U.S. patent application number 12/375678 was filed with the patent office on 2009-12-10 for pegylated, extended insulins.
This patent application is currently assigned to Novo Nordisk A/S. Invention is credited to Palle Jakobsen, Thomas Borglum Kjeldsen, Peter Madsen, Tina Moller Tagmose.
Application Number | 20090306337 12/375678 |
Document ID | / |
Family ID | 38904644 |
Filed Date | 2009-12-10 |
United States Patent
Application |
20090306337 |
Kind Code |
A1 |
Madsen; Peter ; et
al. |
December 10, 2009 |
Pegylated, Extended Insulins
Abstract
PEGylated, extended insulins are insulins which, compared with
human insulin, has one or more extensions extended from the A1, B1,
A21 and/or B30 position(s), said extension(s) consist(s) of amino
acid residue(s) and wherein a PEG moiety, via a linker, is attached
to one or more of the amino acid residues in the extension(s). PEG
is polyethyleneglycol. Such PEGylated, extended insulins have
higher bioavailability and a longer time-action profile than
regular insulin and are in particular suited for pulmonary
administration and can, conveniently, be used to treat
diabetes.
Inventors: |
Madsen; Peter; (Ulvebjerg,
DK) ; Kjeldsen; Thomas Borglum; (Virum, DK) ;
Tagmose; Tina Moller; (Ballerup, DK) ; Jakobsen;
Palle; (Vaerlose, DK) |
Correspondence
Address: |
NOVO NORDISK, INC.;INTELLECTUAL PROPERTY DEPARTMENT
100 COLLEGE ROAD WEST
PRINCETON
NJ
08540
US
|
Assignee: |
Novo Nordisk A/S
Bagsvaerd
DK
|
Family ID: |
38904644 |
Appl. No.: |
12/375678 |
Filed: |
July 16, 2007 |
PCT Filed: |
July 16, 2007 |
PCT NO: |
PCT/EP07/57321 |
371 Date: |
February 27, 2009 |
Current U.S.
Class: |
530/303 |
Current CPC
Class: |
A61P 3/08 20180101; A61K
47/60 20170801; A61P 5/48 20180101; A61P 3/10 20180101; A61K 38/28
20130101 |
Class at
Publication: |
530/303 |
International
Class: |
C07K 14/62 20060101
C07K014/62 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 31, 2006 |
EP |
06118171.5 |
Sep 15, 2006 |
EP |
06120735.3 |
Claims
1. A PEGylated insulin analogue which, compared with human insulin,
has one or more PEG-containing extensions extended from the A1, B1,
A21 and/or B30 position(s), said extension(s) consist(s) of amino
acid residue(s) and wherein the PEG moiety, via a linker, is
attached to an amino acid residue in the extension and with the
proviso that, preferably, the parent insulin analogue contains only
one lysine residue.
2. The PEGylated insulin analogue according to claim 1, wherein
only one of the extensions carries a PEG moiety and, preferably,
there is only one extension.
3. The PEGylated insulin analogue according to claim 1, wherein the
extension carrying a PEG moiety is situated in a position
C-terminally to the A21 position.
4. The PEGylated insulin analogue according to claim 1, wherein the
extension carrying a PEG moiety is situated in a position
C-terminally to the B30 position.
5. The PEGylated insulin analogue according to claim 1, wherein the
parent insulin analogue deviates from human insulin in one or more
of the following extensions: G in position A-3, G in position A-2,
K or R in position A-1, G or K in position A22, G or K in position
A23, G or K in position A24, K in position A25, and K in position
B31 and, compared with human insulin, there is, optionally, up to
12, preferably up to 8, more preferred up to 4, more mutations
among deletion, substitution and addition of an amino acid residue
and, preferably, there are no further mutations in said insulin
analogue and with the proviso that, preferably, the parent insulin
analogue contains only one lysine residue.
6. The PEGylated insulin analogue according to claim 1, wherein the
extension consists of one or more of the following formulae wherein
the PEG moiety is attached to side chain(s) of lysine or cysteine
residue(s) when present or to the N-terminal amino group(s) (or
both): -AA.sub.x1K (for C-terminal extensions), wherein X1 is 0, 1,
2, 3, 4, 5, 6, 7, or 8 (and wherein K is lysine), K-AA.sub.x2- (for
N-terminal extensions), wherein x2 is 0, 1, 2, 3, 4, 5, 6, 7, or 8
(and wherein K is lysine), -AA.sub.x3C (for C-terminal extensions),
wherein x3 is 0, 1, 2, 3, 4, 5, 6, 7, or 8 (and wherein C is
cysteine), C-AA.sub.x4- (for N-terminal extensions), wherein x4 is
0, 1, 2, 3, 4, 5, 6, 7, or 8 (and wherein C is cysteine),
AA.sub.x5-R.sub.y- (for N-terminal extensions), wherein x5 is 0, 1,
2, 3, 4, 5, 6, 7, or 8, and y is 0 or 1 (and wherein R is
arginine), and wherein AA is a residue of a peptide chain wherein
each of the amino acid residues are the same or different and each
is any codable amino acid except Lys and Cys.
7. The PEGylated insulin analogue according to claim 1, wherein AA
is a peptide residue consisting of amino acid residues of glycine,
alanine or glutamine.
8. The PEGylated insulin analogue according to claim 7, wherein AA
is a residue of glycine.
9. The PEGylated insulin analogue according to claim 7, wherein AA
is a residue of alanine.
10. The PEGylated insulin analogue according to claim 7, wherein AA
is a residue of glutamine.
11. The PEGylated insulin analogue according to claim 1 wherein the
parent insulin, optionally contains one or more of the following
mutations: A14E/D, A18Q, A21G/A/Q, desB1, B1G/Q, B3Q/S/T, B13Q,
desB25, B25H, desB27, B28D/E/R, desB29, B29P/Q/R or desB30.
12. The PEGylated insulin analogue according to claim 1, wherein
the parent insulin analogue deviates from human insulin in having
A22K, B29R and desB30 and, preferably, there are no further
mutations in said insulin analogue.
13. The PEGylated insulin analogue according to claim 1, wherein
the parent insulin analogue deviates from human insulin in having
A22G, A23K, B29R and desB30 and, preferably, there are no further
mutations in said insulin analogue.
14. The A PEGylated insulin analogue according to claim 1,
comprising the moiety --(OCH.sub.2CH.sub.2).sub.n--, wherein n is
in integer in the range from 2 to about 1000, preferably from 2 to
about 500, preferably from 2 to about 250, preferably from 2 to
about 125, preferably from 2 to about 50, and preferably from 2 to
about 25.
15. The PEGylated insulin analogue according to claim 1, which is
one of the following PEGylated insulin analogues: a) a PEGylated
insulin analogue wherein the parent insulin analogue deviates from
human insulin in having A22G, A23G, A24K, B29R and desB30 and,
preferably, there are no further mutations in said insulin
analogue, b) a PEGylated insulin analogue wherein the parent
insulin analogue deviates from human insulin in having A22G, A23G,
A24G, A25K, B29R and desB30 and, preferably, there are no further
mutations in said insulin analogue, c) a PEGylated insulin analogue
wherein the parent insulin analogue deviates from human insulin in
having A21Q, A22K, B29R and desB30 and, preferably, there are no
further mutations in said insulin analogue, d) a PEGylated insulin
analogue wherein the parent insulin analogue deviates from human
insulin in having A21Q, A22G, A23K, B29R and desB30 and,
preferably, there are no further mutations in said insulin
analogue, e) a PEGylated insulin analogue wherein the parent
insulin analogue deviates from human insulin in having A21A, A22K,
B29R and desB30 and, preferably, there are no further mutations in
said insulin analogue, f) a PEGylated insulin analogue wherein the
parent insulin analogue deviates from human insulin in having A21G,
A22K, B29R and desB30 and, preferably, there are no further
mutations in said insulin analogue, g) a PEGylated insulin analogue
comprising a group of the general formula
-Q.sup.1-(OCH.sub.2CH.sub.2).sub.n--R.sup.1, wherein Q.sup.1 is a
linker connecting the polyethylene glycol moiety to an .alpha.- or
.gamma.-NH-group of an amino acid in the extension, preferably via
an amide or a carbamate bond, n is an integer in the range from 2
to about 1000, and R.sup.1 is alkoxy or hydroxyl, preferably
methoxy, and h) a PEGylated insulin analogue wherein the parent
insulin analogue deviates from human insulin in having A14E, A22K,
B25H, B29R and desB30 and, preferably, there are no further
mutations in said insulin analogue.
Description
FIELD OF THIS INVENTION
[0001] The present invention is related to PEGylated, extended
insulins which have insulin activity and can be used for the
treatment of diabetes. The PEGylated, extended insulins have higher
bioavailability and a longer time-action profile than regular
insulin and are in particular suited for pulmonary administration.
They will also have a high physical stability and a low tendency to
fibrillation and will be soluble at neutral pH. This invention is
also related to pharmaceutical compositions containing the
PEGylated, extended insulins.
BACKGROUND OF THIS INVENTION
[0002] The inherited physical and chemical stability of the insulin
molecule is a basic condition for insulin therapy of diabetes
mellitus. These basic properties are fundamental for insulin
formulation and for applicable insulin administration methods, as
well as for shelf-life and storage conditions of pharmaceutical
preparations. Use of solutions in administration of insulin exposes
the molecule to a combination of factors, e.g., elevated
temperature, variable air-liquid-solid interphases as well as shear
forces, which may result in irreversible conformation changes,
e.g., fibrillation.
[0003] Unfortunately, many diabetics are unwilling to undertake
intensive therapy due to the discomfort associated with the many
injections required to maintain close control of glucose levels.
This type of therapy can be both psychologically and physically
painful. Upon oral administration, insulin is rapidly degraded in
the gastro intestinal tract and is not absorbed into the blood
stream. Therefore, many investigators have studied alternate routes
for administering insulin, such as oral, rectal, transdermal, and
nasal routes. Thus far, however, these routes of administration
have not resulted in effective insulin absorption.
[0004] Efficient pulmonary delivery of a protein is dependent on
the ability to deliver the protein to the deep lung alveolar
epithelium. Proteins that are deposited in the upper airway
epithelium are not absorbed to a significant extent. This is due to
the overlying mucus which is approximately 30-40 .mu.m thick and
acts as a barrier to absorption. In addition, proteins deposited on
this epithelium are cleared by mucociliary transport up the airways
and then eliminated via the gastrointestinal tract. This mechanism
also contributes substantially to the low absorption of some
protein particles. The extent to which proteins are not absorbed
and instead eliminated by these routes depends on their solubility,
their size, as well as other less understood characteristics.
[0005] It is, however, well recognised that the properties of
peptides can be enhanced by grafting organic chain-like molecules
onto them. Such grafting can improve pharmaceutical properties such
as half life in serum, stability against proteolytical degradation
and reduced immunogenicity.
[0006] The organic chain-like molecules often used to enhance
properties are polyethylene glycolbased chains, i.e., chains that
are based on the repeating unit --CH.sub.2CH.sub.2O--. Hereinafter,
the abbreviation "PEG" is used for polyethyleneglycol.
[0007] Classical PEG technology takes advantage of providing
polypeptides with increased size (Stoke radius) by attaching a
soluble organic molecule to the polypeptide (Kochendoerfer, G., et
al., Science (299) 884 et seq., 2003). This technology leads to
reduced clearance in man and animals of a hormone polypeptide
compared to the native polypeptide. However, this technique is
often hampered by reduced potency of the hormone polypeptides
subjected to this technique (Hinds, K., et al., Bioconjugate Chem.
(11), 195-201, 2000).
[0008] Insulin compositions for pulmonary administration comprising
a conjugate of two-chain insulin covalently coupled to one or more
molecules of non-naturally hydrophilic polymers including
polyalkylene glycols and methods for their preparation are
disclosed in WO 02/094200 and WO 03/022996.
OBJECTS OF THIS INVENTION
[0009] There is still a need for insulins having a more prolonged
profile of action than the insulin derivatives known up till now
and which at the same time are soluble at physiological pH values
and have a potency which is comparable to that of human insulin.
Furthermore, there is need for further insulin formulations which
are well suited for pulmonary application.
[0010] An aspect of this invention deals with furnishing of a
medicament which can conveniently be administered pulmonary to
treat diabetic patients.
[0011] Another aspect of this invention deals with the furnishing
of a medicament which can conveniently be administered pulmonary to
treat diabetic patients and to reduce the risk of some of or all of
the late complications often associated with diabetes.
[0012] Another aspect of this invention deals with the furnishing
of a medicament which can conveniently be administered pulmonary to
treat diabetic patients and which is more convenient to use for
many patients that the use of injections.
[0013] Another aspect of this invention deals with the furnishing
of a medicament which can conveniently be administered pulmonary to
treat diabetic patients and which has a sufficient chemical
stability.
[0014] Another aspect of this invention deals with the furnishing
of a medicament which can conveniently be administered pulmonary to
treat diabetic patients and which has a sufficient physical
stability.
[0015] Another aspect of this invention deals with the furnishing
of a medicament having a sufficiently high insulin receptor
affinity.
[0016] The object of this invention is to overcome or ameliorate at
least one of the disadvantages of the prior art, or to provide a
useful alternative.
DEFINITIONS
[0017] Insulin is a polypeptide hormone secreted by .beta.-cells of
the pancreas and consists of two polypeptide chains designated the
A and B chains which are linked together by two inter-chain
disulphide bridges. The hormone is synthesized as a single-chain
precursor proinsulin (preproinsulin) consisting of a prepeptide of
24 amino acid 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, and A and B are the A and B
chains, respectively, of insulin. Arg-Arg and Lys-Arg are cleavage
sites for cleavage of the connecting peptide between the A and B
chains to form the two-chain insulin molecule. Insulin is essential
in maintaining normal metabolic regulation.
[0018] Herein, the term insulin covers natural occurring insulins,
e.g., human insulin, as well as insulin analogues thereof.
[0019] Herein the term amino acid residue covers an amino acid from
which a hydrogen atom has been removed from an amino group and/or a
hydroxy group has been removed from a carboxy group and/or a
hydrogen atom has been removed from a mercapto group. Imprecise, an
amino acid residue may be designated an amino acid.
[0020] Herein, the term insulin analogue covers a polypeptide which
has a molecular structure which formally can be derived from the
structure of a naturally occurring insulin, e.g., human insulin, by
deleting and/or substituting (replacing) one or more amino acid
residue occurring in the natural insulin and/or by adding one or
more amino acid residue. The added and/or substituted amino acid
residues can either be codable amino acid residues or other
naturally occurring amino acid residues or purely synthetic amino
acid residues.
[0021] Herein, the term extended insulin covers an insulin analogue
wherein there (compared with human insulin) is added one or more
amino acid residue either C- or N-terminally to the A- or B-chain
of insulin. For example, the A chain may be extended at its
C-terminal end, e.g., by 1, 2, 3 or 4 amino acid residues (compared
with human insulin) which extensions are denoted A22, A23, A24 and
A25, respectively. For example, when the amino acid residue in
position A23 is PEGylated, then the amino acid in position A22 may
be any amino acid residue except Cys and Lys, and so forth. For
example, the A chain may be extended at its N-terminal end, e.g.,
by 1, 2, 3 or 4 amino acid residues (compared with human insulin)
which extensions are denoted A-1, A-2, A-3 and A-4, respectively.
For example, when the amino acid residue in position A-2 is
PEGylated, then the amino acid in position A-1 may be any amino
acid residue except Cys and Lys, and so forth. Even though the
extended insulin has an extension at one of the four termini, there
may be deletions at other positions in said extended insulin.
Similarly as with human insulin, the extended insulin consists of
two chains, i.e., the A chain and B chain. In the extended insulin,
there are six cysteine residues, two of which are present in the A
chain forming an intra-chain disulphide bridge (corresponding to A6
and A11 in human insulin) and four of which form two inter-chain
disulphide bridges (corresponding to positions A7, A20, B7 and B19
in human insulin). Herein, the last mentioned four cysteine
residues are designated inter-chain cysteine residues. In each
chain (A and B chain), one of the inter-chain cysteine residues is
closest to the N terminal end of each chain and the other
inter-chain cysteine residues is closest to the C terminal end of
each chain and, herein, such inter-chain cysteine residues are
designated an N terminal inter-chain cysteine residue and a C
terminal inter-chain cysteine residue, respectively. When
determining whether an insulin analogue is an extended insulin, one
has to count the number of amino acid residues present in each
chain on the N terminal side of the N terminal inter-chain cysteine
residue and to count the number of amino acid residues present in
each chain on the C terminal side of the C terminal inter-chain
cysteine residue. If one of these numbers (figures) is larger that
the corresponding number for human insulin, that insulin is
considered an extended insulin. In human insulin, there are six
amino acid residues present on the N terminal side of the N
terminal inter-chain cysteine residue in the A chain, one amino
acid residue present on the C terminal side of the C terminal
inter-chain cysteine residue in the A chain, six amino acid
residues present on the N terminal side of the N terminal
inter-chain cysteine residue in the B chain, and eleven amino acid
residues present on the C terminal side of the C terminal
inter-chain cysteine residue in the B chain.
[0022] Herein the term parent insulin means the extended insulin
without appended PEG moieties.
[0023] Herein, the term mutation covers any change in amino acid
sequence (substitutions and insertions with codable amino acids as
well as deletions).
[0024] Herein, the term analogues of the A chain and analogues of
the B chains of human insulin covers A and B chains of human
insulin, respectively, having one or more substitutions, deletions
and or extensions (additions) of the A and B amino acid chains,
respectively, relative to the A and B chains, respectively, of
human insulin.
[0025] Herein terms like A1, A2, A3 etc. indicates the position 1,
2 and 3, respectively, in the A chain of insulin (counted from the
N-terminal end). Similarly, terms like B1, B2, B3 etc. indicates
the position 1, 2 and 3, respectively, in the B chain of insulin
(counted from the N-terminal end). Using the one letter codes for
amino acids, terms like A21A, A21G and A21Q designates that the
amino acid in the A21 position is A, G and Q, respectively. Using
the three letter codes for amino acids, the corresponding
expressions are AlaA21, GlyA21 and GInA21, respectively.
[0026] Herein terms like A-1, B-1, etcetera, indicates the
positions of the first amino acids N-terminally to the A1 and B1
positions, respectively, and so forth.
[0027] Herein terms like desB29 and desB30 indicate an insulin
analogue lacking the B29 or B30 amino acid residue,
respectively.
[0028] Herein the term single chain insulin covers a polypeptide
sequence of the general structure BC-A, wherein A is the A chain of
human insulin or an analogue thereof, B is the B chain of human
insulin or an analogue thereof, and C is a bond or the so-called
connecting peptide, e.g., a peptide chain of about 1-35 amino acid
residues connecting the C-terminal amino acid residue in the
B-chain, e.g., B30, with the N-terminal amino acid residue in the
A-chain, e.g., A1. If the B chain is a desB30 chain, the connecting
peptide (C) will connect B29 with A1. The single-chain insulin will
contain the three, correctly positioned disulphide bridges as in
human insulin, i.e., between Cys.sup.A7 and Cys.sup.B7, between
Cys.sup.A20 and Cys.sup.B19 and between Cys.sup.A6 and
Cys.sup.A11.
[0029] The term connecting peptide covers a peptide chain which can
connect the C-terminal amino acid residue of the B-chain with the
N-terminal amino acid residue of the A-chain in insuin. Herein the
expression B'A means a single chain insulin wherein the connecting
peptide does not consist an any amino acids but simply is a bond,
i.e. there is a bond between the B-chain C-terminal and the A-chain
N-terminal.
[0030] With fast acting insulin is meant an insulin having a faster
onset of action than normal or regular human insulin.
[0031] With long acting insulin is meant an insulin having a longer
duration of action than normal or regular human insulin.
[0032] The numbering of the positions in insulin analogues,
extended insulins and A and B chains is done so that the parent
compound is human insulin with the numbering used for it.
[0033] The term basal insulin as used herein means an insulin
peptide which has a time-action of more than 8 hours, in
particularly of at least 9 hours. Preferably, the basal insulin has
a time-action of at least 10 hours. The basal insulin may thus have
a time-action in the range from about 8 to 24 hours, preferably in
the range from about 9 to about 15 hours.
[0034] Herein the term linker covers a chemical moiety which
connects an --HN-- group of the extended insulin with the --O--
group of the PEG moiety. The linker does not have any influence on
the desired action of the final PEGylated extended insulin,
especially it does not have any adverse influence.
[0035] With "PEG" or polyethylene glycol, as used herein is meant
any water soluble poly(ethylene glycole) or poly(ethylene oxide).
The expression PEG will comprise the structure
--(CH.sub.2CH.sub.2O).sub.n--, where n is an integer from 2 to
about 1000. A commonly used PEG is end-capped PEG, wherein one end
of the PEG termini is end-capped with a relatively inactive group
such as alkoxy, while the other end is a hydroxyl group that may be
further modified by linker moieties. An often used capping group is
methoxy and the corresponding end-capped PEG is often denoted mPEG.
Hence, mPEG is CH.sub.3O(CH.sub.2CH.sub.2O).sub.n--, where n is an
integer from 2 to about 1000 sufficient to give the average
molecular weight indicated for the whole PEG moiety, e.g., for mPEG
Mw 2,000, n is approximately 44 (a number that is subject for
batch-to-batch variation). The notion PEG is often used instead of
mPEG. "PEG" followed by a number (not being a subscript) indicates
a PEG moiety with the approximate molecular weight equal the
number. Hence, "PEG2000" is a PEG moiety having an approximate
molecular weight of 2000.
[0036] Specific PEG forms of this invention are branched, linear,
forked, dumbbell PEGs, and the like and the PEG groups are
typically polydisperse, possessing a low polydispersity index of
less than about 1.05. The PEG moieties present in an extended
insulin will for a given molecular weight typically consist of a
range of ethyleneglycol (or ethyleneoxide) monomers. For example, a
PEG moiety of molecular weight 2000 will typically consist of
44.+-.10 monomers, the average being around 44 monomers. The
molecular weight (and number of monomers) will typically be subject
to some batch-to-batch variation.
[0037] Other specific PEG forms are monodisperse that can be
branched, linear, forked, or dumbbell shaped as well. Being
monodisperse means that the length (or molecular weight) of the PEG
polymer is specifically defined and is not a mixture of various
lengths (or molecular weights). Herein the notion mdPEG is used to
indicate that the mPEG moiety is monodisperse, using "d" for
"discrete". The number in subscript after mdPEG, for example "12"
in mdPEG.sub.12, indicates the number of ethyleneglycol monomers
within the monodisperse polymer (oligomer).
[0038] The term PEGylation covers modification of insulin by
attachment of one or more PEG moieties via a linker. The PEG moiety
can either be attached by nucleophilic substitution (acylation) on
N-terminal alpha-amino groups or on lysine residue(s) on the
gamma-positions, e.g., with OSu-activated esters, or PEG moieties
can be attached by reductive alkylation--also on amino groups
present in the extended insulin molecule--using PEG-aldehyde
reagents and a reducing agent, such as sodium cyanoborohydride, or,
alternatively, PEG moieties can be attached to the sidechain of an
unpaired cysteine residue in a Michael addition reaction using eg.
PEG maleimide reagents.
[0039] By PEGylated, extended insulin having insulin activity is
meant a PEGylated, extended insulin with either the ability to
lower the blood glucose in mammalians as measured in a suitable
animal model, which may be a rat, rabbit, or pig model, after
suitable administration e.g., by intravenous, subcutaneous, or
pulmonary administration, or an insulin receptor binding
affinity.
[0040] Herein the term alkyl covers a saturated, branched or
straight hydrocarbon group.
[0041] Herein the term alkoxy covers the radical "alkyl-O--".
Representative examples are methoxy, ethoxy, propoxy (e.g.,
1-propoxy and 2-propoxy), butoxy (e.g., 1-butoxy, 2-butoxy and
2-methyl-2-propoxy), pentoxy (1-pentoxy and 2-pentoxy), hexoxy
(1-hexoxy and 3-hexoxy), and the like.
[0042] Herein the term alkylene covers a saturated, branched or
straight bivalent hydrocarbon group having from 1 to 12 carbon
atoms. Representative examples include, but are not limited to,
methylene, 1,2-ethylene, 1,3-propylene, 1,2-propylene,
1,3-butylene, 1,4-butylene, 1,4-pentylene, 1,5-pentylene,
1,5-hexylene, 1,6-hexylene, and the like.
[0043] By high physical stability is meant a tendency to
fibrillation being less than 50% of that of human insulin.
Fibrillation may be described by the lag time before fibril
formation is initiated at a given conditions.
[0044] A polypeptide with insulin receptor and IGF-1 receptor
affinity is a polypeptide which is capable of interacting with an
insulin receptor and a human IGF-1 receptor in a suitable binding
assay. Such receptor assays are well-know within the field and are
further described in the examples. The present PEGylated, extended
insulin will not bind to the IGF-1 receptor or will have a rather
low affinity to said receptor. More precisely, the PEGylated,
extended insulins of this invention will have an affinity towards
the IGF-1 receptor of substantially the same magnitude or less as
that of human insulin
[0045] The terms treatment and treating as used herein means the
management and care of a patient for the purpose of combating a
disease, disorder or condition. The term is intended to include the
delaying of the progression of the disease, disorder or condition,
the alleviation or relief of symptoms and complications, and/or the
cure or elimination of the disease, disorder or condition. The
patient to be treated is preferably a mammal, in particular a human
being.
[0046] The term treatment of a disease as used herein means the
management and care of a patient having developed the disease,
condition or disorder. The purpose of treatment is to combat the
disease, condition or disorder. Treatment includes the
administration of the active compounds to eliminate or control the
disease, condition or disorder as well as to alleviate the symptoms
or complications associated with the disease, condition or
disorder.
[0047] The term prevention of a disease as used herein is defined
as the management and care of an individual at risk of developing
the disease prior to the clinical onset of the disease. The purpose
of prevention is to combat the development of the disease,
condition or disorder, and includes the administration of the
active compounds to prevent or delay the onset of the symptoms or
complications and to prevent or delay the development of related
diseases, conditions or disorders.
[0048] The term effective amount as used herein means a dosage
which is sufficient in order for the treatment of the patient to be
effective compared with no treatment.
[0049] POT is the Schizosaccharomyces pombe triose phosphate
isomerase gene, and TPI1 is the S. cerevisiae triose phosphate
isomerase gene.
[0050] By a leader is meant an amino acid sequence consisting of a
pre-peptide (the signal peptide) and a pro-peptide.
[0051] The term signal peptide is understood to mean a pre-peptide
which is present as an N-terminal sequence on the precursor form of
a protein. The function of the signal peptide is to allow the
heterologous protein to facilitate translocation into the
endoplasmic reticulum. The signal peptide is normally cleaved off
in the course of this process. The signal peptide may be
heterologous or homologous to the yeast organism producing the
protein. A number of signal peptides which may be used with the DNA
construct of this invention including yeast aspartic protease 3
(YAP3) signal peptide or any functional analog (Egel-Mitani et al.
(1990) YEAST 6:127-137 and U.S. Pat. No. 5,726,038) and the
.alpha.-factor signal of the MF.alpha.1 gene (Thorner (1981) in The
Molecular Biology of the Yeast Saccharomyces cerevisiae, Strathern
et al., eds., pp 143-180, Cold Spring Harbor Laboratory, NY and
U.S. Pat. No. 4,870,00.
[0052] The term pro-peptide means a polypeptide sequence whose
function is to allow the expressed polypeptide to be directed from
the endoplasmic reticulum to the Golgi apparatus and further to a
secretory vesicle for secretion into the culture medium (i.e.
exportation of the polypeptide across the cell wall or at least
through the cellular membrane into the periplasmic space of the
yeast cell). The pro-peptide may be the yeast .alpha.-factor
pro-peptide, vide U.S. Pat. Nos. 4,546,082 and 4,870,008.
Alternatively, the pro-peptide may be a synthetic pro-peptide,
which is to say a pro-peptide not found in nature. Suitable
synthetic pro-peptides are those disclosed in U.S. Pat. Nos.
5,395,922; 5,795,746; 5,162,498 and WO 98/32867. The pro-peptide
will preferably contain an endopeptidase processing site at the
C-terminal end, such as a Lys-Arg sequence or any functional
analogue thereof.
[0053] In the present context, the three-letter or one-letter
indications of the amino acids have been used in their conventional
meaning as indicated in the following. Unless indicated explicitly,
the amino acids mentioned herein are L-amino acids. Further, the
left and right ends of an amino acid sequence of a peptide are,
respectively, the N- and C-termini, unless otherwise specified.
[0054] Abbreviations for Amino Acids
TABLE-US-00001 Amino acid Three-letter code One-letter code Glycine
Gly G Proline Pro P Alanine Ala A Valine Val V Leucine Leu L
Isoleucine Ile I Methionine Met M Cysteine Cys C Phenylalanine Phe
F Tyrosine Tyr Y Tryptophan Trp W Histidine His H Lysine Lys K
Arginine Arg R Glutamine Gln Q Asparagine Asn N Glutamic Acid Glu E
Aspartic Acid Asp D Serine Ser S Threonine Thr T
The amino acids present in the PEGylated insulins of this invention
are, preferably, amino acids which can be coded fro by a nucleic
acid.
[0055] The following abbreviations have been used in the
specification and examples: Da is Dalton (molecular weight), kDa is
kilo-Dalton (=1000 Da), mPEG-SBA is
mPEG-CH.sub.2CH.sub.2CH.sub.2--CO--OSu (N-hydroxysuccinimidyl ester
of mPEG-butanoic acid), mPEG-SMB is
mPEG-CH.sub.2CH.sub.2CH(CH.sub.3)--CO--OSu (N-hydroxysuccinimidyl
ester of mPEG-.alpha.-methylbutanoic acid), mPEG-SPA is
mPEG-CH.sub.2CH.sub.2--CO--OSu (N-hydroxysuccinimidyl ester of
mPEG-propionic acid), Mw is molecular weight, OSu is
1-succinimidyloxy=2,5-dioxopyrrolidin-1-yloxy, R is room
temperature, SA is sinapinic acid and Su is
1-succinimidyl=2,5-dioxopyrrolidin-1-yl.
SUMMARY OF THIS INVENTION
[0056] In one aspect, this invention is related to a PEGylated
insulin analogue which, compared with human insulin, has one or
more extensions extended from the A1, B1, A21 and/or B30
position(s), said extension(s) consist(s) of amino acid residue(s)
and wherein the PEG moiety, via a linker, is attached to one or
more of the amino acid residues in the extension(s).
[0057] Via a suitable linker group, a PEG group can be attached to
side chain(s) of lysine or cysteine residue(s) when present or
attached to the N-terminal amino group(s) or at both places in the
parent insulin. The linker is typically a derivative of a
carboxylic acid, where the carboxylic acid functionality is used
for attachment to the parent insulin via an amide bond. The linker
may be an acetic acid moiety with the linking motif:
--CH.sub.2CO--, a propionic acid moiety with the linking motif:
--CH.sub.2CH.sub.2CO-- or --CHCH.sub.3CO--, or a butyric acid
moiety with the linking motif: --CH.sub.2CH.sub.2CH.sub.2CO-- or
--CH.sub.2CHCH.sub.3CO--. Alternatively, the linker may be a --CO--
group.
[0058] Since PEGylation of the lysine group present in position B29
in the human insulin B-chain is unwanted, this amino acid residue
shall be replaced by another amino acid residue. Suitable
replacement amino acid residues are Ala, Arg, Gln and His.
Furthermore, it is desirable that there is no Lys present in any of
the positions 1 through 21 in the A chain (A1-A21) and no Lys
present in any of the positions 1 through 30 in the B chain
(B1-B30).
[0059] The parent insulin molecule may have a limited number of the
naturally occurring amino acid residues substituted with other
amino acid residues as explained in the detailed part of the
specification.
[0060] In one embodiment, this invention relates to a PEGylated,
extended insulin, wherein the parent insulin analogue deviates from
human insulin in one or more of the following deletions or
substitutions: E or D in position A14, Q in position A18, A, G or Q
in position A21, G or Q in position B1 or no amino acid residue in
position B1, Q, S or T in position B3 or no amino acid residue in
position B3, Q in position B13, H in position B25 or no amino acid
residue in position B25, no amino acid residue in position B27, D,
E or R in position B28, P, Q or R in position B29 or no amino acid
residue in position B29, no amino acid residue in position B30.
[0061] The PEG group may vary in size within a large range as is
well known within the art. However, too large PEG groups may
interfere in a negative way with the biological activity of the
PEGylated, extended insulin molecule.
[0062] In still a further aspect, this invention is related to
pharmaceutical preparations comprising the PEGylated, extended
insulin of this invention and suitable adjuvants and additives such
as one or more agents suitable for stabilization, preservation or
isotoni, e.g., zinc ions, phenol, cresol, a parabene, sodium
chloride, glycerol or mannitol. The zinc content of the present
formulations may be between 0 and about 6 zinc atoms per insulin
hexamer. The pH value of the pharmaceutical preparation may be
between about 4 and about 8.5, between about 4 and about 5 or
between about 6.5 and about 7.5.
[0063] In a further embodiment, this invention is related to the
use of the PEGylated, extended insulin as a pharmaceutical for the
reducing of blood glucose levels in mammalians, in particularly for
the treatment of diabetes.
[0064] In a further aspect, this invention is related to the use of
the PEGylated, extended insulin for the preparation of a
pharmaceutical preparation for the reducing of blood glucose level
in mammalians, in particularly for the treatment of diabetes.
[0065] In a further embodiment, this invention is related to a
method of reducing the blood glucose level in mammalians by
administrating a therapeutically active dose of a PEGylated,
extended insulin of this invention to a patient in need of such
treatment.
[0066] In a further aspect of this invention, the PEGylated,
extended insulins are administered in combination with one or more
further active substances in any suitable ratios. Such further
active agents may be selected from human insulin, fast acting
insulin analogues, antidiabetic agents, antihyperlipidemic agents,
antiobesity agents, antihypertensive agents and agents for the
treatment of complications resulting from or associated with
diabetes.
[0067] In one embodiment, the two active components are
administered as a mixed pharmaceutical preparation. In another
embodiment, the two components are administered separately either
simultaneously or sequentially.
[0068] In one embodiment, the PEGylated, extended insulins of this
invention may be administered together with fast acting human
insulin or human insulin analogues. Such fast acting insulin
analogue may be such wherein the amino acid residue in position B28
is Asp, Lys, Leu, Val, or Ala and the amino acid residue in
position B29 is Lys or Pro, des(B28-B30), des(B27) or des(B30)
human insulin, and an analogue wherein the amino acid residue in
position B3 is Lys and the amino acid residue in position B29 is
Glu or Asp. The PEGylated, extended insulin of this invention and
the rapid acting human insulin or human insulin analogue can be
mixed in a ratio from about 90/10%; about 70/30% or about
50/50%.
[0069] The PEGylated, extended insulins of this invention may also
be used on combination treatment together with an antidiabetic
agent.
[0070] Antidiabetic agents will include insulin, GLP-1 (1-37)
(glucagon like peptide-1) described in WO 98/08871, WO 99/43706,
U.S. Pat. No. 5,424,286 and WO 00/09666, GLP-2, exendin-4(1-39),
insulinotropic fragments thereof, insulinotropic analogues thereof
and insulinotropic derivatives thereof. Insulinotropic fragments of
GLP-1 (1-37) are insulinotropic peptides for which the entire
sequence can be found in the sequence of GLP-1 (1-37) and where at
least one terminal amino acid has been deleted.
[0071] The PEGylated, extended insulins of this invention may also
be used on combination treatment together with an oral antidiabetic
such as a thiazolidindione, metformin and other type 2 diabetic
pharmaceutical preparation for oral treatment.
[0072] Furthermore, the PEGylated, extended insulin of this
invention may be administered in combination with one or more
antiobesity agents or appetite regulating agents.
[0073] In one embodiment this invention is related to a pulmonal
pharmaceutical preparation comprising the PEGgylated extended
insulin of this invention and suitable adjuvants and additives such
as one or more agents suitable for stabilization, preservation or
isotoni, e.g., zinc ions, phenol, cresol, a parabene, sodium
chloride, glycerol, propyleneglycol or mannitol.
[0074] It should be understood that any suitable combination of the
PEGylated, extended insulins with diet and/or exercise, one or more
of the above-mentioned compounds and optionally one or more other
active substances are considered to be within the scope of this
invention.
DETAILED DESCRIPTION OF THIS INVENTION
[0075] The stability and solubility properties of insulin are
important underlying aspects for current insulin therapy. This
invention is addressed to these issues by providing stable,
PEGylated, extended insulin analogues wherein the PEGylation in the
extension decreases molecular flexibility and concomitantly reduce
the fibrillation propensity and limit or modify the pH
precipitation zone.
[0076] The PEGylated, extended insulins of this invention are in
particularly intended for pulmonal administration due to their
relatively high bioavailability compared to, e.g., human insulin.
Furthermore, the PEGylated, extended insulins will have a
protracted insulin activity.
[0077] Because virtually all PEG polymers are mixtures of many
large molecules, one must resort to averages to describe molecular
weight. Among many possible ways of reporting averages, three are
commonly used: the number average, weight average, and z-average
molecular weights. The weight average is probably the most useful
of the three, because it fairly accounts for the contributions of
different sized chains to the overall behaviour of the polymer, and
correlates best with most of the physical properties of
interest.
Number average M W ( M _ n ) . .SIGMA. ( M i N i ) .SIGMA. ( M i N
i ) Weight average M W ( M _ w ) . .SIGMA. ( M i 2 N i ) .SIGMA. (
M i N i ) Z average M W ( M _ z ) . .SIGMA. ( M i 3 N i ) .SIGMA. (
M i 2 N i ) ##EQU00001##
where N.sub.i is the mole-fraction (or the number-fraction) of
molecules with molecular weight M.sub.i in the polymer mixture. The
ratio of M.sub.w to M.sub.n is known as the polydispersity index
(PDI), and provides a rough indication of the breadth of the
distribution. The PDI approaches 1.0 (the lower limit) for special
polymers with very narrow MW distributions.
[0078] While lower molecular weight PEG groups may be preferred for
increasing bioavailability, high molecular weight PEG chains, e.g.,
having an average molecular weight of 4000-6000 daltons or greater,
although generally found to decrease the bioactivity of the insulin
molecule, may be preferred for increasing half-life, e.g., in the
case of formulations for pulmonary administration.
[0079] The PEG groups of this invention will typically comprise a
number of (--OCH.sub.2CH.sub.2--) subunits.
[0080] The PEG groups of the invention will for a given molecular
weight typically consist of a range of ethyleneglycol (or
ethyleneoxide) monomers. For example, a PEG group of molecular
weight 2000 dalton will typically consist of 43.+-.10 monomers, the
average being around 43-44 monomers.
[0081] The parent insulin molecule which is PEGylated in this
invention is an extended insulin molecule, i.e., an insulin
molecule having one or more amino acid residues attached to the
N-terminal end of the parent A and/or B chain, e.g., to A1 and/or
B1, and/or attached to the C-terminal end of the parent A and/or B
chain, e.g., A21 and/or B30, referring to human insulin.
Preferably, the extended insulin molecule, i.e., the parent
insulin, contains at least 52 amino acid residues.
[0082] The PEGgylated extended insulins of this invention may be
mono-substituted having only one PEG group attached to a lysine
amino acid residue in the parent insulin molecule or to a
N-terminal amino acid residue. Alternatively, the PEGylated,
extended insulins of this invention may comprise two, three- or
four PEG groups. If the extended insulin comprises more than one
PEG group, it will typically have the same PEG moiety attached to
each lysine group or to the N-terminal amino acid residue. However,
the individual PEG groups may also vary from each other in size and
length.
[0083] For example, an extended insulin having the following
deviations as compared to human insulin: A22K, B29R, desB30 and
being PEGylated in the lysine residue in position A22 with
mPEG-propionic acid, 2 kDa, e.g., using mPEG-SPA is named
A22K(N.sup..epsilon.mPEG2000-propionyl) B29R desB30 human insulin.
It is obvious that if any of the corresponding other PEGylation
reagents (Mw 2000 Da), containing other linkers, e.g. the butyric
acid linkers, were used for preparation of that particular
compound, the "exact" name of that particular compound would be
different, but the small molecular differences will not result in
any differences in biological properties. In this application, the
PEGylated extended insulins are, to a great extent, named as if the
linking moiety is a propionic acid linker, irrespective of the
actual linker. In fact, within protein PEGylation literature, it is
rarely specified which linking groups are used. The important
variables are, with respect to biological properties: Size (in
Daltons) and shape of the PEG moiety and position of the PEG
attachment within the protein.
[0084] The parent insulins are produced by expressing a DNA
sequence encoding the extended insulin in question in a suitable
host cell by well known technique as disclosed in, e.g., U.S. Pat.
No. 6,500,645. The parent insulin is either expressed directly or
as a precursor molecule which has an N-terminal extension on the
B-chain. This N-terminal extension may have the function of
increasing the yield of the directly expressed product and may be
of up to 15 amino acid residues long. The N-terminal extension is
to be cleaved of in vitro after isolation from the culture broth
and will therefore have a cleavage site next to B1. N-terminal
extensions of the type suitable in this invention are disclosed in
U.S. Pat. No. 5,395,922, and European Patent No. 765,395A.
[0085] The polynucleotide sequence coding for the parent insulin
may be prepared synthetically by established standard methods,
e.g., the phosphoamidite method described by Beaucage et al. (1981)
Tetrahedron Letters 22:1859-1869, or the method described by
Matthes et al. (1984) EMBO Journal 3: 801-805. According to the
phosphoamidite method, oligonucleotides are synthesized, e.g., in
an automatic DNA synthesizer, 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).
[0086] The polynucleotide sequences may also be of mixed genomic,
cDNA, and synthetic origin. For example, a genomic or cDNA sequence
encoding a leader peptide may be joined to a genomic or cDNA
sequence encoding the A and B chains, after which the DNA sequence
may be modified at a site by inserting synthetic oligonucleotides
encoding the desired amino acid sequence for homologous
recombination in accordance with well-known procedures or
preferably generating the desired sequence by PCR using suitable
oligonucleotides.
[0087] 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
parent insulin. 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.
[0088] The recombinant expression vector is 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.
[0089] 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 of which provides for biocide or viral
resistance, resistance to heavy metals, prototrophy to auxotrophs,
and the like. Examples of bacterial selectable markers are the dal
genes from Bacillus subtilis or Bacillus lichenifonnis, 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 (or nithine carbamoyltransferase), pyrG
(orotidine-5'-phosphate decarboxylase) and trpC (anthranilate
synthase. 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 (1985)
Gene 40:125-130).
[0090] 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.
[0091] 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
lichenifonnis alpha-amylase gene (amyL), Bacillus
stearothermophilus maltogenic amylase gene (amyM), Bacillus
amyloliquefaciens alpha-amylase gene (amyQ), and Bacillus
lichenifonnis penicillinase gene (penP). Examples of suitable
promoters for directing 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 or PGK promoters.
[0092] The polynucleotide sequence encoding the parent insulin will
also typically be operably connected to a suitable terminator. In
yeast a suitable terminator is the TPI terminator (Alber et al.
(1982) J. Mol. Appl. Genet. 1:419-434).
[0093] The procedures used to ligate the polynucleotide sequence
encoding the parent insulin, 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 extended
insulins of this 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, pro-peptide, connecting
peptide, A and B chains) followed by ligation.
[0094] The vector comprising the polynucleotide sequence encoding
the parent insulin 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,
Streptomyces cell, or gram negative bacteria such as E. coli and
Pseudomonas sp. Eukaryote cells may be mammalian, insect, plant, or
fungal cells. In one embodiment, the host cell is a yeast cell. The
yeast organism may be any suitable yeast organism which, on
cultivation, produces large amounts of the single chain insulin of
the invention. Examples of suitable yeast organisms are strains
selected from the yeast species 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.
[0095] The transformation of the yeast cells may for instance be
effected by protoplast formation followed by transformation in a
manner known per se. The medium used to cultivate the cells may be
any conventional medium suitable for growing yeast organisms. The
secreted extended insulin, a significant proportion of which will
be present in the medium in correctly processed form, may be
recovered from the medium by conventional procedures including
separating the yeast cells from the medium by centrifugation,
filtration or catching the insulin precursor by an ion exchange
matrix or by a reverse phase absorption matrix, precipitating the
proteinaceous components of the supernatant or filtrate 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, or the like.
Pharmaceutical Compositions
[0096] The PEGylated, extended insulins of this invention may be
administered subcutaneously, orally, or pulmonary.
[0097] For subcutaneous administration, the PEGylated, extended
insulins of this invention are formulated analogously with the
formulation of known insulins. Furthermore, for subcutaneous
administration, the PEGylated, extended insulins of this invention
are administered analogously with the administration of known
insulins and, generally, the physicians are familiar with this
procedure.
[0098] PEGylated, extended insulins of this invention may be
administered by inhalation in a dose effective to increase
circulating insulin levels and/or to lower circulating glucose
levels. Such administration can be effective for treating disorders
such as diabetes or hyperglycemia. Achieving effective doses of
insulin requires administration of an inhaled dose of more than
about 0.5 .mu.g/kg to about 50 .mu.g/kg of PEGylated, extended
insulins of this invention. A therapeutically effective amount can
be determined by a knowledgeable practitioner, who will take into
account factors including insulin level, blood glucose levels, the
physical condition of the patient, the patient's pulmonary status,
or the like.
[0099] The PEGylated, extended insulins of this invention may be
delivered by inhalation to achieve slow absorption and/or reduced
systemical clearance thereof. Different inhalation devices
typically provide similar pharmacokinetics when similar particle
sizes and similar levels of lung deposition are compared.
[0100] The PEGylated, extended insulins of this invention may be
delivered by any of a variety of inhalation devices known in the
art for administration of a therapeutic agent by inhalation. These
devices include metered dose inhalers, nebulizers, dry powder
generators, sprayers, and the like. Preferably, the PEGylated,
extended insulins of this are delivered by a dry powder inhaler or
a sprayer. There are a several desirable features of an inhalation
device for administering PEGylated, extended insulins of this
invention. For example, delivery by the inhalation device is
advantageously reliable, reproducible, and accurate. The inhalation
device should deliver small particles or aerosols, e.g., less than
about 10 .mu.m, for example about 1-5 .mu.m, for good
respirability. Some specific examples of commercially available
inhalation devices suitable for the practice of this invention are
Cyclohaler, Turbohaler.TM. (Astra), Rotahaler.RTM. (Glaxo),
Diskus.RTM. (Glaxo), Spiros.TM. inhaler (Dura), devices marketed by
Inhale Therapeutics, AERx.TM. (Aradigm), the Ultravent.RTM.
nebulizer (Mallinckrodt), the Acorn II.RTM. nebulizer (Marquest
Medical Products), the Ventolin.RTM. metered dose inhaler (Glaxo),
the Spinhaler.RTM. powder inhaler (Fisons), or the like.
[0101] As those skilled in the art will recognize, the formulation
of PEGylated, extended insulins of this invention, the quantity of
the formulation delivered and the duration of administration of a
single dose depend on the type of inhalation device employed. For
some aerosol delivery systems, such as nebulizers, the frequency of
administration and length of time for which the system is activated
will depend mainly on the concentration of PEGylated, extended
insulins in the aerosol. For example, shorter periods of
administration can be used at higher concentrations of PEGylated,
extended insulins in the nebulizer solution. Devices such as
metered dose inhalers can produce higher aerosol concentrations,
and can be operated for shorter periods to deliver the desired
amount of the PEGylated, extended insulins. Devices such as powder
inhalers deliver active agent until a given charge of agent is
expelled from the device. In this type of inhaler, the amount of
insulin PEGylated, extended insulins of this invention in a given
quantity of the powder determines the dose delivered in a single
administration.
[0102] The particle size of PEGylated, extended insulins of this
invention in the formulation delivered by the inhalation device is
critical with respect to the ability of insulin to make it into the
lungs, and preferably into the lower airways or alveoli.
Preferably, the PEGylated, extended insulins of this invention ion
is formulated so that at least about 10% of the PEGylated, extended
insulins delivered is deposited in the lung, preferably about 10 to
about 20%, or more. It is known that the maximum efficiency of
pulmonary deposition for mouth breathing humans is obtained with
particle sizes of about 2 .mu.m to about 3 .mu.m. When particle
sizes are above about 5 .mu.m, pulmonary deposition decreases
substantially. Particle sizes below about 1 .mu.m cause pulmonary
deposition to decrease, and it becomes difficult to deliver
particles with sufficient mass to be therapeutically effective.
Thus, particles of the PEGylated, extended insulins delivered by
inhalation have a particle size preferably less than about 10
.mu.m, more preferably in the range of about 1 .mu.m to about 5
.mu.m. The formulation of the PEGylated, extended insulins is
selected to yield the desired particle size in the chosen
inhalation device.
[0103] Advantageously for administration as a dry powder a
PEGylated, extended insulin of this invention is prepared in a
particulate form with a particle size of less than about 10 .mu.m,
preferably about 1 to about 5 .mu.m. The preferred particle size is
effective for delivery to the alveoli of the patient's lung.
Preferably, the dry powder is largely composed of particles
produced so that a majority of the particles have a size in the
desired range. Advantageously, at least about 50% of the dry powder
is made of particles having a diameter less than about 10 .mu.m.
Such formulations can be achieved by spray drying, milling,
micronisation, or critical point condensation of a solution
containing the PEGylated, extended insulin of this invention and
other desired ingredients. Other methods also suitable for
generating particles useful in the current invention are known in
the art.
[0104] The particles are usually separated from a dry powder
formulation in a container and then transported into the lung of a
patient via a carrier air stream. Typically, in current dry powder
inhalers, the force for breaking up the solid is provided solely by
the patient's inhalation. In another type of inhaler, air flow
generated by the patient's inhalation activates an impeller motor
which deagglomerates the particles.
[0105] Formulations of PEGylated, extended insulins of this
invention for administration from a dry powder inhaler typically
include a finely divided dry powder containing the derivative, but
the powder can also include a bulking agent, carrier, excipient,
another additive, or the like. Additives can be included in a dry
powder formulation of PEGylated, extended insulin, e.g., to dilute
the powder as required for delivery from the particular powder
inhaler, to facilitate processing of the formulation, to provide
advantageous powder properties to the formulation, to facilitate
dispersion of the powder from the inhalation device, to stabilize
the formulation (for example, antioxidants or buffers), to provide
taste to the formulation, or the like. Advantageously, the additive
does not adversely affect the patient's airways. The PEGylated,
extended insulin can be mixed with an additive at a molecular level
or the solid formulation can include particles of the PEGylated,
extended insulin mixed with or coated on particles of the additive.
Typical additives include mono-, di-, and polysaccharides; sugar
alcohols and other polyols, such as, e.g., lactose, glucose,
raffinose, melezitose, lactitol, maltitol, trehalose, sucrose,
mannitol, starch, or combinations thereof; surfactants, such as
sorbitols, diphosphatidyl choline, or lecithin; or the like.
Typically an additive, such as a bulking agent, is present in an
amount effective for a purpose described above, often at about 50%
to about 90% by weight of the formulation. Additional agents known
in the art for formulation of a protein such as insulin analogue
protein can also be included in the formulation.
[0106] A spray including the PEGylated, extended insulins of this
invention can be produced by forcing a suspension or solution of
the PEGylated, extended insulin through a nozzle under pressure.
The nozzle size and configuration, the applied pressure, and the
liquid feed rate can be chosen to achieve the desired output and
particle size. An electrospray can be produced, e.g., by an
electric field in connection with a capillary or nozzle feed.
Advantageously, particles of insulin conjugate delivered by a
sprayer have a particle size less than about 10 .mu.m, preferably
in the range of about 1 .mu.m to about 5 .mu.m.
[0107] Formulations of PEGylated, extended insulins of this
invention suitable for use with a sprayer will typically include
the PEGylated, extended insulins in an aqueous solution at a
concentration of from about 1 mg to about 500 mg of the PEGylated,
extended insulin per ml of solution. Depending on the PEGylated
insulin chosen and other factors known to the medical advisor, the
upper limit may be lower, e.g., 450, 400, 350, 300, 250, 200, 150,
120, 100 or 50 mg of the PEGylated insulin per ml of solution. The
formulation can include agents such as an excipient, a buffer, an
isotonicity agent, a preservative, a surfactant, and, preferably,
zinc. The formulation can also include an excipient or agent for
stabilization of the PEGylated, extended insulin, such as a buffer,
a reducing agent, a bulk protein, or a carbohydrate. Bulk proteins
useful in formulating insulin conjugates include albumin,
protamine, or the like. Typical carbohydrates useful in formulating
the PEGylated, extended insulin include sucrose, mannitol, lactose,
trehalose, glucose, or the like. The PEGylated, extended insulins
formulation can also include a surfactant, which can reduce or
prevent surface-induced aggregation of the insulin conjugate caused
by atomization of the solution in forming an aerosol. Various
conventional surfactants can be employed, such as polyoxyethylene
fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty
acid esters. Amounts will generally range between about 0.001 and
about 4% by weight of the formulation.
[0108] Pharmaceutical compositions containing a PEGylated, extended
insulin of this invention may also be administered parenterally to
patients in need of such a treatment. Parenteral administration may
be performed by subcutaneous, intramuscular or intravenous
injection by means of a syringe, optionally a pen-like syringe.
Alternatively, parenteral administration can be performed by means
of an infusion pump.
[0109] Injectable compositions of the PEGylated, extended insulins
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, a PEGylated, extended insulin is
dissolved in an amount of water which is somewhat less than the
final volume of the composition to be prepared. Zink, an isotonic
agent, a preservative and/or a buffer is/are added as required and
the pH value of the solution is adjusted--if necessary--using an
acid, e.g., hydrochloric acid, or a base, e.g., aqueous sodium
hydroxide as needed. Finally, the volume of the solution is
adjusted with water to give the desired concentration of the
ingredients.
[0110] In a further embodiment of this invention the buffer is
selected from the group consisting of sodium acetate, sodium
carbonate, citrate, glycylglycine, histidine, glycine, lysine,
arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate,
sodium phosphate, and tris(hydroxymethyl)aminomethan, bicine,
tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric
acid, aspartic acid or mixtures thereof. Each one of these specific
buffers constitutes an alternative embodiment of this
invention.
[0111] In a further embodiment of this invention the formulation
further comprises a pharmaceutically acceptable preservative which
may be selected from the group consisting of phenol, o-cresol,
m-cresol, p-cresol, methyl p-hydroxybenzoate, propyl
p-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate,
2-phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal,
bronopol, benzoic acid, imidurea, chlorohexidine, sodium
dehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethonium
chloride, chlorphenesine (3-(4-chlorophenoxy)-1,2-propanediol) or
mixtures thereof. In a further embodiment of this invention the
preservative is present in a concentration from about 0.1 mg/ml to
20 mg/ml. In a further embodiment of this invention the
preservative is present in a concentration from about 0.1 mg/ml to
5 mg/ml. In a further embodiment of this invention the preservative
is present in a concentration from about 5 mg/ml to 10 mg/ml. In a
further embodiment of this invention the preservative is present in
a concentration from about 10 mg/ml to 20 mg/ml. Each one of these
specific preservatives constitutes an alternative embodiment of
this invention. The use of a preservative in pharmaceutical
compositions is well-known to the skilled person. For convenience
reference is made to Remington: The Science and Practice of
Pharmacy, 1 gth edition, 1995.
[0112] In a further embodiment of this invention, the formulation
further comprises an isotonic agent which may be selected from the
group consisting of a salt (e.g., sodium chloride), a sugar or
sugar alcohol, an amino acid (for example, L-glycine, L-histidine,
arginine, lysine, isoleucine, aspartic acid, tryptophan or
threonine), an alditol (e.g. glycerol (glycerine), 1,2-propanediol
(propyleneglycol), 1,3-propanediol or 1,3-butanediol),
polyethyleneglycol (e.g., PEG400) or mixtures thereof. Any sugar
such as mono-, di-, or polysaccharides, or water-soluble glucans,
including for example fructose, glucose, mannose, sorbose, xylose,
maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin,
cyclodextrin, soluble starch, hydroxyethyl starch and
carboxymethylcellulose-Na may be used. In one embodiment the sugar
additive is sucrose. Sugar alcohol is defined as a C4-C8
hydrocarbon having at least one--OH group and includes, e.g.,
mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and
arabitol. In one embodiment the sugar alcohol additive is mannitol.
The sugars or sugar alcohols mentioned above may be used
individually or in combination. There is no fixed limit to the
amount used, as long as the sugar or sugar alcohol is soluble in
the liquid preparation and does not adversely effect the
stabilizing effects achieved using the methods of this invention.
In one embodiment, the sugar or sugar alcohol concentration is
between about 1 mg/ml and about 150 mg/ml. In a further embodiment
of this invention the isotonic agent is present in a concentration
from about 1 mg/ml to 50 mg/ml. In a further embodiment of this
invention the isotonic agent is present in a concentration from
about 1 mg/ml to 7 mg/ml. In a further embodiment of this invention
the isotonic agent is present in a concentration from about 8 mg/ml
to 24 mg/ml. In a further embodiment of this invention the isotonic
agent is present in a concentration from about 25 mg/ml to 50
mg/ml. Each one of these specific isotonic agents constitutes an
alternative embodiment of this invention. The use of an isotonic
agent in pharmaceutical compositions is well-known to the skilled
person. For convenience reference is made to Remington: The Science
and Practice of Pharmacy, 19.sup.th edition, 1995.
[0113] Typical isotonic agents are sodium chloride, mannitol,
dimethyl sulfone and glycerol and typical preservatives are phenol,
m-cresol, methyl p-hydroxybenzoate and benzyl alcohol.
[0114] Examples of suitable buffers are sodium acetate,
glycylglycine, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid) and sodium phosphate.
[0115] A composition for nasal administration of a PEGylated,
extended insulins of this invention may, e.g., be prepared as
described in European Patent No. 272097.
[0116] Compositions containing PEGylated, extended insulins of this
invention can be used in the treatment of states which are
sensitive to insulin. Thus, they can be used in the treatment of
type 1 diabetes, type 2 diabetes and hyperglycaemia for example as
sometimes seen in seriously injured persons and persons who have
undergone major surgery. The optimal dose level for any patient
will depend on a variety of factors including the efficacy of the
specific insulin derivative employed, the age, body weight,
physical activity, and diet of the patient, on a possible
combination with other drugs, and on the severity of the state to
be treated. It is recommended that the daily dosage of the
PEGylated, extended insulin of this invention be determined for
each individual patient by those skilled in the art in a similar
way as for known insulin compositions.
PREFERRED FEATURES OF THIS INVENTION
[0117] To sum up, the features of this invention are as follows:
[0118] 1. A PEGylated insulin analogue which, compared with human
insulin, has one or more PEG-containing extensions extended from
the A1, B1, A21 and/or B30 position(s), said extension(s)
consist(s) of amino acid residue(s) and wherein the PEG moiety, via
a linker, is attached to one or more of the amino acid residues in
the extension(s). [0119] 2. A PEGylated insulin analogue, according
to clause 1, wherein only one of the amino acid residues in one of
the extensions carries a PEG moiety. [0120] 3. A PEGylated insulin
analogue, according to clause 1, wherein only two of the extensions
carries a PEG moiety, and, preferably, there are only two PEG
moieties. [0121] 4. A PEGylated insulin analogue, according to
clause 1, wherein the extension carrying a PEG moiety is situated
in a position N-terminally to the A1 position. [0122] 5. A
PEGylated insulin analogue, according to clause 1, wherein the
extension carrying a PEG moiety is situated in a position
N-terminally to the B1 position. [0123] 6. A PEGylated insulin
analogue, according to clause 1, wherein the extension carrying a
PEG moiety is situated in a position C-terminally to the A21
position. [0124] 7. A PEGylated insulin analogue, according to
clause 1, wherein the extension carrying a PEG moiety is situated
in a position C-terminally to the B30 position. [0125] 8. A
PEGylated insulin analogue, according to any one of the preceding,
possible clauses, wherein the number of extensions per insulin
molecule is four. [0126] 9. A PEGylated insulin analogue, according
to any one of the preceding, possible clauses, wherein the number
of extensions per insulin molecule is three. [0127] 10. A PEGylated
insulin analogue, according to any one of the preceding, possible
clauses, wherein the number of extensions per insulin molecule is
two. [0128] 11. A PEGylated insulin analogue, according to any one
of the preceding, possible clauses, wherein the number of
extensions per insulin molecule is only one. [0129] 12. A PEGylated
insulin analogue, according to any one of the preceding, possible
clauses, wherein the parent insulin analogue deviates from human
insulin in one or more of the following extensions: G in position
A-3, G in position A-2, K or R in position A-1, G or K in position
A22, G or K in position A23, G or K in position A24, K in position
A25, and K in position B31 and, compared with human insulin, there
is, optionally, up to 12 more mutations among deletion,
substitution and addition of an amino acid residue and, preferably,
there are no further mutations in said insulin analogue. [0130] 13.
A PEGylated insulin analogue, according to any one of the
preceding, possible clauses, wherein the extension consists of one
or more of the following formulae wherein the PEG moiety is
attached to side chain(s) of lysine or cysteine residue(s) when
present or to the N-terminal amino group(s) (or both): -AA.sub.x1K
(for C-terminal extensions), wherein X1 is 0, 1, 2, 3, 4, 5, 6, 7,
or 8 (and wherein K is lysine), K-AA.sub.x2- (for N-terminal
extensions), wherein x2 is 0, 1, 2, 3, 4, 5, 6, 7, or 8 (and
wherein K is lysine), -AA.sub.x3C (for C-terminal extensions),
wherein x3 is 0, 1, 2, 3, 4, 5, 6, 7, or 8 (and wherein C is
cysteine), C-AA.sub.x4- (for N-terminal extensions), wherein x4 is
0, 1, 2, 3, 4, 5, 6, 7, or 8 (and wherein C is cysteine),
AA.sub.x5-R.sub.y- (for N-terminal extensions), wherein x5 is 0, 1,
2, 3, 4, 5, 6, 7, or 8, and y is 0 or 1 (and wherein R is
arginine), and wherein AA is the residue of any codable amino acid
except Lys and Cys, and, preferably, AA is a peptide chain wherein
each of the codable amino acid residues are the same or different.
[0131] 14. A PEGylated insulin analogue, according to any one of
the preceding clauses, wherein AA is a residue of glycine, alanine
or glutamine, and, preferably, AA is a peptide chain wherein each
of the codable amino acid residues are the same or different.
[0132] 15. A PEGylated insulin analogue, according to the preceding
clause, wherein AA is a residue of glycine. [0133] 16. A PEGylated
insulin analogue, according to the preceding clause, wherein AA is
a residue of alanine. [0134] 17. A PEGylated insulin analogue,
according to the preceding clause but one, wherein AA is a residue
of glutamine. [0135] 18. A PEGylated insulin analogue, according to
any one of the preceding, possible clauses, wherein the extension
consists of one or more of the following formulae wherein the PEG
moiety is attached to side chain(s) of lysine or cysteine
residue(s) when present or to the N-terminal amino group(s) (or
both): -G.sub.x1K (for C-terminal extensions), wherein X1 is 0, 1,
2, 3, 4, 5, 6, 7, or 8 (and wherein G and K are glycine and lysine,
respectively), K-G.sub.x2- (for N-terminal extensions), wherein x2
is 0, 1, 2, 3, 4, 5, 6, 7, or 8 (and wherein G and K are glycine
and lysine, respectively), -G.sub.x3C (for C-terminal extensions),
wherein x3 is 0, 1, 2, 3, 4, 5, 6, 7, or 8 (and wherein G and C are
glycine and cysteine, respectively), C-G.sub.x4- (for N-terminal
extensions), wherein x4 is 0, 1, 2, 3, 4, 5, 6, 7, or 8 (and
wherein G and C are glycine and cysteine, respectively),
G.sub.x5-R.sub.y- (for N-terminal extensions), wherein x5 is 0, 1,
2, 3, 4, 5, 6, 7, or 8, and y is 0 or 1 (and wherein G and R are
glycine and arginine, respectively). [0136] 19. PEGylated insulin
according to anyone of the preceding, possible clauses wherein the
parent insulin, optionally contains one or more of the following
mutations: A14E/D, A18Q, A21G/A/Q, desB1, B1G/Q, B3Q/S/T, B13Q,
desB25, B25H, desB27, B28D/E/R, des B29, B29P/Q/R or desB30. [0137]
20. A PEGylated insulin analogue, according to any one of the
preceding, possible clauses, wherein the parent insulin analogue
deviates from human insulin in having A22K, B29R and desB30 and,
preferably, there are no further mutations in said insulin
analogue. [0138] 21. A PEGylated insulin analogue, according to any
one of the preceding, possible clauses, wherein the parent insulin
analogue deviates from human insulin in having A22G, A23K, B29R and
desB30 and, preferably, there are no further mutations in said
insulin analogue. [0139] 22. A PEGylated insulin analogue,
according to any one of the preceding, possible clauses, wherein
the parent insulin analogue deviates from human insulin in having
A22G, A23G, A24K, B29R and desB30 and, preferably, there are no
further mutations in said insulin analogue. [0140] 23. A PEGylated
insulin analogue, according to any one of the preceding, possible
clauses, wherein the parent insulin analogue deviates from human
insulin in having A22G, A23G, A24G, A25K, B29R and desB30 and,
preferably, there are no further mutations in said insulin
analogue. [0141] 24. A PEGylated insulin analogue, according to any
one of the preceding, possible clauses, wherein the parent insulin
analogue deviates from human insulin in having A22K, B3Q, B29R and
desB30 and, preferably, there are no further mutations in said
insulin analogue. [0142] 25. A PEGylated insulin analogue,
according to any one of the preceding, possible clauses, wherein
the parent insulin analogue deviates from human insulin in having
A22K, B3S, B29R and desB30 and, preferably, there are no further
mutations in said insulin analogue. [0143] 26. A PEGylated insulin
analogue, according to any one of the preceding, possible clauses,
wherein the parent insulin analogue deviates from human insulin in
having A22K, B3T, B29R and desB30 and, preferably, there are no
further mutations in said insulin analogue. [0144] 27. A PEGylated
insulin analogue, according to any one of the preceding, possible
clauses, wherein the parent insulin analogue deviates from human
insulin in having A22K, B1Q, B29R and desB30 and, preferably, there
are no further mutations in said insulin analogue. [0145] 28. A
PEGylated insulin analogue, according to any one of the preceding,
possible clauses, wherein the parent insulin analogue deviates from
human insulin in having A18Q, A22K, B29R and desB30 and,
preferably, there are no further mutations in said insulin
analogue. [0146] 29. A PEGylated insulin analogue, according to any
one of the preceding, possible clauses, wherein the parent insulin
analogue deviates from human insulin in having A22K, B3Q, B29R,
desB1 and desB30 and, preferably, there are no further mutations in
said insulin analogue. [0147] 30. A PEGylated insulin analogue,
according to any one of the preceding, possible clauses, wherein
the parent insulin analogue deviates from human insulin in having
B29Q and B31K and, preferably, there are no further mutations in
said insulin analogue. [0148] 31. A PEGylated insulin analogue,
according to any one of the preceding, possible clauses, wherein
the parent insulin analogue deviates from human insulin in having
A21G, B29Q and B31K and, preferably, there are no further mutations
in said insulin analogue. [0149] 32. A PEGylated insulin analogue,
according to any one of the preceding, possible clauses, wherein
the parent insulin analogue deviates from human insulin in having
A21A, B29Q and B31K and, preferably, there are no further mutations
in said insulin analogue. [0150] 33. A PEGylated insulin analogue,
according to any one of the preceding, possible clauses, wherein
the parent insulin analogue deviates from human insulin in having
A21Q, B29Q and B31K and, preferably, there are no further mutations
in said insulin analogue. [0151] 34. A PEGylated insulin analogue,
according to any one of the preceding, possible clauses, wherein
the parent insulin analogue deviates from human insulin in having
A-1K and desB30 and, preferably, there are no further mutations in
said insulin analogue. [0152] 35. A PEGylated insulin analogue,
according to any one of the preceding, possible clauses, wherein
the parent insulin analogue deviates from human insulin in having
A-1K, B29R and desB30 and, preferably, there are no further
mutations in said insulin analogue. [0153] 36. A PEGylated insulin
analogue, according to any one of the preceding, possible clauses,
wherein the parent insulin analogue deviates from human insulin in
having A-3G, A-2G, A-1R and desB30 and, preferably, there are no
further mutations in said insulin analogue. [0154] 37. A PEGylated
insulin analogue, according to any one of the preceding, possible
clauses, wherein the parent insulin analogue deviates from human
insulin in having A22K, B28E, B29R and desB30 and, preferably,
there are no further mutations in said insulin analogue. [0155] 38.
A PEGylated insulin analogue, according to any one of the
preceding, possible clauses, wherein the parent insulin analogue
deviates from human insulin in having A22K, B28D, B29R and desB30
and, preferably, there are no further mutations in said insulin
analogue. [0156] 39. A PEGylated insulin analogue, according to any
one of the preceding, possible clauses, wherein the parent insulin
analogue deviates from human insulin in having A22K, B28E, B29R,
desB27 and desB30 and, preferably, there are no further mutations
in said insulin analogue. [0157] 40. A PEGylated insulin analogue,
according to any one of the preceding, possible clauses, wherein
the parent insulin analogue deviates from human insulin in having
B28E, B29Q and B31K and, preferably, there are no further mutations
in said insulin analogue. [0158] 41. A PEGylated insulin analogue,
according to any one of the preceding, possible clauses, wherein
the parent insulin analogue deviates from human insulin in having
B28E, B29Q, B31K and desB27 and, preferably, there are no further
mutations in said insulin analogue. [0159] 42. A PEGylated insulin
analogue, according to any one of the preceding, possible clauses,
wherein the parent insulin analogue deviates from human insulin in
having A22K, B28R, desB29 and desB30 and, preferably, there are no
further mutations in said insulin analogue. [0160] 43. A PEGylated
insulin analogue, according to any one of the preceding, possible
clauses, wherein the parent insulin analogue deviates from human
insulin in having B28R, B29P and B31K and, preferably, there are no
further mutations in said insulin analogue. [0161] 44. A PEGylated
insulin analogue, according to any one of the preceding, possible
clauses, wherein the parent insulin analogue deviates from human
insulin in having A22K, B3Q, B28E, B29R and desB30 and, preferably,
there are no further mutations in said insulin analogue. [0162] 45.
A PEGylated insulin analogue, according to any one of the
preceding, possible clauses, wherein the parent insulin analogue
deviates from human insulin in having A21G, B3Q, B28E, B29Q and
B31K and, preferably, there are no further mutations in said
insulin analogue. [0163] 46. A PEGylated insulin analogue,
according to any one of the preceding, possible clauses, wherein
the parent insulin analogue deviates from human insulin in having
A22K, B13Q, B29R and desB30 and, preferably, there are no further
mutations in said insulin analogue. [0164] 47. A PEGylated insulin
analogue, according to any one of the preceding, possible clauses,
wherein the parent insulin analogue deviates from human insulin in
having A22K, B29R, desB1 and desB30 and, preferably, there are no
further mutations in said insulin analogue. [0165] 48. A PEGylated
insulin analogue, according to any one of the preceding, possible
clauses, wherein the parent insulin analogue deviates from human
insulin in having A14E, A22K, B25H and desB30 and, preferably,
there are no further mutations in said insulin analogue. [0166] 49.
A PEGylated insulin analogue, according to any one of the
preceding, possible clauses, wherein the parent insulin analogue
deviates from human insulin in having A14E, B25H, B29Q and B31K
and, preferably, there are no further mutations in said insulin
analogue. [0167] 50. A PEGylated insulin analogue, according to any
one of the preceding, possible clauses, wherein the parent insulin
analogue deviates from human insulin in having A13E, A22K, B25H and
desB30 and, preferably, there are no further mutations in said
insulin analogue. [0168] 51. A PEGylated insulin analogue,
according to any one of the preceding, possible clauses, wherein
the parent insulin analogue deviates from human insulin in having
A21Q, A22K, B29R and desB30 and, preferably, there are no further
mutations in said insulin analogue. [0169] 52. A PEGylated insulin
analogue, according to any one of the preceding, possible clauses,
wherein the parent insulin analogue deviates from human insulin in
having A21Q, A22G, A23K, B29R and desB30 and, preferably, there are
no further mutations in said insulin analogue. [0170] 53. A
PEGylated insulin analogue, according to any one of the preceding,
possible clauses, wherein the parent insulin analogue deviates from
human insulin in having A21Q, A22G, A23G, A24K, B29R and desB30
and, preferably, there are no further mutations in said insulin
analogue. [0171] 54. A PEGylated insulin analogue, according to any
one of the preceding, possible clauses, wherein the parent insulin
analogue deviates from human insulin in having A21Q, A22G, A23G,
A24G, A25K, B29R and desB30 and, preferably, there are no further
mutations in said insulin analogue.
[0172] 55. A PEGylated insulin analogue, according to any one of
the preceding, possible clauses, wherein the parent insulin
analogue deviates from human insulin in having A21A, A22K, B29R and
desB30 and, preferably, there are no further mutations in said
insulin analogue. [0173] 56. A PEGylated insulin analogue,
according to any one of the preceding, possible clauses, wherein
the parent insulin analogue deviates from human insulin in having
A21A, A22G, A23K, B29R and desB30 and, preferably, there are no
further mutations in said insulin analogue. [0174] 57. A PEGylated
insulin analogue, according to any one of the preceding, possible
clauses, wherein the parent insulin analogue deviates from human
insulin in having A21A, A22G, A23G, A24K, B29R and desB30 and,
preferably, there are no further mutations in said insulin
analogue. [0175] 58. A PEGylated insulin analogue, according to any
one of the preceding, possible clauses, wherein the parent insulin
analogue deviates from human insulin in having A21A, A22G, A23G,
A24G, A25K, B29R and desB30 and, preferably, there are no further
mutations in said insulin analogue. [0176] 59. A PEGylated insulin
analogue, according to any one of the preceding, possible clauses,
wherein the parent insulin analogue deviates from human insulin in
having A21G, A22K, B29R and desB30 and, preferably, there are no
further mutations in said insulin analogue. [0177] 60. A PEGylated
insulin analogue, according to any one of the preceding, possible
clauses, wherein the parent insulin analogue deviates from human
insulin in having A21G, A22G, A23K, B29R and desB30 and,
preferably, there are no further mutations in said insulin
analogue. [0178] 61. A PEGylated insulin analogue, according to any
one of the preceding, possible clauses, wherein the parent insulin
analogue deviates from human insulin in having A21G, A22G, A23G,
A24K, B29R and desB30 and, preferably, there are no further
mutations in said insulin analogue. [0179] 62. A PEGylated insulin
analogue, according to any one of the preceding, possible clauses,
wherein the parent insulin analogue deviates from human insulin in
having A21G, A22G, A23G, A24G, A25K, B29R and desB30 and,
preferably, there are no further mutations in said insulin
analogue. [0180] 63. A PEGylated insulin analogue, according to any
one of the preceding, possible clauses, wherein the parent insulin
analogue deviates from human insulin in having A21Q, A22K, B3Q,
B29R and desB30 and, preferably, there are no further mutations in
said insulin analogue. [0181] 64. A PEGylated insulin analogue,
according to any one of the preceding, possible clauses, wherein
the parent insulin analogue deviates from human insulin in having
A21G, A22K, B3Q, B29R and desB30 and, preferably, there are no
further mutations in said insulin analogue. [0182] 65. A PEGylated
insulin analogue, according to any one of the preceding, possible
clauses, wherein the parent insulin analogue deviates from human
insulin in having A21A, A22K, B3Q, B29R and desB30 and, preferably,
there are no further mutations in said insulin analogue. [0183] 66.
A PEGylated insulin analogue, according to any one of the
preceding, possible clauses, wherein the parent insulin analogue
deviates from human insulin in having A14E, A22K, B25H, B29R and
desB30 and, preferably, there are no further mutations in said
insulin analogue. [0184] 67. A PEGylated insulin analogue,
according to any one of the preceding, possible clauses, wherein
said PEG containing group is attached to an --NH-- group of a
lysine residue and/or to a cysteine residue present in the
extension(s) and/or attached N-terminally to the extension(s).
[0185] 68. A PEGylated insulin analogue, according to any one of
the preceding, possible clauses, comprising the moiety
--(OCH.sub.2CH.sub.2).sub.n--, wherein n is in integer in the range
from 2 to about 1000, preferably from 2 to about 500, preferably
from 2 to about 250, preferably from 2 to about 125, preferably
from 2 to about 50, and preferably from 2 to about 25. [0186] 69. A
PEGylated insulin analogue, according to any one of the preceding,
possible clauses, wherein the polyethylene glycol moiety has a
nominal molecular weight in the range from about 200 to about
40,000, preferably from about 200 to about 30,000, preferably from
about 200 to about 20,000, preferably from about 200 to about
10,000, preferably from about 200 to about 5,000, preferably from
about 200 to about 2,000, preferably from about 200 to about 1,000,
and preferably from about 200 to about 750. [0187] 70. A PEGylated
insulin analogue, according to any one of the preceding, possible
clauses, wherein the polyethylene glycol moiety is monodisperse.
[0188] 71. A PEGylated insulin analogue, according to the preceding
clause, wherein the polyethylene glycol moiety has the general
formula --(CH.sub.2CH.sub.2O).sub.n--, wherein n is in an integer
which is at least about 6, preferably at least about 10, and not
more than about 110, preferably not more than about 75, and even
more preferred n is in the range from about 6 to about 30,
preferably in the range from about 10 to about 48. [0189] 72. A
PEGylated insulin analogue, according to any one of the preceding
possible clauses, wherein the polyethylene glycol moiety is
polydisperse. [0190] 73. A PEGylated insulin analogue, according to
any one of the preceding, possible clauses, wherein the
polyethylene glycol moiety is linear, branched, forked or dumbbell
shaped. [0191] 74. A PEGylated insulin analogue, according to any
one of the preceding, possible clauses, comprising a group of the
general formula -Q.sup.1-(OCH.sub.2CH.sub.2).sub.n--R.sup.1 wherein
Q.sup.1 is a linker connecting the polyethylene glycol moiety to an
.alpha.- or .gamma.-NH-group of an amino acid in the extension,
preferably via an amide or a carbamate bond, n is an integer in the
range from 2 to about 1000, and R.sup.1 is alkoxy or hydroxyl,
preferably methoxy. [0192] 75. A PEGylated insulin analogue,
according to the preceding clause, wherein n is an integer in the
range from 2 to about 500, preferably from 2 to about 500,
preferably from 2 to about 250, preferably from 2 to about 125,
preferably from 2 to about 50, and preferably from 2 to about 25.
[0193] 76. A PEGylated insulin analogue, according to any one of
the preceding, possible clauses, wherein Q.sup.1 is -alkylene-CO--,
which is connected to the --NH-- residue of the extended insulin
via the carbonyl group. [0194] 77. A PEGylated insulin analogue,
according to the preceding clause, wherein Q.sup.1 is ethylene
carbonyl ((CH.sub.2).sub.2--CO--), which is connected to the --NH--
residue via the carbonyl group. [0195] 78. A PEGylated insulin
analogue, according to any one of the preceding, possible clauses
except the two last, wherein Q.sup.1 is
-alkylene-NHCO-alkylene-CO--, which is connected to the --NH--
residue of the extended insulin via the carbonyl group. [0196] 79.
A PEGylated insulin analogue, according to any one of the
preceding, possible clauses except the three last, wherein Q.sup.1
is --CO-alkylene-CO--. [0197] 80. A PEGylated insulin analogue,
according to any one of the preceding, possible clauses except the
four last, wherein Q.sup.1 is --CO-(4-nitrophenoxy). [0198] 81. A
PEGylated insulin analogue, according to any one of the preceding,
possible clauses except the five last, wherein Q.sup.1 is
(-alkylene-NHCO-alkylene-O-alkylene-).sub.pCH.sub.q--NHCO-alkylene-(OCH.s-
ub.2CH.sub.2).sub.r--NHCO-alkylene-CO--, wherein p is 1, 2 or 3, q
is 0, 1 or 2, p+q is 3, and r is an integer in the range from 1 to
about 12, which is connected to the --NH-- residue of the extended
insulin via the carbonyl group. [0199] 82. A PEGylated insulin
analogue, according to any on of the preceding, possible clauses,
wherein Q.sup.1 is --CH.sub.2CO--, --CH.sub.2CH.sub.2CO--,
--CH.sub.2CH.sub.2CH.sub.2CO--, --CH.sub.2CH(CH.sub.3)CO--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CO--,
--CH.sub.2CH.sub.2CH(CH.sub.3)CO--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CO--,
--CH.sub.2CH.sub.2NH--COCH.sub.2CH.sub.2CO--,
--CH.sub.2CH.sub.2NH--COCH.sub.2CH.sub.2CH.sub.2CO--,
--CH.sub.2CH.sub.2CH.sub.2NH--COCH.sub.2CH.sub.2CO--,
--CH.sub.2CH.sub.2CH.sub.2NH--COCH.sub.2CH.sub.2CH.sub.2CO--,
--COCH.sub.2CH.sub.2CO--, --COCH.sub.2CH.sub.2CH.sub.2CO--,
--CO-(4-nitrophenoxy),
(--CH.sub.2CH.sub.2NHCOCH.sub.2CH.sub.2O--CH.sub.2).sub.3CNHCOCH.sub.2CH.-
sub.2(OCH.sub.2CH.sub.2).sub.4NHCOCH.sub.2CH.sub.2CO-- or
(--CH.sub.2CH.sub.2NHCOCH.sub.2CH.sub.2OCH.sub.2).sub.3CNH--COCH.sub.2CH.-
sub.2(OCH.sub.2CH.sub.2).sub.4NHCOCH.sub.2CH.sub.2CH.sub.2CO--.
[0200] 83. A PEGylated insulin analogue, according to any one of
the preceding, possible clauses, wherein R.sup.1 is alkoxy. [0201]
84. A PEGylated insulin analogue, according to the preceding
clause, wherein R.sup.1 is methoxy. [0202] 85. A compound according
to any one of the preceding product clauses, which is any one of
the compounds mentioned specifically in the above specification
such as in the specific examples, especially any one of the
examples 1 et seq. above [0203] 86. The use of a compound according
to any one of the preceding product clauses for the preparation of
a pharmaceutical composition for the treatment of diabetes. [0204]
87. The use of a compound according to any one of the preceding
product clauses for the preparation of a pharmaceutical composition
which can be administered pulmonary for the treatment of diabetes.
[0205] 88. The use of a compound according to any one of the
preceding product clauses for the preparation of a pharmaceutical
composition which can be administered pulmonary for the treatment
of diabetes and which gives a long acting effect. [0206] 89. The
use of a compound according to any one of the preceding product
clauses for the preparation of a powder pharmaceutical composition
which can be administered pulmonary for the treatment of diabetes.
[0207] 90. The use of a compound according to any one of the
preceding product clauses for the preparation of a liquid
pharmaceutical composition which can be administered pulmonary for
the treatment of diabetes. [0208] 91. A method of treatment of
diabetes, the method comprising administering to a subject in need
thereof a therapeutically effective amount of a compound according
to any one of the preceding product clauses. [0209] 92. A
composition containing human insulin as well as a PEGylated insulin
analogue according to any one of the preceding clauses. [0210] 93.
A composition containing insulin aspart as well as a PEGylated
insulin analogue according to any one of the preceding clauses.
[0211] 94. A composition containing insulin Lispro as well as a
PEGylated insulin analogue according to any one of the preceding
clauses. [0212] 95. A composition containing insulin Glulisine as
well as a PEGylated insulin analogue according to any one of the
preceding clauses. [0213] 96. Any novel feature or combination of
features described herein.
General Comments
[0214] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference in
their entirety and to the same extent as if each reference were
individually and specifically indicated to be incorporated by
reference and were set forth in its entirety herein (to the maximum
extent permitted by law).
[0215] All headings and sub-headings are used herein for
convenience only and should not be construed as limiting this
invention in any way.
[0216] The use of any and all examples, or exemplary language
(e.g., "such as") provided herein, is intended merely to better
illuminate this invention and does not pose a limitation on the
scope of this invention unless otherwise claimed. No language in
the specification should be construed as indicating any non-claimed
element as essential to the practice of this invention.
[0217] The citation and incorporation of patent documents herein is
done for convenience only and does not reflect any view of the
validity, patentability, and/or enforceability of such patent
documents. The mentioning herein of references is no admission that
they constitute prior art.
[0218] Herein, the word "comprise" is to be interpreted broadly
meaning "include", "contain" or "comprehend" (EPO guidelines C
4.13).
[0219] This invention includes all modifications and equivalents of
the subject matter recited in the claims appended hereto as
permitted by applicable law.
[0220] Combining one or more of the embodiments described herein,
optionally also with one or more of the claims below, results in
further embodiments and this invention relates to all possible
combinations of said embodiments and claims.
[0221] For the sake of completeness, it is to be noted that this
invention does not relate to PEGylated insulin analogues wherein
the parent insulin analogue is so-called single-chain insulin
(Danish appl. No.: 2005/00400 and WO appl. No.: EP2006/060816; our
ref.: 7148). Hence, in this invention, we are hereby disclaiming
the content thereof which is incorporated by reference.
[0222] The following examples are offered by way of illustration,
not by limitation.
[0223] In the following list, selected PEGylation reagents are
listed as activated N-hydroxysuccinimide esters (OSu). Obviously,
other active esters may be employed, such as 4-nitrophenoxy and
many other active esters known to those skilled in the art. The PEG
(or mPEG) moiety, CH.sub.3O--(CH.sub.2CH.sub.2O).sub.n--, can be of
any size up to Mw 40.000 Da, e.g., 750 Da, 2000 Da, 5000 Da, 20.000
Da and 40.000 Da. The mPEG moiety can be polydisperse but also
monodisperse consisting of mPEG's with well defined chain lengths
(and, thus, molecular weights) of, e.g., 12 or 24 repeating
ethylene glycol units--denoted mdPEGx for m: methyl/methoxy
end-capped, d: discrete and x for the number of repeating ethylene
glucol residues, e.g., 12 or 24. The PEG moiety can be either
straight chain or branched. The structure/sequence of the
PEG-residue on the extended insulin can formally be obtained by
replacing the leaving group (e.g., "--OSu") from the various
PEGylation reagents with "NH-insulin", where the insulin is
PEGylated either in an epsilon position in a lysine residue or in
the alpha-amino position in the A- or B-chain (or both):
mPEG-COCH.sub.2CH.sub.2CO--OSu,
mPEG-COCH.sub.2CH.sub.2CH.sub.2CO--OSu, mPEG-CH.sub.2CO--OSu,
mPEG-CH.sub.2CH.sub.2CO--OSu, mPEG-CH.sub.2CH.sub.2CH.sub.2CO--OSu,
mPEG-CH.sub.2CH.sub.2CH.sub.2CH.sub.2CO--OSu,
mPEG-CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CO--OSu,
mPEG-CH.sub.2CH(CH.sub.3)CO--OSu,
mPEG-CH.sub.2CH.sub.2CH(CH.sub.3)CO--OSu,
mPEG-CH.sub.2CH.sub.2NH--COCH.sub.2CH.sub.2CO--OSu,
mPEG-CH.sub.2CH.sub.2CH.sub.2NH--COCH.sub.2CH.sub.2CH.sub.2CO--OSu,
mPEG-CH.sub.2CH.sub.2CH.sub.2NH--COCH.sub.2CH.sub.2CO--OSu,
mPEG-CH.sub.2CH.sub.2NH--COCH.sub.2CH.sub.2CH.sub.2CO--OSu,
mPEG-CO-(4-nitrophenoxy),
(mdPEG.sub.12-CH.sub.2CH.sub.2NHCOCH.sub.2CH.sub.2OCH.sub.2).sub.3CNHCOCH-
.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.4NHCOCH.sub.2CH.sub.2CO--OSu
(or, in short: (mdPEG.sub.12)-3-dPEG.sub.4-OSu),
(mdPEG.sub.12-CH.sub.2CH.sub.2NHCOCH.sub.2CH.sub.2OCH.sub.2).sub.3CNHCOCH-
.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.4NHCOCH.sub.2CH.sub.2CH.sub.2CO--OSu
(or, in short: (mdPEG.sub.12)-3-dPEG.sub.4-OSu),
mdPEGx-COCH.sub.2CH.sub.2CO--OSu,
mdPEGx-COCH.sub.2CH.sub.2CH.sub.2CO--OSu, mdPEGx-CH.sub.2CO--OSu,
mdPEGx-CH.sub.2CH.sub.2CO--OSu,
mdPEGx-CH.sub.2CH.sub.2CH.sub.2CO--OSu,
mdPEGx-CH.sub.2CH.sub.2CH.sub.2CH.sub.2CO--OSu,
mdPEGx-CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CO--OSu,
mdPEGx-CH.sub.2CH(CH.sub.3)CO--OSu,
mdPEGx-CH.sub.2CH.sub.2CH(CH.sub.3)CO--OSu,
mdPEGx-CH.sub.2CH.sub.2NH--COCH.sub.2CH.sub.2CO--OSu,
mdPEGx-CH.sub.2CH.sub.2CH.sub.2NH--COCH.sub.2CH.sub.2CH.sub.2CO--OSu,
mdPEGx-CH.sub.2CH.sub.2CH.sub.2NH--COCH.sub.2CH.sub.2CO--OSu,
mdPEGx-CH.sub.2CH.sub.2NH--COCH.sub.2CH.sub.2CH.sub.2CO--OSu,
mdPEGx-CO-(4-nitrophenoxy), wherein x is an integer in the range
from about 6 to about 48, e.g., 12 or 24.
[0224] In addition, larger PEGylation reagents can be prepared by
assembling two or more smaller PEG reagents. For example,
end-capped PEG reagents as N-hydroxysuccinimide esters like any of
the ones above can be coupled to--optionally protected--PEG
moieties that are functionalised by amino-groups in one end and
carboxylic acid (esters) in the other end. After deprotection of
the carboxylic acid (if necessary) the carboxylic acid is activated
eg. as the N-hydroxysuccinimide ester to furnish a longer
PEGylation reagent. If desired, the obtained PEGylation reagent can
be further extended by repeating the cycle one or more times. This
principle and methodology is illustrated in the examples.
[0225] This methodology enables construction of larger monodisperse
(and polydisperse) PEGylation reagents of tailored sizes.
[0226] Examples of PEG residues of the invention includes:
mPEG750 (where "750" indicates the average molecular weight in Da),
mPEG2000, mPEG5000, mPEG10000, mPEG20000, mPEG30000, mPEG40000,
mdPEG.sub.12, (wherein "12" in subscript indicates the number of
PEG monomers--as defined herein and eg. by Quanta BioDesign Ltd.)
mdPEG.sub.24, mdPEG.sub.3x12 (wherein "3.times.12" in subscript
indicates that PEG is branched and composed of 3 arms each composed
of 12 PEG monomers--as defined herein and eg. by Quanta BioDesign
Ltd.), mdPEG.sub.12-dPEG.sub.12, mdPEG.sub.12-dPEG.sub.24,
mdPEG.sub.24-dPEG.sub.12, mdPEG.sub.24-dPEG.sub.24,
mdPEG.sub.24-dPEG.sub.24-dPEG.sub.24, mdPEG.sub.3x12-dPEG.sub.12,
mdPEG.sub.3x12-dPEG.sub.24-dPEG.sub.24
[0227] In the following, selected PEGylation reagents are listed as
maleimide derivatives. Obviously, as alternatives to the maleimide
group, other Michael acceptors may be employed, such as
vinylsulfones and many other Michael acceptors known to those
skilled in the art. The PEG (or mPEG) moiety,
CH.sub.3O--(CH.sub.2CH.sub.2O).sub.n--, can be of any size up to Mw
about 40.000 Da. The structure/sequence of the PEG-residue on the
extended insulin can formally be obtained by replacing the
maleimide "MAL" from the various PEGylation reagents with
"3-thio-succinimidyl-Ala-insulin", where the insulin is PEGylated
at a free cysteine residue according to the scheme below:
##STR00001##
[0228] This scheme illustrates PEGylation on a terminal Cys.
Obviously, Cys need not be placed terminally to enable
PEGylation.
Example of PEG-MAL: mPEG-MAL.
[0229] The PEGylated, extended insulins of this invention have in
the following all been named as if the linker connecting the PEG
moiety to the insulin in all cases is a (3-)propionyl linker
(--CH.sub.2--CH.sub.2--CO--). It is evident from the foregoing that
many types of linkers are commercially available and since it is
not the exact structure/composition of the linker that governs the
beneficial effects of placing the PEG moiety at residues outside
the sequence of regular insulin, it is to be understood that all
types of linkers (cf. above) are within the scope of this
invention.
[0230] Parent extended insulins of the invention comprise the
following:
A22K, B29R, desB30 human insulin; A21Q, A22G, A23K, B29R, desB30
human insulin; A21G, A22G, A23K, B29R, desB30 human insulin; A22G,
A23K, B29R, desB30 human insulin; A21Q, A22G, A23G, A24K, B29R,
desB30 human insulin; A21G, A22G, A23G, A24K, B29R, desB30 human
insulin; A21Q, A22G, A23G, A24G, A25K, B29R, desB30 human insulin;
A21G, A22G, A23G, A24G, A25K, B29R, desB30 human insulin; A21G,
A22K, B29R, desB30 human insulin; A21G, A22G, A23K, B29R, desB30
human insulin; A21G, A22G, A23G, A24K, B29R, desB30 human insulin;
A21G, A22G, A23G, A24G, A25K, B29R, desB30 human insulin; A21Q,
A22K, B29R, desB30 human insulin; A21Q, A22G, A23K, B29R, desB30
human insulin; A21Q, A22G, A23G, A24K, B29R, desB30 human insulin;
A21Q, A22G, A23G, A24G, A25K, B29R, desB30 human insulin; A14E,
A22K, B25H, B29R, desB30 human insulin; A14E, A21Q, A22K, B25H,
B29R, desB30 human insulin; A14E, A21G, A22K, B25H, B29R, desB30
human insulin; A14E, A21Q, A22G, A23K, B25H, B29R, desB30 human
insulin; A14E, A21G, A22G, A23K, B25H, B29R, desB30 human insulin;
A14E, A21Q, A22G, A23G, A24K, B25H, B29R, desB30 human insulin;
A14E, A21G, A22G, A23G, A24K, B25H, B29R, desB30 human insulin;
A14E, A21Q, A22G, A23G, A24G, A25K, B25H, B29R, desB30 human
insulin; A14E, A21G, A22G, A23G, A24G, A25K, B25H, B29R, desB30
human insulin; B29Q, B31K human insulin; A22K, B3Q, B29R, desB30
human insulin; A22K, B3S, B29R, desB30 human insulin; A22K, B3T,
B29R, desB30 human insulin; A22K, B1Q, B29R, desB30 human insulin;
A18Q, A22K, B29R, desB30 human insulin; A22K, desB1, B3Q, B29R,
desB30 human insulin; A21G, B29Q, B31K human insulin; A21A, B29Q,
B31K human insulin; A21Q, B29Q, B31K human insulin; A-1K, desB30
human insulin; A-1K, B29R, desB30 human insulin; A-3G, A-2G, A-1R
desB30 human insulin; (for N-terminal A-3-PEGylation) A22K, B28E,
B29R, desB30 human insulin; A22K, B28D, B29R, desB30 human insulin;
A22K, desB27, B28E, B29R, desB30 human insulin; B28E, B29Q, B31K
human insulin; desB27, B28E, B29Q, B31K human insulin; A22K, B28R,
desB29, desB30 human insulin; B28R, B29P, B31K human insulin; A22K,
B3Q, B28E, B29R, desB30 human insulin; A21G, B3Q, B28E, B29Q, B31K
human insulin; A22K, B13Q, B29R, desB30 human insulin; A22K, desB1,
B29R, desB30 human insulin; A14E, A22K, B25H, desB30 human insulin;
A14E, B25H, B29Q, B31K human insulin; A13E, A22K, B25H, desB30
human insulin; A21Q, A22G, A23K, B29R, desB30 human insulin; A21Q,
A22G, A23G, A24K, B29R, desB30 human insulin; A21Q, A22G, A23G,
A24G, A25K, B29R, desB30 human insulin; A21A, A22K, B29R, desB30
human insulin; A21A, A22G, A23K, B29R, desB30 human insulin; A21G,
A22G, A23K, B29R, desB30 human insulin; A21A, A22G, A23G, A24K,
B29R, desB30 human insulin; A21G, A22G, A23G, A24K, B29R, desB30
human insulin; A21G, A22G, A23G, A24G, A25K, B29R, desB30 human
insulin; A21A, A22G, A23G, A24G, A25K, B29R, desB30 human insulin;
A21Q, A22K, B3Q, B29R, desB30 human insulin; A21A, A22K, B3Q, B29R,
desB30 human insulin; A21G, A22K, B3Q, B29R, desB30 human
insulin.
EXAMPLES
General Procedures
Construction of Expression Vectors, Transformation of the Yeast
Cells, and Expression of the Insulin Precursors of the
Invention
[0231] All expressions plasmids are of the C-POT type, similar to
those described in EP 171142, which are characterized by containing
the Schizosaccharomyces pombe triose phosphate isomerase gene (POT)
for the purpose of plasmid selection and stabilization in S.
cerevisiae. The plasmids also contain the S. cerevisiae triose
phosphate isomerase promoter and terminator. These sequences are
similar to the corresponding sequences in plasmid pKFN1003
(described in WO 90/10075) as are all sequences except the sequence
of the EcoRI-XbaI fragment encoding the fusion protein of the
leader and the insulin product. In order to express different
fusion proteins, the EcoRI-XbaI fragment of pKFN1003 is simply
replaced by an EcoRI-XbaI fragment encoding the leader-insulin
fusion of interest. Such EcoRI-XbaI fragments may be synthesized
using synthetic oligonucleotides and PCR according to standard
techniques.
[0232] Yeast transformants were prepared by transformation of the
host strain S. cerevisiae strain MT663 (MATa/MAT.alpha.
pep4-3/pep4-3 HIS4/his4 tpi::LEU2/tpi::LEU2 Cir.sup.+). The yeast
strain MT663 was deposited in the Deutsche Sammlung von
Mikroorganismen und Zellkulturen in connection with filing WO
92/11378 and was given the deposit number DSM 6278.
[0233] MT663 was grown on YPGaL (1% Bacto yeast extract, 2% Bacto
peptone, 2% galactose, 1% lactate) to an O.D. at 600 nm of 0.6. 100
ml of culture was harvested by centrifugation, washed with 10 ml of
water, recentrifuged and resuspended in 10 ml of a solution
containing 1.2 M sorbitol, 25 mM Na.sub.2EDTA pH=8.0 and 6.7 mg/ml
dithiotreitol. The suspension was incubated at 30.degree. C. for 15
minutes, centrifuged and the cells resuspended in 10 ml of a
solution containing 1.2 M sorbitol, 10 mM Na.sub.2EDTA, 0.1 M
sodium citrate, pH 05.8, and 2 mg Novozym.RTM.234. The suspension
was incubated at 30.degree. C. for 30 minutes, the cells collected
by centrifugation, washed in 10 ml of 1.2 M sorbitol and 10 ml of
CAS (1.2 M sorbitol, 10 mM CaCl.sub.2, 10 mM Tris HCl (pH=7.5) and
resuspended in 2 ml of CAS. For transformation, 1 ml of
CAS-suspended cells was mixed with approx. 0.1 mg of plasmid DNA
and left at room temperature for 15 minutes. 1 ml of (20%
polyethylene glycol 4000, 10 mM CaCl.sub.2, 10 mM Tris HCl, pH=7.5)
was added and the mixture left for a further 30 minutes at room
temperature. The mixture was centrifuged and the pellet resuspended
in 0.1 ml of SOS (1.2 M sorbitol, 33% v/v YPD, 6.7 mM CaCl.sub.2)
and incubated at 30.degree. C. for 2 hours. The suspension was then
centrifuged and the pellet resuspended in 0.5 ml of 1.2 M sorbitol.
Then, 6 ml of top agar (the SC medium of Sherman et al. (1982)
Methods in Yeast Genetics, Cold Spring Harbor Laboratory)
containing 1.2 M sorbitol plus 2.5% agar) at 52.degree. C. was
added and the suspension poured on top of plates containing the
same agarsolidified, sorbitol containing medium. S. cerevisiae
strain MT663 transformed with expression plasmids was grown in YPD
for 72 h at 30.degree. C.
Production, Purification and Characterization of the PEGylated
Insulin Derivatives of the Invention
[0234] A number of insulin precursors were produced as described
above and isolated from the culture medium and purified. The
insulin precursors were PEGylated and processed as described in the
examples below to produce the final insulin derivatives (General
Procedure (A)). Optionally, the precursors can be processed by
trypsin prior to PEGylation (General Procedure (B)). These insulin
derivatives were tested for biological insulin activity as measured
by binding affinity to the human insulin receptor relative to that
of human insulin as described below.
[0235] The following examples refer to intermediate compounds and
final products identified in the specification and in the examples.
The preparation of the insulin derivatives of this invention is
described in detail using the following examples, but the chemical
reactions and purification schemes described are disclosed in terms
of their general applicability to the preparation of the insulin
derivatives of the invention. Occasionally, the reaction may not be
applicable as described to each compound included within the
disclosed scope of the invention. The compounds for which this
occurs will be readily recognised by those skilled in the art. In
these cases the reactions can be successfully performed by
conventional modifications known to those skilled in the art, that
is, by appropriate protection of interfering groups, by changing to
other conventional reagents, or by routine modification of reaction
conditions. Alternatively, other reactions disclosed herein or
otherwise conventional will be applicable to the preparation of the
corresponding compounds of the invention. In all preparative
methods, all starting materials are known or may easily be prepared
from known starting materials. All temperatures are set forth in
degrees Celsius and unless otherwise indicated, all parts and
percentages are by weight when referring to yields and all parts
are by volume when referring to solvents and eluents.
[0236] The insulin derivatives of this invention can be purified by
employing one or more of the following procedures which are typical
within the art. These procedures can--if needed--be modified with
regard to gradients, pH, salts, concentrations, flow, columns and
so forth. Depending on factors such as impurity profile, solubility
of the insulins in question etcetera, these modifications can
readily be recognised and made by a person skilled in the art.
General Procedure (A) for Preparation of PEGylated, Extended
Insulins of this Invention
[0237] The general procedure (A) is outlined below and illustrated
in the first example:
Example 1
General Procedure (A)
[0238] A22K(N.sup..epsilon.-mPEG2000-Propionyl), B29R, desB30 Human
Insulin
##STR00002##
Step 1: Preparation and Purification of the Insulin Precursor
LysA22 ArqB29 B29R desB30 B'A
[0239] The insulin precursor A22K, B29R, desB30, B'A single chain
insulin can be purified as described in the purification steps A to
C below.
Purification step A: Capture
[0240] In step A, 10.75 litres of cleared culture media is diluted
by addition of 4.5 litres of 99% ethanol, to give a total volume of
15.25 litres containing 30 vol % ethanol (conductivity 2.7 mS/cm,
pH=3.4). A 300 ml SP Big Beads Sepharose column (100-300 .mu.m,
Amersham Biosciences) was equilibrated with 1 litre of 0.1 M citric
acid pH 3.5 (flow app. 20 ml/min), before loading the 15.25 litres
of prepared culture media over night (flow app. 10 ml/min). After
loading the column was again washed with 1 litre of 0.1 M citric
acid pH 3.5 followed by 1 liter of 40 vol % ethanol (flow app. 20
ml/min). The bound insulin precursor A22K, B29R, desB30, B'A single
chain insulin was then eluted with 1.5 litres of 0.2 M sodium
acetate, 35 vol % ethanol, pH 5.75 (flow: 1.5 ml/min, volume of
eluted precursor: 400 ml, amount of precursor: 220 mg).
Purification Step B: Reverse-Phase HPLC
[0241] In step B the eluate was evaporated to dryness and the
pellet re-dissolved in 0.25 M acetic acid. The pH was lowered
further to 1.5 immediately before purification by reverse-phase
HPLC on a C18 column (ODDMS C18, 20.times.250 mm, 200 .ANG., 10
.mu.m, FeF Chemicals A/S). Before application to the column the
precursor solution was sterile filtrated (22 .mu.m, Low Protein
Binding Durapore.RTM. (PVDF), Millipore). A gradient from 15% B to
50% B was run over the column, where Buffer A: 0.2 M
(NH.sub.4).sub.2SO.sub.4, 0.04 M ortho-phosphoric acid, 10 vol %
ethanol, pH 2.5 and Buffer B: 70 vol % ethanol. The gradient was
run over 120 min with a flow of 5 ml/min, column temperature at
40.degree. C. The insulin precursor A22K, B29R, desB30, B'A single
chain insulin was eluted and pooled (total volume 75 ml).
Purification Step C: De-Salting by Gelfiltration
[0242] In step C the ethanol content in the eluate from
reverse-phase HPLC was lowered to less than 5 vol % using a rotary
evaporator (new volume: .about.50 ml). A 1000 ml G25 Sephadex
column (5.times.55 cm, Amersham Biosciences) was washed in 0.5 M
acetic acid and the insulin precursor A22K, B29R, desB30, B'A
single chain insulin was then applied to the column and thereby
de-salted by gelfiltration in 0.5 M acetic acid. The insulin
precursor was followed by UV detection at 280 nm, while the salt
was followed by conductivity measurement. Immediately after
de-salting, the insulin precursor was lyophilized.
Step 2: Synthesis of A22K(N.sup..epsilon.-mPEG2000-propionyl),
B29R, desB30 B'A human insulin precursor 0.15 mmol of lyophilized
insulin precursor LysA22 ArgB29 desB30 B'A is dissolved in aqueous
sodium carbonate (3 ml, 100 mM). A solution of the PEGylation
reagent mPEG2000-SPA-OSu in acetonitrile (0.15 mmol in 3 ml) is
added to the solution of the precursor, and the mixture is gently
stirred for 1 hour. The mixture is lyophilised, purified by HPLC
and lyophilised to afford the PEGylated precursor. Step 3:
Conversion to A22K(N.sup..epsilon.-mPEG2000-Propionyl), B29R,
desB30 Human Insulin
[0243] The PEGylated insulin precursor
A22K(N.sup..epsilon.-mPEG2000-propionyl), desB30 B'A single chain
human insulin precursor (3.9 .mu.mol) is dissolved in 4.2 ml 50 mM
glycine, 20 vol % ethanol pH 10.0. 3.6 mg of lyophilized porcine
trypsin (Novo Nordisk A/S) is also dissolved in 3.5 ml 50 mM
glycine, 20 vol % ethanol pH 10.0. Of this trypsin solution 0.5 ml
is then added to the insulin precursor solution (hereby the insulin
precursor is in 200 times excess). The mixture is then incubated at
room temperature for 15 minutes, after which the trypsin activity
is stopped by lowering the pH<3 (pH=2.08 with 0.5 M acetic
acid). The PEGylated insulin analogue
A22K(N.sup..epsilon.-mPEG2000-propionyl), B29R, desB30 human
insulin is then purified (removing trypsin and any un-acylated,
doubly-acylated etc. or un-cleaved insulin molecules) by
reverse-phase HPLC an lyophilised to afford the title insulin.
General Procedure (B) for Preparation of PEGylated, Extended
Insulins of this Invention
[0244] This procedure is quite similar to General Procedure (A).
The order of the individual steps has been changed, so that the
B'A-precursors are cleaved by trypsin prior to PEGylation. The
general procedure (B) is illustrated in the first example.
Example 2
General Procedure (B)
[0245] A22K(N.sup..epsilon.mPEG2000-propionyl), B29R, desB30 Human
Insulin
##STR00003##
[0246] A22K, B29R, desB30 human insulin (125 mg) was dissolved in
0.1 M Na.sub.2CO.sub.3 (2.8 ml). mPEG-SPA 2000 (50 mg) dissolved in
acetonitrile (1.25 ml) was added. pH was adjusted from 10.2 to 10.4
with 0.1 N NaOH. After 50 min more mPEG-SPA 2000 (25 mg) dissolved
in acetonitrile (1.25 ml) was added. After slow stirring for 80
min, water (4.5 ml) was added and pH was adjusted to 5 with 1 N
HCl. The mixture was lyophilized. The title compound was obtained
by preparative HPLC purification. Column: C4, 2 cm. A-Buffer: 0.1%
TFA in MiliQ Water; B-buffer: 0.1% TFA in acetonitrile. Gradient
30-65% B over 30 min. Yield 43 mg.
[0247] MALDI-MS (matrix: sinapinic acid); m/z: 8114.
Example 3
General Procedure (B)
[0248] A22K(N.sup..epsilon.mPEG750-Propionyl, B29R, desB30 Human
Insulin
##STR00004##
[0249] MALDI-MS (matrix: sinapinic acid); m/z: 5862.
Example 4
General Procedure (B)
[0250] A22K(N.sup..epsilon.mdPEG12-Propionyl, B29R, desB30 Human
Insulin
##STR00005##
[0251] MALDI (matrix: sinapinic acid); m/z: 6432.
Example 5
General Procedure (B)
[0252] A22K(N.sup..epsilon.mdPEG.sub.24-Propionyl, B29R, desB30
Human Insulin
##STR00006##
[0253] MALDI (matrix: sinapinic acid); m/z: 6962.
Example 6
General Procedure (B)
[0254] A22K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl),
B29R, desB30 Human Insulin
##STR00007##
[0255] MALDI-MS (matrix: sinapinic acid); m/z: 8167.
Example 7
General Procedure (B)
[0256] A22G, A23G, A24G, A25K(N.sup..epsilon.mPEG750-Propionyl),
B29R, desB30 Human Insulin
##STR00008##
[0257] MALDI-MS (matrix: sinapinic acid); m/z: 6807
Example 8
General Procedure (B)
[0258] A22G, A23K(N.sup..epsilon.mPEG2000-Propionyl), B29R, desB30
human insulin
##STR00009##
[0259] MALDI-MS (matrix: sinapinic acid); m/z: 8170
Example 9
General Procedure (B)
[0260] A22K(N.sup..epsilon.mdPEG.sub.8-Propionyl), B29R, desB30
Human Insulin
##STR00010##
[0261] MALDI-MS (matrix: sinapinic acid); m/z: 6258.
Example 10
General Procedure (B)
[0262] A22K(N.sup..epsilon.mdPEG.sub.4-Propionyl), B29R, desB30
Human Insulin
##STR00011##
[0263] MALDI-MS (matrix: sinapinic acid); m/z: 6082.
Example 11
General Procedure (B)
[0264] A22K(N.sup..epsilon.mPEG5000-Propionyl), B29R, desB30 human
insulin
##STR00012##
[0265] MALDI-MS (matrix: sinapinic acid); m/z: around 11600.
Example 12
General Procedure (B)
[0266] A22K(N.sup..epsilon.mPEG20000-Propionyl), B29R, desB30 human
insulin
##STR00013##
[0267] MALDI-MS (matrix: sinapinic acid); m/z: around 21500.
Example 13
General Procedure (B)
[0268] A14E, A22K(N.sup..epsilon.mdPEG.sub.12-Propionyl), B25H,
B29R, desB30 Human Insulin
##STR00014##
[0269] MALDI-MS (matrix: sinapinic acid); m/z: 7520.
Example 14
General Procedure (B)
[0270] A14E,
A22K(N.sup..epsilon.mdPEG.sub.12-dPEG.sub.24-Propionyl), B25H,
B29R, desB30 human insulin
##STR00015##
[0271] MALDI-MS (matrix: sinapinic acid); m/z: 7520.
[0272] The PEGylation reagent was prepared as described in the
following:
Preparation of
omega-(methoxy-PEG.sub.11-propanoylamino)-PEG.sub.24-Propanoic Acid
(mdPEG.sub.12-dPEG.sub.24 Acid)
##STR00016##
[0274] mdPEG.sub.12 NHS ester (0.457 mmol, Quanta BioDesign Ltd.
Product No 10262) and amino-dPEG.sub.24 tert-butylester (0.416
mmol, Quanta BioDesign, Product No 10311) were dissolved separately
in acetonitrile (each 10 mL) and then the two solutions were mixed,
pH was adjusted with DIPEA to pH 8 (measurement of pH was done
using wet indicator strips). The resulting mixture was stirred at
RT overnight, and subsequently evaporated to dryness, followed by
treatment with TFA/DCM (1/1), 10 mL for 1 h at RT. The mixture was
then evaporated to dryness and stripped twice with DCM. The residue
was purified by HPLC (2 cm, C18 column) using acetonitrile
(AcCN)/0, 1% TFA and water/0, 1% TFA as eluents. Gradient: 10-80%
AcCN/TFA from 5-20 min. Fractions containing the desired compound
were collected, combined and evaporated to dryness resulting in
omega-(methoxy-PEGL-Dropanoylamino)PEG.sub.23-propanoic acid as an
oil (249 mg, 35%).
[0275] LCMS: m/z: 1718 (M+1).sup.+.
Preparation of
omega-(methoxy-PEG.sub.11-propanoylamino)-PEG.sub.24-Propanoic Acid
N-hydroxysuccinimide Ester (mdPEG.sub.12-dPEG.sub.24-NHS or
mdPEG.sub.12-dPEG.sub.24-propanoic Acid OSu Ester)
##STR00017##
[0277]
Omega-(methoxy-PEG.sub.11-propanoylamino)PEG.sub.24-propanoic acid
(249 mg, 0.145 mmol) was dissolved in acetonitrile (10 mL) and pH
was adjusted to 8 by addition of DIPEA (measurement of pH was done
using wet indicator strips). TSTU (48 mg, 0.16 mmol) in
acetonitrile (10 mL) was added and the mixture was stirred at room
temperature for 1.5 h, and evaporated to dryness. The residue was
dissolved in DCM and washed with hydrochloric acid (0.01 M), the
organic phase was dried (MgSO.sub.4), filtered and the filtrate was
evaporated to dryness. The resulting
omega-(methoxy-PEG.sub.11-propanoylamino)PEG.sub.24 propanoic acid
N-hydroxysuccinimide ester was used for coupling to insulin without
further purification.
[0278] LCMS: m/z 1813.8 (M+1).sup.+.
Example 15
General Procedure (B)
[0279] A14E,
A22K(N.sup..epsilon.mdPEG.sub.24-dPEG.sub.12-Propionyl), B25H,
B29R, desB30 Human Insulin
##STR00018##
[0280] MALDI-MS (matrix: sinapinic acid); m/z: 7519.
[0281] The N-hydroxysuccinimide activated PEG reagent was prepared
similarly as described above from mdPEG.sub.24 NHS ester (Quanta
BiodDesign Ltd. Product No 10304) and amino-dPEG.sub.12 tert-butyl
ester (Quanta BioDesign Ltd. Product No 10281) via
omega-(methoxy-PEG.sub.23-propanoylamino)PEG.sub.12 propanoic acid
tert-butyl ester,
omega-(methoxy-PEG.sub.23-propanoylamino)PEG.sub.12 propanoic acid,
and omega-(methoxy-PEG.sub.23-propanoylamino)PEG.sub.12 propanoic
acid NHS ester
[0282] LCMS: m/z 1814 (M+1).sup.+.
Example 16
General Procedure (B)
[0283] A14E, A22K(N.sup..epsilon.mPEG2000-Propionyl), B25H, B29R,
desB30 Human Insulin
##STR00019##
[0284] MALDI-MS (matrix: sinapinic acid); m/z: around 8200.
Example 17
General Procedure (B)
[0285] A14E,
A22K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl), B25H,
B29R, desB30 Human Insulin
##STR00020##
[0286] MALDI-MS (matrix: sinapinic acid); m/z: 8123.
[0287] This insulin was prepared using the PEG reagent
NHS-dPEG.sub.4-(m-dPEG.sub.12).sub.3 ester (Quanta BioDesign Ltd.
Product No 10401).
Example 18
General Procedure (B)
[0288] A14E,
A22K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl-dPEG12-yl),
B25H, B29R, desB30 Human insulin
##STR00021##
[0289] MALDI-MS (matrix: sinapinic acid); m/z: 8724.
[0290] This insulin was prepared using the PEG reagent
NHS-dPEG.sub.4-(m-dPEG.sub.12).sub.3 ester (Quanta BioDesign Ltd.
Product No 10401) and amino-dPEG.sub.12 tert-butyl ester (Quanta
BioDesign Product No 10281) similarly as described above.
Example 19
General Procedure (B)
[0291]
A22K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl-dPEG12-yl),
B29R, desB30 Human Insulin
##STR00022##
[0292] MALDI-MS (matrix: sinapinic acid); m/z: 8768.
[0293] This insulin was prepared using the PEG reagent
NHS-dPEG.sub.4-(m-dPEG.sub.12).sub.3 ester (Quanta BioDesign Ltd.
Product No 10401) and amino-dPEG.sub.12 tert-butyl ester (Quanta
BioDesign Product No 10281) similarly as described above.
Example 20
General Procedure (B)
[0294] A22K(N.sup..epsilon.(mdPEG.sub.24-Propionyl), A14E, B25H,
B29R, desB30 human insulin
##STR00023##
MALDI-MS (matrix: sinapinic acid); m/z: 6918.
Example 21
General Procedure (B)
[0295] A22K(N.sup..epsilon.(mPEG5.000-Propionyl)), A14E, B25H,
B29R, desB30 human insulin
##STR00024##
[0296] MALDI-MS (matrix: sinapinic acid); m/z: around 11400.
Example 22
General Procedure (B)
[0297] B29Q, B31K(N/(mPEG2000-Propionyl)) human insulin
##STR00025##
[0298] MALDI-MS (matrix: sinapinic acid); m/z: around 8268.
Example 23
Insulin Receptor Binding of the Insulin Derivatives of this
Invention
[0299] The affinity of the insulin derivatives of this invention
for the human insulin receptor is determined by a SPA assay
(Scintillation Proximity Assay) microtiterplate antibody capture
assay. SPA-PVT antibody-binding beads, anti-mouse reagent (Amersham
Biosciences, Cat No. PRNQ0017) are mixed with 25 ml of binding
buffer (100 mM HEPES pH 7.8; 100 mM sodium chloride, 10 mM
MgSO.sub.4, 0.025% Tween-20). Reagent mix for a single Packard
Optiplate (Packard No. 6005190) is composed of 2.4 .mu.l of a
1:5000 diluted purified recombinant human insulin receptor (either
with or without exon 11), an amount of a stock solution of
A14Tyr[.sup.125]-human insulin corresponding to 5000 cpm per 100
.mu.l of reagent mix, 12 .mu.l of a 1:1000 dilution of F12
antibody, 3 ml of SPA-beads and binding buffer to a total of 12 ml.
A total of 100 .mu.l reagent mix is then added to each well in the
Packard Optiplate and a dilution series of the insulin derivative
is made in the Optiplate from appropriate samples. The samples are
then incubated for 16 hours while gently shaken. The phases are the
then separated by centrifugation for 1 min and the plates counted
in a Topcounter. The binding data were fitted using the nonlinear
regression algorithm in the GraphPad Prism 2.01 (GraphPad Software,
San Diego, Calif.).
Insulin Receptor Binding Affinities of Selected Compounds of this
Invention:
TABLE-US-00002 Insulin receptor binding, A-isoform (without exon
11) Ex. No: Relative to human insulin: 1, 2 90% 3 123% 4 188% 6
120% 5 118% 7 44% 8 58% 9 128% 10 123% 15 20% 13 24% 14 16% 17 19%
18 16% 19 106% 20 24% 22 14% 16 15%
Example 24
Blood Glucose Lowering Effect After i.v. Bolus Injection in Rat of
the Insulin Derivatives of this Invention
[0300] Male Wistar rats, 200-300 g, fasted for 18 h, is
anesthetized using either Hypnorm-Dormicum s.c. (1.25 mg/ml
Dormicum, 2.5 mg/ml fluanisone, 0.079 mg/ml fentanyl citrate) 2
ml/kg as a priming dose (to timepoint -30 min prior to test
substance dosing) and additional 1 ml/kg every 20 minutes.
[0301] The animals are dosed with an intravenous injection (tail
vein), 1 ml/kg, of control and test compounds (usual dose range
0.125-20 nmol/kg). Blood samples for the determination of whole
blood glucose concentration are collected in heparinized 10 .mu.l
glass tubes by puncture of the capillary vessels in the tail tip to
time -20 min and 0 min (before dosing), and to time 10, 20, 30, 40,
60, 80, 120, and 180 min after dosing. Blood glucose concentrations
are measured after dilution in analysis buffer by the immobilized
glucose oxidase method using an EBIO Plus autoanalyzer (Eppendorf,
Germany). Mean plasma glucose concentrations courses (mean.+-.SEM)
are made for each dose and each compound.
Example 25
Potency of the Insulin Derivatives of this Invention Relative to
Human Insulin
[0302] Sprague Dawley male rats weighing 238-383 g on the
experimental day are used for the clamp experiment. The rats have
free access to feed under controlled ambient conditions and are
fasted overnight (from 3 .mu.m) prior to the clamp experiment.
Experimental Protocol:
[0303] The rats are acclimatized in the animal facilities for at
least 1 week prior to the surgical procedure. Approximately 1 week
prior to the clamp experiment, Tygon catheters are inserted under
halothane anaesthesia into the jugular vein (for infusion) and the
carotid artery (for blood sampling) and exteriorised and fixed on
the back of the neck. The rats are given Streptocilin vet.
(Boehringer Ingelheim; 0.15 ml/rat, i.m.) post-surgically and
placed in an animal care unit (25.degree. C.) during the recovery
period. In order to obtain analgesia, Anorphin (0.06 mg/rat, s.c.)
is administered during anaesthesia and Rimadyl (1.5 mg/kg, s.c.) is
administered after full recovery from the anaesthesia (2-3 h) and
again once daily for 2 days.
[0304] At 7 am on the experimental day overnight fasted (from 3
.mu.m the previous day) rats are weighed and connected to the
sampling syringes and infusion system (Harvard 22 Basic pumps,
Harvard, and Perfectum Hypodermic glass syringe, Aldrich) and then
placed into individual clamp cages where they rest for ca. 45 min
before start of experiment. The rats are able to move freely on
their usual bedding during the entire experiment and have free
access to drinking water. After a 30 min basal period during which
plasma glucose levels were measured at 10 min intervals, the
insulin derivative to be tested and human insulin (one dose level
per rat, n=6-7 per dose level) are infused (i.v.) at a constant
rate for 300 min. Plasma glucose levels are measured at 10 min
intervals throughout and infusion of 20% aqueous glucose is
adjusted accordingly in order to maintain euglyceamia. Samples of
re-suspended erythrocytes are pooled from each rat and returned in
about 1/2 ml volumes via the .alpha.-rotid catheter.
[0305] On each experimental day, samples of the solutions of the
individual insulin derivatives to be tested and the human insulin
solution are taken before and at the end of the clamp experiments
and the concentrations of the peptides are confirmed by HPLC.
Plasma concentrations of rat insulin and C-peptide as well as of
the insulin derivative to be tested and human insulin are measured
at relevant time points before and at the end of the studies. Rats
are killed at the end of experiment using a pentobarbital
overdose.
Example 26
Pulmonary Delivery of Insulin Derivatives to Rats
[0306] The test substance will be dosed pulmonary by the drop
instillation method. In brief, male Wistar rats (app.250 g) are
anaesthesized in app. 60 ml fentanyl/dehydrodenzperidol/-dormicum
given as a 6.6 ml/kg sc primingdose and followed by 3 maintenance
doses of 3.3 ml/kg sc with an interval of 30 min. Ten minutes after
the induction of anaesthesia, basal samples are obtained from the
tail vein (t=-20 min) followed by a basal sample immediately prior
to the dosing of test substance (t=0). At t=0, the test substance
is dosed intra tracheally into one lung. A special cannula with
rounded ending is mounted on a syringe containing the 200 ul air
and test substance (1 ml/kg). Via the orifice, the cannula is
introduced into the trachea and is forwarded into one of the main
bronchi--just passing the bifurcature. During the insertion, the
neck is palpated from the exterior to assure intratracheal
positioning. The content of the syringe is injected followed by 2
sec pause. Thereafter, the cannula is slowly drawn back. The rats
are kept anaesthesized during the test (blood samples for up to 4
or 8 hrs) and are euthanized after the experiment.
[0307] The PEGylated extended insulins in the following examples
may be prepared similarly as described above:
Examples 27-419
TABLE-US-00003 [0308] Ex. #: PEGylated, extended insulin: 27
A22K(N.sup..epsilon.mPEG10.000-propionyl) B29R desB30 human
insulin; 28 A22K(N.sup..epsilon.mPEG40.000-propionyl) B29R desB30
human insulin; 29 A22G A23K(N.sup..epsilon.mPEG750-propionyl) B29R
desB30 human insulin; 30 A22G
A23K(N.sup..epsilon.mPEG5.000-propionyl) B29R desB30 human insulin;
31 A22G A23K(N.sup..epsilon.mPEG10.000-propionyl) B29R desB30 human
insulin; 32 A22G A23K(N.sup..epsilon.mPEG20.000-propionyl) B29R
desB30 human insulin; 33 A22G
A23K(N.sup..epsilon.mPEG40.000-propionyl) B29R desB30 human
insulin; 34 A22G A23K(N.sup..epsilon.mdPEG.sub.12-propionyl) B29R
desB30 human insulin; 35 A22G
A23K(N.sup..epsilon.mdPEG.sub.24-propionyl) B29R desB30 human
insulin; 36 A22G A23K((mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B29R
desB30 human insulin; 37 A22G A23G
A24K(N.sup..epsilon.mPEG750-propionyl) B29R desB30 human insulin;
38 A22G A23G A24K(N.sup..epsilon.mPEG2.000-propionyl) B29R desB30
human insulin; 39 A22G A23G
A24K(N.sup..epsilon.mPEG5.000-propionyl) B29R desB30 human insulin;
40 A22G A23G A24K(N.sup..epsilon.mPEG10.000-propionyl) B29R desB30
human insulin; 41 A22G A23G
A24K(N.sup..epsilon.mPEG20.000-propionyl) B29R desB30 human
insulin; 42 A22G A23G A24K(N.sup..epsilon.mPEG40.000-propionyl)
B29R desB30 human insulin; 43 A22G A23G
A24K(N.sup..epsilon.mdPEG.sub.12-propionyl) B29R desB30 human
insulin; 44 A22G A23G A24K(N.sup..epsilon.mdPEG.sub.24-propionyl)
B29R desB30 human insulin; 45 A22G A23G
A24K(N.sup..epsilon.(mdPEG12).sub.3-dPEG.sub.4-yl) B29R desB30
human insulin; 46 A22G A23G A24G
A25K(N.sup..epsilon.mPEG2.000-propionyl) B29R desB30 human insulin;
47 A22G A23G A24G A25K(N.sup..epsilon.mPEG5.000-propionyl) B29R
desB30 human insulin; 48 A22G A23G A24G
A25K(N.sup..epsilon.mPEG10.000-propionyl) B29R desB30 human
insulin; 49 A22G A23G A24G
A25K(N.sup..epsilon.mPEG20.000-propionyl) B29R desB30 human
insulin; 50 A22G A23G A24G
A25K(N.sup..epsilon.mPEG40.000-propionyl) B29R desB30 human
insulin; 51 A22G A23G A24G
A25K(N.sup..epsilon.mdPEG.sub.12-propionyl) B29R desB30 human
insulin; 52 A22G A23G A24G
A25K(N.sup..epsilon.mdPEG.sub.24-propionyl) B29R desB30 human
insulin; 53 A22G A23G A24G
A25K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B29R desB30
human insulin; 54 A22K(N.sup..epsilon.mPEG750-propionyl) B3Q B29R
desB30 human insulin; 55 A22K(N.sup..epsilon.mPEG2.000-propionyl)
B3Q B29R desB30 human insulin; 56
A22K(N.sup..epsilon.mPEG5.000-propionyl) B3Q B29R desB30 human
insulin; 57 A22K(N.sup..epsilon.mPEG10.000-propionyl) B3Q B29R
desB30 human insulin; 58 A22K(N.sup..epsilon.mPEG20.000-propionyl)
B3Q B29R desB30 human insulin; 59
A22K(N.sup..epsilon.mPEG40.000-propionyl) B3Q B29R desB30 human
insulin; 60 A22K(N.sup..epsilon.mdPEG.sub.12-propionyl) B3Q B29R
desB30 human insulin; 61
A22K(N.sup..epsilon.mdPEG.sub.24-propionyl) B3Q B29R desB30 human
insulin; 62 A22K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl)
B3Q B29R desB30 human insulin; 63
A22K(N.sup..epsilon.mPEG750-propionyl) B3S B29R desB30 human
insulin; 64 A22K(N.sup..epsilon.mPEG2.000-propionyl) B3S B29R
desB30 human insulin; 65 A22K(N.sup..epsilon.mPEG5.000-propionyl)
B3S B29R desB30 human insulin; 66
A22K(N.sup..epsilon.mPEG10.000-propionyl) B3S B29R desB30 human
insulin; 67 A22K(N.sup..epsilon.mPEG20.000-propionyl) B3S B29R
desB30 human insulin; 68 A22K(N.sup..epsilon.mPEG40.000-propionyl)
B3S B29R desB30 human insulin; 69
A22K(N.sup..epsilon.mdPEG.sub.12-propionyl) B3S B29R desB30 human
insulin; 70 A22K(N.sup..epsilon.mdPEG.sub.24-propionyl) B3S B29R
desB30 human insulin; 71
A22K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B3S B29R
desB30 human insulin; 72 A22K(N.sup..epsilon.mPEG750-propionyl) B3T
B29R desB30 human insulin; 73
A22K(N.sup..epsilon.mPEG2.000-propionyl) B3T B29R desB30 human
insulin; 74 A22K(N.sup..epsilon.mPEG5.000-propionyl) B3T B29R
desB30 human insulin; 75 A22K(N.sup..epsilon.mPEG10.000-propionyl)
B3T B29R desB30 human insulin; 76
A22K(N.sup..epsilon.mPEG20.000-propionyl) B3T B29R desB30 human
insulin; 77 A22K(N.sup..epsilon.mPEG40.000-propionyl) B3T B29R
desB30 human insulin; 78
A22K(N.sup..epsilon.mdPEG.sub.12-propionyl) B3T B29R desB30 human
insulin; 79 A22K(N.sup..epsilon.mdPEG.sub.24-propionyl) B3T B29R
desB30 human insulin; 80
A22K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B3T B29R
desB30 human insulin; 81 A22K(N.sup..epsilon.mPEG750-propionyl) B1Q
B29R desB30 human insulin; 82
A22K(N.sup..epsilon.mPEG2.000-propionyl) B1Q B29R desB30 human
insulin; 83 A22K(N.sup..epsilon.mPEG5.000-propionyl) B1Q B29R
desB30 human insulin; 84 A22K(N.sup..epsilon.mPEG10.000-propionyl)
B1Q B29R desB30 human insulin; 85
A22K(N.sup..epsilon.mPEG20.000-propionyl) B1Q B29R desB30 human
insulin; 86 A22K(N.sup..epsilon.mPEG40.000-propionyl) B1Q B29R
desB30 human insulin; 87
A22K(N.sup..epsilon.mdPEG.sub.12-propionyl) B1Q B29R desB30 human
insulin; 88 A22K(N.sup..epsilon.mdPEG.sub.24-propionyl) B1Q B29R
desB30 human insulin; 89
A22K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B1Q B29R
desB30 human insulin; 90 A18Q
A22K(N.sup..epsilon.mPEG750-propionyl) B29R desB30 human insulin;
91 A18Q A22K(N.sup..epsilon.mPEG2.000-propionyl) B29R desB30 human
insulin; 92 A18Q A22K(N.sup..epsilon.mPEG5.000-propionyl) B29R
desB30 human insulin; 93 A18Q
A22K(N.sup..epsilon.mPEG10.000-propionyl) B29R desB30 human
insulin; 94 A18Q A22K(N.sup..epsilon.mPEG20.000-propionyl) B29R
desB30 human insulin; 95 A18Q
A22K(N.sup..epsilon.mPEG40.000-propionyl) B29R desB30 human
insulin; 96 A18Q A22K(N.sup..epsilon.mdPEG.sub.12-propionyl) B29R
desB30 human insulin; 97 A18Q
A22K(N.sup..epsilon.mdPEG.sub.24-propionyl) B29R desB30 human
insulin; 98 A18Q
A22K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B29R desB30
human insulin; 99 A22K(N.sup..epsilon.mPEG750-propionyl) desB1 B3Q
B29R desB30 human insulin; 100
A22K(N.sup..epsilon.mPEG2.000-propionyl) desB1 B3Q B29R desB30
human insulin; 101 A22K(N.sup..epsilon.mPEG5.000-propionyl) desB1
B3Q B29R desB30 human insulin; 102
A22K(N.sup..epsilon.mPEG10.000-propionyl) desB1 B3Q B29R desB30
human insulin; 103 A22K(N.sup..epsilon.mPEG20.000-propionyl) desB1
B3Q B29R desB30 human insulin; 104
A22K(N.sup..epsilon.mPEG40.000-propionyl) desB1 B3Q B29R desB30
human insulin; 105 A22K(N.sup..epsilon.mdPEG.sub.12-propionyl)
desB1 B3Q B29R desB30 human insulin; 106
A22K(N.sup..epsilon.mdPEG.sub.24-propionyl) desB1 B3Q B29R desB30
human insulin; 107
A22K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl) desB1 B3Q
B29R desB30 human insulin; 108 B29Q
B31K(N.sup..epsilon.mPEG750-propionyl) human insulin; 109 B29Q
B31K(N.sup..epsilon.mPEG2.000-propionyl) human insulin; 110 B29Q
B31K(N.sup..epsilon.mPEG5.000-propionyl) human insulin; 111 B29Q
B31K(N.sup..epsilon.mPEG10.000-propionyl) human insulin; 112 B29Q
B31K(N.sup..epsilon.mPEG20.000-propionyl) human insulin; 113 B29Q
B31K(N.sup..epsilon.mPEG40.000-propionyl) human insulin; 114 B29Q
B31K(N.sup..epsilon.mdPEG.sub.12-propionyl) human insulin; 115 B29Q
B31K(N.sup..epsilon.mdPEG.sub.24-propionyl) human insulin; 116 B29Q
B31K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl) human
insulin; 117 A21G B29Q B31K(N.sup..epsilon.mPEG750-propionyl) human
insulin; 118 A21G B29Q B31K(N.sup..epsilon.mPEG2.000-propionyl)
human insulin; 119 A21G B29Q
B31K(N.sup..epsilon.mPEG5.000-propionyl) human insulin; 120 A21G
B29Q B31K(N.sup..epsilon.mPEG10.000-propionyl) human insulin; 121
A21G B29Q B31K(N.sup..epsilon.mPEG20.000-propionyl) human insulin;
122 A21G B29Q B31K(N.sup..epsilon.mPEG40.000-propionyl) human
insulin; 123 A21G B29Q B31K(N.sup..epsilon.mdPEG.sub.12-propionyl)
human insulin; 124 A21G B29Q
B31K(N.sup..epsilon.mdPEG.sub.24-propionyl) human insulin; 125 A21G
B29Q B31K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl) human
insulin; 126 A21A B29Q B31K(N.sup..epsilon.mPEG750-propionyl) human
insulin; 127 A21A B29Q B31K(N.sup..epsilon.mPEG2.000-propionyl)
human insulin; 128 A21A B29Q
B31K(N.sup..epsilon.mPEG5.000-propionyl) human insulin; 129 A21A
B29Q B31K(N.sup..epsilon.mPEG10.000-propionyl) human insulin; 130
A21A 29Q B31K(N.sup..epsilon.mPEG20.000-propionyl) human insulin;
131 A21A B29Q B31K(N.sup..epsilon.mPEG40.000-propionyl) human
insulin; 132 A21A B29Q B31K(N.sup..epsilon.mdPEG.sub.12-propionyl)
human insulin; 133 A21A B29Q
B31K(N.sup..epsilon.mdPEG.sub.24-propionyl) human insulin; 134 A21A
B29Q B31K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl) human
insulin; 135 A21Q B29Q B31K(N.sup..epsilon.mPEG750-propionyl) human
insulin; 136 A21Q B29Q B31K(N.sup..epsilon.mPEG2.000-propionyl)
human insulin; 137 A21Q B29Q
B31K(N.sup..epsilon.mPEG5.000-propionyl) human insulin; 138 A21Q
B29Q B31K(N.sup..epsilon.mPEG10.000-propionyl) human insulin; 139
A21Q B29Q B31K(N.sup..epsilon.mPEG20.000-propionyl) human insulin;
140 A21Q B29Q B31K(N.sup..epsilon.mPEG40.000-propionyl) human
insulin; 141 A21Q B29Q B31K(N.sup..epsilon.mdPEG.sub.12-propionyl)
human insulin; 142 A21Q B29Q
B31K(N.sup..epsilon.mdPEG.sub.24-propionyl) human insulin; 143 A21Q
B29Q B31K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl) human
insulin; 144 A-1K(N.sup..epsilon.mPEG750-propionyl) desB30 human
insulin; 145 A-1K(N.sup..epsilon.mPEG2.000-propionyl) desB30 human
insulin; 146 A-1K(N.sup..epsilon.mPEG5.000-propionyl) desB30 human
insulin; 147 A-1K(N.sup..epsilon.mPEG10.000-propionyl) desB30 human
insulin; 148 A-1K(N.sup..epsilon.mPEG20.000-propionyl) desB30 human
insulin; 149 A-1K(N.sup..epsilon.mPEG40.000-propionyl) desB30 human
insulin; 150 A-1K(N.sup..epsilon.mdPEG.sub.12-propionyl) desB30
human insulin; 151 A-1K(N.sup..epsilon.mdPEG.sub.24-propionyl)
desB30 human insulin; 152
A-1K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl) desB30
human insulin; 153 A-1K(N.sup..epsilon.mPEG750-propionyl) B29R
desB30 human insulin; 154 A-1K(N.sup..epsilon.mPEG2.000-propionyl)
B29R desB30 human insulin; 155
A-1K(N.sup..epsilon.mPEG5.000-propionyl) B29R desB30 human insulin;
156 A-1K(N.sup..epsilon.mPEG10.000-propionyl) B29R desB30 human
insulin; 157 A-1K(N.sup..epsilon.mPEG20.000-propionyl) B29R desB30
human insulin; 158 A-1K(N.sup..epsilon.mPEG40.000-propionyl) B29R
desB30 human insulin; 159
A-1K(N.sup..epsilon.mdPEG.sub.12-propionyl) B29R desB30 human
insulin; 160 A-1K(N.sup..epsilon.mdPEG.sub.24-propionyl) B29R
desB30 human insulin; 161
A-1K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B29R desB30
human insulin; 162 A-3G(N.sup..alpha.mPEG750-propionyl) A-2G A-1R
desB30 human insulin; 163 A-3G(N.sup..alpha.mPEG2.000-propionyl)
A-2G A-1R desB30 human insulin; 164
A-3G(N.sup..alpha.mPEG5.000-propionyl) A-2G A-1R desB30 human
insulin; 165 A-3G(N.sup..alpha.mPEG10.000-propionyl) A-2G A-1R
desB30 human insulin; 166 A-3G(N.sup..alpha.mPEG20.000-propionyl)
A-2G A-1R desB30 human insulin; 167
A-3G(N.sup..alpha.mPEG40.000-propionyl) A-2G A-1R desB30 human
insulin; 168 A-3G(N.sup..alpha.mdPEG.sub.12-propionyl) A-2G A-1R
desB30 human insulin; 169 A-3G(N.sup..alpha.mdPEG.sub.24-propionyl)
A-2G A-1R desB30 human insulin; 170
A-3G(N.sup..alpha.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl) A-2G A-1R
desB30 human insulin; 171 A22K(N.sup..epsilon.mPEG750-propionyl)
B28E B29R desB30 human insulin; 172
A22K(N.sup..epsilon.mPEG2.000-propionyl) B28E B29R desB30 human
insulin; 173 A22K(N.sup..epsilon.mPEG5.000-propionyl) B28E B29R
desB30 human insulin; 174 A22K(N.sup..epsilon.mPEG10.000-propionyl)
B28E B29R desB30 human insulin; 175
A22K(N.sup..epsilon.mPEG20.000-propionyl) B28E B29R desB30 human
insulin; 176 A22K(N.sup..epsilon.mPEG40.000-propionyl) B28E B29R
desB30 human insulin; 177
A22K(N.sup..epsilon.mdPEG.sub.12-propionyl) B28E B29R desB30
human
insulin; 178 A22K(N.sup..epsilon.mdPEG.sub.24-propionyl) B28E B29R
desB30 human insulin; 179
A22K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B28E B29R
desB30 human insulin; 180 A22K(N.sup..epsilon.mPEG750-propionyl)
B28D B29R desB30 human insulin; 181
A22K(N.sup..epsilon.mPEG2.000-propionyl) B28D B29R desB30 human
insulin; 182 A22K(N.sup..epsilon.mPEG5.000-propionyl) B28D B29R
desB30 human insulin; 183 A22K(N.sup..epsilon.mPEG10.000-propionyl)
B28D B29R desB30 human insulin; 184
A22K(N.sup..epsilon.mPEG20.000-propionyl) B28D B29R desB30 human
insulin; 185 A22K(N.sup..epsilon.mPEG40.000-propionyl) B28D B29R
desB30 human insulin; 186
A22K(N.sup..epsilon.mdPEG.sub.12-propionyl) B28D B29R desB30 human
insulin; 187 A22K(N.sup..epsilon.mdPEG.sub.24-propionyl) B28D B29R
desB30 human insulin; 188
A22K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B28D B29R
desB30 human insulin; 189 A22K(N.sup..epsilon.mPEG750-propionyl)
desB27 B28E B29R desB30 human insulin; 190
A22K(N.sup..epsilon.mPEG2.000-propionyl) desB27 B28E B29R desB30
human insulin; 191 A22K(N.sup..epsilon.mPEG5.000-propionyl) desB27
B28E B29R desB30 human insulin; 192
A22K(N.sup..epsilon.mPEG10.000-propionyl) desB27 B28E B29R desB30
human insulin; 193 A22K(N.sup..epsilon.mPEG20.000-propionyl) desB27
B28E B29R desB30 human insulin; 194
A22K(N.sup..epsilon.mPEG40.000-propionyl) desB27 B28E B29R desB30
human insulin; 195 A22K(N.sup..epsilon.mdPEG.sub.12-propionyl)
desB27 B28E B29R desB30 human insulin; 196
A22K(N.sup..epsilon.mdPEG.sub.24-propionyl) desB27 B28E B29R desB30
human insulin; 197
A22K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl) desB27 B28E
B29R desB30 human insulin; 198 B28E B29Q
B31K(N.sup..epsilon.mPEG750-propionyl) human insulin; 199 B28E B29Q
B31K(N.sup..epsilon.mPEG2.000-propionyl) human insulin; 200 B28E
B29Q B31K(N.sup..epsilon.mPEG5.000-propionyl) human insulin; 201
B28E B29Q B31K(N.sup..epsilon.mPEG10.000-propionyl) human insulin;
202 B28E B29Q B31K(N.sup..epsilon.mPEG20.000-propionyl) human
insulin; 203 B28E B29Q B31K(N.sup..epsilon.mPEG40.000-propionyl)
human insulin; 204 B28E B29Q
B31K(N.sup..epsilon.mdPEG.sub.12-propionyl) human insulin; 205 B28E
B29Q B31K(N.sup..epsilon.mdPEG.sub.24-propionyl) human insulin; 206
B28E B29Q B31K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl)
human insulin; 207 desB27 B28E B29Q
B31K(N.sup..epsilon.mPEG750-propionyl) human insulin; 208 desB27
B28E B29Q B31K(N.sup..epsilon.mPEG2.000-propionyl) human insulin;
209 desB27 B28E B29Q B31K(N.sup..epsilon.mPEG5.000-propionyl) human
insulin; 210 desB27 B28E B29Q
B31K(N.sup..epsilon.mPEG10.000-propionyl) human insulin; 211 desB27
B28E B29Q B31K(N.sup..epsilon.mPEG20.000-propionyl) human insulin;
212 desB27 B28E B29Q B31K(N.sup..epsilon.mPEG40.000-propionyl)
human insulin; 213 desB27 B28E B29Q
B31K(N.sup..epsilon.mdPEG.sub.12-propionyl) human insulin; 214
desB27 B28E B29Q B31K(N.sup..epsilon.mdPEG.sub.24-propionyl) human
insulin; 215 desB27 B28E B29Q
B31K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl) human
insulin; 216 A22K(N.sup..epsilon.mPEG750-propionyl) B28R desB29
desB30 human insulin; 217 A22K(N.sup..epsilon.mPEG2.000-propionyl)
B28R desB29 desB30 human insulin; 218
A22K(N.sup..epsilon.mPEG5.000-propionyl) B28R desB29 desB30 human
insulin; 219 A22K(N.sup..epsilon.mPEG10.000-propionyl) B28R desB29
desB30 human insulin; 220 A22K(N.sup..epsilon.mPEG20.000-propionyl)
B28R desB29 desB30 human insulin; 221
A22K(N.sup..epsilon.mPEG40.000-propionyl) B28R desB29 desB30 human
insulin; 222 A22K(N.sup..epsilon.mdPEG.sub.12-propionyl) B28R
desB29 desB30 human insulin; 223
A22K(N.sup..epsilon.mdPEG.sub.24-propionyl) B28R desB29 desB30
human insulin; 224
A22K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B28R desB29
desB30 human insulin; 225 B28R B29P
B31K(N.sup..epsilon.mPEG750-propionyl) human insulin; 226 B28R B29P
B31K(N.sup..epsilon.mPEG2.000-propionyl) human insulin; 227 B28R
B29P B31K(N.sup..epsilon.mPEG5.000-propionyl) human insulin; 228
B28R B29P B31K(N.sup..epsilon.mPEG10.000-propionyl) human insulin;
229 B28R B29P B31K(N.sup..epsilon.mPEG20.000-propionyl) human
insulin; 230 B28R B29P B31K(N.sup..epsilon.mPEG40.000-propionyl)
human insulin; 231 B28R B29P
B31K(N.sup..epsilon.mdPEG.sub.12-propionyl) human insulin; 232 B28R
B29P B31K(N.sup..epsilon.mdPEG.sub.24-propionyl) human insulin; 233
B28R B29P B31K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl)
human insulin; 234 A22K(N.sup..epsilon.mPEG750-propionyl) B3Q B28E
B29R desB30 human insulin; 235
A22K(N.sup..epsilon.mPEG2.000-propionyl) B3Q B28E B29R desB30 human
insulin; 236 A22K(N.sup..epsilon.mPEG5.000-propionyl) B3Q B28E B29R
desB30 human insulin; 237 A22K(N.sup..epsilon.mPEG10.000-propionyl)
B3Q B28E B29R desB30 human insulin; 238
A22K(N.sup..epsilon.mPEG20.000-propionyl) B3Q B28E B29R desB30
human insulin; 239 A22K(N.sup..epsilon.mPEG40.000-propionyl) B3Q
B28E B29R desB30 human insulin; 240
A22K(N.sup..epsilon.mdPEG.sub.12-propionyl) B3Q B28E B29R desB30
human insulin; 241 A22K(N.sup..epsilon.mdPEG.sub.24-propionyl) B3Q
B28E B29R desB30 human insulin; 242
A22K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B3Q B28E
B29R desB30 human insulin; 243 A21G B3Q B28E B29Q
B31K(N.sup..epsilon.mPEG750-propionyl) human insulin; 244 A21G B3Q
B28E B29Q B31K(N.sup..epsilon.mPEG2.000-propionyl) human insulin;
245 A21G B3Q B28E B29Q B31K(N.sup..epsilon.mPEG5.000-propionyl)
human insulin; 246 A21G B3Q B28E B29Q
B31K(N.sup..epsilon.mPEG10.000-propionyl) human insulin; 247 A21G
B3Q B28E B29Q B31K(N.sup..epsilon.mPEG20.000-propionyl) human
insulin; 248 A21G B3Q B28E B29Q
B31K(N.sup..epsilon.mPEG40.000-propionyl) human insulin; 249 A21G
B3Q B28E B29Q B31K(N.sup..epsilon.mdPEG.sub.12-propionyl) human
insulin; 250 A21G B3Q B28E B29Q
B31K(N.sup..epsilon.mdPEG.sub.24-propionyl) human insulin; 251 A21G
B3Q B28E B29Q
B31K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl) human
insulin; 252 A22K(N.sup..epsilon.mPEG750-propionyl) B13Q B29R
desB30 human insulin; 253 A22K(N.sup..epsilon.mPEG2.000-propionyl)
B13Q B29R desB30 human insulin; 254
A22K(N.sup..epsilon.mPEG5.000-propionyl) B13Q B29R desB30 human
insulin; 255 A22K(N.sup..epsilon.mPEG10.000-propionyl) B13Q B29R
desB30 human insulin; 256 A22K(N.sup..epsilon.mPEG20.000-propionyl)
B13Q B29R desB30 human insulin; 257
A22K(N.sup..epsilon.mPEG40.000-propionyl) B13Q B29R desB30 human
insulin; 258 A22K(N.sup..epsilon.mdPEG.sub.12-propionyl) B13Q B29R
desB30 human insulin; 259
A22K(N.sup..epsilon.mdPEG.sub.24-propionyl) B13Q B29R desB30 human
insulin; 260
A22K(N.sup..epsilon.((mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B13Q B29R
desB30 human insulin; 261 A22K(N.sup..epsilon.mPEG750-propionyl)
desB1 B29R desB30 human insulin; 262
A22K(N.sup..epsilon.mPEG2.000-propionyl) desB1 B29R desB30 human
insulin; 263 A22K(N.sup..epsilon.mPEG5.000-propionyl) desB1 B29R
desB30 human insulin; 264 A22K(N.sup..epsilon.mPEG10.000-propionyl)
desB1 B29R desB30 human insulin; 265
A22K(N.sup..epsilon.mPEG20.000-propionyl) desB1 B29R desB30 human
insulin; 266 A22K(N.sup..epsilon.mPEG40.000-propionyl) desB1 B29R
desB30 human insulin; 267
A22K(N.sup..epsilon.mdPEG.sub.12-propionyl) desB1 B29R desB30 human
insulin; 268 A22K(N.sup..epsilon.mdPEG.sub.24-propionyl) desB1 B29R
desB30 human insulin; 269
A22K(N.sup..epsilon.((mdPEG.sub.12).sub.3-dPEG.sub.4-yl) desB1 B29R
desB30 human insulin; 270 A14E
A22K(N.sup..epsilon.mPEG750-propionyl) B25H desB30 human insulin;
271 A14E A22K(N.sup..epsilon.mPEG2.000-propionyl) B25H desB30 human
insulin; 272 A14E A22K(N.sup..epsilon.mPEG5.000-propionyl) B25H
desB30 human insulin; 273 A14E
A22K(N.sup..epsilon.mPEG10.000-propionyl) B25H desB30 human
insulin; 274 A14E A22K(N.sup..epsilon.mPEG20.000-propionyl) B25H
desB30 human insulin; 275 A14E
A22K(N.sup..epsilon.mPEG40.000-propionyl) B25H desB30 human
insulin; 276 A14E A22K(N.sup..epsilon.mdPEG.sub.12-propionyl) B25H
desB30 human insulin; 277 A14E
A22K(N.sup..epsilon.mdPEG.sub.24-propionyl) B25H desB30 human
insulin; 278 A14E
A22K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B25H desB30
human insulin; 279 A14E B25H B29Q
B31K(N.sup..epsilon.mPEG750-propionyl) human insulin; 280 A14E B25H
B29Q B31K(N.sup..epsilon.mPEG2.000-propionyl) human insulin; 281
A14E B25H B29Q B31K(N.sup..epsilon.mPEG5.000-propionyl) human
insulin; 282 A14E B25H B29Q
B31K(N.sup..epsilon.mPEG10.000-propionyl) human insulin; 283 A14E
B25H B29Q B31K(N.sup..epsilon.mPEG20.000-propionyl) human insulin;
284 A14E B25H B29Q B31K(N.sup..epsilon.mPEG40.000-propionyl) human
insulin; 285 A14E B25H B29Q
B31K(N.sup..epsilon.mdPEG.sub.12-propionyl) human insulin; 286 A14E
B25H B29Q B31K(N.sup..epsilon.mdPEG.sub.24-propionyl) human
insulin; 287 A14E B25H B29Q
B31K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl) human
insulin; 288 A13E A22K(N.sup..epsilon.mPEG750-propionyl) B25H
desB30 human insulin; 289 A13E
A22K(N.sup..epsilon.mPEG2.000-propionyl) B25H desB30 human insulin;
290 A13E A22K(N.sup..epsilon.mPEG5.000-propionyl) B25H desB30 human
insulin; 291 A13E A22K(N.sup..epsilon.mPEG10.000-propionyl) B25H
desB30 human insulin; 292 A13E
A22K(N.sup..epsilon.mPEG20.000-propionyl) B25H desB30 human
insulin; 293 A13E A22K(N.sup..epsilon.mPEG40.000-propionyl) B25H
desB30 human insulin; 294 A13E
A22K(N.sup..epsilon.mdPEG.sub.12-propionyl) B25H desB30 human
insulin; 295 A13E A22K(N.sup..epsilon.mdPEG.sub.24-propionyl) B25H
desB30 human insulin; 296 A13E
A22K(N.sup..epsilon.(mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B25H desB30
human insulin; 297 A21Q A22K(N.sup..epsilon.mPEG750-propionyl) B29R
desB30 human insulin; 298 A21Q
A22K(N.sup..epsilon.mPEG2.000-propionyl) B29R desB30 human insulin;
299 A21Q A22K(N.sup..epsilon.mPEG5.000-propionyl) B29R desB30 human
insulin; 300 A21Q A22K(N.sup..epsilon.mPEG10.000-propionyl) B29R
desB30 human insulin; 301 A21Q
A22K(N.sup..epsilon.mPEG20.000-propionyl) B29R desB30 human
insulin; 302 A21Q A22K(N.sup..epsilon.mPEG40.000-propionyl) B29R
desB30 human insulin; 303 A21Q
A22K(N.sup..epsilon.mdPEG.sub.12-propionyl) B29R desB30 human
insulin; 304 A21Q A22K(N.sup..epsilon.mdPEG.sub.24-propionyl) B29R
desB30 human insulin; 305 A21Q
A22K((mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B29R desB30 human insulin;
306 A21Q A22G A23K(N.sup..epsilon.mPEG750-propionyl) B29R desB30
human insulin; 307 A21Q A22G
A23K(N.sup..epsilon.mPEG2.000-propionyl) B29R desB30 human insulin;
308 A21Q A22G A23K(N.sup..epsilon.mPEG5.000-propionyl) B29R desB30
human insulin; 309 A21Q A22G
A23K(N.sup..epsilon.mPEG10.000-propionyl) B29R desB30 human
insulin; 310 A21Q A22G A23K(N.sup..epsilon.mPEG20.000-propionyl)
B29R desB30 human insulin; 311 A21Q A22G
A23K(N.sup..epsilon.mPEG40.000-propionyl) B29R desB30 human
insulin; 312 A21Q A22G A23K(N.sup..epsilon.mdPEG.sub.12-propionyl)
B29R desB30 human insulin; 313 A21Q A22G
A23K(N.sup..epsilon.mdPEG.sub.24-propionyl) B29R desB30 human
insulin; 314 A21Q A22G A23K((mdPEG.sub.12).sub.3-dPEG.sub.4-yl)
B29R desB30 human insulin; 315 A21Q A22G A23G
A24K(N.sup..epsilon.mPEG750-propionyl) B29R desB30 human insulin;
316 A21Q A22G A23G A24K(N.sup..epsilon.mPEG2.000-propionyl) B29R
desB30 human insulin; 317 A21Q A22G A23G
A24K(N.sup..epsilon.mPEG5.000-propionyl) B29R desB30 human insulin;
318 A21Q A22G A23G A24K(N.sup..epsilon.mPEG10.000-propionyl) B29R
desB30 human insulin; 319 A21Q A22G A23G
A24K(N.sup..epsilon.mPEG20.000-propionyl) B29R desB30 human
insulin; 320 A21Q A22G A23G
A24K(N.sup..epsilon.mPEG40.000-propionyl) B29R desB30 human
insulin; 321 A21Q A22G A23G
A24K(N.sup..epsilon.mdPEG.sub.12-propionyl) B29R desB30 human
insulin; 322 A21Q A22G A23G
A24K(N.sup..epsilon.mdPEG.sub.24-propionyl) B29R desB30 human
insulin; 323 A21Q A22G A23G
A24K((mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B29R desB30 human insulin;
324 A21Q A22G A23G A24G A25K(N.sup..epsilon.mPEG750-propionyl) B29R
desB30 human insulin; 325 A21Q A22G A23G A24G
A25K(N.sup..epsilon.mPEG2.000-propionyl) B29R desB30 human insulin;
326 A21Q A22G A23G A24G A25K(N.sup..epsilon.mPEG5.000-propionyl)
B29R desB30 human insulin; 327 A21Q A22G A23G A24G
A25K(N.sup..epsilon.mPEG10.000-propionyl) B29R desB30 human
insulin; 328 A21Q A22G A23G A24G
A25K(N.sup..epsilon.mPEG20.000-propionyl) B29R desB30 human
insulin; 329 A21Q A22G A23G A24G
A25K(N.sup..epsilon.mPEG40.000-propionyl) B29R desB30 human
insulin; 330 A21Q A22G A23G A24G
A25K(N.sup..epsilon.mdPEG.sub.12-propionyl) B29R desB30 human
insulin; 331 A21Q A22G A23G A24G
A25K(N.sup..epsilon.mdPEG.sub.24-propionyl) B29R desB30 human
insulin; 332 A21Q A22G A23G A24G
A25K((mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B29R desB30 human insulin;
333 A21A A22K(N.sup..epsilon.mPEG750-propionyl) B29R desB30 human
insulin; 334 A21A A22K(N.sup..epsilon.mPEG2.000-propionyl) B29R
desB30 human insulin; 335 A21A
A22K(N.sup..epsilon.mPEG5.000-propionyl) B29R desB30 human insulin;
336 A21A A22K(N.sup..epsilon.mPEG10.000-propionyl) B29R desB30
human insulin; 337 A21A A22K(N.sup..epsilon.mPEG20.000-propionyl)
B29R desB30 human insulin; 338 A21A
A22K(N.sup..epsilon.mPEG40.000-propionyl) B29R desB30 human
insulin; 339 A21A A22K(N.sup..epsilon.mdPEG.sub.12-propionyl) B29R
desB30 human insulin; 340 A21A
A22K(N.sup..epsilon.mdPEG.sub.24-propionyl) B29R desB30 human
insulin; 341 A21A A22K((mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B29R
desB30 human insulin; 342 A21A A22G
A23K(N.sup..epsilon.mPEG750-propionyl) B29R desB30 human insulin;
343 A21A A22G A23K(N.sup..epsilon.mPEG2.000-propionyl) B29R desB30
human insulin; 344 A21A A22G
A23K(N.sup..epsilon.mPEG5.000-propionyl) B29R desB30 human insulin;
345 A21A A22G A23K(N.sup..epsilon.mPEG10.000-propionyl) B29R desB30
human insulin; 346 A21A A22G
A23K(N.sup..epsilon.mPEG20.000-propionyl) B29R desB30 human
insulin; 347 A21A A22G A23K(N.sup..epsilon.mPEG40.000-propionyl)
B29R desB30 human insulin; 348 A21A A22G
A23K(N.sup..epsilon.mdPEG.sub.12-propionyl) B29R desB30 human
insulin; 349 A21A A22G A23K(N.sup..epsilon.mdPEG.sub.24-propionyl)
B29R desB30 human insulin; 350 A21A A22G
A23K((mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B29R desB30 human insulin;
351 A21A A22G A23G A24K(N.sup..epsilon.mPEG750-propionyl) B29R
desB30 human insulin; 352 A21A A22G A23G
A24K(N.sup..epsilon.mPEG2.000-propionyl) B29R desB30 human insulin;
353 A21A A22G A23G A24K(N.sup..epsilon.mPEG5.000-propionyl) B29R
desB30 human insulin; 354 A21A A22G A23G
A24K(N.sup..epsilon.mPEG10.000-propionyl) B29R desB30 human
insulin; 355 A21A A22G A23G
A24K(N.sup..epsilon.mPEG20.000-propionyl) B29R desB30 human
insulin; 356 A21A A22G A23G
A24K(N.sup..epsilon.mPEG40.000-propionyl) B29R desB30 human
insulin; 357 A21A A22G A23G
A24K(N.sup..epsilon.mdPEG.sub.12-propionyl) B29R desB30 human
insulin; 358 A21A A22G A23G
A24K(N.sup..epsilon.mdPEG.sub.24-propionyl) B29R desB30 human
insulin; 359 A21A A22G A23G
A24K((mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B29R desB30 human insulin;
360 A21A A22G A23G A24G A25K(N.sup..epsilon.mPEG750-propionyl) B29R
desB30 human insulin; 361 A21A A22G A23G A24G
A25K(N.sup..epsilon.mPEG2.000-propionyl) B29R desB30 human insulin;
362 A21A A22G A23G A24G A25K(N.sup..epsilon.mPEG5.000-propionyl)
B29R desB30 human insulin; 363 A21A A22G A23G A24G
A25K(N.sup..epsilon.mPEG10.000-propionyl) B29R desB30 human
insulin; 364 A21A A22G A23G A24G
A25K(N.sup..epsilon.mPEG20.000-propionyl) B29R desB30 human
insulin; 365 A21A A22G A23G A24G
A25K(N.sup..epsilon.mPEG40.000-propionyl) B29R desB30 human
insulin; 366 A21A A22G A23G A24G
A25K(N.sup..epsilon.mdPEG.sub.12-propionyl) B29R desB30 human
insulin; 367 A21A A22G A23G A24G
A25K(N.sup..epsilon.mdPEG.sub.24-propionyl) B29R desB30 human
insulin; 368 A21A A22G A23G A24G
A25K((mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B29R desB30 human insulin;
369 A21G A22K(N.sup..epsilon.mPEG750-propionyl) B29R desB30 human
insulin; 370 A21G A22K(N.sup..epsilon.mPEG2.000-propionyl) B29R
desB30 human insulin; 371 A21G
A22K(N.sup..epsilon.mPEG5.000-propionyl) B29R desB30 human insulin;
372 A21G A22K(N.sup..epsilon.mPEG10.000-propionyl) B29R desB30
human insulin; 373 A21G A22K(N.sup..epsilon.mPEG20.000-propionyl)
B29R desB30 human insulin; 374 A21G
A22K(N.sup..epsilon.mPEG40.000-propionyl) B29R desB30 human
insulin; 375 A21G A22K(N.sup..epsilon.mdPEG.sub.12-propionyl) B29R
desB30 human insulin; 376 A21G
A22K(N.sup..epsilon.mdPEG.sub.24-propionyl) B29R desB30 human
insulin; 377 A21G A22K((mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B29R
desB30 human insulin; 378 A21G A22G
A23K(N.sup..epsilon.mPEG750-propionyl) B29R desB30 human insulin;
379 A21G A22G A23K(N.sup..epsilon.mPEG2.000-propionyl) B29R desB30
human insulin; 380 A21G A22G
A23K(N.sup..epsilon.mPEG5.000-propionyl) B29R desB30 human insulin;
381 A21G A22G A23K(N.sup..epsilon.mPEG10.000-propionyl) B29R desB30
human insulin; 382 A21G A22G
A23K(N.sup..epsilon.mPEG20.000-propionyl) B29R desB30 human
insulin; 383 A21G A22G A23K(N.sup..epsilon.mPEG40.000-propionyl)
B29R desB30 human insulin; 384 A21G A22G
A23K(N.sup..epsilon.mdPEG.sub.12-propionyl) B29R desB30 human
insulin; 385 A21G A22G A23K(N.sup..epsilon.mdPEG.sub.24-propionyl)
B29R desB30 human insulin; 386 A21G A22G
A23K((mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B29R desB30 human insulin;
387 A21G A22G A23G A24K(N.sup..epsilon.mPEG750-propionyl) B29R
desB30 human insulin; 388 A21G A22G A23G
A24K(N.sup..epsilon.mPEG2.000-propionyl) B29R desB30 human insulin;
389 A21G A22G A23G A24K(N.sup..epsilon.mPEG5.000-propionyl) B29R
desB30 human insulin; 390 A21G A22G A23G
A24K(N.sup..epsilon.mPEG10.000-propionyl) B29R desB30 human
insulin; 391 A21G A22G A23G
A24K(N.sup..epsilon.mPEG20.000-propionyl) B29R desB30 human
insulin; 392 A21G A22G A23G
A24K(N.sup..epsilon.mPEG40.000-propionyl) B29R desB30 human
insulin; 393 A21G A22G A23G
A24K(N.sup..epsilon.mdPEG.sub.12-propionyl) B29R desB30 human
insulin; 394 A21G A22G A23G
A24K(N.sup..epsilon.mdPEG.sub.24-propionyl) B29R desB30 human
insulin; 395 A21G A22G A23G
A24K((mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B29R desB30 human insulin;
396 A21G A22G A23G A24G A25K(N.sup..epsilon.mPEG750-propionyl) B29R
desB30 human insulin; 397 A21G A22G A23G A24G
A25K(N.sup..epsilon.mPEG2.000-propionyl) B29R desB30 human insulin;
398 A21G A22G A23G A24G A25K(N.sup..epsilon.mPEG5.000-propionyl)
B29R desB30 human insulin; 399 A21G A22G A23G A24G
A25K(N.sup..epsilon.mPEG10.000-propionyl) B29R desB30 human
insulin; 400 A21G A22G A23G A24G
A25K(N.sup..epsilon.mPEG20.000-propionyl) B29R desB30 human
insulin; 401 A21G A22G A23G A24G
A25K(N.sup..epsilon.mPEG40.000-propionyl) B29R desB30 human
insulin; 402 A21G A22G A23G A24G
A25K(N.sup..epsilon.mdPEG.sub.12-propionyl) B29R desB30 human
insulin; 403 A21G A22G A23G A24G
A25K(N.sup..epsilon.mdPEG.sub.24-propionyl) B29R desB30 human
insulin; 404 A21G A22G A23G A24G
A25K((mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B29R desB30 human insulin;
405 A21Q A22K(N.sup..epsilon.mPEG750-propionyl) B3Q B29R desB30
human insulin; 406 A21Q A22K(N.sup..epsilon.mPEG2.000-propionyl)
B3Q B29R desB30 human insulin; 407 A21Q
A22K(N.sup..epsilon.mPEG5.000-propionyl) B3Q B29R desB30 human
insulin; 408 A21Q A22K(N.sup..epsilon.mPEG10.000-propionyl) B3Q
B29R desB30 human insulin; 409 A21Q
A22K(N.sup..epsilon.mPEG20.000-propionyl) B3Q B29R desB30 human
insulin; 410 A21Q A22K(N.sup..epsilon.mPEG40.000-propionyl) B3Q
B29R desB30 human insulin; 411 A21Q
A22K(N.sup..epsilon.mdPEG.sub.12-propionyl) B3Q B29R desB30 human
insulin; 412 A21Q A22K(N.sup..epsilon.mdPEG.sub.24-propionyl) B3Q
B29R desB30 human insulin; 413 A21Q
A22K((mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B3Q B29R desB30 human
insulin; 414 A21G A22K(N.sup..epsilon.mPEG750-propionyl) B3Q B29R
desB30 human insulin; 415 A21G
A22K(N.sup..epsilon.mPEG2.000-propionyl) B3Q B29R desB30 human
insulin; 416 A21G A22K(N.sup..epsilon.mPEG5.000-propionyl) B3Q B29R
desB30 human insulin; 417 A21G
A22K(N.sup..epsilon.mPEG10.000-propionyl) B3Q B29R desB30 human
insulin; 418 A21G A22K(N.sup..epsilon.mPEG20.000-propionyl) B3Q
B29R desB30 human insulin; 419 A21G
A22K(N.sup..epsilon.mPEG40.000-propionyl) B3Q B29R desB30 human
insulin; 420 A21G A22K(N.sup..epsilon.mdPEG.sub.12-propionyl) B3Q
B29R desB30 human insulin; 421 A21G
A22K(N.sup..epsilon.mdPEG.sub.24-propionyl) B3Q B29R desB30 human
insulin; 422 A21G A22K((mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B3Q B29R
desB30 human insulin; 423 A21A
A22K(N.sup..epsilon.mPEG750-propionyl) B3Q B29R desB30 human
insulin; 424 A21A A22K(N.sup..epsilon.mPEG2.000-propionyl) B3Q B29R
desB30 human insulin; 425 A21A
A22K(N.sup..epsilon.mPEG5.000-propionyl) B3Q B29R desB30 human
insulin; 426 A21A A22K(N.sup..epsilon.mPEG10.000-propionyl) B3Q
B29R desB30 human insulin; 427 A21A
A22K(N.sup..epsilon.mPEG20.000-propionyl) B3Q B29R desB30 human
insulin; 428 A21A A22K(N.sup..epsilon.mPEG40.000-propionyl) B3Q
B29R desB30 human insulin; 429 A21A
A22K(N.sup..epsilon.mdPEG.sub.12-propionyl) B3Q B29R desB30 human
insulin; 430 A21A A22K(N.sup..epsilon.mdPEG.sub.24-propionyl) B3Q
B29R desB30 human insulin; 431 A21A
A22K((mdPEG.sub.12).sub.3-dPEG.sub.4-yl) B3Q B29R desB30 human
insulin.
BRIEF DESCRIPTION OF THE FIGURES
[0309] FIG. 1 and FIG. 2 is the rat intratracheal drop instillation
of the insulin of example 1 and 2.
[0310] FIG. 3 is the rat intratracheal drop instillation of the
insulin of example 6.
[0311] FIG. 4 is the rat intratracheal drop instillation of the
insulin of example 5.
[0312] FIG. 5 is the rat intratracheal drop instillation of the
insulin of example 16.
[0313] FIG. 6 is the rat intratracheal drop instillation of the
insulin of example 18.
[0314] FIG. 7 is the rat intratracheal drop instillation of the
insulin of example 17.
[0315] FIG. 8 is the rat intratracheal drop instillation of the
insulin of example 19.
[0316] FIG. 9 is the rat intratracheal drop instillation of the
insulin of example 22.
[0317] FIG. 10 is the blood glucose profile by pulmonary
administration of a spray dried powder of the insulin of examples 1
and 2 to mini-pigs where the mean dose delivered was 0.037.+-.0.009
mg/kg.
[0318] FIG. 11 is the pharmacokinetic profile by pulmonary
administration of a spray dried powder of the insulin of examples 1
and 2 to mini-pigs where the mean dose delivered was 0.037.+-.0.009
mg/kg.
Sequence CWU 1
1
7121PRTArtificialvariant of insulin chain 1Gly Ile Val Glu Gln Cys
Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu1 5 10 15Glu Asn Tyr Cys Asn
20224PRTArtificialA chain 2Gly Ile Val Glu Gln Cys Cys Thr Ser Ile
Cys Ser Leu Tyr Gln Leu1 5 10 15Glu Asn Tyr Cys Asn Gly Gly Gly
20322PRTArtificialA chain 3Gly Ile Val Glu Gln Cys Cys Thr Ser Ile
Cys Ser Leu Tyr Gln Leu1 5 10 15Glu Asn Tyr Cys Asn Gly
20421PRTArtificialA chain 4Gly Ile Val Glu Gln Cys Cys Thr Ser Ile
Cys Ser Leu Glu Gln Leu1 5 10 15Glu Asn Tyr Cys Asn
20529PRTArtificialB chain 5Phe 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 Arg 20 25629PRTArtificialB chain 6Phe 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 His Tyr Thr Pro Arg 20 25730PRTArtificialB chain
7Phe 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 Gln Thr 20 25
30
* * * * *