by a variety of procedures
known in the art including, but not limited to, chromatography
(e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and
size exclusion), electrophoretic procedures (e.g., preparative
isoelectric focusing (IEF), differential solubility (e.g., ammonium
sulfate precipitation), or extraction (see, e.g., Protein
Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers,
New York, 1989). Preferably, they may be purified by affinity
chromatography on an anti-"[an insulin/insulin analogue/insulin
derivative]" antibody column. Additional purification may be
achieved by conventional chemical purification means, such as high
performance liquid chromatography. Other methods of purification,
including barium citrate precipitation, are known in the art, and
may be applied to the purification of the novel "[an
insulin/insulin analogue/insulin derivative]" described herein
(see, for example, Scopes, R., Protein Purification,
Springer-Verlag, N.Y., 1982).
[0416] Pharmaceutical Formulations
[0417] Injectable compositions containing an insulin derivative of
this 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 of 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, for
example, hydrochloric acid, or a base, for example, aqueous sodium
hydroxide, as needed. Finally, the volume of the solution is
adjusted with water to give the desired concentration of the
ingredients.
[0418] Optionally, an insulin preparation of this invention, for
example a solution or suspension, may be prepared by dissolving an
insulin derivative of the invention in an aqueous medium, for
example, in a concentration in the range from about 240 to about
6000 nmol/ml. The aqueous medium is made isotonic, for example,
with sodium chloride, propylenglycol or glycerol. Furthermore, the
aqueous medium may contain buffers such as acetate or citrate,
preservatives such as m-cresol or phenol and zinc ions, for
example, in a concentration of up to about 12 Zn/6ins. The pH value
of the solution is adjusted towards neutrality without getting too
close to the isoelectric point of the compound of this invention in
order to avoid potential precipitation. The pH value of the final
insulin preparation depends upon which compound of this invention
is used, the concentration of zinc ions and the concentration of
the compound of this invention. The insulin preparation is made
sterile, for example, by sterile filtration.
EMBODIMENTS
[0419] The invention is further described by the following
non-limiting embodiments: [0420] 1. An insulin derivative, wherein
said insulin derivative comprises B5Y or B5F and a substituent
comprising an acyl group, or a pharmaceutically acceptable salt,
amide or ester thereof. [0421] 2. The insulin analogue according to
any of the previous embodiments, wherein said insulin derivative
further comprises one or more amino acid substitutions and/or
deletions. [0422] 3. The insulin derivative according to any of the
previous embodiments, wherein said insulin derivative further
comprises up to 5 additional substitutions and/or deletions. [0423]
4. The insulin derivative according to any of the previous
embodiments, wherein said insulin derivative further comprises 2, 3
or 4 amino acid substitutions. [0424] 5. The insulin derivative
according to any of embodiments 1-4, wherein said insulin
derivative further comprises B26G or B26A. [0425] 6. The insulin
derivative according to embodiment 5, wherein said insulin
derivative further comprises B26G. [0426] 7. The insulin derivative
according to embodiment 5, wherein said insulin derivative further
comprises B26A. [0427] 8. The insulin derivative according to any
one of embodiments 1-7, wherein said substituent has the following
formula (I):
[0427] Acy-L1-L2-L3
wherein: Acy is an acyl group and is represented by lithocholic
acid, or by a functional group of the formulae:
--CO--(CH.sub.2).sub.x--COOH; or Chem. 1:
--CO--(CH.sub.2).sub.x-tetrazolyl; Chem. 2:
wherein x represents an integer in the range of from 12 to 20; and
the tetrazolyl group is 1H-tetrazol-5-yl or a fatty acid of
formula:
--CO--(CH.sub.2).sub.x--CH.sub.3 Chem. 3:
wherein x represents an integer in the range from 8 to 16 [0428] L1
is absent and represents a covalent bond or represents OEG, gGlu,
DgGlu or sulfonimide C-4 [0429] L2 is absent and represents a
covalent bond or represents OEG, gGlu, DgGlu or sulfonimide C-4
[0430] L3 is absent and represents a covalent bond or represents
OEG, gGlu, DgGlu or sulfonimide C-4 wherein gGlu represents a gamma
glutamic acid residue and OEG represents
[2-(2-aminoethoxy)ethoxy]acetyl. [0431] 9. The insulin derivative
according to any of the previous embodiments, wherein said insulin
derivative further comprises A14E. [0432] 10. The insulin
derivative according to any of the previous embodiments, wherein
said insulin derivative further comprises B28K, B26K or B29K.
[0433] 11. The insulin derivative according to embodiment 10,
wherein said insulin derivative further comprises B28K. [0434] 12.
The insulin derivative according to embodiment 10, wherein said
insulin derivative further comprises B26K. [0435] 13. The insulin
derivative according to embodiment 10, wherein said insulin
derivative further comprises B29K. [0436] 14. The insulin
derivative according to any one of embodiments 1-13, wherein said
substituent is attached to the epsilon amino group of the lysine of
B26K, B28K or B29K. [0437] 15. The insulin derivative according to
any one of the previous embodiments, wherein said insulin
derivative further comprises desB30, desB29-30 or desB27-30. [0438]
16. The insulin derivative according to embodiment 15, wherein said
insulin derivative further comprises desB30. [0439] 17. The insulin
derivative according to embodiment 15, wherein said insulin
derivative further comprises desB29-30. [0440] 18. The insulin
derivative according to embodiment 15, wherein said insulin
derivative further comprises desB27-30. [0441] 19. The insulin
derivative according to any of the previous embodiments, wherein
said substitutions are selected from the group consisting of:
[0442] i. A14E, B5Y, B26A, B28K, desB29-30 (SEQ ID NO: 3 and 4)
[0443] ii. A14E, B5Y, B26G, B28K, desB29-30 (SEQ ID NO: 3 and 5)
[0444] iii. B5Y, B26A, B28K, desB29-30 (SEQ ID NO: 1 and 7) [0445]
iv. B5Y, B26G, B28K, desB29-30 (SEQ ID NO: 1 and 8) [0446] v. B5Y,
B28K, desB29-30 (SEQ ID NO: 1 and 9) [0447] vi. A14E, B5F, B26G,
B28K, desB29-30 (SEQ ID NO: 3 and 10) [0448] vii. B5F, B28K,
desB29-30 (SEQ ID NO: 1 and 11) [0449] viii. B5Y, B26K,
desB27-desB30 (SEQ ID 1 and 12) [0450] ix. B5Y, desB30 (SEQ ID 1
and 13) [0451] x. B5Y, B26G, desB30 (SEQ ID 1 and 14) [0452] xi.
B5Y, B26A, desB30 (SEQ ID 1 and 15) [0453] xii. B5F, B26A, B28K,
desB29-30 (SEQ ID 1 and 6) [0454] xiii. B5Y, B26A, B28K, desB29-30
(SEQ ID NO: 1 and 4) [0455] xiv. B5Y, B26G, B28K, desB29-30 (SEQ ID
NO: 1 and 5) [0456] xv. B5F, B26G, B28K, desB29-30 (SEQ ID NO: 1
and 10) [0457] xvi. A14E, B5F, B26A, B28K, desB29-30 (SEQ ID 3 and
6) [0458] xvii. A14E, B5Y, B26A, B28K, desB29-30 (SEQ ID NO: 3 and
7) [0459] xviii. A14E, B5Y, B26G, B28K, desB29-30 (SEQ ID NO: 3 and
8) [0460] xix. A14E, B5Y, B28K, desB29-30 (SEQ ID NO: 3 and 9)
[0461] xx. A14E, B5F, B28K, desB29-30 (SEQ ID NO: 3 and 11) [0462]
xxi. A14E, B5Y, B26K, desB27-desB30 (SEQ ID 3 and 12) [0463] xxii.
A14E, B5Y, desB30 (SEQ ID 3 and 13) [0464] xxiii. A14E, B5Y, B26G,
desB30 (SEQ ID 3 and 14) [0465] A14E, B5Y, B26A, desB30 (SEQ ID 3
and 15) [0466] 20. The insulin derivative according to any of the
previous embodiments, wherein said acyl group is lithocholic acid,
or comprises a functional group of the formulae:
[0466] --CO--(CH.sub.2).sub.x--COOH; Chem. 1:
--CO--(CH.sub.2).sub.x-tetrazolyl; Chem. 2: [0467] wherein x
represents an integer in the range of from 12 to 20; and the
tetrazolyl group is 1H-tetrazol-5-yl. or is a fatty acid of
formula:
[0467] --CO--(CH.sub.2).sub.x--CH.sub.3 Chem. 3:
wherein x represents an integer in the range from 8 to 16. [0468]
21. The insulin derivative according to embodiment 20, wherein Acy
is selected from the group consisting of: lithocholic acid,
1,16-hexadecanedioic acid, 1,18-octadecanedioic acid,
1,20-eicosanedioic acid, tetrazole-C16, tetrazole-C17, tetrazole
C18 and tetradecanoic acid. [0469] 22. The insulin derivative
according to embodiment 21, wherein said substituent comprises
lithocholic acid. [0470] 23. The insulin derivative according to
embodiment 21, wherein said acyl group comprises a fatty diacid
group selected from 1,16-hexadecanedioic acid, 1,18-octadecanedioic
acid, and 1,20-eicosanedioic acid. [0471] 24. The insulin
derivative according to embodiment 23, wherein the at least one
substituent comprise a fatty diacid group 1,16-hexadecanedioic
acid. [0472] 25. The insulin derivative according to embodiment 23,
wherein the at least one substituent comprise a fatty diacid group
1,18-octadecanedioic acid. [0473] 26. The insulin derivative
according to embodiment 23, wherein the at least one substituent
comprise a fatty diacid group 1,20-eicosanedioic acid. [0474] 27.
The insulin derivative according to embodiment 21, wherein said
tetrazole moiety is selected from the group consisting of
tetrazole-C16, tetrazole-C17 and tetrazole C18. [0475] 28. The
insulin derivative according to embodiment 27, wherein said
tetrazole moiety is tetrazole-C16. [0476] 29. The insulin
derivative according to embodiment 27, wherein said tetrazole
moiety is tetrazole-C17. [0477] 30. The insulin derivative
according to embodiment 27, wherein said tetrazole moiety is
tetrazole C18. [0478] 31. The insulin derivative according to
embodiment 20, wherein said fatty acid is tetradecanoic acid or
C14. [0479] 32. The insulin derivative according to any of the
previous embodiments, wherein said substituent comprises a linker
group. [0480] 33. The insulin derivative according to embodiments
1-31, wherein the linker group is absent is represented by a
covalent bond. [0481] 34. The insulin derivative according to any
one of embodiments 1-32, wherein -L1-L2-L3 represents a divalent
linker group selected from DgGlu, gGlu, gGlu-gGlu, gGlu-OEG,
gGlu-OEG-OEG, OEG, sulfonimide-C4 and
sulfonimide-C4-sulfonimide-C4, wherein, gGlu represents a gamma
glutamic acid residue; and OEG represents
[2-(2-aminoethoxy)ethoxy]acetyl. [0482] 35. The insulin derivative
according to embodiment 34, wherein said divalent linking group is
DgGlu. [0483] 36. The insulin derivative according to embodiment
34, wherein said divalent linking group is gGlu. [0484] 37. The
insulin derivative according to embodiment 34, wherein said
divalent linking group is gGlu-gGlu. [0485] 38. The insulin
derivative according to embodiment 34, wherein said divalent
linking group is gGlu-OEG. [0486] 39. The insulin derivative
according to embodiment 34, wherein said divalent linking group is
gGlu-OEG-OEG. [0487] 40. The insulin derivative according to
embodiment 34, wherein said divalent linking group is OEG. [0488]
41. The insulin derivative according to embodiment 34, wherein said
divalent linking group is sulfonimide-C4. [0489] 42. The insulin
derivative according to embodiment 34, wherein said divalent
linking group is sulfonimide-C4-sulfonimide-C4. [0490] 43. The
insulin derivative according to any one of the preceding
embodiments, wherein said substituent is the substituent of the
compounds of examples 1-46, represented independently by: [0491]
Lithocholic acid [0492] Lithocholic acid-gGlu [0493] C14 [0494] C16
diacid [0495] C16 diacid-gGlu [0496] C18 diacid [0497] C18
diacid-gGlu [0498] C18 diacid-2.times.gGlu [0499] C18 diacid-DgGlu
[0500] C18 diacid-gGlu-OEG [0501] C18 diacid-gGlu-2.times.OEG
[0502] C18 diacid-OEG [0503] C18 diacid-sulfonimide-C4 [0504] C20
diacid [0505] C20 diacid-gGlu [0506] C20 diacid-gGlu-OEG [0507] C20
diacid-gGlu-2.times.OEG [0508] Tetrazole-C16 [0509]
Tetrazole-C16-gGlu-OEG [0510] Tetrazole-C16-gGlu-2.times.OEG [0511]
Tetrazole-C16-2.times.sulfonimide-C4 [0512] Tetrazole-C17 [0513]
Tetrazole-C17-sulfonimide-C4 [0514] Tetrazole-C18 [0515]
Tetrazole-C18-sulfonimide-C4 [0516]
Tetrazole-C18-2.times.sulfonimide-C4 [0517] 44. The insulin
derivative according to any one of the previous embodiments,
selected from the group consisting of the compounds of Examples
1-46. [0518] 45. The insulin derivative according to any one of the
previous embodiments, selected from the group consisting of the
compounds of Examples 1-36. [0519] 46. The insulin derivative
according to any one of the previous embodiments, selected from the
group consisting of the compounds of Examples 10-34. [0520] 47. The
insulin derivative according to any one of the previous
embodiments, selected from the group consisting of the compounds of
Examples 3, 4, 12-16, 18-20, 22, 23, 25, 26, 28-30. [0521] 48. The
insulin derivative according to any one of the previous
embodiments, selected from the group consisting of the compounds of
Examples 14-16, 18, 20 and 26. [0522] 49. The insulin derivative
according to any one of the previous embodiments, selected from the
group consisting of the compounds of Examples 37-39 and 46. [0523]
50. The insulin derivative according to any one of the previous
embodiments, selected from the group consisting of the compounds of
Examples 40-45. [0524] 51. The insulin derivative according to any
one of the previous embodiments, represented by compound of Example
15:
N{Epsilon-B28}-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)-
butanoyl]amino]ethoxy]ethoxy]acetyl]-[GluA14,TyrB5,GlyB26,LysB28],des-(B29-
-B30)-Insulin
[0524] ##STR00024## [0525] 52. The insulin derivative according to
any one of the previous embodiments, wherein said insulin
derivative induces a submaximal phosphorylation of insulin receptor
of about or below 65%, when compared to human insulin. [0526] 53.
The insulin derivative according to any one of the previous
embodiments, wherein said insulin derivative induces a submaximal
phosphorylation of insulin receptor of about or below 60%, when
compared to human insulin. [0527] 54. The insulin derivative
according to any one of the previous embodiments, wherein said
insulin derivative induces a submaximal phosphorylation of insulin
receptor of about or below 50%, when compared to human insulin.
[0528] 55. The insulin derivative according to any one of the
previous embodiments, wherein said insulin derivative induces a
submaximal phosphorylation of insulin receptor of about or below
40%, when compared to human insulin. [0529] 56. The insulin
derivative according to any one of the previous embodiments,
wherein said insulin derivative induces a submaximal
phosphorylation of insulin receptor of about or below 30%, when
compared to human insulin. [0530] 57. The insulin derivative
according to any one of the previous embodiments, wherein said
insulin derivative induces a submaximal phosphorylation of insulin
receptor of about or below 20%, when compared to human insulin.
[0531] 58. The insulin derivative according to any one of the
previous embodiments, wherein said insulin derivative induces a
submaximal phosphorylation of insulin receptor of about or below
12%, when compared to human insulin. [0532] 59. The insulin
derivative according to any of the previous embodiments, for use as
a medicament. [0533] 60. The insulin derivative according to any of
embodiments 1-58, for use in a method of treatment. [0534] 61. The
insulin derivative according to any of the previous embodiments
1-58, for use in the prevention or treatment of a cardiovascular
disease. [0535] 62. The insulin derivative according to any of the
previous embodiments 1-58, for use in the prevention or treatment
of atherosclerosis. [0536] 63. The insulin derivative according to
any of the previous embodiments 1-58, for use in preventing or
reducing of endothelial dysfunction. [0537] 64. The insulin
derivative according to any of the previous embodiments 1-58, for
use in improving lipid parameters. [0538] 65. The insulin
derivative according to any of the previous embodiments 1-58, for
use in preventing or reducing liver triglyceride content. [0539]
66. The insulin derivative according to any of the previous
embodiments 1-58, for use in for preventing or reducing body weight
gain. [0540] 67. The insulin derivative according to any of the
previous embodiments 1-58, for use in treatment of: [0541]
improving lipid parameters, such as prevention and/or treatment of
dyslipidemia, lowering total serum lipids, increasing HDL-C,
lowering LDL-C, lowering small, dense LDL-C, lowering VLDL-C,
lowering triglycerides, lowering cholesterol, lowering plasma
levels of lipoprotein a (Lp(a)) or inhibiting generation of
apolipoprotein A (apo(A)); [0542] prevention and/or treatment of
cardiovascular diseases, such as cardiac syndrome X,
atherosclerosis, myocardial infarction, coronary heart disease,
reperfusion injury, stroke, cerebral ischemia, an early cardiac or
early cardiovascular disease, left ventricular hypertrophy,
coronary artery disease, hypertension, essential hypertension,
acute hypertensive emergency, cardiomyopathy, heart insufficiency,
exercise intolerance, acute and/or chronic heart failure,
arrhythmia, cardiac dysrhythmia, syncopy, angina pectoris, cardiac
bypass and/or stent reocclusion, intermittent claudication
(atheroschlerosis oblitterens), diastolic dysfunction, and/or
systolic dysfunction; and/or reduction of blood pressure, such as
reduction of systolic blood pressure; the treatment of
cardiovascular disease. [0543] 68. A pharmaceutical composition
comprising an insulin derivative according to any of the previous
embodiments 1-58, and a pharmaceutically acceptable excipient.
[0544] 69. The pharmaceutical composition according to embodiment
68, for subcutaneous administration. [0545] 70. 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 any one of embodiments 1-58,
together with a pharmaceutically acceptable carrier. [0546] 71. The
pharmaceutical compositions of embodiments 68-70, for use as a
medicament. [0547] 72. The pharmaceutical compositions of
embodiments 68-70, for use in the treatment of patients with
diabetes and high risk of cardiovascular disease. [0548] 73. The
pharmaceutical compositions of embodiments 68-70 and/or prevent or
reduce development of atherosclerosis. [0549] 74. Use of an insulin
derivative according to any one of embodiments 1-58, for the
manufacture of a medicament for the treatment or prevention of
diabetes, diabetes of Type 1, diabetes of Type 2, impaired glucose
tolerance, hyperglycemia, dyslipidemia, obesity, metabolic syndrome
(metabolic syndrome X, insulin resistance syndrome), hypertension,
cognitive disorders, atherosclerosis, myocardial infarction,
stroke, cardiovascular disorders, coronary heart disease, stroke,
inflammatory bowel syndrome, dyspepsia, hypotension or gastric
ulcers. [0550] 75. A method for improving lipid parameters
comprising a step of administering a pharmaceutically active amount
of an insulin derivative according to any of the previous
embodiments 1-58. [0551] 76. A method for improving lipid
parameters comprising a step of administering a pharmaceutically
active amount of an insulin derivative according to any of the
previous embodiments 1-58, wherein improving lipid parameters, is
such as prevention and/or treatment of dyslipidemia, lowering total
serum lipids; increasing HDL; lowering LDL-C; lowering small, dense
LDL-C; lowering VLDL-C; non_HDL-C; lowering triglycerides; lowering
cholesterol; lowering plasma levels of lipoprotein a (Lp(a));
inhibiting generation of apolipoprotein A (apo(A)). [0552] 77. A
method for prevention and/or treatment of a cardiovascular disease
comprising a step of administering a pharmaceutically active amount
of an insulin derivative according to any of the previous
embodiments 1-58. [0553] 78. A method for the treatment or
prevention of diabetes, diabetes of Type 1, diabetes of Type 2,
impaired glucose tolerance, hyperglycemia, dyslipidemia, obesity,
metabolic syndrome (metabolic syndrome X, insulin resistance
syndrome), hypertension, cognitive disorders, atherosclerosis,
myocardial infarction, stroke, cardiovascular disorders, coronary
heart disease, stroke, inflammatory bowel syndrome, dyspepsia, or
gastric ulcers, which method comprises administration to a subject
in need thereof a therapeutically effective amount of a derivative
of human insulin according to any one of embodiments 1-58. [0554]
79. An intermediate product comprising a backbone selected from the
group consisting of: [0555] i. A14E, B5Y, B26A, B28K, desB29-30
(SEQ ID NO: 3 and 4) [0556] ii. A14E, B5Y, B26G, B28K, desB29-30
(SEQ ID NO: 3 and 5) [0557] iii. B5Y, B26A, B28K, desB29-30 (SEQ ID
NO: 1 and 7) [0558] iv. B5Y, B26G, B28K, desB29-30 (SEQ ID NO: 1
and 8) [0559] v. B5Y, B28K, desB29-30 (SEQ ID NO: 1 and 9) [0560]
vi. A14E, B5F, B26G, B28K, desB29-30 (SEQ ID NO: 3 and 10) [0561]
vii. B5F, B28K, desB29-30 (SEQ ID NO: 1 and 11) [0562] viii. B5Y,
B26K, desB27-desB30 (SEQ ID 1 and 12) [0563] ix. B5Y, desB30 (SEQ
ID 1 and 13) [0564] x. B5Y, B26G, desB30 (SEQ ID 1 and 14) [0565]
xi. B5Y, B26A, desB30 (SEQ ID 1 and 15) [0566] xii. B5F, B26A,
B28K, desB29-30 (SEQ ID 1 and 6) [0567] xiii. B5Y, B26A, B28K,
desB29-30 (SEQ ID NO: 1 and 4) [0568] xiv. B5Y, B26G, B28K,
desB29-30 (SEQ ID NO: 1 and 5) [0569] xv. B5F, B26G, B28K,
desB29-30 (SEQ ID NO: 1 and 10) [0570] xvi. A14E, B5F, B26A, B28K,
desB29-30 (SEQ ID 3 and 6) [0571] xvii. A14E, B5Y, B26A, B28K,
desB29-30 (SEQ ID NO: 3 and 7) [0572] xviii. A14E, B5Y, B26G, B28K,
desB29-30 (SEQ ID NO: 3 and 8) [0573] xix. A14E, B5Y, B28K,
desB29-30 (SEQ ID NO: 3 and 9) [0574] xx. A14E, B5F, B28K,
desB29-30 (SEQ ID NO: 3 and 11) [0575] xxi. A14E, B5Y, B26K,
desB27-desB30 (SEQ ID 3 and 12) [0576] xxii. A14E, B5Y, desB30 (SEQ
ID 3 and 13) [0577] xxiii. A14E, B5Y, B26G, desB30 (SEQ ID 3 and
14) [0578] xxiv. A14E, B5Y, B26A, desB30 (SEQ ID 3 and 15) or a
pharmaceutically acceptable salt, amide or ester thereof. [0579]
80. A method for determining selectivity of a compound comprising
the following steps: [0580] measuring the maximal AKT
phosphorylation induced by said compound relative to human insulin
[0581] measuring the maximal ERK activation induced by compound A
relative to human insulin, wherein the ERK/AKT ratio is less than
1. [0582] 81. The method of embodiment 80, wherein said compound is
an insulin compound. [0583] 82. The method of embodiment 81,
wherein said insulin compound is a compound of embodiments
1-58.
EXAMPLES
[0584] Pharmacological Methods
[0585] Assay (I) In Vitro Biology
[0586] Insulin Receptor Binding
[0587] The relative binding affinity of the insulin derivatives of
the invention for the human insulin receptor (IR) was determined by
competition binding in a scintillation proximity assay (SPA)
(according to Glendorf T et al; Biochemistry 2008 47
4743-4751).
[0588] In brief, dilution series of a human insulin standard and an
insulin derivative were performed in 96-well plates followed by the
addition of SPA beads (Anti-Mouse polyvinyltoluene SPA Beads, GE
Healthcare), anti-IR mouse antibody 83-7 (can be purchased from
e.g. Thermo Fisher Scientific), solubilised human IR-A (purified
from Baby Hamster Kidney (BHK) cells overexpressing IR-A), and
[.sup.125I-TyrA14]-human insulin in binding buffer. After
incubation, plates were centrifuged and counted on a TopCount NXT
(Perkin-Elmer Life Sciences).
[0589] Data from the SPA were analysed according to the
four-parameter logistic model (Volund A; Biometrics 1978 34
357-365), and the binding affinities of the analogues were
calculated relative to that of the human insulin standard measured
within the same plate.
[0590] The relative binding affinities of insulin derivatives
representative for the invention are listed in Table 1, below. FIG.
1 shows representative receptor binding curves for human insulin
and Compound of example 15 from a competition binding assay with
solubilised IR-A.
[0591] Insulin Receptor Phosphorylation (IRpY1158)
[0592] The effect of the insulin derivatives of this invention on
activation of the insulin receptor (IR) was assessed by the ability
of the insulin derivatives to phosphorylate the tyrosine residue in
position 1158 of the insulin receptor, as described by Hansen B F
et al; PLOS One May 2012 7 e34274.
[0593] In brief, CHO-hIR cells (Chinese hamster ovarian cells
overexpressing the insulin receptor-A) were stimulated with
increasing concentrations of insulin analogues for 30 min, washed
in ice-cold phosphate buffered saline (PBS), snap-frozen and lysed
in lysis buffer. Equal amounts of protein were loaded into
Phospho-IR-ELISA wells (IRpY1158), and phosphorylation measured
according to the manufacturer's protocol (Invitrogen).
[0594] The maximum response of an insulin derivative of the
invention on insulin receptor phosphorylation level was determined
by measuring the insulin receptor phosphorylation level in the
presence of increasing amounts of said insulin derivative to obtain
a dose-response curve. A standard dose-response curve was defined
by four parameters: the baseline response (Bottom), the maximum
response (Top), the slope, and the drug concentration that provokes
a response halfway between baseline and maximum (EC50).
[0595] Insulin receptor phosphorylation data dose-response curves
was fitted by non-linear regression (four-parameter model
(Y=Bottom+(Top-Bottom)/(1+10{circumflex over (
)}((LogEC50-X)*HillSlope))) using GraphPad Prism 7 from GraphPad
Software Inc. The estimated parameters estimated was used to
calculate the % max value for each ligand as
(Top(analogue)-Bottom(analogue))*100/(Top(insulin)-Bottom(insulin)).
[0596] Calculated % max values of insulin derivatives
representative for the invention are listed in Table 1, below. FIG.
2 shows examples of such dose-response curves for human insulin and
compound of example 15 from an IRpY1158 phosphorylation assay in
CHO-hIR cells overexpressing the IR-A.
[0597] Thus from FIGS. 1 and 2 it can be seen how a compound
representative of the invention, i.e. compound of example 15, was
fully capable of displacing human insulin from the insulin receptor
(FIG. 1), but only induces submaximal phosphorylation of the
insulin receptor (FIG. 2.).
[0598] Insulin Receptor Signalling
[0599] Insulin receptor (IR) signalling of the analogues of this
invention via AKT and ERK pathways was assessed either by
traditional Western blotting technique or by the use of Surefire,
alphascreen technique.
[0600] Western Blotting
[0601] In brief, CHO-hIR cells were stimulated for 10 min with
increasing concentrations of the insulin analogue, washed in
ice-cold PBS, snap-frozen and lysed in lysis buffer (Biosource).
Equal amounts of proteins were loaded on gels and blotted to
nitrocellulose membranes. Phosphorylated AKT and ERK were
visualised with Phospho-AKT (Ser473) (Cell signalling #9271) and
pMAPK 44/42 ERK1/2 rabbit (Cell Signalling #4376) antibodies,
respectively. Band intensities were evaluated using a LAS3000
(Fuji).
[0602] Surefire
[0603] Cells were plated in 96 well tissue culture plate, and the
day after stimulated with insulin or insulin derivatives for 10 min
at 37.degree. C. and analyses according to manufactures protocol
(AKT1/2/3 (p-Ser473) cat # TGRA4S10K and ERK1/2 p-T202/Y204 Cat #
TGRESB10K)
[0604] The responsiveness value (Span) for each analogue was
calculated using non-linear regression in GraphPad Prism 7 from
GraphPad Software Inc and expresses as percent of the
responsiveness of insulin (% max AKT or % max ERK)
[0605] FIGS. 3 and 4 show representative dose-response curves for
human insulin and Compound of example 15 from an AKT
phosphorylation assay and an ERK phosphorylation assay in CHO-hIR
cells overexpressing the IR-A and show how a compound
representative of the invention, i.e. Compound of example 15,
induces a lower submaximal activation of ERK than of AKT compared
to human insulin. Calculated (% max ERK/% max AKT) values of
insulin analogues representative for the invention and reference
insulin derivatives are listed in Table 1.
[0606] The results given in Table 1 show that only the insulin
derivatives which induce insulin receptor phosphorylation less than
65% of the maximum response induced by human insulin have a lower
submaximal effect on ERK activation (phosphorylation) than on AKT
activation (phosphorylation), when compared to the effect of human
insulin.
[0607] Potency in Rat Free Fat Cell Assay (Lipogenesis)
[0608] The metabolic potency of the insulin derivatives of the
invention was determined by lipogenesis using isolated rat
adipocytes. The assays were carried out largely as described by
Moody A J et al; Horm Metab Res 1974 6 12-16 (a modified version of
the assay described in Rodbell M; J Biol Chem 1964 239
375-380).
[0609] In brief, epididymal fat pads were removed from killed
Sprague-Dawley rats and placed in degradation buffer with
collagenase (Worthington) in order to degrade the fat pads into a
single-cell suspension. The cell suspension was washed with PBS and
cells re-suspended in Krebs buffer supplied with 0.1 or 1% HSA
(A-1887, Sigma) and HEPES. Cell suspension aliquots were incubated
with glucose solution containing D-[3-.sup.3H]glucose (PerkinElmer)
and increasing concentrations of human insulin standard or insulin
derivative. The incubation was stopped by addition of MicroScint-E
(PerkinElmer), and the plates counted in a TopCount NXT (Perkin
Elmer).
[0610] The data were analysed according to the four-parameter
logistic model (Volund A; Biometrics 1978 34 357-365) and the
metabolic potencies of the analogues were calculated relative to
that of the human insulin standard measured within the same
plate.
[0611] The metabolic potencies of the insulin derivatives
representative for the invention are listed in Table 1, below. FIG.
5 shows a compound representative of the invention, i.e. compound
of example 15, have the same maximum effect as human insulin on
stimulating lipogenesis in primary rat adipocytes.
[0612] Isolation of Rat Hepatocytes
[0613] Rat hepatocytes were isolated from male Sprague-Dawley rats
by retrograde perfusion of the liver with collagenase (Sigma) using
a modified version of Berry M N and Friend D S; J Cell Biol 1969 43
506-520.
[0614] Hepatocytes were washed in Media 199 (Gibco) supplemented
with human insulin, dexamethasone and foetal calf serum (Gibco),
seeded on collagen-coated plates (BD BioCoat, BD Biosciences) and
allowed to attach for 1-4 hours.
[0615] Stimulation of Glycogen Synthesis in Primary Rat
Hepatocytes
[0616] The effect of the insulin analogues of this invention on
stimulation of glycogen accumulation was determined in primary
hepatocytes (see isolation procedure above) using a specific and
simple enzymatic method named PAS (Periodic Acid-Schiff) assay
adapted from M. Kilcoyne et al., Analytical Biochemistry 416 (2011)
18-26. This method is an adaptation of the PAS reagent staining for
a microtiter plate colorimetric assay. Primary rat hepatocytes were
cultured in 96-well plates with varying human insulin or insulin
derivative concentrations for 18-24 hours. To determine cellular
glycogen content, the hepatocytes were lysed in 1% Triton for 30
minutes in a shaker at room temperature. Periodic acid solution
(0.1% Periodic Acid+7% acetic acid in MQ water) (Periodic Acid
3951, Sigma) was added to each well and mix with shaker for one
min. Plates were then incubated for 90 min. at 37.degree. C.
allowing oxidization of the hydroxyl groups of glucose to
aldehydes. Then Schiff's solution was added and the plate was
protected from the light, shaken for 5 min. before letting them
stand for 25 min. at room temperature. Absorbance at 550 nm was
then measured using a SpectraMax spectrophotometer. Data were
analysed in GraphPad Prism 7 and the relative metabolic potency of
the insulin analogue calculated as the ratio between the estimated
EC50 values for human insulin and the insulin analogue.
[0617] The potencies of the insulin derivatives representative for
the invention on glycogen accumulation in hepatocytes are listed in
Table 1, below. FIG. 6 a compound representative of the invention,
i.e. compound of example 15, has the same maximum effect as human
insulin on the stimulation of glycogen accumulation in primary rat
hepatocytes.
[0618] Gene Expression
[0619] The effect of the insulin derivatives of this invention on
gene expression was determined by quantitative real-time polymerase
chain reaction (RT-PCR) on cDNA isolated from primary rat
hepatocytes (see isolation procedure above).
[0620] One day after isolation, hepatocytes were treated with
glucagon for two hours in assay Media 199 (Gibco) supplemented with
1 .mu.M dexamethasone and 0.1% HSA (A-1887, Sigma). After
pre-incubation, cells were treated with human insulin or insulin
derivative in assay media for 16 hours, changing to fresh media
after 2, 4 and 6 hours.
[0621] RNA was extracted and purified from hepatocytes using RNeasy
Mini Kit (Qiagen). cDNA was produced from RNA using iScript cDNA
Synthesis Kit (Bio-Rad). Gene expression was determined using
TagMan Fast Advanced Master mix and primer/probe on demand (Applied
BioSystems; Ppib #Rn03302274_m1 rFAS #Rn00565347_m1; rG6Pc
#Rn00565347_m1). Gene expression was normalised to expression of
Ppip (cycB, cyclophilin b) and fitted to a sigmoidal dose-response
curve using GraphPad Prism 7 from GraphPad Software Inc.
[0622] FIGS. 7 and 8 show representative dose-response curves for
human insulin and Compound of example 15, from quantitative RT-PCR
assays for fasn and g6pc performed on cDNA isolated from primary
rat hepatocytes. The results given in FIG. 7 show how a compound
representative of the invention, i.e. compound of example 15,
induces a submaximal induction of FAS mRNA, when compared to human
insulin.
[0623] FIG. 8 show a compound representative of the invention, i.e.
compound of example 15, inhibits the expression of G6Pc RNA to the
same level as human insulin in primary rat hepatocytes.
[0624] De Novo Lipogenesis
[0625] The effect of the insulin derivatives of this invention on
de novo synthesis of lipids (DNL) was determined in primary
hepatocytes (see isolation procedure above) using labelled
acetate.
[0626] One day after isolation, hepatocytes were pre-incubated in
assay media (Media 199 (Gibco), HSA (Sigma)) supplemented with
glucose (Sigma) and human insulin or insulin derivative for 24
hours. After pre-incubation, hepatocytes were treated in assay
media with human insulin or insulin derivative and
.sup.14C-labelled acetate (PerkinElmer) for 24 h hours. After
incubation, cells were washed in PBS and lysed with MicroScint-E
(PerkinElmer).
[0627] Incorporation of radioactive acetate into lipids (DNL) was
determined using a TopCount NXT (PerkinElmer Life Sciences) and the
results fitted to a sigmoidal dose-response curve using GraphPad
Prism 7 from GraphPad Software Inc.
[0628] FIG. 9 shows representative dose-response curves for human
insulin and compound of example 15 from a de novo lipogenesis assay
in primary rat hepatocytes and show how a compound representative
of the invention, i.e. compound of example 15, exhibits a
submaximal response on the de novo lipogenesis in primary rat
hepatocytes.
TABLE-US-00004 TABLE 1A In vitro biological activity hIR
Lipogenesis Lipogenesis Elisa hIR binding 0.1% HSA 1% HSA pY1158
Compound of IC50 EC50 EC50 max Example (% of human (% of human (%
of human (% of human % maxERK/ no. insulin) insulin) insulin)
insulin) % maxAKT 1 8 0.82 0.15 32 0.38 2 10 0.86 0.21 43 0.47 3 8
0.04 0.01 17 0.35 4 4 0.14 0.07 19 0.37 5 8 0.14 0.07 31 0.48 6 5
0.14 0.06 36 0.45 7 9 0.20 0.09 39 0.61 8 10 1.76 0.72 25 0.32 9 12
0.40 0.49 14 0.29 10 14 0.68 0.11 10 0.27 11 10 0.05 0.02 3 0.07 12
12 0.10 0.02 10 0.25 13 10 0.26 0.05 11 0.15 14 10 0.26 0.05 16
0.25 15 10 0.29 0.09 16 0.25 16 7 0.08 0.05 20 0.38 17 7 0.18 0.16
37 0.53 18 6 0.32 0.26 14 0.35 19 7 0.04 0.02 15 0.21 20 20 0.34
0.14 23 0.23 21 12 0.39 0.43 37 0.54 22 17 0.21 0.15 26 0.36 23 23
0.29 0.17 28 0.36 24 34 0.39 0.21 30 0.42 25 15 0.14 0.12 26 0.50
26 14 0.50 0.24 21 0.37 27 19 0.11 0.09 35 0.47 28 15 0.24 0.16 12
0.30 29 16 0.37 0.17 17 0.32 30 12 0.17 0.11 14 0.25 31 12 0.26
0.20 7 0.34 32 4 0.12 0.05 32 0.40 33 7 0.08 0.05 6 0.20 34 3 0.09
0.03 27 0.27 35 4 0.02 0.01 10 0.31 36 9 0.03 0.01 10 0.26 37 6
0.01 0.01 2 ND 38 4 0.03 0.01 6 0.33 39 4 0.05 0.01 24 0.41 40 11
1.42 0.16 58 0.84 41 3 0.22 0.16 40 0.66 42 12 2.21 0.41 8 0.19 43
7 1.08 0.32 13 0.30 44 8 0.09 0.02 7 0.36 45 4 0.13 0.08 6 0.29 46
3 ND 0.03 20 0.27 ND: Not determined
TABLE-US-00005 TABLE 1B In vitro biological activity for
comparators hIR Lipogenesis Lipogenesis Elisa hIR binding 0.1% HSA
1% HSA pY1158 IC50 EC50 EC50 max Comparator (% of human (% of human
(% of human (% of human % maxERK/ compound insulin) insulin)
insulin) insulin) % maxAKT Human insulin 100 100 100 100 1
Comparator 47 71 56 93 0.84 no. 1 Comparator 27 44 43 93 0.82 no. 2
Comparator 54 37 35 132 0.85 no. 3 Comparator 45 58 39 95 0.84 no.
4 Comparator 13 ND 0.7 69 0.81 no. 5 PK comparator used in Assay
(II) Insulin lispro 119 ND ND 104 0.85 Insulin aspart 83 ND ND 95
0.79 Insulin 20 3.8 0.8 87 0.97 degludec Insulin glargine 79 ND
68.9 104 0.85
[0629] Assay (II) Subchronic In Vivo Study with Diabetic Mice
[0630] Animals and Compounds
[0631] 12-week old male C57BL/6J-Diet-Induced Obese (DIO) mice from
Jackson Laboratory, on Research Diet D12492 high-fat chow, were
habituated for two weeks in the facilities, and then, under brief
isoflurane gas anaesthesia, made diabetic by subcutaneous (s.c.)
injection of 150 mg/kg streptozotocin (STZ) (Sigma). The animals
were allowed two weeks to develop stable diabetes, before being
assigned to treatment groups based on measurements of body weight,
blood glucose levels, and blood glycosylated haemoglobin (HbA1c)
levels. The animals were housed under standard lighting and
temperature conditions, and had ad lib access to the D12492 diet
and water at all times.
[0632] Animals were assigned to either vehicle treatment (n=16), or
to different doses of either the PK Comparator (comparator no. 5)
or compound of example 15 (n=26 per dose group).
[0633] The test compounds, i.e. the PK Comparator (comparator no.
5) and compound of example 15, were formulated in 5 mM phosphate,
140 mM sodium chloride, and 70 ppm polysorbate-20.
[0634] The PK Comparator (comparator no. 5) is an acylated, but
non-partial insulin derivative.
[0635] Treatment Protocol
[0636] The animals were treated for six weeks, with twice-daily
s.c. injections with their assigned treatment. Injections were
given 12 hours apart, in the time frames 07:30-08:30 and
19:30-20:30 every day. The dosing volume was 2 mL/kg, and the
injections were administered with a NovoPen.RTM. injection
device.
[0637] Throughout the study, the animals were regularly measured
for body weight and blood glucose levels, and blood HbA1c levels
and body composition was measured at start and at completion of the
study. Furthermore, the plasma exposure level of the PK Comparator
(comparator no. 5) and compound of example 15 was assessed.
[0638] Analytical Methods
[0639] Blood glucose concentrations were determined by diluting
whole blood in EBIO buffer solution, followed by measurement on an
EKF Diagnostic Biosen autoanalyser.
[0640] Blood HbA1c levels were determined by diluting whole blood
in hemolysing buffer, followed by measurement on a Hitachi Cobas
6000 autoanalyser, and by using a Roche Diagnostics HbA1c kit.
[0641] Body composition was determined by scanning un-anaesthetised
mice for fat mass and lean mass by quantitative magnetic resonance
(EchoMRI-100.TM., Echo Medical Systems).
[0642] The liver TG content was determined in samples of liver
tissue, collected immediately after euthanasia. After
homogenization, saponification and centrifugation of the liver
sample, the TG concentration was measured in the supernatant on a
Hitachi Cobas 6000 Analyzer (Roche). The TG content was then
divided with the mass of the original homogenized liver sample and
expressed as pmol TG per mg liver tissue.
[0643] Endothelial function was assessed ex vivo in HbA1c matched
groups (vehicle n=12, PK Comparator (comparator no. 5) n=15 and
compound of example 15 (n=15) using ACh-induced vasorelaxation in
mesenteric arteries mounted in a wire myograph. The arteries were
pre-constricted to 70% of maximal tone with phenylephrine followed
by stimulation with ACh in cumulatively increasing doses.
[0644] Plasma exposure levels of the PK Comparator (comparator no.
5) and compound of example 15, were determined by analysis of
plasma samples using in-house luminescent oxygen channelling
immunoassays (LOCI).
[0645] FIG. 10-14 show representative curves for HbA1c levels, body
weight, body fat mass, liver TG and ACh-stimulated vasorelaxation
of mesenteric arteries from a sub-chronic in vivo study in diabetic
STZ-DIO mice dosed subcutaneously twice daily for six weeks with
vehicle, the PK Comparator (comparator no. 5), and an insulin
analogue representative of the invention, i.e. compound of example
15.
[0646] The animal experiments showed that treatment of diabetic
high-fat fed mice with insulin derivatives of the invention can
lower blood glucose levels similarly to the pharmacokinetics (PK)
Comparator (comparator no. 5). The PK Comparator (comparator no. 5)
is an acylated insulin analogue that is not a partial agonist on
insulin receptor phosphorylation, but with a PK profile in mice
similar to that of compound of example 15 of the present
invention.
[0647] Sub-chronic treatment (6 weeks) of diabetic, high-fat fed
mice with an insulin derivative representative of the invention,
i.e. compound of example 15, versus a PK Comparator (comparator no.
5) dosed to reach the same level of glycaemic control (measured by
HbA1c levels), resulted in significantly less weight gain less
increase in body fat mass, increased lowering of liver
triglycerides (TG) and improved acetylcholine (ACh) stimulated
vasorelaxation ex vivo.
[0648] Thus, the present invention provides insulin derivatives,
which are capable of reducing blood glucose levels, but have
improved effects on processes related to lipid metabolism, such as
less weight gain, less increase in body fat mass and increased
lowering of liver TG, as well as improved endothelial function
relative to a PK-matched comparator.
List of Abbreviations
[0649] ACh--Acetylcholine [0650] AKT--Protein Kinase B (PKB) [0651]
BHK--Baby hamster kidney [0652] CHO--Chinese hamster ovarian [0653]
CPM--Counts per minute [0654] CVD--Cardiovascular diseases [0655]
DIO--Diet-induced obese [0656] DNL--de novo lipogenesis [0657]
DPM--Disintegrations per minute [0658] ELISA--Enzyme-linked
immunosorbent assay [0659] ERK--Extracellular regulated kinase
(also known as MAPK) [0660] FAS--Fatty acid synthase [0661]
G6Pc--Glucose-6-phosphatase [0662] HbA1c--glycosylated haemoglobin
[0663] HEPES--4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
[0664] HI--Human insulin [0665] HSA--Human serum albumin [0666]
IR--Insulin receptor [0667] IR-A--Insulin receptor isoform A [0668]
LOCI--luminescent oxygen channelling immunoassays [0669]
MAPK--Mitogen-activated protein kinase (also known as ERK) [0670]
NAFLD--Non-alcoholic fatty liver diseases [0671] PAS--Periodic acid
solution [0672] PBS--Phosphate buffered saline [0673]
PK--pharmacokinetics [0674] PKB--Protein kinase B (also known as
AKT) [0675] Ppip--Cyclophilin b (cycB) [0676] RT-PCR--real time
polymerase chain reaction [0677] SPA--scintillation proximity assay
[0678] SREPB1c--Sterol regulatory element-binding transcription
factor 1c [0679] STZ--streptozotocin [0680]
STZ-DIO--streptozotocin-treated diet-induced obese [0681]
TG--Triglycerides
[0682] Stability
[0683] The stability of the insulin derivatives of this invention
were evaluated using Differential Scanning calorimetry (DSC) and
Size Exclusion Chromatography (SEC)
[0684] Evaluation of Insulin Derivative Self-Association and
Stability by Differential Scanning Calorimetry (DSC)
[0685] Formulation
[0686] The insulin derivatives were dissolved to about 0.2 mM and
pH adjusted to around 7.6 with sodium hydroxide and hydrochloric
acid. Concentration was determined by SEC Waters PROTEIN PAK 125
(250*8 mm) with an eluent containing 2.5 M acetic acid, 4 mM
L-arginine and 20% (V/V) acetonitrile at a flow rate of 1 ml/min.
and ambient temperature. Detection at 280 nm against a human
insulin reference using absorption coefficient correction according
to Pace.
[0687] For a final insulin derivative concentration of 0.1 mM was
added, 16 mM phenol, 7 mM phosphate, 10 mM NaCl and 0.1 mM zinc
acetate. pH was adjusted to 7.4 with hydrochloric acid and sodium
hydroxide.
[0688] Data collection was performed using a MicroCal VP-Capillary
DSC (Malvern Instruments Ltd). All scans were performed with a
vehicle (same composition as the insulin samples but without
insulin and zinc acetate) in the reference cell from 20.degree. C.
to 110.degree. C. at a scan rate of 4.degree. C./min. Instrument
feedback mode set at "low" and data filtering period set at 2
seconds. A buffer-buffer reference scan (performed with the
aforementioned vehicle) was subtracted from each sample scan prior
to concentration normalization and baseline creation. It should be
noted that the insulin zinc complex could be in other association
states than hexameric with regards to insulin, but for the sake of
simplification the zinc stabilized insulin complex is referred to
as hexameric.
[0689] The (T.sub.onset) and the midpoint of the hexamer unfolding
peak (T.sub.m,hexamer) were compared for different insulin
derivatives and values of T.sub.,onset and T.sub.m,hexamer are
shown in Table 2. The results show how B5Tyr and B5Phe mutations
have highly stabilizing effect on the insulin zinc hexamer.
TABLE-US-00006 TABLE 2 Stability data SEC, SEC, Tm, Tm, % .gtoreq.
% .gtoreq. onset hexamer hexamer hexamer Compound (.degree. C.)
(.degree. C.) 3Zn/6ins 6Zn/6ins Compound of Example 1 71.3 83.8 100
100 Compound of Example 2 77.3 92.1 ND ND Compound of Example 3
73.7 88.6 100 100 Compound of Example 4 77.2 91.7 96.7 97.1
Compound of Example 5 67.9 82.2 98.3 97.5 Compound of Example 6
75.4 90.3 86.7 92.4 Compound of Example 7 74.1 89 ND ND Compound of
Example 8 59.1 85 96.1 93 Compound of Example 9 56.5 89.4 85.5 74.1
Compound of Example 10 71.7 82.1 91.5 88.5 Compound of Example 11
67 79.4 95.4 93.2 Compound of Example 12 66 77.3 89.4 76.3 Compound
of Example 13 68.5 79.4 92.8 96.5 Compound of Example 14 71.5 82.3
78.5 87.3 Compound of Example 15 69.9 81 97.1 ND Compound of
Example 16 low signal low signal 78.7 51.4 Compound of Example 17
low signal low signal 21.7 71.1 Compound of Example 18 64.3 76.9
90.4 90.1 Compound of Example 19 60 73.6 80.7 74.2 Compound of
Example 20 low signal low signal 88.1 76.3 Compound of Example 21
ND ND ND ND Compound of Example 22 low signal low signal 93 68.6
Compound of Example 23 low signal low signal 42.5 44.1 Compound of
Example 24 low signal low signal 26.8 26.9 Compound of Example 25
low signal low signal 52 67.1 Compound of Example 26 low signal low
signal ND ND Compound of Example 27 low signal low signal 18 42.5
Compound of Example 28 67 78.5 ND ND Compound of Example 29 low
signal low signal ND ND Compound of Example 30 68.9 79.7 95.4 98.9
Compound of Example 31 67.9 97.7 91.7 86.5 Compound of Example 32
67.2 78.4 76.9 89.7 Compound of Example 33 67.4 98.2 ND ND Compound
of Example 34 67.7 78.9 84.4 89.6 Compound of Example 35 67.6 91.4
91.4 91.4 Compound of Example 36 68.5 80.6 95.9 92 Compound of
Example 37 60.8 74.1 74.2 64.7 Compound of Example 38 70.1 101.8 ND
ND Compound of Example 39 76.3 97.3 ND ND Compound of Example 40
90.7 103.1 ND ND Compound of Example 41 67.8 82.6 ND ND Compound of
Example 42 71.1 84 90.2 97.4 Compound of Example 43 71.8 82.7 89 97
Compound of Example 44 71.2 82.9 89 81 Compound of Example 45 73.6
84.3 86.2 80.5 Compound of Example 46 72 102 82.7 79.9 Insulin
aspart 52 72 ND ND Comparator 1 ND ND ND ND Comparator 2 ND ND ND
ND Comparator 3 ND ND ND ND Comparator 4 ND ND ND ND Comparator 5
ND ND ND ND Comparator 6 56.6 75 100 78 Comparator 7 44.5 68.8 100
90 Comparator 8 59.5 79 100 96 Comparator 9 low signal low signal
45.1 77.6 ND: not determined
[0690] Evaluation of Insulin Derivative Self-Association by Size
Exclusion Chromatography (SEC)
[0691] Size Exclusion Chromatography (SEC) is a common method to
evaluate insulin derivative self-association.
[0692] Formulation
[0693] The insulin analogues were dissolved to about 2 mM and pH
adjusted to 7.6 with sodium hydroxide and hydrochloric acid.
Concentration was determined by SEC Waters PROTEIN PAK 125 (250*8
mm) with an eluent containing 2.5 M acetic acid, 0.065% L-arginine
and 20% (V/V) acetonitrile at a flow rate of 1 ml/min. and ambient
temperature. Detection at 280 nm against a human insulin reference
using absorption coefficient correction according to Pace CN, [Pace
CN, Protein Science (1995) 4, 2411-2433].
[0694] For a final insulin derivative concentration of 0.6 mM was
added 1.6% glycerol, 30 mM phenol, 7 mM
tris(hydroxymethyl)aminomethane and 0.3 mM zinc acetate. pH was
adjusted with hydrochloric acid and sodium hydroxide to pH 7.6.
[0695] In order to simulate insulin derivative formulation
condition the eluent included 16 mM phenol, 20 mM sodium chloride,
and 10 mM tris(hydroxymethyl)aminomethane adjusted to pH 7.3 by
hydrochloric acid. The column used was ACQUITY U PLC.RTM. BEH200
(150*4.6 mm, d=1.7 .mu.m) from Waters Corporation, Milford, Mass.,
USA. Flow was 0.3 mL/min, injection volume 20 .mu.L and ultraviolet
detection at 286 nm. The column temperature was maintained at
23.degree. C.
[0696] FIG. 15-19 show representative chromatograms from SEC
experiments. FIG. 15 shows how a comparator compound i.e.
Comparator 9 was unstable in formulation since the various peaks
are broad and tailing showing several overlapping self-association
states. In contrast the results given in FIG. 16-19 show how
compounds representative of the invention, i.e. compounds of
Examples no. 3, 11, 12 and 15 are stable in formulation as the
chromatograms showed a predominant single sharp peak.
[0697] While certain features of the invention have been
illustrated and described herein, many modifications,
substitutions, changes, and equivalents will now occur to those of
ordinary skill in the art. It is, therefore, to be understood that
the appended embodiments are intended to cover all such
modifications and changes as fall within the true spirit of the
invention.
[0698] General Methods of Preparation
[0699] 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.
[0700] 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, i.e. by
appropriate protection of interfering groups, by changing to other
conventional reagents, or by routine modification of reaction
conditions.
[0701] 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 solids, and all parts
are by volume when referring to solvents and eluents.
[0702] 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, etc.
according to personal the preferences. 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.
[0703] (Method 1) Preparative Example--Backbone Expression and
Purification
[0704] Backbone Expression
[0705] The insulin peptide backbones, i.e. the two-chain
non-acylated insulin analogues, for use according to the invention
are produced recombinantly by expressing a DNA sequence encoding
the insulin backbone in question in a suitable host cell by
well-known techniques, e.g. as disclosed in U.S. Pat. No.
6,500,645. The insulin peptide backbone is either expressed
directly or as a precursor molecule which may have an N-terminal
extension on the B-chain and/or a connecting peptide (C-peptide)
between the B-chain and the A-chain. This N-terminal extension and
C-peptide are cleaved off in vitro by a suitable protease, e.g.
Achromobactor lyticus protease (ALP) or trypsin, and will therefore
have a cleavage site next to position B1 and A1, respectively.
N-terminal extensions and C-peptides of the type suitable for use
according to this invention are disclosed in e.g. U.S. Pat. No.
5,395,922, EP 765395 and WO 9828429 A1.
[0706] The polynucleotide sequence encoding the insulin analogue
peptide backbone precursor for use according to the invention may
be prepared synthetically by established methods, e.g. the
phosphoamidite method described by Beaucage et al; Tetrahedron
Letters 1981 22 1859-1869; or the method described by Matthes et
al; EMBO Journal 1984 3 801-805. According to the phosphoamidite
method, oligonucleotides are synthesised in e.g. an automatic DNA
synthesiser, purified, duplexed, and ligated to form the synthetic
DNA construct. A currently preferred way of preparing the DNA
construct is by polymerase chain reaction (PCR).
[0707] The recombinant method will typically make use of a vector
which is capable of replicating in the selected microorganism or
host cell and which carries a polynucleotide sequence encoding the
insulin peptide backbone precursor for use according to the present
invention. The recombinant vector may be an autonomously
replicating vector, i.e., a vector which exists as an
extra-chromosomal entity, the replication of which is independent
of chromosomal replication, e.g. a plasmid, an extra-chromosomal
element, a mini-chromosome, or an artificial chromosome. The vector
may contain any means for assuring self-replication. Alternatively,
the vector may be one which, when introduced into the host cell, is
integrated into the genome and replicated together with the
chromosome(s) into which it has been integrated. Furthermore, a
single vector or plasmid or two or more vectors or plasmids which
together contain the total DNA to be introduced into the genome of
the host cell, or a transposon may be used. The vector may be
linear or closed circular plasmids and will preferably contain an
element(s) that permits stable integration of the vector into the
host cell's genome or autonomous replication of the vector in the
cell independent of the genome.
[0708] The recombinant expression vector may be one capable of
replicating in yeast. Examples of sequences which enable the vector
to replicate in yeast are the yeast plasmid 2 .mu.m replication
genes REP 1-3 and origin of replication.
[0709] The vector may contain one or more selectable markers, which
permit easy selection of trans-formed cells. A selectable marker is
a gene the product, which provides for biocide or viral
re-sistance, resistance to heavy metals, prototrophy to auxotrophs,
and the like. Examples of bacterial selectable markers are the dal
genes from Bacillus subtilis or Bacillus licheniformis, or markers
which confer antibiotic resistance such as ampicillin, kanamycin,
chloramphenicol or tetracycline resistance. Selectable markers for
use in a filamentous fungal host cell include amdS (acetamidase),
argB (orni-thine carbamoyltransferase), pyrG
(orotidine-5'-phosphate decarboxylase) and trpC (anthranilate
syn-thase. Suitable markers for yeast host cells are ADE2, HIS3,
LEU2, LYS2, MET3, TRP1, and URA3. A well suited selectable marker
for yeast is the Schizosaccharomyces pompe TPI gene (Russell; Gene
1985 40 125-130).
[0710] In the vector, the polynucleotide sequence is operably
connected to a suitable promoter sequence. The promoter may be any
nucleic acid sequence which shows transcriptional activity in the
host cell of choice including mutant, truncated, and hybrid
promoters, and may be obtained from genes encoding extra-cellular
or intra-cellular polypeptides either homologous or heterologous to
the host cell.
[0711] Examples of suitable promoters for directing the
transcription in a bacterial host cell, are the promoters obtained
from the E. coli lac operon, Streptomyces coelicolor agarase gene
(dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus
licheniformis alpha-amylase gene (amyL), Bacillus
stearothermophilus maltogenic amylase gene (amyM), Bacillus
amyloliquefaciens alpha-amylase gene (amyQ), and Bacillus
licheniformis penicillinase gene (penP). Examples of suitable
promoters for di-recting the transcription in a filamentous fungal
host cell are promoters obtained from the genes for Aspergillus
oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase,
Aspergillus niger neutral alpha-amylase, and Aspergillus niger acid
stable alpha-amylase. In a yeast host, useful promoters are the
Saccharomyces cerevisiae Mal, TPI, ADH, TDH3 or PGK promoters.
[0712] The polynucleotide sequence encoding the insulin peptide
backbone for use according to the invention also will typically be
operably connected to a suitable terminator. In yeast, a suitable
terminator is the TPI terminator (Alber et al; J. Mol. Appl. Genet.
1982 1 419-434).
[0713] The procedures used to combine the polynucleotide sequence
encoding the insulin peptide backbone for use according to the
invention, the promoter and the terminator, respectively, and to
insert them into a suitable vector containing the information
necessary for replication in the selected host, are well known to
persons skilled in the art. It will be understood that the vector
may be constructed either by first preparing a DNA construct
containing the entire DNA sequence encoding the insulin backbones
for use according to the invention, and subsequently inserting this
fragment into a suitable expression vector, or by sequentially
inserting DNA fragments containing genetic information for the
individual elements (such as the signal and pro-peptide (N-terminal
extension of the B-chain), C-peptide, A- and B-chains), followed by
ligation.
[0714] The vector comprising the polynucleotide sequence encoding
the insulin backbone for use according to the invention is
introduced into a host cell, so that the vector is maintained as a
chromosomal integrant, or as a self-replicating extra-chromosomal
vector. The term "host cell" encompasses any progeny of a parent
cell that is not identical to the parent cell due to mutations that
occur during replication. The host cell may be a unicellular
microorganism, e.g. a prokaryote, or a non-unicellular
microorganism, e.g. a eukaryote. Useful unicellular cells are
bacterial cells such as gram positive bacteria including, but not
limited to, a Bacillus cell, a Streptomyces cell, or a gram
negative bacteria such as E. coli and Pseudomonas sp. Eukaryote
cells may be mammalian, insect, plant, or fungal cells.
[0715] The host cell may in particular be a yeast cell. The yeast
organism may be any suitable yeast organism which, on cultivation,
secretes the insulin peptide backbone or the precursor hereof into
the culture medium. Examples of suitable yeast organisms are
include strains selected from Saccharomyces cerevisiae,
Saccharomyces kluyveri, Schizosaccharomyces pombe, Sacchoromyces
uvarum, Kluyveromyces lactis, Hansenula polymorpha, Pichia
pastoris, Pichia methanolica, Pichia kluyveri, Yarrowia lipolytica,
Candida sp., Candida utilis, Candida cacaoi, Geotrichum sp., and
Geotrichum fermentans.
[0716] The transformation of the yeast cells may for instance be
effected by protoplast formation followed by transformation by
known methods. The medium used to cultivate the cells may be any
conventional medium suitable for growing yeast organisms.
[0717] Backbone Purification
[0718] The secreted insulin peptide backbone or precursor hereof
may be recovered from the medium by conventional procedures
including separating the yeast cells from the medium by
centrifugation, by filtration or by catching the insulin peptide
backbone or precursor hereof on an ion exchange matrix or on a
reverse phase absorption matrix, precipitating the proteinaceous
components of the supernatant, or by filtration by means of a salt,
e.g. ammonium sulphate, followed by purification by a variety of
chromatographic procedures, e.g. ion exchange chromatography,
affinity chromatography, etc.
[0719] The purification and digestion of the insulin peptide
backbones of this invention is carried out as follows:
[0720] The single-chain insulin peptide backbone precursor, which
may contain an N-terminal extension of the B-chain and a modified
C-peptide between the B-chain and the A-chain is purified and
concentrated from the yeast culture supernatant by cation exchange
(Kjeldsen et al; Prot. Expr. Pur. 1998 14 309-316). The
single-chain insulin peptide backbone precursor is matured into
two-chain insulin peptide backbone by digestion with
lysine-specific immobilised ALP (Kristensen et al; J. Biol. Chem.
1997 20 12978-12983) or by use of trypsin to cleave off the
N-terminal extension of the B-chain, if present, and the
C-peptide.
[0721] ALP Digestion
[0722] The eluate from the cation exchange chromatography step
containing the insulin peptide backbone precursor is diluted with
water to an ethanol concentration of 15-20%. Sodium glutamate is
added to a concentration of 15 mM and pH is adjusted to 9.7 by
NaOH. Immobilised ALP (4 gram/L) is added in a proportion of 1:100
(volume:volume) and digestion is allowed to proceed with mild
stirring in room temperature overnight.
[0723] The digestion reaction is analysed by analytical LC on a
Waters Acquity Ultra-Performance Liquid Chromatography system using
a C18 column and the molecular weight is confirmed by
matrix-assisted laser desorption ionisation time-of-flight
(MALDI-TOF) mass spectrometry (MS) (Bruker Daltonics Autoflex II
TOF/TOF).
[0724] The immobilised ALP is removed by filtration using a 0.2
.mu.m filter. The two-chain insulin peptide backbone is purified by
reversed phase HPLC (Waters 600 system) on a C18 column using an
acetonitrile gradient. The desired insulin peptide backbone, i.e.
B28K desB29-B30 human insulin, is recovered by lyophilisation.
[0725] Trypsin Digestion
[0726] The eluate from the cation exchange chromatography step
containing the insulin peptide backbone precursor is diluted with
water to an ethanol concentration of 15-20%. Glycine is added to a
concentration of 50 mM and pH is adjusted to 9.0-9.5 by NaOH.
Trypsin is added in a proportion of 1:300 (w:w) and digestion is
allowed to proceed at 4 degrees. The digestion is analytically
monitored every 20 minutes until digestion is completed. The
digestion is terminated with addition of 1 M citric acid in a
proportion of 3:100 (volume:volume).
[0727] The digestion reaction is analysed by analytical LC on a
Waters Acquity Ultra-Performance Liquid Chromatography system using
a C18 column and the molecular weight is confirmed by MALDI-TOF MS
(Bruker Daltonics Autoflex II TOF/TOF).
[0728] The two-chain insulin peptide backbone is purified by
reversed phase HPLC (Waters 600 system) on a C18 column using an
acetonitrile gradient. The desired insulin peptide backbone, is
recovered by lyophilisation.
[0729] Purity is determined by analytical LC on a Waters Acquity
Ultra-Performance Liquid Chromatography system using a C18 column,
and the molecular weight is confirmed by MALDI-TOF MS.
[0730] (Method 2) Preparative Example--Acylation and Purification
Procedures
[0731] Purification Procedures
[0732] Purification Method 1
[0733] Column: Waters xBridge PrepC18, 30.times.250 mm
[0734] Flow: 20 ml/min
[0735] Buffer A: 0.1% TFA in water
[0736] Buffer B: 0.1% TFA in acetonitrile
[0737] Gradient: 20-45% B or 30-40% B or 20-60% B or 20-40% B or
25-50% B or 20-55% B or 30-45% B over 40 min
[0738] Purification Method 2
[0739] Column: Phenomenex, 5 u, C18, 110 .ANG., 30.times.250 mm
[0740] Flow: 20 ml/min
[0741] Buffer A: 0.1% TFA in water
[0742] Buffer B: 0.1% TFA in acetonitrile
[0743] Gradient: 10-60% B over 80 min or 0-60% B over 95 min or
25-55% B over 60 min
[0744] General Procedure for the Preparation of Acylation
Reagents
[0745] Simple diacids: NHS-activated monoacids were either prepared
by activating a diacid-mono-t-butyl ester with TSTU and DIPEA in
NMP and used directly or the diacid-mono-NHS ester was prepared and
isolated. When a tert-butyl protected acylation reagent was used
the resulting insulin derivative was deprotected by treatment with
TFA.
[0746] Diacids with OEG linkers: The reagent was made by stepwise
solid phase synthesis on chlorotrityl resin using Fmoc-chemistry,
and cleaved from the resin with HFIP, TFA, a HFIP/DCM mixture, or a
TFA/DCM mixture.
[0747] Some acylation agents were prepared, isolated and used as
NHS-esters without any protecting groups.
[0748] General Acylation Procedure 1
[0749] 100 mg of the appropriate insulin analogue was dissolved in
1.2 ml 0.1 M Na2CO3 and 0.6 ml NMP and pH adjusted to 10.7.+-.0.3.
0.03 mmol acylation reagent in 0.3 ml NMP was added at pH
maintained at 10.7.+-.0.3 by addition of 4 M NaOH if necessary.
After 30 to 60 minutes the reaction was complete, and the product
was precipitated by addition of 40 ml isopropanol, centrifuged,
washed with ether and dried. Alternatively the product could be
isolated by dilution with 30 ml water and adjustment of pH to
4.5-5.0 which lead to isoelectric precipitation. Alternatively the
reaction mixture was acidified with acetic acid or TFA and purified
immediately.
[0750] Subsequently, the product was purified by reverse phase
high-performance liquid chromatography (RP-HPLC) in
TFA-acetonitrile as described above and the pure fractions
lyophilised. The identity of the product was confirmed by
matrix-assisted laser desorption ionisation mass spectrometry
(MALDI-MS or ultra-performance liquid chromatography
(UPLC)/electrospray-MS.
[0751] In some cases the synthesis was scaled to produce larger or
smaller amounts. In that case, all amounts of starting materials,
reagents and solvents were scaled by the same factor
[0752] Modification 1
[0753] When the acylation reagent contained a tert-butyl protecting
group, the product after the isopropanol or isoelectric
precipitation was dissolved in 5 ml TFA or TFA:water (95:5) for 5
minutes, precipitated with 35 ml ether, washed twice with ether and
dried.
[0754] Modification 2
[0755] When the acylation reagent contained a tert-butyl protecting
group, the crude iso-precipitated product was dissolved in 5 ml of
TFA or TFA:water 95:5 or TFA:triisopropylsilane 99:1. The solution
was stirred for 20-30 min, then diluted with water, in some cases
additionally with either DMSO or NMF, and immediately purified by
preparative HPLC.
[0756] Modification 3
[0757] In some cases where the acylation reagent appears to be
poorly soluble in the reaction mixture, more NMP is added (up to 1
ml) and the reaction mixture warmed briefly to 37.degree. C.
[0758] General Acylation Procedure 2
[0759] 100 mg of the appropriate insulin was dissolved in 2 ml DMSO
and 40 ul Bartons Base (2-t-butyl-1,1,3,3-tetramethylguanidine) was
added. 20 umol of the acylating agent was added, and after one
minute the reaction was complete. The reaction mixture was
acidified and diluted into water and purified immediately.
[0760] (Method 3) Chemical Synthesis of Insulin Derivatives
[0761] Insulin A-chain was synthesized by standard Fmoc peptide
synthesis, e.g. on a Prelude or Liberty synthesizer. Deprotection
was performed with 10% or 20% piperidine in DMF. All protecting
groups were standard, except that Cys 6, 11 and 20 had Trt
protecting groups and Cys 7 had Acm. If the C-terminal residue was
Asn, the peptide was synthesized on a PAL or Rink resin with
Fmoc-Asp-OBut as the first amino acid.
[0762] After the synthesis, the resin was washed with DCM, treated
with 1% iodine in DCM/HFIP (4:1) for 1 minute, and incubated in
DCM/HFIP (4:1) for 15 minutes which lead to formation of the A6-A11
disulfide. After washing with DCM and drying, the peptide was
cleaved with TFA/water/DPDS (93:5:2) which provided the insulin
A-chain with pyridylsulfide on Cys20. Ether precipitation, washing,
and drying lead to the crude A-chain which was used in subsequent
chain combination.
[0763] Insulin B-chain was synthesized by standard Fmoc peptide
synthesis, e.g. on a Liberty or Prelude synthesizer. The resin was
e.g. a preloaded Wang resin. All protecting groups were standard,
except that Cys7 had Acm and Cys19 had Trt.
[0764] Cleavage was performed with TFA/water/TIPS (93:4:3). Ether
precipitation, washing, and drying lead to crude B-chain which was
used in subsequent chain combination.
[0765] When an insulin derivative with a side chain modified lysine
was desired, the modification was introduced during the peptide
synthesis. The lysine to be modified was introduced as
Fmoc-Lysine(Mtt)-OH and the N-terminal of the peptide protected
with Boc (either by coupling of a Boc-protected amino acid in the
last coupling step or by reaction of the resin bound peptide with 5
equivalents of Boc-anhydride in DMF). The Mtt group was removed by
treating the resin with DMF/HFIP (1:4) for 30 minutes and the side
chain was synthesized by standard Fmoc chemistry as described for
the peptide backbone.
[0766] Crude A-chain and B-chain (0.14 mmol of each) was dissolved
in 40 ml DMSO. 2 ml 2M Tris buffer pH 8.5 was added and the chain
combination was complete in about 10 minutes. The solution was
diluted with 40 ml DMSO and 200 ml 40% acetonitrile, 1% TFA.
N-chlorosuccinimide was added to a final concentration of 5 mM.
This formed the third disulfide bridge in less than 10 minutes. The
solution was diluted and neutralized with 600 ml 0.2 M Tris pH 7.8
and purified by RP-HPLC on a C18 column running a gradient of
acetonitrile in 20 mM phosphate pH 7.2.
[0767] Fractions containing the desired compound were combined and
repurified on the same column with an acetonitrile gradient in 0.1%
TFA. The pure fractions were combined and lyophilized. The yield
was usually 20-50 mg of the desired insulin derivative which was
subsequently characterized by UPLC and LC-MS.
Examples of Insulin Derivatives of the Invention
Example 1:
N{Epsilon-B28}-15-carboxypentadecanoyl-[TyrB5,LysB28],des-(B29--
B30)-Insulin]
##STR00025##
[0769] Synthesised by Synthesis Method 3.
[0770] Calc. Mass.=5903.8; Found LC-MS m/4=1476.66
Example 2:
N{Epsilon-B28}-[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)-
butanoyl]-[TyrB5,LysB28],des-(B29-B30)-Insulin
##STR00026##
[0771] Synthesised by Synthesis Method 3
[0772] Calc. Mass.=6032.9; Found LC-MS m/4=1509.07
Example 3:
N{Epsilon-B28}-17-carboxyheptadecanoyl-[TyrB5,LysB28],des-(B29--
B30)-Insulin
##STR00027##
[0773] Synthesised by Synthesis Method 3
[0774] Calc. Mass.=5931.8; Found LC-MS m/4=1484
Example 4:
N{Epsilon-B28}-[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)-
butanoyl]-[TyrB5,LysB28],des-(B29-B30)-Insulin
##STR00028##
[0775] Synthesised by Synthesis Method 3
[0776] Calc. Mass.=6060.9; Found LC-MS m/4=1516.73
Example 5:
N{Epsilon-B28}-[2-[2-[2-(17-carboxyheptadecanoylamino)ethoxy]et-
hoxy]acetyl]-[TyrB5, LysB28],des-(B29-B30)-Insulin
##STR00029##
[0777] Synthesised by Synthesis Method 3
[0778] Calc. Mass.=6077.0; Found LC-MS m/5=1216.34
Example 6:
N{Epsilon-B28}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxy-
heptadecanoylamino)butanoyl]amino]butanoyl]-[TyrB5,LysB28],des-(B29-B30)-I-
nsulin
##STR00030##
[0779] Synthesised by Synthesis Method 3
[0780] Calc. Mass.=6190.0; Found LC-MS m/4=1548.37
Example 7:
N{Epsilon-B28}-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadeca-
noylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]-[TyrB5,LysB28],des-(B29-B30-
)-Insulin
##STR00031##
[0781] Synthesised by Synthesis Method 3
[0782] Calc. Mass.=6206.1; Found LC-MS m/4=1552.37
Example 8:
N{Epsilon-B28}-[(4S)-4-carboxy-4-[[(4R)-4-[(3R,10S,13R,17R)-3-h-
ydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H--
cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]butanoyl]-[TyrB5,LysB28],de-
s-(B29-B30)-Insulin
##STR00032##
[0783] Synthesised by Synthesis Method 3
[0784] Calc. Mass.=6123.1; Found LC-MS m/4=1531.7
Example 9:
N{Epsilon-B28}-[(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimeth-
yl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenan-
thren-17-yl]pentanoyl]-[TyrB5,LysB28],des-(B29-B30)-Insulin
##STR00033##
[0785] Synthesised by Synthesis Method 3
[0786] Calc. Mass.=5993.9; Found LC-MS m/4=1499.16
Example 10:
N{Epsilon-B28}-15-carboxypentadecanoyl-[TyrB5,GlyB26,LysB28],des-(B29-B30-
)-Insulin
##STR00034##
[0787] Synthesised by Synthesis Method 3
[0788] Calc. Mass.=5797.7; Found LC-MS m/4=1449.93
Example 11:
N{Epsilon-B28}-17-carboxyheptadecanoyl-[TyrB5,GlyB26,LysB28],des-(B29-B30-
)-Insulin
##STR00035##
[0789] Synthesised by Synthesis Method 3
[0790] Calc. mass=5825.7; Found MALDI-MS=5825.7
Example 12:
N{Epsilon-B28}-17-carboxyheptadecanoyl-[GluA14,TyrB5,GlyB26,LysB28],des-(-
B29-B30)-Insulin
##STR00036##
[0791] Synthesised by Synthesis Method 3
[0792] Calc. mass.=5791.6; Found LC-MS m/3=1931.44
Example 13:
N{Epsilon-B28}-[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]--
[GluA14,TyrB5,GlyB26,LysB28],des-(B29-B30)-Insulin
##STR00037##
[0793] Synthesised by Synthesis Method 3
[0794] Calc. mass.=5920.8; Found LC-MS m/5=1185.04
Example 14:
N{Epsilon-B28}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]butanoyl]-[GluA14,TyrB5,GlyB26,LysB28],des-(B29-B3-
0)-Insulin
##STR00038##
[0795] Synthesised by Synthesis Method 3
[0796] Calc. mass.=6049.9; Found LC-MS m/4=1513.32
Example 15:
N{Epsilon-B28}-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)-
butanoyl]amino]ethoxy]ethoxy]acetyl]-[GluA14,TyrB5,GlyB26,LysB28],des-(B29-
-B30)-Insulin
##STR00039##
[0797] Synthesised by Synthesis Method 3
[0798] Calc. mass.=6065.9; Found LC-MS m/4=1517.34
Example 16:
N{Epsilon-B28}-[2-[2-[2-[[(4S)-4-carboxy-4-(19-carboxynonadecanoylamino)b-
utanoyl]amino]ethoxy]ethoxy]acetyl]-[GluA14,TyrB5,GlyB26,LysB28],des-(B29--
B30)-Insulin
##STR00040##
[0799] Synthesised by Synthesis Method 3
[0800] Calc. mass.=6094.0; Found LC-MS m/4=1524.36
Example 17:
N{Epsilon-B28}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(19-carboxynonadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
GluA14,TyrB5,GlyB26,LysB28],des-(B29-B30)-Insulin
##STR00041##
[0801] Synthesised by Synthesis Method 3
[0802] Calc. mass.=6239.1; Found LC-MS m/4=1560.4
Example 18:
N{Epsilon-B28}-[(4S)-4-carboxy-4-(19-carboxynonadecanoylamino)butanoyl]-[-
GluA14,TyrB5,GlyB26,LysB28],des-(B29-B30)-Insulin
##STR00042##
[0803] Synthesised by Synthesis Method 3
[0804] Calc. mass.=5948.8; Found LC-MS m/5=1190.69
Example 19:
N{Epsilon-B28}-19-carboxynonadecanoyl-[GluA14,TyrB5,GlyB26,LysB28],des-(B-
29-B30)-Insulin
##STR00043##
[0805] Synthesised by Synthesis Method 3
[0806] Calc. mass.=5819.7; Found LC-MS m/4=1455.67
Example 20:
N{Epsilon-B28}-15-(1H-tetrazol-5-yl)pentadecanoyl-[GluA14,TyrB5,GlyB26,Ly-
sB28],des-(B29-B30)-Insulin
##STR00044##
[0807] Synthesised by Synthesis Method 2
[0808] Calc. mass.=5787.6; Found LC-MS m/4=1448.14
Example 21:
N{Epsilon-B28}-17-(1H-tetrazol-5-yl)heptadecanoyl-[GluA14,TyrB5,GlyB26,Ly-
sB28],des-(B29-B30)-Insulin
##STR00045##
[0809] Synthesised by Synthesis Method 2
[0810] Calc. mass.=5815.7; Found LC-MS m/4=1455.19
Example 22:
N{Epsilon-B28}-16-(1H-tetrazol-5-yl)hexadecanoyl-[GluA14,TyrB5,GlyB26,Lys-
B28],des-(B29-B30)-Insulin
##STR00046##
[0811] Synthesised by Synthesis Method 2
[0812] Calc. mass.=5801.7; Found LC-MS m/4=1451.6
Example 23:
N{Epsilon-B28}-4-[16-(1H-tetrazol-5-yl)hexadecanoylsulfamoyl]butanoyl-[Gl-
uA14,TyrB5,GlyB26,LysB28],des-(B29-B30)-Insulin
##STR00047##
[0813] Synthesised by Synthesis Method 2
[0814] Calc. mass.=5950.8; Found LC-MS m/4=1488.81
Example 24:
N{Epsilon-B28}-4-[4-[15-(1H-tetrazol-5-yl)pentadecanoylsulfamoyl]butanoyl-
sulfamoyl]butanoyl-[GluA14,TyrB5,GlyB26,LysB28],des-(B29-B30)-Insulin
##STR00048##
[0815] Synthesised by Synthesis Method 2
[0816] Calc. Mass.=6086.0; Found LC-MS m/4=1522.65
Example 25:
N{Epsilon-B28}-4-[17-(1H-tetrazol-5-yl)heptadecanoylsulfamoyl]butanoyl-[G-
luA14,TyrB5,GlyB26,LysB28],des-(B29-B30)-Insulin
##STR00049##
[0817] Synthesised by Synthesis Method 2
[0818] Calc. mass.=5964.8; Found LC-MS m/4=1492.41
Example 26:
N{Epsilon-B28}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[15-(1H-tetrazol-5-y-
l)pentadecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]etho-
xy]acetyl]-[GluA14,TyrB5,GlyB26,LysB28],des-(B29-B30)-Insulin
##STR00050##
[0819] Synthesised by Synthesis Method 2
[0820] Calc. mass.=6207.0; Found LC-MS m/4=1552.95
Example 27:
N{Epsilon-B28}-4-[4-[17-(1H-tetrazol-5-yl)heptadecanoylsulfamoyl]butanoyl-
sulfamoyl]butanoyl-[GluA14,TyrB5,GlyB26,LysB28],des-(B29-B30)-Insulin
##STR00051##
[0821] Synthesised by Synthesis Method 2
[0822] Calc. Mass.=6114.0; Found LC-MS m/4=1529.62
Example 28:
N{Epsilon-B28}-4-(17-carboxyheptadecanoylsulfamoyl)butanoyl-[GluA14,TyrB5-
,GlyB26,LysB28],des-(B29-B30)-Insulin
##STR00052##
[0823] Synthesised by Synthesis Method 2
[0824] Calc. mass.=5940.8; Found LC-MS m/4=1486.34
Example 29:
N{Epsilon-B28}-[2-[2-[2-[[(4S)-4-carboxy-4-[15-(1H-tetrazol-5-yl)pentadec-
anoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]-[GluA14,TyrB5,GlyB26,LysB2-
8],des-(B29-B30)-Insulin
##STR00053##
[0825] Synthesised by Synthesis Method 2
[0826] Calc. mass.=6061.9; Found LC-MS m/4=1516.63
Example 30:
N{Epsilon-B28}-[(4R)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]--
[GluA14,TyrB5,GlyB26,LysB28],des-(B29-B30)-Insulin
##STR00054##
[0827] Synthesised by Synthesis Method 2
[0828] Calc. mass.=5920.8; Found LC-MS m/4=1481.13
Example 31:
N{Epsilon-B28}-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)-
butanoyl]amino]ethoxy]ethoxy]acetyl]-[TyrB5,GlyB26,LysB28],des-(B29-B30)-I-
nsulin
##STR00055##
[0829] Synthesised by Synthesis Method 2
[0830] Calc. mass.=6100.0; Found LC-MS m/4=1525.8
Example 32:
N{Epsilon-B28}-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)-
butanoyl]amino]ethoxy]ethoxy]acetyl]-[GluA14,TyrB5,AlaB26,LysB28],des-(B29-
-B30)-Insulin
##STR00056##
[0831] Synthesised by Synthesis Method 2
[0832] Calc. mass.=6079.9; Found LC-MS m/4=1520.77
Example 33:
N{Epsilon-B28}-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)-
butanoyl]amino]ethoxy]ethoxy]acetyl]-[TyrB5,AlaB26,LysB28],des-(B29-B30)-I-
nsulin
##STR00057##
[0833] Synthesised by Synthesis Method 2
[0834] Calc. mass.=6114.0; Found LC-MS m/4=1529.2
Example 34:
N{Epsilon-B28}-[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]--
[GluA14,TyrB5,AlaB26,LysB28],des-(B29-B30)-Insulin
##STR00058##
[0835] Synthesised by Synthesis Method 2
[0836] Calc. mass.=5934.8; Found LC-MS m/4=1484.5
Example 35:
N{Epsilon-B28}-17-carboxyheptadecanoyl-[PheB5,LysB28],des-(B29-B30)-Insul-
in
##STR00059##
[0837] Synthesized by Synthesis method 3
[0838] Calc. Mass=5915.8; Found MALDI-MS=5916
Example 36:
N{Epsilon-B28}-17-carboxyheptadecanoyl-[GluA14,PheB5,GlyB26,LysB28],des-(-
B29-B30)-Insulin
##STR00060##
[0839] Synthesized by Synthesis method 3
[0840] Calc. Mass=5775.6; Found LC-MS m/4=1444.8
Example 37:
N{Epsilon-B26}-17-carboxyheptadecanoyl-[TyrB5,LysB26],des-(B27-B30)-Insul-
in
##STR00061##
[0841] Synthesized by Synthesis Method 3
[0842] Calc. mass=5667.6; Found LC-MS m/3=1889.9
Example 38:
N{Epsilon-B26}-[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]--
[TyrB5,LysB26],des-(B27-B30)-Insulin
##STR00062##
[0843] Synthesized by Synthesis Method 3
[0844] Calc. mass=5796.7; Found LC-MS m/3=1933.1
Example 39:
N{Epsilon-B26}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadeca-
noylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]--
[TyrB5,LysB26],des-(B27-B30)-Insulin
##STR00063##
[0845] Synthesized by Synthesis Method 3
[0846] Calc. mass=6087.0; Found LC-MS m/4=1522.5
Example 40:
N{Epsilon-B29}-[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoy]-[-
TyrB5],des-ThrB30-Insulin
##STR00064##
[0847] Synthesized by Synthesis Method 3
[0848] Calc. mass=6130.0; Found LC-MS m/3=2044.3
Example 41:
N{Epsilon-B29}-tetradecanoyl-[TyrB5],des-ThrB30-Insulin
##STR00065##
[0850] Synthsized by Synthesis Method 3
[0851] Calc. mass=5942.9; Found LC-MS m/3=1982
Example 42:
N{Epsilon-B29}-[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]--
[TyrB5,GlyB26],des-ThrB30-Insulin
##STR00066##
[0852] Synthesized by Synthesis Method 3
[0853] Calc. mass=6023.9; Found MALDI-MS=6026
Example 43:
N{Epsilon-B29}-[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]--
[TyrB5,AlaB26],des-ThrB30-Insulin
##STR00067##
[0854] Synthesized by Synthesis Method 3
[0855] Calc. mass=6037.9; Found LC-MS m/4=1510.5
Example 44:
N{Epsilon-B29}-[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]--
[TyrB5,GlyB26],des-ThrB30-Insulin
##STR00068##
[0856] Synthesized by Synthesis Method 3
[0857] Calc. mass=6051.9; Found MALDI-MS=6052
Example 45:
N{Epsilon-B29}-[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]--
[TyrB5,AlaB26],des-ThrB30-Insulin
##STR00069##
[0858] Synthesized by Synthesis Method 3
[0859] Calc. mass=6066.0; Found MALDI-MS=6065
Example 46:
N{Epsilon-B26}-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)-
butanoyl]amino]ethoxy]ethoxy]acetyl]-[TyrB5,LysB26],des-(B27-B30)-Insulin
##STR00070##
[0861] Synthesized by Synthesis Method 3
[0862] Calc. Mass=5941.8; Found LC-MS m/4=1486
Comparators
Comparator 1: [TyrB5,LysB28],des-(B29-B30)-Insulin
##STR00071##
[0863] Synthesised by Synthesis Method 1
[0864] Calc. Mass.=5635.4; Found MALDI-MS 5635
Comparator 2: [TyrB5,GlyB26],des-ThrB30-Insulin
##STR00072##
[0865] Synthesised by Synthesis Method 1
[0866] Calc. Mass.=5626.4; Found MALDI-MS=5626
Comparator 3: [TyrB5,AlaB26],des-ThrB30-Insulin
##STR00073##
[0867] Synthesised by Synthesis Method 1
[0868] Calc. Mass.=5640.4; Found MALDI-MS=5641
Comparator 4:
[GluA14,TyrB5,GlyB26,LysB28],des-(B29-B30)-Insulin
##STR00074##
[0869] Synthesised by Synthesis Method 1
[0870] Calc. mass.=5495.2; Found LC-MS m/4=1374.83
Comparator 5:
N{Epsilon-B29}-[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]--
Insulin
##STR00075##
[0871] Comparator 6:
N{Epsilon-B28}-17-carboxyheptadecanoyl-[LeuB5,LysB28],des-(B29-B30)-Insul-
in
##STR00076##
[0872] Synthesized by Synthesis Method 3
[0873] Calc. Mass=5881.8; Found MALDI-MS=5882
Comparator 7:
N{Epsilon-B28}-17-carboxyheptadecanoyl-[SerB5,LysB28],des-(B29-B30)-Insul-
in
##STR00077##
[0874] Synthesized by Synthesis Method 3
[0875] Calc. Mass=5875.7; Found MALDI-MS=5876
Comparator 8:
N{Epsilon-B28}-17-carboxyheptadecanoyl-[AlaB5,LysB28],des-(B29-B30)-Insul-
in
##STR00078##
[0876] Synthesized by Synthesis Method 3
[0877] Calc. Mass=5839.7; Found MALDI-MS=5840
Comparator 9:
N{Epsilon-B28}-17-carboxyheptadecanoyl-[GlyB26,LysB28],des-(B29-B30)-Insu-
lin
##STR00079##
[0878] Synthesized by Synthesis Method 3
[0879] Calc. Mass=5799.7; Found MALDI-MS=5800
Sequence CWU 1
1
15121PRTHomo sapiens 1Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys
Ser Leu Tyr Gln Leu1 5 10 15Glu Asn Tyr Cys Asn 20230PRTHomo
sapiens 2Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala
Leu Tyr1 5 10 15Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys
Thr 20 25 30321PRTArtificial SequenceSynthetic 3Gly Ile Val Glu Gln
Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu1 5 10 15Glu Asn Tyr Cys
Asn 20428PRTArtificial SequenceSynthetic 4Phe Val Asn Gln Tyr Leu
Cys Gly Ser His Leu Val Glu Ala Leu Tyr1 5 10 15Leu Val Cys Gly Glu
Arg Gly Phe Phe Ala Thr Lys 20 25528PRTArtificial SequenceSynthetic
5Phe Val Asn Gln Tyr Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr1 5
10 15Leu Val Cys Gly Glu Arg Gly Phe Phe Gly Thr Lys 20
25628PRTArtificial SequenceSynthetic 6Phe Val Asn Gln Phe Leu Cys
Gly Ser His Leu Val Glu Ala Leu Tyr1 5 10 15Leu Val Cys Gly Glu Arg
Gly Phe Phe Ala Thr Lys 20 25728PRTArtificial SequenceSynthetic
7Phe Val Asn Gln Tyr Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr1 5
10 15Leu Val Cys Gly Glu Arg Gly Phe Phe Ala Thr Lys 20
25828PRTArtificial Sequencesynthetic 8Phe Val Asn Gln Tyr Leu Cys
Gly Ser His Leu Val Glu Ala Leu Tyr1 5 10 15Leu Val Cys Gly Glu Arg
Gly Phe Phe Gly Thr Lys 20 25928PRTArtificial SequenceSynthetic
9Phe Val Asn Gln Tyr Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr1 5
10 15Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Lys 20
251028PRTArtificial SequenceSynthetic 10Phe Val Asn Gln Phe Leu Cys
Gly Ser His Leu Val Glu Ala Leu Tyr1 5 10 15Leu Val Cys Gly Glu Arg
Gly Phe Phe Gly Thr Lys 20 251128PRTArtificial SequenceSynthetic
11Phe Val Asn Gln Phe Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr1
5 10 15Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Lys 20
251226PRTArtificial SequenceSynthethic sequence 12Phe Val Asn Gln
Tyr Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr1 5 10 15Leu Val Cys
Gly Glu Arg Gly Phe Phe Lys 20 251329PRTArtificial
SequenceSynthethic 13Phe Val Asn Gln Tyr Leu Cys Gly Ser His Leu
Val Glu Ala Leu Tyr1 5 10 15Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr
Thr Pro Lys 20 251429PRTArtificial SequenceSynthethic 14Phe Val Asn
Gln Tyr Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr1 5 10 15Leu Val
Cys Gly Glu Arg Gly Phe Phe Gly Thr Pro Lys 20 251529PRTArtificial
SequenceSynthethic 15Phe Val Asn Gln Tyr Leu Cys Gly Ser His Leu
Val Glu Ala Leu Tyr1 5 10 15Leu Val Cys Gly Glu Arg Gly Phe Phe Ala
Thr Pro Lys 20 25
* * * * *