U.S. patent application number 16/354651 was filed with the patent office on 2019-07-04 for growth hormones with prolonged in-vivo efficacy.
The applicant listed for this patent is Novo Nordisk Healthcare AG. Invention is credited to Henrik Sune Andersen, Carsten Behrens, Jens Buchardt, Nils Langeland Johansen, Leif Noerskov-Lauritsen.
Application Number | 20190203213 16/354651 |
Document ID | / |
Family ID | 42075933 |
Filed Date | 2019-07-04 |
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United States Patent
Application |
20190203213 |
Kind Code |
A1 |
Behrens; Carsten ; et
al. |
July 4, 2019 |
Growth Hormones with Prolonged In-Vivo Efficacy
Abstract
The invention relates to growth hormone compounds with a
protracted profile. The effect is obtained by linking an albumin
binding residue via a hydrophilic spacer to growth hormone
variants. Further described are methods of preparing and using such
compounds. These growth hormone compounds are based on there
althered profile considered particular useful in therapy.
Inventors: |
Behrens; Carsten;
(Koebenhavn, DK) ; Johansen; Nils Langeland;
(Koebenhavn OE, DK) ; Andersen; Henrik Sune;
(Holte, DK) ; Noerskov-Lauritsen; Leif;
(Tappernoeje, DK) ; Buchardt; Jens; (Gentofte,
DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novo Nordisk Healthcare AG |
Zurich |
|
CH |
|
|
Family ID: |
42075933 |
Appl. No.: |
16/354651 |
Filed: |
March 15, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15586960 |
May 4, 2017 |
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16354651 |
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13522390 |
Aug 24, 2012 |
9695226 |
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PCT/EP2011/050923 |
Jan 24, 2011 |
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15586960 |
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61297305 |
Jan 22, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 5/00 20180101; A61P
3/00 20180101; A61K 47/542 20170801; A61K 47/54 20170801; A61P 5/06
20180101; G01N 2030/027 20130101; A61K 47/545 20170801; A61P 43/00
20180101; C07K 14/61 20130101; C12N 15/70 20130101 |
International
Class: |
C12N 15/70 20060101
C12N015/70; A61K 47/54 20060101 A61K047/54; C07K 14/61 20060101
C07K014/61 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 22, 2010 |
EP |
10151405.7 |
Claims
1. A growth hormone conjugate which comprises a growth hormone
compound (GH) having a) a single Cys mutation, b) an additional
disulfide bridge, or c) a single Cys mutation and an additional
disulfide bridge, wherein an albumin binding residue via a
hydrophilic spacer is linked to said GH, or a pharmaceutically
acceptable salt thereof.
2. The conjugate of claim 1 wherein the growth hormone conjugate
has the formula (I): A-W--B-GH (I) wherein GH represents the growth
hormone compound; B represents a hydrophilic spacer; W is a
chemical group linking A and B; and A represent an albumin binding
residue; and pharmaceutically acceptable salts thereof.
3. The conjugate of claim 1 wherein the hydrophilic spacer is
linked to a) the N-terminal or Gln40 or Gln141 of the growth
hormone compound or b) the sulphur residue of a single Cys mutation
present in the growth hormone compound selected from the group
consisting of T3C, P5C, S7C, D11C, H18C, Q29C, E30C, E33C, A34C,
Y35C, K38C, E39C, Y42C, S43C, D47C, P48C, S55C, S57C, P59C, S62,
E65C, Q69C, E88C, Q91C, S95C, A98C, N99C, S100C, L101C, V102C,
Y103C, D107C, S108C, D112C, Q122C, G126C, E129C, D130C, G131C,
P133C, T135C, G136C, T142C, D147C, N149C, D154C, A155C, L156C,
R178C, E186C, G187C and G190C of SEQ ID NO: 1.
4. The conjugate of claim 1, wherein the GH has an additional
disulfide bridge between at least one of the amino acid pairs in
the positions selected from the group consisting of R16C/L117C,
A17C/E174C, H21C/M170C, D26C/V102C, D26C/Y103C, N47C/T50C,
Q49C/G161C, F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C,
S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C,
P61C/E66C, P61C/T67C, S71C/S132C, L73C/S132C, L73C/F139C,
R77C/I38C, R77C/F139C, L81C/Q141C, L81C/Y143C, Q84C/Y143C,
Q84C/S144C, S85C/Y143C, S85C/S144C, P89C/F146C, F92C/F146C,
F92C/T148C, R94C/D107C, V102C/A105C, L156C/F146C, L156C/T148C and
V185C/S188C in SEQ ID NO: 1.
5. The conjugate of claim 2, wherein A is selected from the group
consisting of: ##STR00170## ##STR00171## wherein * denotes the
attachment to B through W.
6. The conjugate of claim 2, wherein W has the formula
--W.sub.7--Y--, wherein Y is
--(CH.sub.2).sub.l7--C.sub.3-10-cycloalkyl-W.sub.8-- or a valence
bond; l7 is 0-6; W.sub.7 is selected from --C(O)NH--, --NHC(O)--,
--C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--, --C(O)NHS(O).sub.2--,
--S(O).sub.2NHC(O)--, --OC(O)NH--, --NHC(O)O--, --C(O)CH.sub.2--,
--CH.sub.2C(O)--, --C(O)CH.dbd.CH--, --CH.dbd.CHC(O)--,
--(CH.sub.2).sub.s3--, --C(O)--, --C(O)O--, --OC(O)--, or a valence
bond; wherein s3 is 0 or 1; W.sub.8 is selected from --C(O)NH--,
--NHC(O)--, --C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--, --OC(O)NH--,
--NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--, --C(O)CH.dbd.CH--,
--CH.dbd.CHC(O)--, --(CH.sub.2).sub.s4--, --C(O)--, --C(O)O--,
--OC(O)--, or a valence bond; wherein s4 is 0 or 1.
7. The conjugate of claim 2, wherein B has the formula
--X.sub.1--X.sub.2--X.sub.3--X.sub.4-- wherein X.sub.1 is
--W.sub.1--[(CHR.sup.1).sub.l1--W.sub.2].sub.m1--{[(CH.sub.2).sub.n1E1].s-
ub.m2--[(CHR.sup.2).sub.l2--W.sub.3].sub.m3}.sub.n2--; X.sub.2 is
--[(CHR.sup.3).sub.l3--W.sub.4].sub.m4--{[(CH.sub.2).sub.n3E2].sub.m5--[(-
CHR.sup.4).sub.l4--W.sub.5].sub.m6}.sub.n4--, X.sub.3 is
--[(CHR.sup.5).sub.l5--W.sub.6].sub.m7--; X.sub.4 is
F-D1-(CH.sub.2).sub.l6-D2-; l1, l2, l3, l4, l5 and l6 independently
are selected from 0-16, such as from 0-6 m1, m3, m4, m6 and m7
independently are selected from 0-10, such as from 0-6 m2 and m5
independently are selected from 0-25, such as from 0-10 n1, n2, n3
and n4 independently are selected from 0-16, such as from 0-10 F is
aryl, hetaryl, pyrrolidine-2,5-dione or a valence bond, wherein the
aryl and hetaryl groups are optionally substituted with halogen,
--CN, --OH, --C(O)OH, --C(O)NH.sub.2, --S(O).sub.2OH or
C.sub.1-6-alkyl; R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5
independently are selected from hydrogen, --C(O)OH, --C(O)NH.sub.2,
--S(O)OH, --S(O).sub.2OH, --NH--C(.dbd.NH)--NH.sub.2,
C.sub.1-6-alkyl, aryl or hetaryl, wherein the alkyl, aryl and
hetaryl groups optionally are substituted with halogen, --C(O)OH,
--C(O)NH.sub.2, --S(O)OH, --S(O).sub.2OH, --CN or --OH; D1, D2, E1
and E2 independently are selected from --O--, --N(R.sup.6)--,
--N(C(O)R.sup.7)-- or a valence bond, wherein R.sup.6 and R.sup.7
independently represent hydrogen or C.sub.1-6-alkyl; W.sub.1 to
W.sub.5 independently are selected from --C(O)NH--, --NHC(O)--,
--C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--, --C(O)NHS(O).sub.2--,
--S(O).sub.2NHC(O)--, --OC(O)NH--, --NHC(O)O--, --C(O)CH.sub.2--,
--CH.sub.2C(O)--, --C(O)CH.dbd.CH--, --CH.dbd.CHC(O)--,
--(CH.sub.2).sub.s2--, --C(O)--, --C(O)O--, --OC(O)--, or a valence
bond, wherein s2 is 0 or 1; W.sub.6 is selected from --C(O)NH--,
--NHC(O)--, --C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--, --OC(O)NH--,
--NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--, --C(O)CH.dbd.CH--,
--CH.dbd.CHC(O)--, --(CH.sub.2).sub.s1--, --C(O)--, --C(O)O--,
--OC(O)--, --NHC(O)C.sub.1-6-alkyl, --C(O)NHC.sub.1-6-alkyl or a
valence bond, wherein s1 is 0 or 1 and the C.sub.1-6-alkyl group is
optionally substituted with oxo, pyrrolidine-2,5-dione,
--NHC(O)CH*CH.sub.2COOH or --NHC(O)CH.sub.2CH*COOH, wherein (*)
indicates the attachment point from the carbon atom of CH to
X.sub.4.
8. The conjugate of claim 7, wherein D1 and D2 are independently
selected from --O-- or --N(R.sup.6) or a valence bond.
9. The conjugate of claim 7, wherein E1 and E2 are independently
selected from --O-- or --N(R.sup.6) or a valence bond.
10. The conjugate of claim 2, wherein W.sub.1 through W.sub.8
independently are selected from the group consisting of --C(O)NH--,
--NHC(O)--, --CH.sub.2NHC(O)--, --C(O)NHS(O).sub.2--,
--S(O).sub.2NHC(O)--, --NHC(O)C.sub.1-6-alkyl or
--C(O)NHC.sub.1-6-alkyl or a valence bond, wherein the alkyl group
is optionally substituted with oxo, pyrrolidine-2,5-dione,
--NHC(O)CH*CH.sub.2COOH or --NHC(O)CH.sub.2CH*COOH, wherein (*)
indicates the attachment point from the carbon atom of CH to
X.sub.4.
11. The conjugate of claim 7, wherein R.sup.1, R.sup.2, R.sup.3,
R.sup.4 and R.sup.5 independently are selected from hydrogen,
--C(O)OH, --C(O)NH.sub.2, --S(O).sub.2OH or C.sub.1-6-alkyl;
wherein the alkyl group optionally is substituted with --C(O)OH,
--C(O)NH.sub.2 or --S(O).sub.2OH.
12. The conjugate of claim 7, wherein X.sub.4 is a valence bond and
W.sub.6 is selected from either pyrrolidine-2,5-dione,
--NHC(O)CH*CH.sub.2COOH or --NHC(O)CH.sub.2CH*COOH, wherein (*)
indicates the attachment point from the carbon atom of CH to
GH.
13. The conjugate of claim 2, wherein B is selected from the group
consisting of: ##STR00172## ##STR00173##
14. The conjugate of claim 1, wherein the growth hormone compound
(GH) comprising an amino acid sequence having at least 85% identity
to SEQ ID NO: 1.
15. The conjugate of claim 1, wherein the growth hormone compound
(GH) comprising an amino acid sequence having at least 90% identity
to SEQ ID NO: 1.
16. The conjugate of claim 1, wherein the growth hormone compound
(GH) comprising an amino acid sequence having at least 95% identity
to SEQ ID NO: 1.
17. A compound of formula (III) A-W--B1-U (III) wherein A represent
an albumin binding residue; B1 represents a hydrophilic spacer; W
is a chemical group linking A and B1, and U represent a conjugating
moiety wherein the conjugating moiety, U, a) comprises or consists
of an aryl, an heteraryl, a substituted malimide or a
pyrrolidine-2,5-dione, b) comprises D1-(CH.sub.2).sub.l6-D2,
wherein D1 and D2 are independently selected from --O--, --N(R6)-,
--NC(O)R7- or a valence bond, wherein R6 and R7 independently
represent hydrogen or C.sub.1-6-alkyl, c) comprises or consists of
a leaving group, such as Cl, Br, I, --OH, --OS(O).sub.2Me,
--OS(O).sub.2CF.sub.3, --OTs, d) comprises or consists of an allyl
amine (H.sub.2C.dbd.CH--CH.sub.2--NH.sub.2), or e) comprises an
amine.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/586,960, filed May 4, 2017, which is a continuation of U.S.
application Ser. No. 13/522,390, filed Aug. 24, 2012 (now U.S. Pat.
No. 9,695,226, issued Jul. 4, 2017), which is a 35 U.S.C. .sctn.
371 national stage application of International Patent Application
PCT/EP2011/050923 (published as WO 2011/089255), filed Jan. 24,
2011, which claims priority of European Patent Application
10151405.7, filed Jan. 22, 2010; this application further claims
priority under 35 U.S.C. .sctn. 119 of U.S. Provisional Application
61/297,305, filed Jan. 22, 2010, all of which are hereby
incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to a growth hormone compound
linked to an albumin binding residue via a hydrophilic spacer, and
to methods of preparing and using such compounds. These growth
hormone conjugates have increased resistance to proteolytic
degradation in combination with a protracted profile of action and
are useful in therapy.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Mar. 11, 2019, is named 8069US03_SeqListing.txt and is 3
kilobytes in size.
BACKGROUND OF THE INVENTION
[0004] Growth hormone is a polypeptide hormone secreted by the
anterior pituitary in mammals. Dependent on species growth hormone
is a protein composed of approximately 190 amino acid residues
corresponding to a molecular weight of approximately 22 kDa. Growth
hormone binds to and signals through cell surface receptors, the
growth hormone receptors (GHR). Growth hormone plays a key role in
promoting growth, maintaining normal body composition, anabolism
and lipid metabolism. It also has direct effects on intermediate
metabolism, such as decreased glucose uptake, increased lipolysis,
increased amino acid uptake and protein synthesis. The hormone also
exerts effects on other tissues including adipose tissue, liver,
intestine, kidney, skeleton, connective tissue and muscle.
Recombinant human growth hormone (hGH) has been produced and
commercially available as, for ex: Genotropin.TM. (Pharmacia
Upjohn), Nutropin.TM. and Protropin.TM. (Genentech), Humatrope.TM.
(Eli Lilly), Serostim.TM. (Serono), Norditropin (Novo Nordisk),
Omnitrope (Sandoz), Nutropin Depot (Genentech and Alkermes).
Additionally, an analogue with an additional methionine residue at
the N-terminal end is also marketed as, for ex: Somatonorm.TM.
(Pharmacia Upjohn/Pfizer).
[0005] Growth hormone shares a common topology with the other
members of the growth hormone family of proteins, Prolactin (PRL)
and Placental Lactogen (PL). Growth hormone is classified as a
four-helix bundle protein (FIG. 1) exhibiting an "up-up-down-down"
topology with two conserved disulphide linkages. Specifically,
wild-type human Growth hormone (hGH) is composed of 191 amino acid
residues and has four cysteine residues at positions 53, 165, 182
and 189, which stabilizes the three dimensional structure of the
protein by forming two intramolecular disulphide bonds connecting
C53 with C165 and C182 with C189, respectively (FIG. 1). The
structure of hGH has been experimentally determined by X-ray
crystallography in the free form (Chantalet L. et al Protein and
Peptide Letters 3, 333-340, (1995)) and in complex with its binding
protein (the extra cellular domain of the human GHR (hGHR)) (Devos,
A. M. et al Science 255, 306-312, (1992)). These structures have
been deposited in the Protein Data Bank (PDB) and are publicly
available (PDB accession codes 1HGU and 1HWG, respectively). Thus,
from the published hGH structures residues important for hGH
binding to hGHR can be identified. Furthermore, the dynamic
properties of hGH has been studied by Nuclear Magnetic Resonance
(NMR) spectroscopy (Kasimova M. R. et al. J. Mol. Biol. 318,
679-695, (2002)). In combination, the X-ray and NMR data can
distinguish regions of hGH which are well structured and well
defined from regions which are less structured and dynamic. Less
structured and dynamic regions of hGH are expected to be
particularly susceptible to proteolytic cleavage and proper
stabilization of such regions would lead to improved proteolytic
stability.
[0006] hGH has been subject to extensive mutagenesis in attempts to
produce hGH analogues with desired chemical or biological
properties. Specifically, cysteine mutants for several purposes
have been described.
[0007] US 2003/0162949 disclose cysteine variants of members of the
GH supergene family. A general method is provided for creating
site-specific, biologically active conjugates of these proteins.
The method involves adding cysteine residues to non-essential
regions of the proteins or substituting cysteine residues for
non-essential amino acids in the proteins using site-directed
mutagenesis and then covalently coupling a cysteine-reactive
polymer or other type of cysteine-reactive moiety to the proteins
via the added cysteine residue
[0008] WO 02/055532 describes genetically engineered hGH mutants
having at least one non-polypeptide moiety covalently attached,
particularly hGH mutants where a introduced cysteine residue was
used for pegylation.
[0009] U.S. Pat. No. 5,951,972 describes physiologically active
derivatized natural and recombinant mammalian and human proteins
and polypeptides wherein at least one-naturally-occurring or
incorporated cysteine residue within the protein is derivatized
with various substituents.
[0010] The proteolytic cleavage of hGH has been studied in detail.
The long loop composed of residues 128 to 154 has putative cleavage
sites for several proteases, such as thrombin, plasmin,
collagenase, subtilisin and chymotrypsin-like serine proteases.
Accordingly, this part of hGH has been shown to be particularly
susceptible to proteolytic cleavage (Lewis, U. J. Ann. Rev.
Physiol. 46, 33-42, (1984)). Enzymes reported to degrade hGH
include thrombin, plasmin, subtilisin, chymotrypsin-like serine
proteinases and kallikreins.
[0011] The degradation of hGH in rat tissue has been investigated
(Garcia-Barros et al. J. Endocrinol. Invest. 23, 748-754,
(2000)).
[0012] In rat thyroid gland chymotrypsin-like proteases, favouring
cleavage at bulky and lipophilic amino acid residues, were found
initially to cleave the peptide bond between Y143 and S144
resulting in a two chain molecule, followed by cleavage between Y42
and S43, liberating the N-terminal peptide F1-Y42. The split loop
in the two chain molecule is processed further by cleavage between
F146 and D147 by chymotrypsin-like proteases and further by the
action of carboxypeptidases.
[0013] Several methods to produce hGH analogues stabilized towards
proteolytic degradation have been reported.
[0014] Alam et al., J. Biotech. 65, 183-190, (1998) designed hGH
mutants resistant to thrombin and plasmin by specific point
mutations. Thrombin cleaves hGH specifically between R134 and T135,
and the double mutant R134D, T135P yielded a hGH variant resistant
to cleavage by thrombin, and the triple mutant R134D, T135P, K140A
resulted in resistance to plasmin. Furthermore, the latter hGH
mutant was resistant to proteolysis by human plasma over a period
of 7 days.
[0015] EP 534568 describes hGH mutants stabilized towards
proteolytic degradation by mutating R134 to alanine, leucine,
threonine, phenylalanine, proline or histidine.
[0016] WO 2004/022593/Nautilus describes general high through-put
directed evolution methods to produce modified cytokines, including
GH variants, with increased proteolytic stability.
[0017] WO 2006/048777/Nautilus specifically describes modified hGH
analogues with improved proteolytic stability. The analogues
contain one to five mutations at positions 1-55, 57, 58, 60-63,
67-87, 89-91, 93, 95-100, 102-128, 131-132, 135-139, 141, 142, 144,
148-182, 184, 185 and 187-191. Introduction of cysteine residues
can potentially lead to the formation of undesired disulfide linked
dimers and in WO 2006/048777 the substitution of amino acid
residues by cysteine is specifically excluded from the scope; in WO
2006/048777 (p. 65) it is stated: "The replacement of amino acids
by cysteine residues is explicitly avoided since this change would
potentially lead to the formation of intermolecular disulfide
bonds".
[0018] There is an obvious need to develop hGH compounds which are
resistant to proteolytic degradation. Such stabilized compounds
should exhibit increased stability towards proteolytic cleavage
while retaining the desired biological properties of hGH. Such GH
molecules would have increased stability, slower clearance and/or
prolong in vivo half-life.
[0019] Furthermore it is well-known to modify the properties and
characteristics of peptides by conjugating groups to the peptide
which duly changes the properties of the peptide. Such conjugation
generally requires some functional group in the peptide to react
with another functional group in a conjugating group. Typically,
amino groups, such as the N-terminal amino group or the s-amino
group in lysines, have been used in combination with a suitable
acylating reagent. Alternatively, polyethylene glycol (PEG) or
derivatives thereof may be attached to proteins. For a review, see
Exp. Opion. Ther. Patent. 14, 859-894, (2004). It has been shown
that the attachment of PEG to growth hormone may have a positive
effect on the plasma half-life of growth hormone, WO 03/044056.
[0020] The use of carboxypeptidases to modify the C-terminal of
peptides has been described earlier. WO 92/05271 discloses the use
of carboxypeptidases and nucleophilic compounds to amidate the
C-terminal carboxy group, and WO 98/38285 discloses variants of
carboxypeptidase Y particular suitable for this purpose.
[0021] EP 243 929 discloses the use of carboxypeptidase to
incorporate polypeptides, reporter groups or cytotoxic agents into
the C-terminal of proteins or polypeptides.
[0022] WO 2005/035553 describes methods for selective conjugation
of peptides by enzymatically incorporating a functional group at
the C-terminal of a peptide.
[0023] Activated halogen derivatives and maleimides represent some
of the most common used functional groups when incorporateing
conjugates to sulfhydryl groups in peptides (G. T. Hermanson in
Bioconjugate Techniques 2. Ed. 2008, Elsevier).
[0024] Transglutaminase has previously been used to alter the
properties of peptides. In the food industry and particular in the
diary industry many techniques are available to e.g. cross-bind
peptides using transglutaminases. Other documents disclose the use
of transglutaminase to alter the properties of physiologically
active peptides. EP 950665, EP 785276 and Sato, Adv. Drug Delivery
Rev. 54, 487-504, (2002) disclose the direct reaction between
peptides comprising at least one Gin and amine-functionalised PEG
or similar ligands in the presence of transglutaminase, and Wada,
Biotech. Lett. 23, 1367-1372, (2001) discloses the direct
conjugation of .beta.-lactoglobulin with fatty acids by means of
transglutaminase. The international patent application published as
WO 2005/070468 discloses the use of transglutaminase to incorporate
a handle whereto conjugating groups can be attached.
[0025] Growth hormone is a key hormone involved in the regulation
of not only somatic growth, but also in the regulation of
metabolism of proteins, carbohydrates and lipids. The major effect
of growth hormone is to promote growth. Human growth hormone is a
191 amino acid residue protein with the sequence:
TABLE-US-00001 (SEQ ID NO: 1) FPTIPLSRLFDNAMLRAHR-
LHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELL-
RISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRT-
GQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF.
[0026] Administration of human growth hormone and closely related
variants thereof is used to treat a variety of growth hormone
deficiency related diseases. Being a polypeptide, growth hormone is
administered parenterally, i.e., by means of a needle. Growth
hormone is, furthermore, characterised by a relative short
half-life, hence frequent administrations are required with the
corresponding pain and inconvenience for the patient. Hence, there
is still a need for the provision of growth hormone compounds with
improved pharmacological properties, such as e.g. prolonged
half-life.
[0027] The present invention provides novel growth hormone compound
conjugates with improved pharmakinetic and pharmacological
properties as well as methods for their production.
SUMMARY OF THE INVENTION
[0028] The bioavailability of a subcutaneously administed
pharmaceutical compound may be related to the absorption rate. The
ability of a compound to pass the tight junctions of the
subcutaneous capillaries may in part be related to their physical
and chemical properties as well as the molecular size or the
hydrodynamic volume of the compound. A protein conjugate such as a
pegylated hGH (PEG-hGH) with a 40 kDa PEG has an apparent molecular
weight of 150-250 kDa. A hGH molecule with covalent bound albumin
has a molecular weight of 87 kDa, whereas a hGH molecule with a
non-covalent bound albumin will be dissociated from albumin part of
the time and thus have a molecular weight of 22 kDa.
[0029] It is contemplated that the time spend in the dissociated
state depends, at least partly, on the affinity of the albumin
binding moiety. Thus the absorption rate of a hGH molecule with a
non-covalent bound albumin may be faster than for a PEG-hGH. An
increased rate of absorption may be obtained when using albumin
binding moieties having lower affinity for albumin.
[0030] Additionally, the physical and chemical properties of the
linker and/or the spacer providing the attachment of the albumin
binding moiety to hGH will influence the functionalities of the
compounds.
[0031] The present inventors have surprisingly found that growth
hormone compounds (GH) with a single Cys mutation and/or an
additional disulfide bridge may be selectively linked to an albumin
binding residue--via a hydrophilic spacer that separates the GH and
the albumin binding residue, typically with a chemical moiety
having a m Log P<0--or a c Log P<0.5 to obtain GH conjugates
with improved properties, such as high in vitro potency, or such as
an increase in vivo half life, or such as increased resistant to
proteolytic degradation possibly in combination with a protracted
in vivo profile of action. By linking an albumin binding residue
via a hydrophilic spacer to the single Cys mutation the biological
activity may be retained and one or more of the above mention
improvements may be obtained. Such improvements are also obtained
when an albumin binding residue via a hydrophilic spacer is linked
to the growth hormone having an additional disulfide bridge, such
as to the N-terminal, position 40 or position 141 of hGH. The
growth hormone compound may also comprise both a single Cys
mutation and an additional disulfide bridge, in which aspect the
albumin binding residue via a hydrophilic spacer is linked to the
single Cys mutation.
[0032] In a broad aspect the present invention relates to a growth
hormone conjugate which comprises a growth hormone compound (GH)
having
[0033] a) a single Cys mutation,
[0034] b) an additional disulfide bridge, or
[0035] c) a single Cys mutation and an additional disulfide
bridge,
wherein an albumin binding residue via a hydrophilic spacer is
linked to said GH, or a pharmaceutically acceptable salt
thereof.
[0036] In one embodiment of the present invention the stable hGH
compounds have additional disulphide bond(s). The disulphide bonds
are formed between pairs of cysteines of which one or both are
introduced by point mutations in the wild type hGH sequence.
[0037] In another embodiment of the present invention the stable
hGH compounds have additional cysteines. The cysteines are
introduced by point mutations in the wild type hGH sequence.
[0038] In a further embodiment of the present invention the stable
hGH compounds have additional disulphide bond(s) and one or more
additional cysteines. The additional disulphide bond(s) formed
between pairs of additional cysteines and the additional cysteines
are introduced by point mutations in the wild type hGH
sequence.
[0039] Furthermore, the present invention is based on the
observation that introducing an albumin binding residue via a
hydrophilic spacer in human growth hormone (hGH) can be done
selectively wherein a large proportion of the activity has been
retained. Preferably, an albumin binding residue via a hydrophilic
spacer is introduced at the position(s) corresponding to the
introduced cystein(s) and/or in position glutamine 40 and/or in
position glutamine 141 and/or the N-terminal in hGH having the
sequence of SEQ ID NO: 1. The use of transglutaminase (TGase), and
in particular TGase from Streptoverticillium mobaraenae or
Streptomyces lydicus allows a selective introduction of an albumin
binding residue via a hydrophilic spacer at position 40 or position
141, and the remaining 11 glutamine residues are left untouched
despite the fact that glutamine is a substrate for
transglutaminase.
[0040] Thus, in one embodiment of the present invention the growth
hormone compound (GH) is linked to one albumin binding residue via
a hydrophilic spacer. Typically, the albumin binding residue is
attached to the N-terminal, or to position 18, 30, 40, 42, 62, 69,
88, 95, 98, 99, 100, 101, 102, 108, 135, 141 or 154 of hGH via a
hydrophilic spacer. In further embodiments two albumin binding
residues are attached to the single Cys mutation and any one of the
above positions, such as the N-terminal, position 40 or position
141 of hGH via a hydrophilic spacer.
[0041] The growth hormone compound conjugates of the present
invention have faster subcutaneous absorption compared to PEGylated
hGH, and thus, provides less or no lipoathrophy. Furthermore, the
albumin binding residue and the hydrophilic spacer are
biodegradable in contrast to PEG.
[0042] It is a still further objective of the present invention to
provide a method for improving the properties of a GH by
conjugation said protein according to the methods of the present
invention.
[0043] In further aspects the invention relates to isolated growth
hormone compounds (GH) comprising a single cys mutation, an
additional disulfide bond or growth hormone compounds comprising a
single cys mutation and an additional disulfide bond. In a further
object of the invention such compounds are soluble.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] FIG. 1 is a depiction of the three dimensional structure of
wild-type human growth hormone. The depiction shows the
intramolecular disulphide bonds connecting C53 with C165 and C182
with C189.
[0045] FIG. 2 is a depiction of the sequence of human growth
hormone and designates loop and helical segments.
DEFINITIONS
[0046] In the present context, the term "growth hormone compound"
as used herein means growth hormone of mammalian origin, such as
human, bovine, or porcine growth hormone, and recombinant growth
hormone, such as recombinant human, bovine, or porcine growth
hormone, and variants as well as mutants of such growth hormones.
As used herein "GH" and "growth hormone compound" are
interchangeable. When GH is a variant of growth hormone of
mammalian origin, such as hGH and recombinant hGH, said variant is
understood to be the compound obtained by substituting one or more
amino acid residues in the growth hormone, e.g. hGH, sequence with
another natural or unnatural amino acid; and/or by adding one or
more natural or unnatural amino acids to the growth hormone, e.g.
hGH, sequence; and/or by deleting one or more amino acid residue
from the growth hormone, e.g. hGH, sequence, wherein any of these
steps may optionally be followed by further derivatization of one
or more amino acid residues. Typically, the GH has at least 80%
identity with hGH, and typically, at least 10% of the growth
hormone activity of hGH as determined in assay (I) (Example 46)
herein.
[0047] In the present context, the term "albumin binding residue"
as used herein means a residue which binds noncovalently to human
serum albumin. The albumin binding residue attached to the growth
hormone compound (GH) typically has a binding affinity towards
human serum albumin that is below about 10 .mu.M or even below
about 1 .mu.M. A range of albumin binding residues are known among
linear and branched lipohophillic moieties containing 12-40 carbon
atoms, compounds with a cyclopentanophenanthrene skeleton, and/or
peptides having 10-45 amino acid residues etc. Albumin binding
properties can be measured by surface plasmon resonance as
described in J. Biol. Chem. 277(38), 35035-35042, (2002).
[0048] The term "hydrophilic spacer" as used herein means a spacer
that separates a growth hormone compound and an albumin binding
residue with a chemical moiety which comprises at least 5
nonhydrogen atoms where 30-50% of these are either N or O.
[0049] In the present context, the term "transamination" and
related terms are intended to indicate a reaction wherein the amide
nitrogen in the side chain of glutamine is exchanged with nitrogen
from another compound, in particular nitrogen from another nitrogen
containing nucelophile.
[0050] Transglutaminase (E.C.2.3.2.13) is also known as
protein-glutamine-yglutamyltransferase and catalyses the general
reaction
##STR00001##
Q-C(O)--NH.sub.2 (amine acceptor) may represent a glutamine residue
containing peptide or protein and Q'-NH.sub.2 (amine donor)
represents an amine-containing nucleophile. Alternatively,
QC--(O)--NH.sub.2 and Q'-NH.sub.2 may represent an amine acceptor
and a lysine-containing peptide or protein, respectively. In the
present invention, however, Q-C(O)--NH.sub.2 represents a glutamine
residue containing growth hormone and Q'-NH.sub.2 represents an
amine-containing nucleophile as indicated above.
[0051] Examples of useful transglutaminases include microbial
transglutaminases, such as e.g. those from Streptomyces mobaraense,
Streptomyces cinnamoneum and Streptomyces griseocarneum (all
disclosed in U.S. Pat. No. 5,156,956, which is incorporated herein
by reference), and from Streptomyces lavendulae (disclosed in U.S.
Pat. No. 5,252,469, which is incorporated herein by reference) and
Streptomyces ladakanum (JP 2003/199569, which is incorporated
herein by reference). It should be noted that members of the former
genus Streptoverticillium are now included in the genus
Streptomyces (Kaempfer, J. Gen. Microbiol. 137, 1831-1892, (1991)).
Other useful microbial transglutaminases have been isolated from
Bacillus subtilis (disclosed in U.S. Pat. No. 5,731,183, which is
incorporated herein by reference) and from various Myxomycetes.
Other examples of useful microbial transglutaminases are those
disclosed in WO 96/06931 (e.g. transglutaminase from Bacillus
lydicus) and WO 96/22366, both of which are incorporated herein by
reference. Useful non-microbial transglutaminases include
guinea-pig liver transglutaminase, and transglutaminases from
various marine sources like the flat fish Pagrus major (disclosed
in EP-0555649, which is incorporated herein by reference), and the
Japanese oyster Crassostrea gigas (disclosed in U.S. Pat. No.
5,736,356, which is incorporated herein by reference).
[0052] In the present context, the term "not accessible" is
intended to indicate that something is absent or de facto absent in
the sense that it cannot be reached. When it is stated that
functional groups are not accessible in a protein to be conjugated
it is intended to indicate that said functional group is absent
from the protein or, if present, in some way prevented from taking
part in reactions. By way of example, said functional group could
be buried deep in the structure of the protein so that it is
shielded from participating in the reaction. It is recognised that
whether or not a functional group is accessible depends on the
reaction conditions. It may be envisaged that, e.g. in the presence
of denaturing agents or at elevated temperatures the protein may
unfold to expose otherwise not accessible functional groups. It is
to be understood that "not accessible" means "not accessible at the
reaction condition chosen for the particular reaction of
interest".
[0053] The term "alkane" or "alkyl" is intended to indicate a
saturated, linear, branched and/or cyclic hydrocarbon. Unless
specified with another number of carbon atoms, the term is intended
to indicate hydrocarbons with from 1 to 30 (both included) carbon
atoms, such as 1 to 20 (both included), such as from 1 to 10 (both
included), e.g. from 1 to 5 (both included). The terms alkyl and
alkylene refer to the corresponding radical and bi-radical,
respectively.
[0054] The term "C.sub.1-6 alkyl" refers to a straight chained or
branched saturated hydrocarbon having from one to six carbon atoms
inclusive. Examples of such groups include, but are not limited to,
methyl, 2-propyl, 1-butyl, 2-butyl, 2-methyl-2-propyl,
2-methyl-1-butyl and n-hexyl.
[0055] The term "C.sub.3-10 cycloalkyl" typically refers to
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
cyclooctyl, cyclononyl, and cyclodecanyl.
[0056] The term "alkene" is intended to indicate linear, branched
and/or cyclic hydrocarbons comprising at least one carbon-carbon
double bond. Unless specified with another number of carbon atoms,
the term is intended to indicate hydrocarbons with from 2 to 30
(both included) carbon atoms, such as 2 to 20 (both included), such
as from 2 to 10 (both included), e.g. from 2 to 5 (both included).
The terms alkenyl and alkenylene refer to the corresponding radical
and bi-radical, respectively.
[0057] The term "alkyne" is intended to indicate linear, branched
and/or cyclic hydrocarbons comprising at least one carbon-carbon
triple bond, and it may optionally comprise one or more
carbon-carbon double bonds. Unless specified with another number of
carbon atoms, the term is intended to indicate hydrocarbons with
from 2 to 30 (both included) carbon atoms, such as from 2 to 20
(both included), such as from 2 to 10 (both included), e.g. from 2
to 5 (both included). The terms alkynyl and alkynylene refer to the
corresponding radical and bi-radical, respectively.
[0058] The term "homocyclic aromatic compound" is intended to
indicate aromatic hydrocarbons, such as benzene and
naphthalene.
[0059] The term "heterocyclic compound" is intended to indicate a
cyclic compound comprising 5, 6 or 7 ring atoms from which 1, 2, 3
or 4 are hetero atoms selected from N, O and/or S. Examples include
heterocyclic aromatic compounds, such as thiophene, furan, pyran,
pyrrole, imidazole, pyrazole, isothiazole, isooxazole, pyridine,
pyrazine, pyrimidine, pyridazine, as well as their partly or fully
hydrogenated equivalents, such as piperidine, pirazolidine,
pyrrolidine, pyroline, imidazolidine, imidazoline, piperazine and
morpholine.
[0060] The terms "hetero alkane", "hetero alkene" and "hetero
alkyne" are intended to indicate alkanes, alkenes and alkynes as
defined above, in which one or more hetero atom or group have been
inserted into the structure of said moieties. Examples of hetero
groups and atoms include --O--, --S--, --S(O)--, --S(O).sub.2--,
--C(O)--C(S)-- and --N(R*)--, wherein R* represents hydrogen or
C.sub.1-C.sub.6-alkyl. Examples of heteroalkanes include.
##STR00002##
[0061] The term "radical" or "biradical" is intended to indicate a
compound from which one or two, respectively, hydrogen atoms have
been removed. When specifically stated, a radical may also indicate
the moiety formed by the formal removal of a larger group of atoms,
e.g. hydroxyl, from a compound.
[0062] The term "halogen" is intended to indicate members of the
seventh main group of the periodic table, e.g. F, Cl, Br and I.
[0063] In the present context, the term "aryl" is intended to
indicate a carbocyclic aromatic ring radical or a fused aromatic
ring system radical wherein at least one of the rings are aromatic.
Typical aryl groups include phenyl, biphenylyl, naphthyl, and the
like.
[0064] The term "heteroaryl" or "hetaryl", as used herein, alone or
in combination, refers to an aromatic ring radical with for
instance 5 to 7 member atoms, or to a fused aromatic ring system
radical with for instance from 7 to 18 member atoms, wherein at
least one ring is aromatic, containing one or more heteroatoms as
ring atoms selected from nitrogen, oxygen, or sulfur heteroatoms,
wherein N-oxides and sulfur monoxides and sulfur dioxides are
permissible heteroaromatic substitutions. Examples include furanyl,
thienyl, thiophenyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl,
tetrazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl,
thiadiazolyl, isothiazolyl, pyridinyl, pyridazinyl, pyrazinyl,
pyrimidinyl, quinolinyl, isoquinolinyl, benzofuranyl,
benzothiophenyl, indolyl, and indazolyl, and the like.
[0065] The term "conjugate" as a noun is intended to indicate a
modified protein, i.e. a protein with a moiety bonded to it in
order to modify the properties of said protein. As a verb, the term
is intended to indicate the process of bonding a moiety to a
protein to modify the properties of said protein.
[0066] The term "single cys" or a "free cysteine" refers to a
cysteine residue, which is not engaged in double bond. A protein,
may thus include one or more single cys residues in addition to one
or more additional disulfide bridge(s), as long as said single
cys's do not lead to internal disulfide bridge(s).
[0067] As used herein, the term "prodrug" indicates biohydrolyzable
amides and biohydrolyzable esters and also encompasses a) compounds
in which the biohydrolyzable functionality in such a prodrug is
encompassed in the compound according to the present invention, and
b) compounds which may be oxidized or reduced biologically at a
given functional group to yield drug substances according to the
present invention. Examples of these functional groups include
1,4-dihydropyridine, N-alkylcarbonyl-1,4-dihydropyridine,
1,4-cyclohexadiene, tert-butyl, and the like.
[0068] As used herein, the term "biohydrolyzable ester" is an ester
of a drug substance (in casu, a compound according to the
invention) which either a) does not interfere with the biological
activity of the parent substance but confers on that substance
advantageous properties in vivo such as duration of action, onset
of action, and the like, or b) is biologically inactive but is
readily converted in vivo by the subject to the biologically active
principle. The advantage is, for example increased solubility or
that the biohydrolyzable ester is orally absorbed from the gut and
is transformed to a compound according to the present invention in
plasma. Many examples of such are known in the art and include by
way of example lower alkyl esters (e.g., C.sub.1-C.sub.4), lower
acyloxyalkyl esters, lower alkoxyacyloxyalkyl esters, alkoxyacyloxy
esters, alkyl acylamino alkyl esters, and choline esters.
[0069] As used herein, the term "biohydrolyzable amide" is an amide
of a drug substance (in casu, a compound according to the present
invention) which either a) does not interfere with the biological
activity of the parent substance but confers on that substance
advantageous properties in vivo such as duration of action, onset
of action, and the like, or b) is biologically inactive but is
readily converted in vivo by the subject to the biologically active
principle. The advantage is, for example increased solubility or
that the biohydrolyzable amide is orally absorbed from the gut and
is transformed to a compound according to the present invention in
plasma. Many examples of such are known in the art and include by
way of example lower alkyl amides, .alpha.-amino acid amides,
alkoxyacyl amides, and alkylaminoalkylcarbonyl amides.
[0070] In the present context, the term "pharmaceutically
acceptable salt" is intended to indicate salts which are not
harmful to the patient. Such salts include pharmaceutically
acceptable acid addition salts, pharmaceutically acceptable metal
salts, ammonium and alkylated ammonium salts. Acid addition salts
include salts of inorganic acids as well as organic acids.
Representative examples of suitable inorganic acids include
hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, nitric
acids and the like. Representative examples of suitable organic
acids include formic, acetic, trichloroacetic, trifluoroacetic,
propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic,
maleic, malic, malonic, mandelic, oxalic, picric, pyruvic,
salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric,
ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic,
gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic,
p-aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic acids
and the like. Further examples of pharmaceutically acceptable
inorganic or organic acid addition salts include the
pharmaceutically acceptable salts listed in J. Pharm. Sci. 66, 2,
(1977) which is incorporated herein by reference. Examples of metal
salts include lithium, sodium, potassium, magnesium salts and the
like. Examples of ammonium and alkylated ammonium salts include
ammonium, methylammonium, dimethylammonium, trimethylammonium,
ethylammonium, hydroxyethylammonium, diethylammonium,
butylammonium, tetramethylammonium salts and the like.
[0071] A "therapeutically effective amount" of a compound as used
herein means an amount sufficient to cure, alleviate or partially
arrest the clinical manifestations of a given disease and its
complications. An amount adequate to accomplish this is defined as
"therapeutically effective amount". Effective amounts for each
purpose will depend on the severity of the disease or injury as
well as the weight and general state of the subject. It will be
understood that determining an appropriate dosage may be achieved
using routine experimentation, by constructing a matrix of values
and testing different points in the matrix, which is all within the
ordinary skills of a trained physician or veterinary.
[0072] The term "treatment" and "treating" as used herein means the
management and care of a patient for the purpose of combating a
condition, such as a disease or a disorder. The term is intended to
include the full spectrum of treatments for a given condition from
which the patient is suffering, such as administration of the
active compound to alleviate the symptoms or complications, to
delay the progression of the disease, disorder or condition, to
alleviate or relief the symptoms and complications, and/or to cure
or eliminate the disease, disorder or condition as well as to
prevent the condition, wherein prevention is to be understood as
the management and care of a patient for the purpose of combating
the disease, condition, or disorder and includes the administration
of the active compounds to prevent the onset of the symptoms or
complications. The patient to be treated is preferably a mammal; in
particular a human being, but it may also include animals, such as
dogs, cats, cows, sheep and pigs.
DESCRIPTION OF THE INVENTION
[0073] In a broad aspect the present invention relates to a stable
growth hormone conjugate which comprises a growth hormone compound
(GH) having
a) a single Cys mutation, b) an additional disulfide bridge, or c)
a single Cys mutation and an additional disulfide bridge, wherein
an albumin binding residue via a hydrophilic spacer is linked to
said GH, or a pharmaceutically acceptable salt thereof.
[0074] When a single Cys mutation is present, an albumin binding
residue via a hydrophilic spacer is linked to the sulphur residue
of the Cys. When an additional disulphide bridge is present (but no
single Cys mutation) then an albumin binding residue via a
hydrophilic spacer is linked to a position in the growth hormone
compound, such as position 40, position 141 or the N-terminal of
hGH, as described herein. When two or more albumin binding residues
are linked to the growth hormone compound via a hydrophilic spacer,
then such albumin binding residues are linked to a single Cys
mutation if such mutation is present or if only an additional
disulphide bridge is present then an albumin binding residue via a
hydrophilic spacer is linked to a position in the growth hormone
compound as described herein.
[0075] In one embodiment the growth hormone compound has one single
Cys mutation.
[0076] In another embodiment the growth hormone compound has two
single Cys mutations.
[0077] In a further embodiment the growth hormone compound has an
additional disulfide bridge.
[0078] In a further embodiment the growth hormone compound has one
single Cys mutation and one additional disulfide bridge.
[0079] In a further embodiment GH represents a growth hormone
compound comprising an amino acid sequence having at least 90%
identity to the amino acid sequence of human growth hormone (hGH)
(SEQ ID NO: 1). In further embodiments, GH has at least 80%, such
as at least 85%, such as at least 95% identity with hGH, such at at
least 96%, such as at least 97%, such as at least 98% or such as at
least 99% identity with SEQ ID NO: 1. In further embodiments, said
identities to hGH is coupled to at least 10%, such as at least 20%,
such as at least 40%, such as at least 60%, such as at least 80% of
the growth hormone activity of hGH as determined in assay I herein.
Any one of the sequence identity embodiments may be combined with
any one of the activity embodiments, such as a GH having at least
80% identity with hGH and coupled to at least 60% of the growth
hormone activity of hGH; a GH having at least 90% identity with hGH
and coupled to at least 40% of the growth hormone activity of hGH;
a GH having at least 95% identity with hGH and coupled to at least
80% of the growth hormone activity of hGH, and so forth. As
described herein HG may be expressed as MetHG which indicates that
the sequence comprise an additional N-terminal methionine.
[0080] In an embodiment GH is a growth hormone variant wherein a
single Cys mutation is introduced. In further embodiments GH
represents a growth hormone compound containing one to five
mutations in addition to a single Cys mutation.
[0081] In a further embodiment the albumin binding residue via a
hydrophilic spacer is linked to the single Cys mutation. In an
embodiment the single Cys mutation is position in the N-terminal,
H1, H2, L2 or H3 of GH. In further embodiments, the single Cys
mutation is positioned in the N-terminal, the mutation being such
as any one of T3C, P5C, S7C, or in H1 (corresponding to AA 9-35),
the mutation being such as any one of D11C, H18C, Q29C, E30C, E33C,
A34C, Y35C, or in L1 (corresponding to AA36-71), the muation being
such as any one of K38C, E39C, Y42C, S43C, D47C, P48C, 555, S57C,
P59C, S62C, E65C, Q69C or preferably any one of Y42C, S55C, S57C,
S62C, Q69C or in H2, L2 or H3 (corresponding to AA 72-98, AA 99-106
and AA 107-127), the mutation being such as any one of E88C, Q91C,
S95C, A98C, N99C, S100C, L101C, V102C, Y103C, D107C, S108C, D112C,
Q122C and G126C of hGH (SEQ ID NO: 1) or in L3 or H4 (corresponding
to AA128-154 and AA155-184) In L3 and H4 (128-154 and AA155-184)
the muation being such as any one of E129C, D130C, G131C, P133C,
T135C, G136C, T142C, D147C, N149C, D154C, A155C, L156C, R178C,
V180C or in the C-terminal the muation being such as any one of
E186C G187C G190C.
[0082] If the single Cys mutation is present in a hGH variant the
mutation is located in corresponding amino acid residues.
[0083] In particular embodiment the GH the single Cys mutation has
been introduced in a position equivalent to a parent GH that is
equivalent to a position of hGH (SEQ ID NO: 1) selected from the
group consisting of: T3, P5, S7, D11, H18, Q29, E30, E33, A34, Y35,
K38, E39, Y42, S43, D47, P48, S55, S57, P59, S62, E65, Q69, E88,
Q91, S95, A98, N99, S100, L101, V102, Y103, D107, S108, D112, Q122,
G126, E129, D130, G131, P133, T135, G136, T142, D147, N149, D154,
A155, L156, R178, E186, G187 and G190, such as the group consisting
of: T3, P5, S7, D11, H18, Q29, E30, E33, A34, Y35, E88, Q91, S95,
A98, N99, S100, L101, V102, Y103, D107, S108, D112, Q122 and G126
the GH conjugate further comprising an albumin binding moiety at
the side chain of said single cysteine residue.
[0084] In further embodiments the single Cys mutation is located
within AA 93-106 in hGH or corresponding residues in hGH variants.
In further specified embodiments the single Cys mutation is located
within L2, such as within AA 99-106 or AA 99-103 or corresponding
residues.
[0085] When a single Cys mutation is present in the growth hormone
compound conjugate of the present invention, a typical single Cys
mutation is E30C. A further typical single Cys mutation is Y42C. A
further typical single Cys mutation is S55C. A further typical
single Cys mutation is S57C. A further typical single Cys mutation
is S62C. A further typical single Cys mutation is Q69C. A further
typical single Cys mutation is S95C. A further typical single Cys
mutation is A98C. A further typical single Cys mutation is N99C. A
further typical single Cys mutation is S100C. A further typical
single Cys mutation is L101C. A further typical single Cys mutation
is V102C. A further typical single Cys mutation is S108C.
[0086] According to the crystal structure of the rhGH/receptor
complex (PDB: 3HHR) the bundle consists of four major helices:
first helix (A) from residue 9 to 34, second helix (B) from residue
72 to 92 and from residue 94 to 100, third helix (C) from residue
106 to 128, and fourth helix (D) from residue 155 to 184 (M. R.
Kasimova et. al. J. Mol. Biol. 318, 679-695, (2002)). The four main
helices are referred to as the core of the protein. Residues that
are not part of the helical regions are defined as loop residues,
and may be part of flexible regions, loops, .beta.-turns, hairpins
and coils. A slightly different localization of helix's is obtained
when hGH is in complex with its binding protein (PDB: 1 HWG), which
is the helix defintion referred to above.
[0087] Moreover, the invention relates to a GH conjugate comprising
at least one introduced cysteine residue which residue has been
introduced in a position equivalent to a position in a helix or
loop region of hGH. In particular the amino acid residues may be
introduced in a surface exposed position in a helix or loop region
that has more than 25% of its side chain exposed at the surface,
preferably more than 50% of its side chain exposed at the surface,
e.g. in a model structure of hGH alone or in a model structure of
hGH complexed to its two receptor molecules. In a preferred
embodiment, the position in the helix or the loop is equivalent to
a position outside a receptor binding site of hGH. Surface exposed
residues may be identified using computational chemistry
algorithms. For example, relative surface accessibilities can be
calculated with the computer program Quanta 2005 from Accelrys Inc.
using the atomic coordinates from the publically available
structures (PDB accession codes 1HGU and 1HWG structure) and
default parameter settings. A description of the underlying
principle behind the algorithm can be found in B. Lee and F. M.
Richards, "The Interpretation of Protein Structures: Estimation of
Static Accessibility" J. Mol. Biol. 55, 379-400, (1971).
[0088] In a further embodiment the albumin binding residue via a
hydrophilic spacer is linked to the GH having an additional
disulfide bridge. Typically, the albumin binding residue via a
hydrophilic spacer is linked to the N-terminal, position 40 or
position 141 of hGH.
[0089] In a further embodiment the GH comprises additional
disulfide bonds between a loop segment and a helical segment or
within loop segment or between loop segments or between helical
segments.
[0090] In a further embodiment the GH comprises an additional
disulfide bond wherein at least one of the cysteines is present in
a loop segment, such from amino acid residues 128-154 (L3).
[0091] In a further embodiment the GH comprises an additional
disulfide bond wherein the additional disulfide bond connects a
loop segment with a helical segment.
[0092] In a further embodiment the GH comprises an additional
disulfide bond wherein the additional disulfide bond connects a
loop segment with helix B or helix 2 (corresponding to AA
72-98).
[0093] In a further embodiment the GH comprises and additional
disulfide bond linking helix 2 (corresponding to AA 72-98) with
loop 3 (corresponding to AA 128-154).
[0094] In a further embodiment the GH comprise an addition
disulfide bond between one of the amino acid pairs in positions
corresponding to R16C/L117C, A17C/E174C, H21C/M170C, D26C/V102C,
D26C/Y103C, N47C/T50C, Q49C/G161C, F54C/Y143C, F54C/S144C,
F54C/F146C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C,
I58C/S144C, P59C/Q137C, P61C/E66C, P61C/T67C, S71C/S132C,
L73C/S132C, L73C/F139C, R77C/I138C, R77C/F139C, L81C/Q141C,
L81C/Y143C, Q84C/Y143C, Q84C/S144C, S85C/Y143C, S85C/S144C,
P89C/F146C, F92C/F146C, F92C/T148C, R94C/D107C, V102C/A105C,
L156C/F146C, L156C/T148C and/or V185C/S188C in SEQ ID NO: 1.
[0095] In a further embodiment the additional disulfide bridge of
GH is between at least one of the amino acid pairs in the positions
corresponding to R16C/L117C, A17C/E174C, H18C/Y143C, H21C/M170C,
N47C/T50C, Q49C/G161C, F54C/S144C, F54C/F146C, I58C/Q141C,
I58C/S144C, P59C/Q137C, P61C/E66C, P61C/T67C, S71C/S132C,
L73C/S132C, L73C/F139C, R77C/I138C, R77C/F139C, L81C/Q141C,
L81C/Y143C, Q84C/Y143C, S85C/Y143C, P89C/F146C, F92C/F146C,
F92C/T148C, R94C/D107C, V102C/A105C, L156C/F146C, L156C/T148C
and/or V185C/S188C in hGH (SEQ ID NO: 1).
[0096] In a further embodiment the additional disulfide bond is
between one of the amino acid pairs in positions corresponding to
A17C/E174C, H21C/M170C, D26C/V102C, D26C/Y103C, F54C/Y143C,
F54C/S144C, F54C/F146C, S55C/Y143C, S57C/Y143C, I58C/Q141C,
I58C/Y143C, I58C/S144C, P59C/Q137C, S71C/S132C, L81C/Y143C,
Q84C/Y143C, S85C/Y143C, S85C/S144C, F92C/T148C and/or R94C/D107C in
SEQ ID NO: 1.
[0097] In a further embodiment the additional disulfide bond is
between one of the amino acid pairs in positions corresponding to
D26C/V102C, D26C/Y103C, S57C/Y143C, I58C/S144C, P59C/Q137C,
S71C/S132C, Q84C/Y143C, S85C/Y143C, S85C/S144C, F92C/T148C and/or
R94C/D107C in SEQ ID NO: 1.
[0098] In a further embodiment the additional disulfide bond is
between one of the amino acid pairs in positions corresponding to
H21C/M170C, D26C/V102C, D26C/Y103C, F54C/Y143C, F54C/S144C,
S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C,
P59C/Q137C, S71C/S132C, L81C/Y143C, Q84C/Y143C, S85C/Y143C and/or
S85C/S144C in SEQ ID NO: 1.
[0099] In a further embodiment the additional disulfide bond is
between one of the amino acid pairs in positions corresponding to
S57C/Y143C, Q84C/Y143C, S85C/Y143C and/or S85C/S144C in SEQ ID NO:
1. Typically, the additional disulfide bridge is Q84C/Y143C.
[0100] In a further embodiment the albumin binding residue via a
hydrophilic spacer is linked to the GH having a single Cys mutation
and an additional disulfide bridge. Typically, the albumin binding
residue via a hydrophilic spacer is linked to the single Cys
mutation. In a particular embodiment the GH has an additional
disulfide bridge Q84C/Y143C and a single Cys mutation L101C where
to the albumin binding residue via a hydrophilic spacer is
linked.
[0101] In further embodiments the GH has an additional disulfide
bond and a single Cys mutation selected from any one of: T3C, P5C,
S7C, D11C, H18C, Q29C, E30C, E33C, A34C, Y35C, K38C, E39C, Y42C,
S43C, D47C, P48C, S55C, S57C, P59C, S62, E65C, Q69C, E88C, Q91C,
S95C, A98C, N99C, S100C, L101C, V102C, Y103C, D107C, S108C, D112C,
Q122C, G126C, E129C, D130C, G131C, P133C, T135C, G136C, T142C,
D147C, N149C, D154C, A155C, L156C, R178C, E186C, G187C and G190C,
such as any one of; T3C, P5C, S7C, D11C, H18C, Q29C, E30C, E33C,
A34C, Y35C, E88C, Q91C, S95C, A98C, N99C, S100C, L101C, V102C,
Y103C, D107C, S108C, D112C, Q122C and G126C of hGH (SEQ ID NO:1) or
corresponding residues in a hGH variant.
[0102] In a particular embodiment the GH has an additional
disulfide bond and a single Cys mutation, and said single Cys
mutation has been introduced in a position equivalent to a parent
GH that is equivalent to a position of hGH (SEQ ID NO: 1) selected
from the group consisting of: T3, P5, S7, D11, H18, Q29, E30, E33,
A34, Y35, K38, E39, Y42, S43, D47, P48, S55, S57, P59, S62, E65,
Q69, E88, Q91, S95, A98, N99, S100, L101, V102, Y103, D107, S108,
D112, Q122, G126, E129, D130, G131, P133, T135, G136, T142, D147,
N149, D154, A155, L156, R178, E186, G187 and G190, preferably the
group; T3, P5, S7, D11, H18, Q29, E30, E33, A34, Y35, E88, Q91,
S95, A98, N99, S100, L101, V102, Y103, D107, S108, D112, Q122 and
G126. The GH conjugate further comprise an albumin binding moiety
at the side chain of said single cysteine residue.
[0103] In a further embodiment the GH comprises a single cys
mutation and additional disulfide bonds between a loop segment and
a helical segment or within loop segment or between loop segments
or between helical segments.
[0104] In a further embodiment the GH comprises a single cys
mutation and an additional disulfide bond wherein at least one of
the cysteines is present in a loop segment, such from amino acid
residues 128-154 (L3).
[0105] In a further embodiment the GH comprises a single cys
mutation and an additional disulfide bond wherein the additional
disulfide bond which connects a loop segment, such from amino acid
residues 128-154, with a helical segment, such as helix B or helix
2 (corresponding to AA 72-98).
[0106] In a further embodiment the GH comprises a single cys
mutation and additional disulfide bond linking helix 2
(corresponding to AA 72-98) with loop 3 (corresponding to AA
128-154).
[0107] In a further embodiment the GH comprises a single cys
mutation and an addition disulfide bond between one of the amino
acid pairs in positions corresponding to R16C/L117C, A17C/E174C,
H21C/M170C, D26C/V102C, D26C/Y103C, N47C/T50C, Q49C/G161C,
F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C, S57C/Y143C,
I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, P61C/E66C,
P61C/T67C, S71C/S132C, L73C/S132C, L73C/F139C, R77C/I138C,
R77C/F139C, L81C/Q141C, L81C/Y143C, Q84C/Y143C, Q84C/S144C,
S85C/Y143C, S85C/S144C, P89C/F146C, F92C/F146C, F92C/T148C,
R94C/D107C, V102C/A105C, L156C/F146C, L156C/T148C and/or
V185C/S188C in SEQ ID NO: 1.
[0108] In a further embodiment the GH comprises a single cys
mutation and an additional disulfide bridge between at least one of
the amino acid pairs in the positions corresponding to R16C/L117C,
A17C/E174C, H18C/Y143C, H21C/M170C, N47C/T50C, Q49C/G161C,
F54C/S144C, F54C/F146C, I58C/Q141C, I58C/S144C, P59C/Q137C,
P61C/E66C, P61C/T67C, S71C/S132C, L73C/S132C, L73C/F139C,
R77C/I138C, R77C/F139C, L81C/Q141C, L81C/Y143C, Q84C/Y143C,
S85C/Y143C, P89C/F146C, F92C/F146C, F92C/T148C, R94C/D107C,
V102C/A105C, L156C/F146C, L156C/T148C and/or V185C/S188C in hGH
(SEQ ID NO: 1).
[0109] In a further embodiment the GH comprises a single cys
mutation and an additional disulfide bond between one of the amino
acid pairs in positions corresponding to A17C/E174C, H21C/M170C,
D26C/V102C, D26C/Y103C, F54C/Y143C, F54C/S144C, F54C/F146C,
S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C,
P59C/Q137C, S71C/S132C, L81C/Y143C, Q84C/Y143C, S85C/Y143C,
S85C/S144C, F92C/T148C and/or R94C/D107C in SEQ ID NO: 1.
[0110] In a further embodiment the additional disulfide bond is
between one of the amino acid pairs in positions corresponding to
D26C/V102C, D26C/Y103C, S57C/Y143C, I58C/S144C, P59C/Q137C,
S71C/S132C, Q84C/Y143C, S85C/Y143C, S85C/S144C, F92C/T148C and/or
R94C/D107C in SEQ ID NO: 1.
[0111] In a further embodiment the GH comprises a single cys
mutation and an additional disulfide bond between one of the amino
acid pairs in positions corresponding to H21C/M170C, D26C/V102C,
D26C/Y103C, F54C/Y143C, F54C/S144C, S55C/Y143C, 557C/Y143C,
I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, S71C/S132C,
L81C/Y143C, Q84C/Y143C, S85C/Y143C and/or S85C/S144C in SEQ ID NO:
1.
[0112] In a further embodiment the GH comprises a single cys
mutation and an additional disulfide bond between one of the amino
acid pairs in positions corresponding to S57C/Y143C, Q84C/Y143C,
S85C/Y143C and/or S85C/S144C in SEQ ID NO: 1.
[0113] Solubility of a hydrophilic spacer (B) can be described by
its log P value. Log P, also known as the partition coefficient, is
the logarithm of the ratio of concentrations of a compound in the
two phases of a mixture of two immiscible solvents at equilibrium.
Typically one of the solvents is water while the second is selected
from octan-1-ol, chloroform, cyclohexane and propylene glycol
dipelargonate (PGDP). Log P values measured in these different
solvents show differences principally due to hydrogen bonding
effects. Octanol can donate and accept hydrogen bonds whereas
cyclohexane is inert. Chloroform can donate hydrogen bonds whereas
PGDP can only accept them. Log P values may be measured by standard
methods know in the art.
[0114] In one embodiment of the invention, the hydrophilic spacer
has a Log P below 0, such as below 0.5 in either octan-1-ol,
chloroform, cyclohexane and propylene glycol dipelargonate
(PGDP).
[0115] In a further embodiment, the hydrophilic spacer has a log P
below -1 in either octan-1-ol, chloroform, cyclohexane and
propylene glycol dipelargonate (PGDP).
[0116] Alternatively, the Log P value can be calculated as m Log P
and/or c Log P for the albumin binder part or hydrophilic spacer
part using published algorithms (T. Fujita; J. Iwasa and C. Hansch,
J. Am. Chem. Soc. 86, 5175-5180, (1964) "A New Substituent
Constant, Pi, Derived from Partition Coefficients", C. A. Lipinski
et al. Advanced Drug Delivery Reviews, 23, 3-25, (1997)
"Experimental and Computational Approaches to Estimate Solubility
and Permeability in Drug Discovery and Development Settings" and I.
Moriguchi, S. Hirono, I. Nakagome, H. Hirano, Chem. Pharm. Bull.
42, 976-978, (1994) "Comparison of Reliability of log P Values for
Drugs Calculated by Several Methods").
[0117] In one embodiment of the present invention the hydrophilic
spacer (B) has a m Log P<0.
[0118] In a further embodiment the growth hormone compound (GH) is
linked to one albumin binding residue via a hydrophilic spacer
(B).
[0119] In a further embodiment the growth hormone compound (GH) is
linked to an albumin binding residue via a hydrophilic spacer (B)
coupled to a free cysteine in the growth hormone compound (GH).
[0120] In another embodiment the growth hormone compound (GH) is
linked to two albumin binding residues via one or two hydrophilic
spacer(s). Thus, in one example one albumin binding residue is
linked via one hydrophilic spacer (B) to the single Cys mutation
and the other albumin binding residue is linked via one hydrophilic
spacer (B') to glutamine in position 40 or position 141; or
alternatively two albumin binding residues are linked via one
hydrophilic spacer (B) to the single Cys mutation or to glutamine
in position 40, position 141 or the N-terminal. In still another
embodiment the growth hormone compound (GH) is linked to three
albumin binding residues via one or more hydrophilic spacer(s).
[0121] In an embodiment the hydrophilic spacer comprise at least
one OEG motif, the radical 8-amino-3,6-dioxaoctanic acid, i.e.
--NH--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--CH.sub.2--C(O)--.
In a further specified embodiment the hydrophilic spacer comprise
at least two OEG motifs. The orientation of such OEG motif(s) is in
one embodiment so that the --C(O)-- is closest to the growth
hormone compound but not connecting the growth hormone compound and
the albumin binder linker and the --NH-- is closest to the albumin
binding residue. In additional embodiments comprising two OEG
motifs the two motifs have identical orientation or different
orientation. In an embodiment two such OEG motifs are located
adjactant to each other whereas in alternative embodiments such OEG
mofifs are separated by one or more covalently linked atoms.
[0122] In an embodiment the hydrophilic spacer comprise at lease
one glutamic acid residue. The amino acid glutamic acid comprises
two carboxylic acid groups. Its gamma-carboxy group may be used for
forming an amide bond with the epsilon-amino group of lysine, or
with an amino group of an OEG molecule, if present, or with the
amino group of another Glu residue, if present. The alfa-carboxy
group may alternatively be used for forming a similar amide bond
with the epsilon-amino group of lysine, or with an amino group of
an OEG molecule, if present, or with the amino group of another Glu
residue, if present. The amino group of Glu may in turn form an
amide bond with the carboxy group of the albumin binding residue,
or with the carboxy group of an OEG motif, if present, or with the
gamma-carboxy group or alfa carboxy group of another Glu, if
present. The linkage of the amino group of one Glu to a
gamma-carboxy group of a second Glu may be referred to as a
"gamma-Glu" motif.
[0123] In an embodiment the hydrophilic spacer comprise at least
one combined OEG-Glu motif
(--NH--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--CH.sub.2--C(O)NH--CH(C(O-
)OH)--(CH.sub.2).sub.2--C(O)--) or at least one combinde Glu-OEG
motif
(--NH--CH(C(O)OH)--(CH.sub.2).sub.2--C(O)NH--(CH.sub.2).sub.2--O--(CH.sub-
.2).sub.2--O--CH.sub.2--C(O)--) or combinations here of, where in
such Glu-OEG and OEG-Glu motifs may be separated by one or more
covalently linked atoms or directly bond to each other by an amide
bond of the Glu's foming a gammal-Glu.
[0124] In a further aspect the present invention relates to a
growth hormone conjugate wherein the growth hormone conjugate has
the formula (I):
A-W--B-GH (I)
Wherein
[0125] GH represents a growth hormone compound having a single Cys
mutation, B represents a hydrophilic spacer linked to the sulphur
residue of the Cys mutation, W is a chemical group linking A and B,
and A represent an albumin binding residue; and pharmaceutically
acceptable salts thereof.
[0126] In a further embodiment GH represents a growth hormone
compound comprising an amino acid sequence having at least 90%
identity to the amino acid sequence of human growth hormone (hGH)
(SEQ ID NO:1). In further embodiments, GH has at least 80%, such as
at least 85%, such as at least 95%, such as at least 96%, such as
at least 97%, such as at least 98% or such as at least 99% identity
with hGH (SEQ ID NO: 1). In further embodiments, said identities to
hGH are coupled to at least 10%, such as at least 20%, such as at
least 40%, such as at least 60%, such as at least 80% of the growth
hormone activity of hGH as determined in assay I herein. Any one of
the sequence identity embodiments may be combined with any one of
the activity embodiments, such as a GH having at least 80% identity
with hGH and coupled to at least 60% of the growth hormone activity
of hGH; a GH having at least 90% identity with hGH and coupled to
at least 40% of the growth hormone activity of hGH; a GH having at
least 95% identity with hGH and coupled to at least 80% of the
growth hormone activity of hGH, and so forth.
[0127] In further embodiments the GH of the conjugate has a single
Cys mutation selected from any one of a single Cys mutation in the
N-terminal, H1, H2, L2 or H3 regions of GH. In further such
embodiments, the single Cys mutation is positioned in the
N-terminal, the mutation being such as any one of T3C, P5C, S7C, or
in H1 (corresponding to AA 9-35), the mutation being such as any
one of D11C, H18C, Q29C, E30C, E33C, A34C, Y35C, or in L1
(corresponding to AA36-71), the muation being such as any one of
K38C, E39C, Y42C, S43C, D47C, P48C, S55, S57C, P59C, S62C, E65C,
Q69C or preferably any one of Y42C, S55C, S57C, S62C, Q69C or in
H2, L2 or H3 (corresponding to AA 72-98, AA 99-106 and AA 107-127),
the mutation being such as any one of E88C, Q91C, S95C, A98C, N99C,
S100C, L101C, V102C, Y103C, D107C, S108C, D112C, Q122C and G126C of
hGH (SEQ ID NO: 1) or in L3 or H4 (corresponding to AA128-154 and
AA155-184) In L3 and H4 (128-154 and AA155-184) the muation being
such as any one of E129C, D130C, G131C, P133C, T135C, G136C, T142C,
D147C, N149C, D154C, A155C, L156C, R178C, V180C or in the
C-terminal the muation being such as any one of E186C G187C
G190.
[0128] If the single Cys mutation is present in a hGH variant the
mutation is located in corresponding amino acid residues.
[0129] Further embodiments includes GH conjugates wherein the
single cys mutation in GH is selected from any one of: T3C, P5C,
S7C, D11C, H18C, Q29C, E30C, E33C, A34C, Y35C, K38C, E39C, Y42C,
S43C, D47C, P48C, S55C, S57C, P59C, S62, E65C, Q69C, E88C, Q91C,
S95C, A98C, N99C, S100C, L101C, V102C, Y103C, D107C, S108C, D112C,
Q122C, G126C, E129C, D130C, G131C, P133C, T135C, G136C, T142C,
D147C, N149C, D154C, A155C, L156C, R178C, E186C, G187C and G190C,
such as any one of; T3C, P5C, S7C, D11C, H18C, Q29C, E30C, E33C,
A34C, Y35C, E88C, Q91C, S95C, A98C, N99C, S100C, L101C, V102C,
Y103C, D107C, S108C, D112C, Q122C and G126C of hGH (SEQ ID NO:
1).
[0130] In even further embodiments the single Cys mutation is
located within AA 93-106 in hGH or corresponding residues in hGH
variants. In further specified embodiments the single Cys mutation
is located within L2, such as within AA 99-106 or AA 99-103 or
corresponding residues.
[0131] In a further embodiment A is selected from
##STR00003## ##STR00004##
[0132] wherein * denotes the attachment to B through W.
[0133] In a further embodiment W has the formula
--W.sub.7--Y--,
wherein [0134] Y is
--(CH.sub.2).sub.l7--C.sub.3-10-cycloalkyl-W.sub.8-- or a valence
bond, [0135] l7 is 0-6, [0136] W.sub.7 is selected from --C(O)NH--,
--NHC(O)--, --C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--, --OC(O)NH--,
--NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--, --C(O)CH.dbd.CH--,
--CH.dbd.CHC(O)--, --(CH.sub.2).sub.s3--, --C(O)--, --C(O)O--,
--OC(O)--, or a valence bond; wherein s3 is 0 or 1, [0137] W.sub.8
is selected from --C(O)NH--, --NHC(O)--, --C(O)NHCH.sub.2--,
--CH.sub.2NHC(O)--, --C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--,
--OC(O)NH--, --NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--,
--C(O)CH.dbd.CH--, --CH.dbd.CHC(O)--, --(CH.sub.2).sub.s4--,
--C(O)--, --C(O)O--, --OC(O)--, or a valence bond; wherein s4 is 0
or 1.
[0138] In further embodiments B comprise or consist of one or more
OEG, and/or gamma-Glu motiv as described above.
[0139] In a further embodiment B has the formula
--X.sub.1--X.sub.2--X.sub.3--X.sub.4--
wherein [0140] X.sub.1 is
--W.sub.1--[(CHR.sup.1).sub.l1--W.sub.2].sub.m1--{[(CH.sub.2).sub.n1E1].s-
ub.m2--[(CHR.sup.2).sub.l2--W.sub.3].sub.m3}.sub.n2--, [0141]
X.sub.2 is
--[(CHR.sup.3).sub.l3--W.sub.4].sub.m4--{[(CH.sub.2).sub.n3E2].sub.m5--[(-
CHR.sup.4).sub.l4--W.sub.5].sub.m6}.sub.n4--, [0142] X.sub.3 is
--[(CHR.sup.5).sub.l5--W.sub.6].sub.m7--, [0143] X.sub.4 is
F-D1-(CH.sub.2).sub.l6-D2-, [0144] l1, l2, l3, l4, l5 and l6
independently are selected from 0-16, [0145] m1, m3, m4, m6 and m7
independently are selected from 0-10, [0146] m2 and m5
independently are selected from 0-25, [0147] n1, n2, n3 and n4
independently are selected from 0-16, [0148] F is aryl, hetaryl,
pyrrolidine-2,5-dione or a valence bond, wherein the aryl and
hetaryl groups are optionally substituted with halogen, --CN, --OH,
--C(O)OH, --C(O)NH.sub.2, --S(O).sub.2OH or C.sub.1-6-alkyl, [0149]
R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 independently are
selected from hydrogen, --C(O)OH, --C(O)NH.sub.2, --S(O)OH,
--S(O).sub.2OH, --NH--C(.dbd.NH)--NH.sub.2, C.sub.1-6-alkyl, aryl
or hetaryl; wherein the alkyl, aryl and hetaryl groups optionally
are substituted with halogen, --C(O)OH, --C(O)NH.sub.2, --S(O)OH,
--S(O).sub.2OH, --CN or --OH, [0150] D1, D2, E1 and E2
independently are selected from --O--, --N(R.sup.6)--,
--N(C(O)R.sup.7)-- or a valence bond; wherein R.sup.6 and R.sup.7
independently represent hydrogen or C.sub.1-6-alkyl, [0151] W.sub.1
to W.sub.5 independently are selected from --C(O)NH--, --NHC(O)--,
--C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--, --C(O)NHS(O).sub.2--,
--S(O).sub.2NHC(O)--, --OC(O)NH--, --NHC(O)O--, --C(O)CH.sub.2--,
--CH.sub.2C(O)--, --C(O)CH.dbd.CH--, --CH.dbd.CHC(O)--,
--(CH.sub.2).sub.s2--, --C(O)--, --C(O)O--, --OC(O)--, or a valence
bond; wherein s2 is 0 or 1, [0152] W.sub.6 is selected from
--C(O)NH--, --NHC(O)--, --C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--, --OC(O)NH--,
--NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--, --C(O)CH.dbd.CH--,
--CH.dbd.CHC(O)--, --(CH.sub.2).sub.s1--, --C(O)--, --C(O)O--,
--OC(O)--, --NHC(O)C.sub.1-6-alkyl, --C(O)NHC.sub.1-6-alkyl or a
valence bond; wherein s1 is 0 or 1 and the C.sub.1-6-alkyl group is
optionally substituted with oxo, pyrrolidine-2,5-dione,
--NHC(O)CH*CH.sub.2COOH or --NHC(O)CH.sub.2CH*COOH; wherein (*)
indicates the attachment point from the carbon atom of CH to
X.sub.4.
[0153] In a further embodiment l1, l2, l3, l4, l5 and l6
independently are 0-6.
[0154] In a further embodiment m1, m3, m4, m6 and m7 independently
are 0-6.
[0155] In a further embodiment m2 and m5 independently are
0-10.
[0156] In a further embodiment n1, n2, n3 and n4 independently are
0-10.
[0157] In a further embodiment D1 and D2 are independently selected
from --O-- or --N(R.sup.6)-- or a valence bond.
[0158] In a further embodiment E1 and E2 are independently selected
from --O-- or --N(R.sup.6)-- or a valence bond.
[0159] In a further embodiment W.sub.1 through W.sub.8
independently are selected from the group consisting of --C(O)NH--,
--NHC(O)--, --CH.sub.2NHC(O)--, --C(O)NHS(O).sub.2--,
--S(O).sub.2NHC(O)--, --NHC(O)C.sub.1-6-alkyl,
--C(O)NHC.sub.1-6-alkyl or a valence bond; wherein the alkyl group
is optionally substituted with oxo, pyrrolidine-2,5-dione,
--NHC(O)CH*CH.sub.2COOH or --NHC(O)CH.sub.2CH*COOH; wherein (*)
indicates the attachment point from the carbon atom of CH to
X.sub.4.
[0160] In a further embodiment R.sup.1, R.sup.2, R.sup.3, R.sup.4
and R.sup.5 independently are selected from hydrogen, --C(O)OH,
--C(O)NH.sub.2, --S(O).sub.2OH or C.sub.1-6-alkyl; wherein the
C.sub.1-6-alkyl group optionally is substituted with --C(O)OH,
--C(O)NH.sub.2 or --S(O).sub.2OH.
[0161] In a further embodiment
--{[(CH.sub.2).sub.n1E1].sub.m2--[(CHR.sup.2).sub.l2--W.sub.3].sub.m3}.su-
b.n2-- and
--{[(CH.sub.2).sub.n3E2].sub.m5--[(CHR.sup.4).sub.l4--W.sub.5].-
sub.m6}.sub.n4--, wherein E1 and E2 are --O--, are selected
from
##STR00005##
[0162] wherein * is intended to denote a point of attachment, ie,
an open bond.
[0163] In a further embodiment X.sub.4 is a valence bond and
W.sub.6 is selected from either pyrrolidine-2,5-dione,
--NHC(O)CH*CH.sub.2COOH or --NHC(O)CH.sub.2CH*COOH wherein (*)
indicates the attachment point from the carbon atom of CH to
GH.
[0164] In a further embodiment B is selected from
##STR00006## ##STR00007##
[0165] In a further embodiment the GH conjugate is selected
from
##STR00008## ##STR00009## ##STR00010## ##STR00011## ##STR00012##
##STR00013## ##STR00014## ##STR00015## ##STR00016##
[0166] In a further aspect the present invention relates to a
growth hormone conjugate having the formula (I):
A-W--B-GH (I)
Wherein
[0167] GH represents a growth hormone compound having an additional
disulfide bridge, B represents a hydrophilic spacer, W is a
chemical group linking A and B, and A represent an albumin binding
residue; and pharmaceutically acceptable salts thereof.
[0168] In a further embodiment GH represents a growth hormone
compound comprising an amino acid sequence having at least 90%
identity to the amino acid sequence of human growth hormone (hGH)
(SEQ ID NO: 1). In further embodiments, GH has at least 80%, such
as at least 85%, such as at least 95%, such as at least 96%, such
as at least 97%, such as at least 98% or such as at least 99%
identity with hGH (SEQ ID NO: 1). In further embodiments, said
identities to hGH are coupled to at least 10%, such as at least
20%, such as at least 40%, such as at least 60%, such as at least
80% of the growth hormone activity of hGH as determined in assay I
herein. Any one of the sequence identity embodiments may be
combined with any one of the activity embodiments, such as a GH
having at least 80% identity with hGH and coupled to at least 60%
of the growth hormone activity of hGH; a GH having at least 90%
identity with hGH and coupled to at least 40% of the growth hormone
activity of hGH; a GH having at least 95% identity with hGH and
coupled to at least 80% of the growth hormone activity of hGH, and
so forth.
[0169] In a further embodiment the GH of the conjugate comprises
additional disulfide bonds between a loop segment and a helical
segment or within loop segment or between loop segments or between
helical segments.
[0170] In a further embodiment the GH of the conjugate comprises an
additional disulfide bond wherein at least one of the cysteines is
present in a loop segment, such from amino acid residues
128-154.
[0171] In a further embodiment the GH of the conjugate comprises an
additional disulfide bond wherein the additional disulfide bond
which connects a loop segment, such from amino acid residues
128-154 (H3), with a helical segment, such as helix B or helix 2
(corresponding to AA 72-98).
[0172] In a further embodiment the GH of the conjugate comprises
and additional disulfide bond linking helix 2 (corresponding to AA
72-98) with loop 3 (corresponding to AA 128-154).
[0173] In a further embodiment the GH of the conjugate comprise an
addition disulfide bond between one of the amino acid pairs in
positions corresponding to R16C/L117C, A17C/E174C, H21C/M170C,
D26C/V102C, D26C/Y103C, N47C/T50C, Q49C/G161C, F54C/Y143C,
F54C/S144C, F54C/F146C, S55C/Y143C, S57C/Y143C, I58C/Q141C,
I58C/Y143C, I58C/S144C, P59C/Q137C, P61C/E66C, P61C/T67C,
S71C/S132C, L73C/S132C, L73C/F139C, R77C/I138C, R77C/F139C,
L81C/Q141C, L81C/Y143C, Q84C/Y143C, S85C/Y143C, S85C/S144C,
P89C/F146C, F92C/F146C, F92C/T148C, R94C/D107C, V102C/A105C,
L156C/F146C, L156C/T148C and/or V185C/S188C in SEQ ID NO: 1.
[0174] In a further embodiment the additional disulfide bridge is
between at least one of the amino acid pairs in the positions
corresponding to R16C/L117C, A17C/E174C, H18C/Y143C, H21C/M170C,
N47C/T50C, Q49C/G161C, F54C/S144C, F54C/F146C, I58C/Q141C,
I58C/S144C, P59C/Q137C, P61C/E66C, P61C/T67C, S71C/S132C,
L73C/S132C, L73C/F139C, R77C/I138C, R77C/F139C, L81C/Q141C,
L81C/Y143C, Q84C/Y143C, S85C/Y143C, P89C/F146C, F92C/F146C,
F92C/T148C, R94C/D107C, V102C/A105C, L156C/F146C, L156C/T148C
and/or V185C/S188C in hGH (SEQ ID NO: 1).
[0175] In a further embodiment the GH of the conjugate comprises an
additional disulfide bond between one of the amino acid pairs in
positions corresponding to A17C/E174C, H21C/M170C, D26C/V102C,
D26C/Y103C, F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C,
S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C,
S71C/S132C, L81C/Y143C, Q84C/Y143C, S85C/Y143C, S85C/S144C,
F92C/T148C and/or R94C/D107C in SEQ ID NO: 1.
[0176] In a further embodiment the additional disulfide bond is
between one of the amino acid pairs in positions corresponding to
D26C/V102C, D26C/Y103C, S57C/Y143C, I58C/S144C, P59C/Q137C,
S71C/S132C, Q84C/Y143C, S85C/Y143C, S85C/S144C, F92C/T148C and/or
R94C/D107C in SEQ ID NO: 1.
[0177] In a further embodiment the GH of the conjugate comprise an
additional disulfide bond between one of the amino acid pairs in
positions corresponding to H21C/M170C, D26C/V102C, D26C/Y103C,
F54C/Y143C, F54C/S144C, S55C/Y143C, S57C/Y143C, I58C/Q141C,
I58C/Y143C, I58C/S144C, P59C/Q137C, S71C/S132C, L81C/Y143C,
Q84C/Y143C, S85C/Y143C and/or S85C/S144C in SEQ ID NO: 1.
[0178] In a further embodiment the GH of the conjugate comprises an
additional disulfide bond between one of the amino acid pairs in
positions corresponding to S57C/Y143C, Q84C/Y143C, S85C/Y143C
and/or S85C/S144C in SEQ ID NO: 1.
[0179] In a further embodiment A is selected from
##STR00017## ##STR00018##
wherein * denotes the attachment to B through W.
[0180] In a further embodiment W has the formula
--W.sub.7--Y--,
wherein [0181] Y is
--(CH.sub.2).sub.l7--C.sub.3-10-cycloalkyl-W.sub.8-- or a valence
bond, [0182] l7 is 0-6, [0183] W.sub.7 is selected from --C(O)NH--,
--NHC(O)--, --C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--, --OC(O)NH--,
--NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--, --C(O)CH.dbd.CH--,
--CH.dbd.CHC(O)--, --(CH.sub.2).sub.s3--, --C(O)--, --C(O)O--,
--OC(O)--, or a valence bond; wherein s3 is 0 or 1, [0184] W.sub.8
is selected from --C(O)NH--, --NHC(O)--, --C(O)NHCH.sub.2--,
--CH.sub.2NHC(O)--, --C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--,
--OC(O)NH--, --NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--,
--C(O)CH.dbd.CH--, --CH.dbd.CHC(O)--, --(CH.sub.2).sub.s4--,
--C(O)--, --C(O)O--, --OC(O)--, or a valence bond; wherein s4 is 0
or 1.
[0185] In further embodiments B comprise or consist of one or more
OEG, and/or gamma-Glu motiv(s) as described above.
[0186] In a further embodiment B has the formula
--X.sub.1--X.sub.2--X.sub.3--X.sub.4--
wherein [0187] X.sub.1 is
--W.sub.1--[(CHR.sup.1).sub.l1--W.sub.2].sub.m1--{[(CH.sub.2).sub.n1E1].s-
ub.m2--[(CHR.sup.2).sub.l2--W.sub.3].sub.m3}.sub.n2--, [0188]
X.sub.2 is
--[(CHR.sup.3).sub.l3--W.sub.4].sub.m4--{[(CH.sub.2).sub.n3E2].sub.m5--[(-
CHR.sup.4).sub.l4--W.sub.5].sub.m6}.sub.n4--, [0189] X.sub.3 is
--[(CHR.sup.5).sub.l5--W.sub.6].sub.m7--, [0190] X.sub.4 is
F-D1-(CH.sub.2).sub.l6-D2-, [0191] l1, l2, l3, l4, l5 and l6
independently are selected from 0-16, [0192] m1, m3, m4, m6 and m7
independently are selected from 0-10, [0193] m2 and m5
independently are selected from 0-25, [0194] n1, n2, n3 and n4
independently are selected from 0-16, [0195] F is aryl, hetaryl,
pyrrolidine-2,5-dione or a valence bond, wherein the aryl and
hetaryl groups are optionally substituted with halogen, --CN, --OH,
--C(O)OH, --C(O)NH.sub.2, --S(O).sub.2OH or C.sub.1-6-alkyl, [0196]
R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 independently are
selected from hydrogen, --C(O)OH, --C(O)NH.sub.2, --S(O)OH,
--S(O).sub.2OH, --NH--C(.dbd.NH)--NH.sub.2, C.sub.1-6-alkyl, aryl
or hetaryl; wherein the alkyl, aryl and hetaryl groups optionally
are substituted with halogen, --C(O)OH, --C(O)NH.sub.2, --S(O)OH,
--S(O).sub.2OH, --CN or --OH, [0197] D1, D2, E1 and E2
independently are selected from --O--, --N(R.sup.6)--,
--N(C(O)R.sup.7)-- or a valence bond; wherein R.sup.6 and R.sup.7
independently represent hydrogen or C.sub.1-6-alkyl, [0198] W.sub.1
to W.sub.5 independently are selected from --C(O)NH--, --NHC(O)--,
--C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--, --C(O)NHS(O).sub.2--,
--S(O).sub.2NHC(O)--, --OC(O)NH--, --NHC(O)O--, --C(O)CH.sub.2--,
--CH.sub.2C(O)--, --C(O)CH.dbd.CH--, --CH.dbd.CHC(O)--,
--(CH.sub.2).sub.s2--, --C(O)--, --C(O)O--, --OC(O)--, or a valence
bond; wherein s2 is 0 or 1, [0199] W.sub.6 is selected from
--C(O)NH--, --NHC(O)--, --C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--, --OC(O)NH--,
--NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--, --C(O)CH.dbd.CH--,
--CH.dbd.CHC(O)--, --(CH.sub.2).sub.s1--, --C(O)--, --C(O)O--,
--OC(O)--, --NHC(O)C.sub.1-6-alkyl, --C(O)NHC.sub.1-6-alkyl or a
valence bond; wherein s1 is 0 or 1 and the C.sub.1-6-alkyl group is
optionally substituted with oxo, pyrrolidine-2,5-dione,
--NHC(O)CH*CH.sub.2COOH or --NHC(O)CH.sub.2CH*COOH; wherein (*)
indicates the attachment point from the carbon atom of CH to
X.sub.4.
[0200] In a further embodiment l1, l2, l3, l4, l5 and l6
independently are 0-6.
[0201] In a further embodiment m1, m3, m4, m6 and m7 independently
are 0-6.
[0202] In a further embodiment m2 and m5 independently are
0-10.
[0203] In a further embodiment n1, n2, n3 and n4 independently are
0-10.
[0204] In a further embodiment D1 and D2 are independently selected
from --O-- or --N(R.sup.6)-- or a valence bond.
[0205] In a further embodiment E1 and E2 are independently selected
from --O-- or --N(R.sup.6)-- or a valence bond.
[0206] In a further embodiment W.sub.1 through W.sub.8
independently are selected from the group consisting of --C(O)NH--,
--NHC(O)--, --CH.sub.2NHC(O)--, --C(O)NHS(O).sub.2--,
--S(O).sub.2NHC(O)--, --NHC(O)C.sub.1-6-alkyl,
--C(O)NHC.sub.1-6-alkyl or a valence bond; wherein the alkyl group
is optionally substituted with oxo, pyrrolidine-2,5-dione,
--NHC(O)CH*CH.sub.2COOH or --NHC(O)CH.sub.2CH*COOH; wherein (*)
indicates the attachment point from the carbon atom of CH to
X.sub.4.
[0207] In a further embodiment R.sup.1, R.sup.2, R.sup.3, R.sup.4
and R.sup.5 independently are selected from hydrogen, --C(O)OH,
--C(O)NH.sub.2, --S(O).sub.2OH or C.sub.1-6-alkyl; wherein the
C.sub.1-6-alkyl group optionally is substituted with --C(O)OH,
--C(O)NH.sub.2 or --S(O).sub.2OH.
[0208] In a further embodiment
--{[(CH.sub.2).sub.n1E1].sub.m2--[(CHR.sup.2).sub.l2--W.sub.3].sub.m3}.su-
b.n2-- and
--{[(CH.sub.2).sub.n3E2].sub.m5--[(CHR.sup.4).sub.l4--W.sub.5].-
sub.m6}.sub.n4--, wherein E1 and E2 are --O--, are selected
from
##STR00019##
[0209] wherein * is intended to denote a point of attachment, ie,
an open bond.
[0210] In a further embodiment B is selected from
##STR00020## ##STR00021##
[0211] In a further embodiment A via B is attached to the glutamine
residue in the position corresponding to position 40, position 141
in SEQ ID NO: 1, or the N-terminal residue of the growth hormone
compound.
[0212] In a further embodiment the GH conjugate is selected
from
##STR00022##
[0213] In a further aspect the present invention relates to a
growth hormone conjugate wherein the growth hormone conjugate has
the formula (I):
A-W--B-GH (I)
wherein GH represents a growth hormone compound having a single Cys
mutation and an additional disulfide bridge, B represents a
hydrophilic spacer linked to the sulphur residue of the Cys
mutation, W is a chemical group linking A and B, and A represent an
albumin binding residue; and pharmaceutically acceptable salts
thereof.
[0214] In a further embodiment GH represents a growth hormone
compound comprising an amino acid sequence having at least 90%
identity to the amino acid sequence of human growth hormone (hGH)
(SEQ ID NO: 1). In further embodiments, GH has at least 80%, such
as at least 85%, such as at least 95%, such as at least 96%, such
as at least 97%, such as at least 98% or such as at least 99%
identity with hGH (SEQ ID NO: 1). In further embodiments, said
identities to hGH are coupled to at least 10%, such as at least
20%, such as at least 40%, such as at least 60%, such as at least
80% of the growth hormone activity of hGH as determined in assay I
herein. Any one of the sequence identity embodiments may be
combined with any one of the activity embodiments, such as a GH
having at least 80% identity with hGH and coupled to at least 60%
of the growth hormone activity of hGH; a GH having at least 90%
identity with hGH and coupled to at least 40% of the growth hormone
activity of hGH; a GH having at least 95% identity with hGH and
coupled to at least 80% of the growth hormone activity of hGH, and
so forth.
[0215] In further embodiments the GH of the conjugate has an
additional disulfide bond an a single Cys mutation selected from
any one of a single Cys mutation in the N-terminal, H1, H2, L2 or
H3 regions of GH. In further such embodiments, the single Cys
mutation is positioned in the N-terminal, the mutation being such
as any one of T3C, P5C, S7C, or in H1 (corresponding to AA 9-35),
the mutation being such as any one of D11C, H18C, Q29C, E30C, E33C,
A34C, Y35C, or in L1 (corresponding to AA36-71), the muation being
such as any one of K38C, E39C, Y42C, S43C, D47C, P48C, S55, S57C,
P59C, S62C, E65C, Q69C or preferably any one of Y42C, S55C, S57C,
S62C, Q69C or in H2, L2 or H3 (corresponding to AA 72-98, AA 99-106
and AA 107-127), the mutation being such as any one of E88C, Q91C,
S95C, A98C, N99C, S100C, L101C, V102C, Y103C, D107C, S108C, D112C,
Q122C and G126C of hGH (SEQ ID NO: 1), or in L3 or H4
(corresponding to AA128-154 and AA155-184) In L3 and H4 (128-154
and AA155-184) the muation being such as any one of E129C, D130C,
G131C, P133C, T135C, G136C, T142C, D147C, N149C, D154C, A155C,
L156C, R178C, V180C or in the C-terminal the muation being such as
any one of E186C G187C G190C.
[0216] If the single Cys mutation is present in a hGH variant the
mutation is located in corresponding amino acid residues.
[0217] Further embodiments includes GH conjugates having an
additional disulfide bond and a single cys mutation in GH is
selected from any one of: T3C, P5C, S7C, D11C, H18C, Q29C, E30C,
E33C, A34C, Y35C, K38C, E39C, Y42C, S43C, D47C, P48C, 55C, S57C,
P59C, S62, E65C, Q69C, E88C, Q91C, S95C, A98C, N99C, S100C, L101C,
V102C, Y103C, D107C, S108C, D112C, Q122C, G126C, E129C, D130C,
G131C, P133C, T135C, G136C, T142C, D147C, N149C, D154C, A155C,
L156C, R178C, E186C, G187C and G190C, such as any one of; T3C, P5C,
S7C, D11C, H18C, Q29C, E30C, E33C, A34C, Y35C, E88C, Q91C, S95C,
A98C, N99C, S100C, L101C, V102C, Y103C, D107C, S108C, D112C, Q122C
and G126C of hGH (SEQ ID NO: 1).
[0218] In even further embodiments the single Cys mutation is
located within AA 93-106 in hGH or corresponding residues in hGH
variants. In further specified embodiments the single Cys mutation
is located within L2, such as within AA 99-106 or AA 99-103 or
corresponding residues.
[0219] In further embodiment the additional disulfide bond may be
an additional disulfide bonds between a loop segment and a helical
segment or within loop segment or between loop segments or between
helical segments.
[0220] In a further embodiment the GH comprises a single cys mutant
and an additional disulfide bond wherein at least one of the
cysteines is present in a loop segment, such from amino acid
residues 128-154 (L3).
[0221] In a further embodiment the GH comprises a single cys mutant
and an additional disulfide bond wherein the additional disulfide
bond which connects a loop segment, such from amino acid residues
128-154, with a helical segment, such as helix B or helix 2
(corresponding to AA 72-98).
[0222] In a further embodiment the GH of the conjugate comprises a
single cys mutant and an additional disulfide bond linking helix 2
(corresponding to AA 72-98) with loop 3 (corresponding to AA
128-154).
[0223] In a further embodiment the GH of the conjugate comprise a
single cys mutant and an addition disulfide bond between one of the
amino acid pairs in positions corresponding to R16C/L117C,
A17C/E174C, H21C/M170C, D26C/V102C, D26C/Y103C, N47C/T50C,
Q49C/G161C, F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C,
S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C,
P61C/E66C, P61C/T67C, S71C/S132C, L73C/S132C, L73C/F139C,
R77C/I138C, R77C/F139C, L81C/Q141C, L81C/Y143C, Q84C/Y143C,
Q84C/S144C, S85C/Y143C, S85C/S144C, P89C/F146C, F92C/F146C,
F92C/T148C, R94C/D107C, V102C/A105C, L156C/F146C, L156C/T148C
and/or V185C/S188C in SEQ ID NO: 1.
[0224] In a further embodiment the additional disulfide bridge is
between at least one of the amino acid pairs in the positions
corresponding to R16C/L117C, A17C/E174C, H18C/Y143C, H21C/M170C,
N47C/T50C, Q49C/G161C, F54C/S144C, F54C/F146C, I58C/Q141C,
I58C/S144C, P59C/Q137C, P61C/E66C, P61C/T67C, S71C/S132C,
L73C/S132C, L73C/F139C, R77C/I138C, R77C/F139C, L81C/Q141C,
L81C/Y143C, Q84C/Y143C, S85C/Y143C, P89C/F146C, F92C/F146C,
F92C/T148C, R94C/D107C, V102C/A105C, L156C/F146C, L156C/T148C
and/or V185C/S188C in hGH (SEQ ID NO: 1).
[0225] In a further embodiment the GH of the conjugate comprises a
singly cys mutant and an additional disulfide bond between one of
the amino acid pairs in positions corresponding to A17C/E174C,
H21C/M170C, D26C/V102C, D26C/Y103C, F54C/Y143C, F54C/S144C,
F54C/F146C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C,
I58C/S144C, P59C/Q137C, S71C/S132C, L81C/Y143C, Q84C/Y143C,
S85C/Y143C, S85C/S144C, F92C/T148C and/or R94C/D107C in SEQ ID NO:
1.
[0226] In a further embodiment the additional disulfide bond is
between one of the amino acid pairs in positions corresponding to
D26C/V102C, D26C/Y103C, S57C/Y143C, I58C/S144C, P59C/Q137C,
S71C/S132C, Q84C/Y143C, S85C/Y143C, S85C/S144C, F92C/T148C and/or
R94C/D107C in SEQ ID NO: 1.
[0227] In a further embodiment the additional disulfide bond
between one of the amino acid pairs in positions corresponding to
H21C/M170C, D26C/V102C, D26C/Y103C, F54C/Y143C, F54C/S144C,
S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C,
P59C/Q137C, S71C/S132C, L81C/Y143C, Q84C/Y143C, S85C/Y143C and/or
S85C/S144C in SEQ ID NO: 1.
[0228] In a further embodiment the additional disulfide bond
between one of the amino acid pairs in positions corresponding to
S57C/Y143C, Q84C/Y143C, S85C/Y143C and/or S85C/S144C in SEQ ID NO:
1.
[0229] In a further embodiment the GH comprise a single cysteine
mutation in L2 and an additional disulfide bond which connects a
loop segment, such as from amino acid residues 128-154 (H3), with a
helical segment, such as helix B or helix 2 (corresponding to AA
72-98).
[0230] In an embodiment the GH comprise a combination of mutations
selected from the following group: A98C/Q84C/Y143C,
A98C/S85C/Y143C, A98C/S85C/S144C, N99C/Q84C/Y143C, N99C/S85C/Y143C,
N99C/S85C/S144C, S101C/Q84C/Y143C, S101C/S85C/Y143C,
S101C/S85C/S144C, L101C/Q84C/Y143C, L101C/S85C/Y143C,
L101C/S85C/S144C, C102C/Q84C/Y143C, C102C/S85C/Y143C and
C102C/S85C/S144C.
[0231] In a further embodiment A is selected from
##STR00023## ##STR00024##
[0232] wherein * denotes the attachment to B through W.
[0233] In a further embodiment W has the formula
--W.sub.7--Y--,
wherein [0234] Y is
--(CH.sub.2).sub.l7--C.sub.3-10-cycloalkyl-W.sub.8-- or a valence
bond, [0235] l7 is 0-6, [0236] W.sub.7 is selected from --C(O)NH--,
--NHC(O)--, --C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--, --OC(O)NH--,
--NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--, --C(O)CH.dbd.CH--,
--CH.dbd.CHC(O)--, --(CH.sub.2).sub.s3--, --C(O)--, --C(O)O--,
--OC(O)--, or a valence bond; wherein s3 is 0 or 1, [0237] W.sub.8
is selected from --C(O)NH--, --NHC(O)--, --C(O)NHCH.sub.2--,
--CH.sub.2NHC(O)--, --C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--,
--OC(O)NH--, --NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--,
--C(O)CH.dbd.CH--, --CH.dbd.CHC(O)--, --(CH.sub.2).sub.s4--,
--C(O)--, --C(O)O--, --OC(O)--, or a valence bond; wherein s4 is 0
or 1.
[0238] In further embodiments B comprise or consist of one or more
OEG, and/or gamma-Glu motiv(s) as described above.
[0239] In a further embodiment B has the formula
--X.sub.1--X.sub.2--X.sub.3--X.sub.4--
wherein [0240] X.sub.1 is
--W.sub.1--[(CHR.sup.1).sub.l1--W.sub.2].sub.m1--{[(CH.sub.2).sub.n1E1].s-
ub.m2--[(CHR.sup.2).sub.l2--W.sub.3].sub.m3}.sub.n2--, [0241]
X.sub.2 is
--[(CHR.sup.3).sub.l3--W.sub.4].sub.m4--{[(CH.sub.2).sub.n3E2].sub.m5--[(-
CHR.sup.4).sub.l4--W.sub.5].sub.m6}.sub.n4--, [0242] X.sub.3 is
--[(CHR.sup.5).sub.l5--W.sub.6].sub.m7--, [0243] X.sub.4 is
F-D1-(CH.sub.2).sub.l6-D2-, [0244] l1, l2, l3, l4, l5 and l6
independently are selected from 0-16, [0245] m1, m3, m4, m6 and m7
independently are selected from 0-10, [0246] m2 and m5
independently are selected from 0-25, [0247] n1, n2, n3 and n4
independently are selected from 0-16, [0248] F is aryl, hetaryl,
pyrrolidine-2,5-dione or a valence bond, wherein the aryl and
hetaryl groups are optionally substituted with halogen, --CN, --OH,
--C(O)OH, --C(O)NH.sub.2, --S(O).sub.2OH or C.sub.1-6-alkyl, [0249]
R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 independently are
selected from hydrogen, --C(O)OH, --C(O)NH.sub.2, --S(O)OH,
--S(O).sub.2OH, --NH--C(.dbd.NH)--NH.sub.2, C.sub.1-6-alkyl, aryl
or hetaryl; wherein the alkyl, aryl and hetaryl groups optionally
are substituted with halogen, --C(O)OH, --C(O)NH.sub.2, --S(O)OH,
--S(O).sub.2OH, --CN or --OH, [0250] D1, D2, E1 and E2
independently are selected from --O--, --N(R.sup.6)--,
--N(C(O)R.sup.7)-- or a valence bond; wherein R.sup.6 and R.sup.7
independently represent hydrogen or C.sub.1-6-alkyl, [0251] W.sub.1
to W.sub.5 independently are selected from --C(O)NH--, --NHC(O)--,
--C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--, --C(O)NHS(O).sub.2--,
--S(O).sub.2NHC(O)--, --OC(O)NH--, --NHC(O)O--, --C(O)CH.sub.2--,
--CH.sub.2C(O)--, --C(O)CH.dbd.CH--, --CH.dbd.CHC(O)--,
--(CH.sub.2).sub.s2--, --C(O)--, --C(O)O--, --OC(O)--, or a valence
bond; wherein s2 is 0 or 1, [0252] W.sub.6 is selected from
--C(O)NH--, --NHC(O)--, --C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--, --OC(O)NH--,
--NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--, --C(O)CH.dbd.CH--,
--CH.dbd.CHC(O)--, --(CH.sub.2).sub.s1--, --C(O)--, --C(O)O--,
--OC(O)--, --NHC(O)C.sub.1-6-alkyl, --C(O)NHC.sub.1-6-alkyl or a
valence bond; wherein s1 is 0 or 1 and the C.sub.1-6-alkyl group is
optionally substituted with oxo, pyrrolidine-2,5-dione,
--NHC(O)CH*CH.sub.2COOH or --NHC(O)CH.sub.2CH*COOH; wherein (*)
indicates the attachment point from the carbon atom of CH to
X.sub.4.
[0253] In a further embodiment l1, l2, l3, l4, l5 and l6
independently are 0-6.
[0254] In a further embodiment m1, m3, m4, m6 and m7 independently
are 0-6.
[0255] In a further embodiment m2 and m5 independently are
0-10.
[0256] In a further embodiment n1, n2, n3 and n4 independently are
0-10.
[0257] In a further embodiment D1 and D2 are independently selected
from --O-- or --N(R.sup.6)-- or a valence bond.
[0258] In a further embodiment E1 and E2 are independently selected
from --O-- or --N(R.sup.6)-- or a valence bond.
[0259] In a further embodiment W.sub.1 through W.sub.8
independently are selected from the group consisting of --C(O)NH--,
--NHC(O)--, --CH.sub.2NHC(O)--, --C(O)NHS(O).sub.2--,
--S(O).sub.2NHC(O)--, --NHC(O)C.sub.1-6-alkyl,
--C(O)NHC.sub.1-6-alkyl or a valence bond; wherein the alkyl group
is optionally substituted with oxo, pyrrolidine-2,5-dione,
--NHC(O)CH*CH.sub.2COOH or --NHC(O)CH.sub.2CH*COOH; wherein (*)
indicates the attachment point from the carbon atom of CH to
X.sub.4.
[0260] In a further embodiment R.sup.1, R.sup.2, R.sup.3, R.sup.4
and R.sup.5 independently are selected from hydrogen, --C(O)OH,
--C(O)NH.sub.2, --S(O).sub.2OH or C.sub.1-6-alkyl; wherein the
C.sub.1-6-alkyl group optionally is substituted with --C(O)OH,
--C(O)NH.sub.2 or --S(O).sub.2OH.
[0261] In a further embodiment
--{[(CH.sub.2).sub.n1E1].sub.m2--[(CHR.sup.2).sub.l2--W.sub.3].sub.m3}.su-
b.n2-- and
--{[(CH.sub.2).sub.n3E2].sub.m5--[(CHR.sup.4).sub.l4--W.sub.5].-
sub.m6}.sub.n4--, wherein E1 and E2 are --O--, are selected
from
##STR00025##
[0262] wherein * is intended to denote a point of attachment, ie,
an open bond.
[0263] In a further embodiment X.sub.4 is a valence bond and
W.sub.6 is selected from either pyrrolidine-2,5-dione,
--NHC(O)CH*CH.sub.2COOH or --NHC(O)CH.sub.2CH*COOH wherein (*)
indicates the attachment point from the carbon atom of CH to
GH.
[0264] In a further embodiment B is selected from
##STR00026## ##STR00027##
[0265] In a further embodiment the GH conjugate is selected
from
##STR00028## ##STR00029## ##STR00030## ##STR00031##
[0266] Typically, the conjugate of the present invention has one
albumin binding residue (A) linked via a hydrophilic spacer (B),
such as one hydrophilic spacer (B), to the growth hormone compound
(GH).
[0267] However, the growth hormone compound (GH) may be linked to
two albumin binding residues via a hydrophilic spacer.
[0268] Thus, in a still further aspect the present invention
relates to a growth hormone conjugate of the formula (II):
A-W--B-GH-B'--W'-A' (II)
wherein
[0269] GH represents a growth hormone compound having a single Cys
mutation,
[0270] B and B' independently are hydrophilic spacers linked to the
sulphur residue of the Cys mutation,
[0271] W is a chemical group linking A and B,
[0272] W' is a chemical group linking A' and B',
[0273] A and A' independently represents an albumin binding
residue, and
[0274] pharmaceutically acceptable salts thereof.
[0275] Further, another aspect the present invention relates to a
growth hormone conjugate of the formula (II):
A-W--B-GH-B'--W'-A' (II)
wherein
[0276] GH represents a growth hormone compound having an additional
disulfide bridge,
[0277] B and B' independently are hydrophilic spacers,
[0278] W is a chemical group linking A and B,
[0279] W' is a chemical group linking A' and B',
[0280] A and A' independently represents an albumin binding
residue, and pharmaceutically acceptable salts thereof.
[0281] A still further aspect the present invention relates to a
growth hormone conjugate of the formula (II):
A-W--B-GH-B'--W'-A' (II)
wherein
[0282] GH represents a growth hormone compound having a single Cys
mutation and an additional disulfide bridge,
[0283] B and B' independently are hydrophilic spacers linked to the
sulphur residue of the
[0284] Cys mutation,
[0285] W is a chemical group linking A and B,
[0286] W' is a chemical group linking A' and B',
[0287] A and A' independently represents an albumin binding
residue, and
[0288] pharmaceutically acceptable salts thereof.
[0289] In the conjugate of formula (II) as described above W' is
selected from the same groups as W, A' is selected from the same
groups as A and B' is selected from the same groups as B, and it
should be understood that W and W', A and A', and B and B' are
independently selected from any one of the respective groups as
defined herein. Thus, any embodiments of W, A, and B herein are
also embodiments of W', A', and B'. Furthermore, any one of the
embodiments described herein refers independently to both of the
conjugates of formula (I) and (II), as well as the broad aspect and
embodiments thereof when suitable.
[0290] The above embodiments as well as the embodiments to be
described hereunder should be seen as referring to any one of the
aspects described herein as well as any one of the embodiments
described herein unless it is specified that an embodiment relates
to a certain aspect or aspects of the present invention.
[0291] In one embodiment GH is a variant of hGH, wherein a variant
is understood to be the compound obtained by substituting one or
more amino acid residues in the hGH sequence with another natural
or unnatural amino acid; and/or by adding one or more natural or
unnatural amino acids to the hGH sequence; and/or by deleting one
or more amino acid residue from the hGH sequence, wherein any of
these steps may optionally be followed by further derivatization of
one or more amino acid residues.
[0292] In a further embodiment GH represents a growth hormone
compound comprising an amino acid sequence having at least 90%
identity to the amino acid sequence of human growth hormone (hGH)
(SEQ ID NO: 1). In further embodiments, GH has at least 80%, such
as at least 85%, such as at least 95%, such as at least 96%, such
as at least 97%, such as at least 98% or such as at least 99%
identity with hGH (SEQ ID NO: 1). In further embodiments, said
identities to hGH are coupled to at least 10%, such as at least
20%, such as at least 40%, such as at least 60%, such as at least
80% of the growth hormone activity of hGH as determined in assay I
herein. Any one of the sequence identity embodiments may be
combined with any one of the activity embodiments, such as a GH
having at least 80% identity with hGH and coupled to at least 60%
of the growth hormone activity of hGH; a GH having at least 90%
identity with hGH and coupled to at least 40% of the growth hormone
activity of hGH; a GH having at least 95% identity with hGH and
coupled to at least 80% of the growth hormone activity of hGH, and
so forth.
[0293] In a still further embodiment GH is hGH (SEQ ID NO: 1).
[0294] In further embodiments B comprise or or consist of one or
more OEG, and/or gamma-Glu motiv(s) as described above.
[0295] In a further embodiment of the conjugate of formula (I) or
(II), the hydrophilic spacer B has the formula
--X.sub.1--X.sub.2--X.sub.3--X.sub.4--
wherein [0296] X.sub.1 is
--W.sub.1--[(CHR.sup.1).sub.l1--W.sub.2].sub.m1--{[(CH.sub.2).sub.n1E1].s-
ub.m2--[(CHR.sup.2).sub.l2--W.sub.3].sub.m3}.sub.n2--, [0297]
X.sub.2 is
--[(CHR.sup.3).sub.l3--W.sub.4].sub.m4--{[(CH.sub.2).sub.n3E2].sub.m5--[(-
CHR.sup.4).sub.l4--W.sub.5].sub.m6}.sub.n4--, [0298] X.sub.3 is
--[(CHR.sup.5).sub.l5--W.sub.6].sub.m7--, [0299] X.sub.4 is
F-D1-(CH.sub.2).sub.l6-D2-, [0300] l1, l2, l3, l4, l5 and l6
independently are selected from 0-16, [0301] m1, m3, m4, m6 and m7
independently are selected from 0-10, [0302] m2 and m5
independently are selected from 0-25, [0303] n1, n2, n3 and n4
independently are selected from 0-16, [0304] F is aryl, hetaryl,
pyrrolidine-2,5-dione or a valence bond, wherein the aryl and
hetaryl groups are optionally substituted with halogen, --CN, --OH,
--C(O)OH, --C(O)NH.sub.2, --S(O).sub.2OH or C.sub.1-6-alkyl, [0305]
R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 independently are
selected from hydrogen, --C(O)OH, --C(O)NH.sub.2, --S(O)OH,
--S(O).sub.2OH, --NH--C(.dbd.NH)--NH.sub.2, C.sub.1-6-alkyl, aryl
or hetaryl; wherein the alkyl, aryl and hetaryl groups optionally
are substituted with halogen, --C(O)OH, --C(O)NH.sub.2, --S(O)OH,
--S(O).sub.2OH, --CN or --OH, [0306] D1, D2, E1 and E2
independently are selected from --O--, --N(R.sup.6)--,
--N(C(O)R.sup.7)-- or a valence bond; wherein R.sup.6 and R.sup.7
independently represent hydrogen or C.sub.1-6-alkyl, [0307] W.sub.1
to W.sub.5 independently are selected from --C(O)NH--, --NHC(O)--,
--C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--, --C(O)NHS(O).sub.2--,
--S(O).sub.2NHC(O)--, --OC(O)NH--, --NHC(O)O--, --C(O)CH.sub.2--,
--CH.sub.2C(O)--, --C(O)CH.dbd.CH--, --CH.dbd.CHC(O)--,
--(CH.sub.2).sub.s2--, --C(O)--, --C(O)O--, --OC(O)--, or a valence
bond; wherein s2 is 0 or 1, [0308] W.sub.6 is selected from
--C(O)NH--, --NHC(O)--, --C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--, --OC(O)NH--,
--NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--, --C(O)CH.dbd.CH--,
--CH.dbd.CHC(O)--, --(CH.sub.2).sub.s1--, --C(O)--, --C(O)O--,
--OC(O)--, --NHC(O)C.sub.1-6-alkyl, --C(O)NHC.sub.1-6-alkyl or a
valence bond; wherein s1 is 0 or 1 and the alkyl group is
optionally substituted with oxo, pyrrolidine-2,5-dione,
--NHC(O)CH*CH.sub.2COOH or --NHC(O)CH.sub.2CH*COOH; wherein (*)
indicates the attachment point from the carbon atom of CH to
X.sub.4.
[0309] In a further embodiment W.sub.1 is selected from --C(O)NH--,
--NHC(O)--, --C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)-- or a valence bond.
Typically, W.sub.1 is selected from --C(O)NH--, --NHC(O)-- or
--C(O)NHS(O).sub.2--.
[0310] In a further embodiment W.sub.2 is selected from --C(O)NH--,
--NHC(O)--, --C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)-- or a valence bond.
Typically, W.sub.2 is selected from --C(O)NH--, --NHC(O)-- or
--C(O)NHS(O).sub.2--.
[0311] In a further embodiment W.sub.3 is selected from --C(O)NH--,
--NHC(O)--, --C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)-- or a valence bond.
Typically, W.sub.3 is selected from --C(O)NH--, --NHC(O)-- or
--C(O)NHS(O).sub.2--.
[0312] In a further embodiment W.sub.4 is selected from --C(O)NH--,
--NHC(O)--, --C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)-- or a valence bond.
Typically, W.sub.4 is selected from --C(O)NH--, --NHC(O)-- or
--C(O)NHS(O).sub.2--.
[0313] In a further embodiment W.sub.5 is selected from --C(O)NH--,
--NHC(O)--, --C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)-- or a valence bond.
Typically, W.sub.5 is selected from --C(O)NH--, --NHC(O)-- or
--C(O)NHS(O).sub.2--.
[0314] In a further embodiment W.sub.6 is selected from --C(O)NH--,
--NHC(O)--, --CH.sub.2NHC(O)--, --C(O)NHS(O).sub.2--,
--S(O).sub.2NHC(O)--, --NHC(O)C.sub.1-6-alkyl,
--C(O)NHC.sub.1-6-alkyl or a valence bond; wherein the alkyl group
is optionally substituted with pyrrolidine-2,5-dione,
--NHC(O)CH*CH.sub.2COOH or --NHC(O)CH.sub.2CH*COOH; wherein (*)
indicates the attachment point from the carbon atom of CH to
X.sub.4. Typically, W.sub.6 is selected from --C(O)NH--,
--NHC(O)--, --C(O)NHS(O).sub.2-- or --NHC(O)C.sub.1-6-alkyl.
[0315] In a further embodiment, D1, D2, F are all valence bonds, l6
is O, and W.sub.6 is selected from either pyrrolidine-2,5-dione,
--NHC(O)CH*CH.sub.2COOH or --NHC(O)CH.sub.2CH*COOH wherein (*)
indicates the attachment point from the carbon atom of CH to
GH.
[0316] In a further embodiment R.sup.1 selected from hydrogen,
--C(O)OH, --C(O)NH.sub.2, --S(O).sub.2OH or C.sub.1-6-alkyl;
wherein the alkyl group optionally is substituted with --C(O)OH,
--C(O)NH.sub.2, --S(O).sub.2OH. Typically, R.sup.1 is selected from
--C(O)OH, --C(O)NH.sub.2, or C.sub.1-6-alkyl; wherein the alkyl
group optionally is substituted with --C(O)OH, --C(O)NH.sub.2, or
--S(O).sub.2OH.
[0317] In a further embodiment R.sup.2 is selected from hydrogen,
--C(O)OH, --C(O)NH.sub.2, --S(O).sub.2OH or C.sub.1-6-alkyl;
wherein the alkyl group optionally is substituted with --C(O)OH,
--C(O)NH.sub.2, --S(O).sub.2OH. Typically, R.sup.2 is selected from
--C(O)OH, --C(O)NH.sub.2, or C.sub.1-6-alkyl; wherein the alkyl
group optionally is substituted with --C(O)OH, --C(O)NH.sub.2, or
--S(O).sub.2OH.
[0318] In a further embodiment R.sup.3 is selected from hydrogen,
--C(O)OH, --C(O)NH.sub.2, --S(O).sub.2OH or C.sub.1-6-alkyl;
wherein the alkyl group optionally is substituted with --C(O)OH,
--C(O)NH.sub.2, --S(O).sub.2OH. Typically, R.sup.3 is selected from
--C(O)OH, --C(O)NH.sub.2, or C.sub.1-6-alkyl; wherein the alkyl
group optionally is substituted with --C(O)OH, --C(O)NH.sub.2, or
--S(O).sub.2OH.
[0319] In a further embodiment R.sup.4 is selected from hydrogen,
--C(O)OH, --C(O)NH.sub.2, --S(O).sub.2OH or C.sub.1-6-alkyl;
wherein the alkyl group optionally is substituted with --C(O)OH,
--C(O)NH.sub.2, --S(O).sub.2OH. Typically, R.sup.4 is selected from
--C(O)OH, --C(O)NH.sub.2, or C.sub.1-6-alkyl; wherein the alkyl
group optionally is substituted with --C(O)OH, --C(O)NH.sub.2, or
--S(O).sub.2OH.
[0320] In a further embodiment R.sup.5 is selected from hydrogen,
--C(O)OH, --C(O)NH.sub.2, --S(O).sub.2OH or C.sub.1-6-alkyl;
wherein the alkyl group optionally is substituted with --C(O)OH,
--C(O)NH.sub.2, --S(O).sub.2OH. Typically, R.sup.5 is selected from
--C(O)OH, --C(O)NH.sub.2, or C.sub.1-6-alkyl; wherein the alkyl
group optionally is substituted with --C(O)OH, --C(O)NH.sub.2, or
--S(O).sub.2OH.
[0321] In a further embodiment E1 is selected from --O-- or
--N(R.sup.6)--, or a valence bond. Typically, E1 is selected from
--O--.
[0322] In a further embodiment E2 is selected from --O-- or
--N(R.sup.6)--, or a valence bond. Typically, E2 is selected from
--O--.
[0323] In a further embodiment E1 and E2 are both --O--.
[0324] In a further embodiment E1 and E2 are both
--N(R.sup.6)--.
[0325] In a further embodiment F is phenyl, pyrrolidine-2,5-dione
or a valence bond.
[0326] In a further embodiment D1 is selected from --O-- or
--N(R.sup.6)--, or a valence bond. Typically, D1 is selected from
--N(R.sup.6)--.
[0327] In a further embodiment D2 is selected from --O-- or
--N(R.sup.6)--, or a valence bond. Typically, D1 is selected from
--N(R.sup.6)--.
[0328] In a further embodiment l1 is 0-6, such as 0, 1, 2, 3, 4, 5
or 6.
[0329] In a further embodiment l2 is 0-6, such as 0, 1, 2, 3, 4, 5
or 6.
[0330] In a further embodiment l3 is 0-6, such as 0, 1, 2, 3, 4, 5
or 6.
[0331] In a further embodiment l4 is 0-6, such as 0, 1, 2, 3, 4, 5
or 6. In a further embodiment l5 is 0-6, such as 0, 1, 2, 3, 4, 5
or 6.
[0332] In a further embodiment 16 is 0-6, such as 0, 1, 2, 3, 4, 5
or 6.
[0333] In a further embodiment m1 is 0-6, such as 0, 1, 2, 3, 4, 5
or 6.
[0334] In a further embodiment m2 is 0-10, such as 0, 1, 2, 3, 4,
5, 6, 7, 8, 9, or 10.
[0335] In a further embodiment m3 is 0-5, such as 0, 1, 2, 3, 4, 5
or 6.
[0336] In a further embodiment m4 is 0-5, such as 0, 1, 2, 3, 4, 5
or 6.
[0337] In a further embodiment m5 is 0-10, such as 0, 1, 2, 3, 4,
5, 6, 7, 8, 9, or 10.
[0338] In a further embodiment m6 is 0-5, such as 0, 1, 2, 3, 4, 5
or 6.
[0339] In a further embodiment m7 is 0-5, such as 0, 1, 2, 3, 4, 5
or 6.
[0340] In a further embodiment n1 is 0-10, such as 0, 1, 2, 3, 4, 5
or 6.
[0341] In a further embodiment n2 is 0-10, such as 0, 1, 2, 3, 4, 5
or 6.
[0342] In a further embodiment n3 is 0-10, such as 0, 1, 2, 3, 4, 5
or 6.
[0343] In a further embodiment n4 is 0-10, such as 0, 1, 2, 3, 4, 5
or 6.
[0344] In a further embodiment X.sub.1 is
--W.sub.1--[(CHR.sup.1).sub.l1--W.sub.2].sub.m1--{[(CH.sub.2).sub.n1O].su-
b.m2--[(CHR.sup.2).sub.l2--W.sub.3].sub.m3}.sub.n2-- and X.sub.2 is
--[(CHR.sup.3).sub.l3--W.sub.4].sub.m4--{[(CH.sub.2).sub.n3O].sub.m5--[(C-
HR.sup.4).sub.l4--W.sub.5].sub.m6}.sub.n4--, wherein
--{[(CH.sub.2).sub.n1O].sub.m2--[(CHR.sup.2).sub.l2--W.sub.3].sub.m3}.sub-
.n2-- and
--{[(CH.sub.2).sub.n3O].sub.m5--[(CHR.sup.4).sub.l4--W.sub.5].su-
b.m6}.sub.n4-- are selected from,
##STR00032##
[0345] wherein * is intended to denote a point of attachment, ie,
an open bond.
[0346] In a further embodiment the molar weight of said hydrophilic
spacer is in the range from 80 Daltons (D) to 1500 D or in the
range from 300 D to 1100 D.
[0347] In a still further embodiment W has the formula
--W.sub.7--Y--,
wherein [0348] Y is
--(CH.sub.2).sub.l7--C.sub.3-10-Cycloalkyl-W.sub.8-- or a valence
bond, [0349] l7 is 0-6, [0350] W.sub.7 is selected from --C(O)NH--,
--NHC(O)--, --C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--, --OC(O)NH--,
--NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--, --C(O)CH.dbd.CH--,
--CH.dbd.CHC(O)--, --(CH.sub.2).sub.s3--, --C(O)--, --C(O)O--,
--OC(O)--, or a valence bond; wherein s3 is 0 or 1, [0351] W.sub.8
is selected from --C(O)NH--, --NHC(O)--, --C(O)NHCH.sub.2--,
--CH.sub.2NHC(O)--, --C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--,
--OC(O)NH--, --NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--,
--C(O)CH.dbd.CH--, --CH.dbd.CHC(O)--, --(CH.sub.2).sub.s4--,
--C(O)--, --C(O)O--, --OC(O)--, or a valence bond; wherein s4 is 0
or 1.
[0352] In an embodiment of W Y is
--(CH.sub.2).sub.l7-cyclohexyl-W.sub.8--.
[0353] In a further embodiment Y is a valence bond.
[0354] In an embodiment W.sub.7 is selected from --C(O)NH--,
--NHC(O)--, --C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)-- or a valence bond.
Typically, W.sub.7 is selected from --C(O)NH--, --NHC(O)--, or
--C(O)NHS(O).sub.2.
[0355] In a further embodiment W.sub.8 is selected from --C(O)NH--,
--NHC(O)--, --C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)-- or a valence bond.
Typically, W.sub.8 is selected from --C(O)NH--, --NHC(O)--, or
--C(O)NHS(O).sub.2.
[0356] In a further embodiment l7 is 0 or 1.
[0357] In a further embodiment the hydrophilic spacer B of the
present invention is selected from
##STR00033## ##STR00034## ##STR00035## ##STR00036##
wherein * is intended to denote a point of attachment, ie, an open
bond.
[0358] The albumin binding residue (substituent A in formula (I) or
(II) above) attached to the growth hormone compound of the present
invention is a lipophilic residue, which binds non-covalently to
albumin. Typically, albumin binding residue is negatively charged
at physiological pH, and has a binding affinity towards human serum
albumin that is below about 10 .mu.M or even below about 1
.mu.M.
[0359] In a further embodiment of the growth hormone compound of
the present invention the albumin binding residue is selected from
a straight chain alkyl group, a branched alkyl group, a group which
has a .omega.-carboxylic acid group or a .omega.-carboxylic acid
isoster. Typically, the albumin binding residue has from 6 to 40
carbon atoms. In a further embodiment the albumin binding residue
has from 8 to 26 carbon atoms. In a further embodiment the albumin
binding residue has from 8 to 20 carbon atoms.
[0360] In a further embodiment A has 14 to 26 carbon atoms and
comprises an .omega.-carboxylic acid group. In a further embodiment
A has 14 to 26 carbon atoms and comprises an .omega.-carboxylic
acid isoster, such as a tetrazol.
[0361] In a further embodiment A is selected from
##STR00037## ##STR00038##
wherein * denotes the attachment to B through W.
[0362] The hydrophilic spacer (B) is preferably introduced in a
position of the growth hormone compound (GH) in a selective manner
in order to be able to control whether one or two albumin binding
residues (A) should be incorporated in the growth hormone compound.
The hydrophilic spacer (B) may be attached to an amino acid
side-chain of the GH compound. Such amino acid side-chain may be a
chemically modified amino acid side-chain of the GH compound.
Another, such amino acid side-chain may be an enzymatically
modified amino acid side-chain of the GH compound. Preferably, a
transglutaminase is used to introduce a hydrophilic spacer in the
glutamine residue in the position corresponding to position 40 or
position 141 in SEQ ID NO: 1. Another way of selectively
introducing a hydrophilic spacer is in the N-terminal residue of
the growth hormone compound, such as hGH (SEQ ID NO: 1).
[0363] In the growth hormone conjugate of the formula (I) the
fragment A-W--B may be linear or branched. In one embodiment,
A-W--B is not a linear peptide.
[0364] In a further embodiment the albumin binding residue via a
hydrophilic spacer is attached to the glutamine residue in the
position corresponding to position 40 in SEQ ID NO: 1.
[0365] In a further embodiment the albumin binding residue via a
hydrophilic spacer is attached to the glutamine residue in the
position corresponding to position 141 in SEQ ID NO: 1.
[0366] In a further embodiment the albumin binding residue via a
hydrophilic spacer is attached to the N-terminal residue of the
growth hormone compound, such as hGH (SEQ ID NO: 1).
[0367] In a further embodiment the albumin binding residue via a
hydrophilic spacer is attached to the glutamine residue in the
position corresponding to position 40, position 141 and to the
N-terminal residue of the growth hormone compound, such as hGH (SEQ
ID NO: 1).
[0368] The growth hormone conjugates of the present invention are
selected from,
##STR00039## ##STR00040## ##STR00041## ##STR00042## ##STR00043##
##STR00044## ##STR00045## ##STR00046## ##STR00047##
##STR00048##
[0369] In a further aspect the present invention relates to a
growth hormone variants such as the growth hormone compounds (GH)
described herein. The growth hormone variants may be useful as
therapeutic agents or as intermediates in the preparation of growth
hormone conjugates. The growth hormone compounds may be produces by
recombinant methods known in the art or as described herein. In an
embodiment the growth hormone variant is soluble.
[0370] In a further aspect the invention relates to a composition
comprising a growth hormone variant as described herein.
[0371] In an embodiment the composition comprises a growth hormone
variant comprising a single cys mutation selected from the group
of: P5C, S7C, D11C, H18C, Q29C, E30C, E33C, A34C, Y35C, E88C, Q91C,
S95C, A98C, N99C, S100C, L101C, V102C, Y103C, D107C, S108C, D112C,
Q122C and G126C.
[0372] In an embodiment of the invention the growth hormone variant
of the composition has a single Cys mutation in the N-terminal (AA
1-8), in Helix 1, in Loop 1, in Helix 2, in Loop 2 or in Helix 3 of
the growth hormone compound.
[0373] In an embodiment the the single Cys mutation is positioned
in the N-terminal, the mutation being such as any one of P5C, S7C.
In an embodiment the single Cys mutation is positioned in H1
(corresponding to AA 9-35), such as any one of D11C, H18C, Q29C,
E30C, E33C, A34C, Y35C. In an embodiment the the single Cys
mutation is positioned in L1 (corresponding to AA36-71), the
muation being such as any one of K38C, E39C, Y42C, S43C, D47C,
P48C, S55, S57C, P59C, S62C, E65C, Q69C or preferably any one of
Y42C, S55C, S57C, S62C, Q69C. In an embodiment the single Cys
mutation is positioned in H2 (AA 72-98), such as any one of E88C,
Q91C, S95C and A98C. In an embodiment the single Cys mutation is
positioned within AA 99-127. In an embodiment the single Cys
mutation is positioned in L2 (AA 99-106), such as any one of N99C,
S100C, L101C, V102C and Y103C. In an embodiment the single Cys
mutation is positioned in H3 (AA 107-127), the mutation being such
as any one of D107C, S108C, D112C, Q122C and G126C of hGH (SEQ ID
NO: 1) or in L3 or H4 (corresponding to AA128-154 and AA155-184)
the muation being such as any one of E129C, D130C, G131C, P133C,
T135C, G136C, T142C, D147C, N149C, D154C, A155C, L156C, R178C,
V180C or in the C-terminal the muation being such as any one of
E186C G187C and G190C. If the single Cys mutation is present in a
hGH variant the mutation is located in corresponding amino acid
residues. In even further embodiments the single Cys mutation is
located within AA 93-106 in hGH or corresponding residues in hGH
variants. In further specified embodiments the single Cys mutation
is located within L2, such as within AA 99-106 or AA 99-104 or
corresponding residues.
[0374] In an embodiment the composition according to the invention
comprises a growth hormone variant comprising a single cys mutation
and an additional disulfide bond. In an embodiment the single cys
mutation is any one of the above described single cys mutations. In
an embodiment the the single cys mutation in GH is selected from
any one of: T3C, P5C, S7C, D11C, H18C, Q29C, E30C, E33C, A34C,
Y35C, K38C, E39C, Y42C, S43C, D47C, P48C, S55C, S57C, P59C, S62,
E65C, Q69C, E88C, Q91C, S95C, A98C, N99C, S100C, L101C, V102C,
Y103C, D107C, S108C, D112C, Q122C, G126C, E129C, D130C, G131C,
P133C, T135C, G136C, T142C, D147C, N149C, D154C, A155C, L156C,
R178C, E186C, G187C and G190C, such as any one of; P5C, S7C, D11C,
H18C, Q29C, E30C, E33C, A34C, Y35C, E88C, Q91C, S95C, A98C, N99C,
S100C, L101C, V102C, Y103C, D107C, S108C, D112C, Q122C and G126C of
hGH (SEQ ID NO: 1) or corresponding residues. In an embodiment the
additional disulfide bond is selected from the following group of
pairs of cystein muations: R16C/L117C, A17C/E174C, H21C/M170C,
D26C/V102C, D26C/Y103C, N47C/T50C, Q49C/G161C, F54C/Y143C,
F54C/S144C, F54C/F146C, S55C/Y143C, S57C/Y143C, I58C/Q141C,
I58C/Y143C, I58C/S144C, P59C/Q137C, P61C/E66C, P61C/T67C,
S71C/S132C, L73C/S132C, L73C/F139C, R77C/I38C, R77C/F139C,
L81C/Q141C, L81C/Y143C, Q84C/Y143C, S85C/Y143C, S85C/S144C,
P89C/F146C, F92C/F146C, F92C/T148C, R94C/D107C, V102C/A105C,
L156C/F146C, L156C/T148C and/or V185C/S188C. In an embodiment the
growth hormone variant comprise a single cystein and an additional
disulfide bond selected from the following pairs of cystein
mutations: S57C/Y143C, Q84C/Y143C, S85C/Y143C and/or
S85C/S144C.
[0375] In an embodiment the growth hormone variant comprise a
single cysteine mutation in L2 and an additional disulfide bond
which connects a loop segment, such as from amino acid residues
128-154 (H3), with a helical segment, such as helix B or helix 2
(corresponding to AA 72-98). In an embodiment the growth hormone
variant comprise a combination of mutations selected from the
following group: A98C/Q84C/Y143C, A98C/S85C/Y143C, A98C/S85C/S144C,
N99C/Q84C/Y143C, N99C/S85C/Y143C, N99C/S85C/S144C,
S101C/Q84C/Y143C, S101C/S85C/Y143C, S101C/S85C/S144C,
L101C/Q84C/Y143C, L101C/S85C/Y143C, L101C/S85C/S144C,
C102C/Q84C/Y143C, C102C/S85C/Y143C and C102C/S85C/S144C. In an
embodiment the growth hormone variant comprise a combination of
mutations selected from the following group: A98C/Q84C/Y143C,
N99C/Q84C/Y143C, S101C/Q84C/Y143C, L101C/Q84C/Y143C and
C102C/Q84C/Y143C. In an embodiment the growth hormone variant
comprise the mutations L101C, Q84C and Y143C.
[0376] In a further aspect the present invention relates to a
growth hormone conjugate which comprises a growth hormone compound
(GH) linked to an albumin binding residue via a hydrophilic spacer,
or a pharmaceutically acceptable salt thereof for use in therapy.
Furthermore, in the growth hormone conjugate of the present
invention GH, the albumin binding residue, and the hydrophilic
spacer are selected from any one of the above embodiments, in
particular the growth hormone conjugate has the formula (I) or
(II).
[0377] In a further aspect the present invention relates to a
pharmaceutical composition comprising a growth hormone conjugate
which comprises a growth hormone compound (GH) linked to an albumin
binding residue via a hydrophilic spacer, or a pharmaceutically
acceptable salt optionally in combination with a pharmaceutical
acceptable excipient.
[0378] The term "identity" as known in the art, refers to a
relationship between the sequences of two or more proteins, as
determined by comparing the sequences. In the art, "identity" also
means the degree of sequence relatedness between proteins, as
determined by the number of matches between strings of two or more
amino acid residues. "Identity" measures the percent of identical
matches between the smaller of two or more sequences with gap
alignments (if any) addressed by a particular mathematical model or
computer program (i.e., "algorithms"). Identity of related proteins
can be readily calculated by known methods. Such methods include,
but are not limited to, those described in Computational Molecular
Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.
and Devereux, J., eds., M. Stockton Press, New York, 1991; and
Carillo et al., SIAM J. Applied Math., 48, 1073, (1988).
[0379] Preferred methods to determine identity are designed to give
the largest match between the sequences tested. Methods to
determine identity are described in publicly available computer
programs. Preferred computer program methods to determine identity
between two sequences include the GCG program package, including
GAP (Devereux et al., Nucl. Acid. Res., 12, 387, (1984)); Genetics
Computer Group, University of Wisconsin, Madison, Wis.), BLASTP,
BLASTN, and FASTA (Altschul et al., J. Mol. Biol., 215, 403-410,
(1990)). The BLASTX program is publicly available from the National
Center for Biotechnology Information (NCBI) and other sources
(BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894;
Altschul et al., supra). The well known Smith Waterman algorithm
may also be used to determine identity.
[0380] For example, using the computer algorithm GAP (Genetics
Computer Group, University of Wisconsin, Madison, Wis.), two
proteins for which the percent sequence identity is to be
determined are aligned for optimal matching of their respective
amino acids (the "matched span", as determined by the algorithm). A
gap opening penalty (which is calculated as 3.times. the average
diagonal; the "average diagonal" is the average of the diagonal of
the comparison matrix being used; the "diagonal" is the score or
number assigned to each perfect amino acid match by the particular
comparison matrix) and a gap extension penalty (which is usually
1/10 times the gap opening penalty), as well as a comparison matrix
such as PAM 250 or BLOSUM 62 are used in conjunction with the
algorithm. A standard comparison matrix (see Dayhoff et al., Atlas
of Protein Sequence and Structure, vol. 5, suppl. 3, (1978) for the
PAM 250 comparison matrix; Henikoff et al., Proc. Natl. Acad. Sci
USA, 89, 10915-10919, (1992) for the BLOSUM 62 comparison matrix)
is also used by the algorithm.
[0381] Preferred parameters for a protein sequence comparison
include the following: Algorithm: Needleman et al., J. Mol. Biol,
48, 443-453, (1970); Comparison matrix: BLOSUM 62 from Henikoff et
al., Proc. Natl. Acad. Sci. USA, 89, 10915-10919, (1992); Gap
Penalty: 12, Gap Length Penalty: 4, Threshold of Similarity: 0.
[0382] The GAP program is useful with the above parameters. The
aforementioned parameters are the default parameters for protein
comparisons (along with no penalty for end gaps) using the GAP
algorithm.
[0383] The compounds of the present invention have improved
pharmacological properties compared to the corresponding
un-conjugated growth hormone, which is also referred to as the
parent compound. Examples of such pharmacological properties
include functional in vivo half-life, immunogencity, renal
filtration, protease protection and albumin binding.
[0384] The term "functional in vivo half-life" is used in its
normal meaning, i.e., the time at which 50% of the biological
activity of the GH or GH conjugate is still present in the
body/target organ, or the time at which the activity of the GH or
GH conjugate is 50% of its initial value. As an alternative to
determining functional in vivo half-life, "in vivo plasma
half-life" may be determined, i.e., the time at which 50% of the GH
or GH conjugate circulate in the plasma or bloodstream prior to
being cleared. Determination of plasma half-life is often more
simple than determining functional half-life and the magnitude of
plasma half-life is usually a good indication of the magnitude of
functional in vivo half-life. Alternative terms to plasma half-life
include serum half-life, circulating half-life, circulatory
half-life, serum clearance, plasma clearance, and clearance
half-life.
[0385] The term "increased" as used in connection with the
functional in vivo half-life or plasma half-life is used to
indicate that the relevant half-life of the GH conjugate is
statistically significantly increased relative to that of the
parent GH, as determined under comparable conditions. For instance
the relevant half-life may be increased by at least about 25%, such
as by at lest about 50%, e.g., by at least about 100%, 150%, 200%,
250%, or 500%. In one embodiment, the compounds of the present
invention exhibit an increase in half-life of at least about 5 h,
preferably at least about 24 h, more preferably at least about 72
h, and most preferably at least about 7 days, relative to the
half-life of the parent GH.
[0386] Measurement of in vivo plasma half-life can be carried out
in a number of ways as described in the literature. An increase in
in vivo plasma half-life may be quantified as a decrease in
clearance (CL) or as an increase in mean residence time (MRT).
Conjugated GH of the present invention for which the CL is
decreased to less than 70%, such as less than 50%, such than less
than 20%, such than less than 10% of the CL of the parent GH as
determined in a suitable assay is said to have an increased in vivo
plasma half-life. Conjugated GH of the present invention for which
MRT is increased to more than 130%, such as more than 150%, such as
more than 200%, such as more than 500% of the MRT of the parent GH
in a suitable assay is said to have an increased in vivo plasma
half-life. Clearance and mean residence time can be assessed in
standard pharmacokinetic studies using suitable test animals. It is
within the capabilities of a person skilled in the art to choose a
suitable test animal for a given protein. Tests in human, of
course, represent the ultimate test. Suitable test animals include
normal, Sprague-Dawley male rats, mice and cynomolgus monkeys.
Typically the mice and rats are in injected in a single
subcutaneous bolus, while monkeys may be injected in a single
subcutaneous bolus or in a single iv dose. The amount injected
depends on the test animal. Subsequently, blood samples are taken
over a period of one to five days as appropriate for the assessment
of CL and MRT. The blood samples are conveniently analysed by ELISA
techniques.
[0387] The term "Immunogenicity" of a compound refers to the
ability of the compound, when administered to a human, to elicit a
deleterious immune response, whether humoral, cellular, or both. In
any human sub-population, there may be individuals who exhibit
sensitivity to particular administered proteins. Immunogenicity may
be measured by quantifying the presence of growth hormone
antibodies and/or growth hormone responsive T-cells in a sensitive
individual, using conventional methods known in the art. In one
embodiment, the conjugated GH of the present invention exhibit a
decrease in immunogenicity in a sensitive individual of at least
about 10%, preferably at least about 25%, more preferably at least
about 40% and most preferably at least about 50%, relative to the
immunogenicity for that individual of the parent GH. The term
"protease protection" or "protease protected" as used herein is
intended to indicate that the conjugated GH of the present
invention is more resistant to the plasma peptidase or proteases
than is the parent GH. Protease and peptidase enzymes present in
plasma are known to be involved in the degradation of circulating
proteins, such as e.g. circulating peptide hormones, such as growth
hormone. Such protease protection may be measured by the method of
Example A described herein.
[0388] Growth hormone may be susceptible to degradation by for
instance thrombin, plasmin, subtilisin, and chymotrypsin-like
serine proteinase. Assays for determination of degradation of these
proteases are described in J. Biotech., 65, 183, (1998). In one
embodiment, the rate of hydrolysis of the GH conjugate is less than
70%, such as less than 40%, such as less than 10% of that of the
parent GH.
[0389] The most abundant protein component in circulating blood of
mammalian species is serum albumin, which is normally present at a
concentration of approximately 3 to 4.5 grams per 100 milliters of
whole blood. Serum albumin is a blood protein of approximately
65,000 daltons which has several important functions in the
circulatory system. It functions as a transporter of a variety of
organic molecules found in the blood, as the main transporter of
various metabolites such as fatty acids and bilirubin through the
blood, and, owing to its abundance, as an osmotic regulator of the
circulating blood. Serum albumin has a half-life of more than one
week, and one approach to increasing the plasma half-life of
proteins has been to conjugate to the protein a group that binds to
serum albumin. Albumin binding property may be determined as
described in J. Med. Chem., 43, 1986, (2000) which is incorporated
herein by reference.
[0390] The growth hormone conjugates of formula (I) or (II) exert
growth hormone activity and may as such be used in the treatment of
diseases or states which will benefit from an increase in the
amount of circulating growth hormone. In particular, the invention
provides a method for the treatment of growth hormone deficiency
(GHD); Turner Syndrome; Prader-Willi syndrome (PWS); Noonan
syndrome; Down syndrome; chronic renal disease, juvenile rheumatoid
arthritis; cystic fibrosis, HIV-infection in children receiving
HAART treatment (HIV/HALS children); short children born short for
gestational age (SGA); short stature in children born with very low
birth weight (VLBW) but SGA; skeletal dysplasia; hypochondroplasia;
achondroplasia; idiopathic short stature (ISS); GHD in adults;
fractures in or of long bones, such as tibia, fibula, femur,
humerus, radius, ulna, clavicula, matacarpea, matatarsea, and
digit; fractures in or of spongious bones, such as the scull, base
of hand, and base of food; patients after tendon or ligament
surgery in e.g. hand, knee, or shoulder; patients having or going
through distraction oteogenesis; patients after hip or discus
replacement, meniscus repair, spinal fusions or prosthesis
fixation, such as in the knee, hip, shoulder, elbow, wrist or jaw;
patients into which osteosynthesis material, such as nails, screws
and plates, have been fixed; patients with non-union or mal-union
of fractures; patients after osteatomia, e.g. from tibia or
1.sup.st toe; patients after graft implantation; articular
cartilage degeneration in knee caused by trauma or arthritis;
osteoporosis in patients with Turner syndrome; osteoporosis in men;
adult patients in chronic dialysis (APCD); malnutritional
associated cardiovascular disease in APCD; reversal of cachexia in
APCD; cancer in APCD; chronic abstractive pulmonal disease in APCD;
HIV in APCD; elderly with APCD; chronic liver disease in APCD,
fatigue syndrome in APCD; Crohn's disease; impaired liver function;
males with HIV infections; short bowel syndrome; central obesity;
HIV-associated lipodystrophy syndrome (HALS); male infertility;
patients after major elective surgery, alcohol/drug detoxification
or neurological trauma; aging; frail elderly; osteo-arthritis;
traumatically damaged cartilage; erectile dysfunction;
fibromyalgia; memory disorders; depression; traumatic brain injury;
subarachnoid haemorrhage; very low birth weight; metabolic
syndrome; glucocorticoid myopathy; or short stature due to
glucucorticoid treatment in children, the method comprising
administering to a patient in need thereof a therapeutically
effective amount of a growth hormone conjugate according to formula
(I) or (II).
[0391] In a further aspect, the invention provides a method for the
acceleration of the healing of muscle tissue, nervous tissue or
wounds; the acceleration or improvement of blood flow to damaged
tissue; or the decrease of infection rate in damaged tissue, the
method comprising administration to a patient in need thereof an
effective amount of a therapeutivcally effective amount of a growth
hormone conjugate of formula (I) or (II).
[0392] In a further embodiment, the invention relates to the use of
a growth hormone conjugate of formula (I) or (II) in the
manufacture of diseases benefiting from an increase in the growth
hormone plasma level, such as the disease mentioned above.
[0393] A typical parenteral dose is in the range of 10.sup.-9 mg/kg
to about 100 mg/kg body weight per administration. Typical
administration doses are from about 0.0000001 to about 10 mg/kg
body weight per administration. The exact dose will depend on e.g.
indication, medicament, frequency and mode of administration, the
sex, age and general condition of the subject to be treated, the
nature and the severity of the disease or condition to be treated,
the desired effect of the treatment and other factors evident to
the person skilled in the art.
[0394] Typical dosing frequencies are twice daily, once daily,
bi-daily, twice weekly, once weekly or with even longer dosing
intervals. Due to the prolonged half-lifes of the fusion proteins
of the present invention, a dosing regime with long dosing
intervals, such as twice weekly, once weekly or with even longer
dosing intervals is a particular embodiment of the invention.
[0395] Many diseases are treated using more than one medicament in
the treatment, either concomitantly administered or sequentially
administered. It is therefore within the scope of the present
invention to use a growth hormone conjugate of formula (I) or (II)
in therapeutic methods for the treatment of one of the above
mentioned diseases in combination with one or more other
therapeutically active compound(s) normally used in the treatment
said diseases. By analogy, it is also within the scope of the
present invention to use a growth hormone conjugate of formula (I)
or (II) in combination with other therapeutically active compounds
normally used in the treatment of one of the above mentioned
diseases in the manufacture of a medicament for said disease.
General Methods
Enzyme Conjugation:
[0396] In the preparation of a growth hormone conjugate of the
present invention, typically at least one of the covalent bonds
established in the preparation of a A-W--B-GH conjugate of formula
(I) is prepared by use of an enzyme as illustrated in the examples
below. Such an enzyme may for instance be selected from the group
consisting of transglutaminases, serine proteases and cysteine
proteases. Typically, said enzyme is a transglutaminase. Such
transglutaminase may for instance be selected from the group
consisting of microbial transglutaminases, tissue transglutaminases
and factor XIII and variants thereof. In another embodiment, said
enzyme is a cysteine protease. The growth hormone conjugate of the
present invention may be prepared by many different methods,
non-limiting examples are shown below.
[0397] The present invention also provides methods for preparing
A-W--B-GH conjugates of formula (I).
Transglutaminase
[0398] As stated above, at least one of the covalent bonds
established in the preparation of a A-W--B-GH conjugate of the
present invention may be prepared by use of a transglutaminase.
Transglutaminases may include microbial transglutaminases such as
that isolated from the Streptomyces species; S. mobaraense, S.
cinnamoneum, S. griseocarneum (U.S. Pat. No. 5,156,956 incorporated
herein by reference), S. lavendulae (U.S. Pat. No. 5,252,469
incorporated herein by reference) and S. ladakanum (JP2003/199569
incorporated herein by reference). Other useful microbial
transglutaminases have been isolated from Bacillus subtilis
(disclosed in U.S. Pat. No. 5,731,183, which is incorporated herein
by reference) and from various Myxomycetes. Other examples of
useful microbial transglutaminases are those disclosed in
WO1996/06931 (e.g. transglutaminase from Bacillus lydicus) and
WO1996/22366, both of which are incorporated herein by reference.
Useful non-microbial transglutaminases include guinea-pig liver
transglutaminase, and transglutaminases from various marine sources
like the flat fish Pagrus major (disclosed in EP0555649, which is
incorporated herein by reference), and the Japanese oyster
Crassostrea gigas (disclosed in U.S. Pat. No. 5,736,356, which is
incorporated herein by reference). Functional analogues and
derivatives thereof may also be useful.
[0399] Typically, the TGase used in the methods of the invention is
a microbial transglutaminase. In one embodiment, the TGase is from
S. mobaraense or a variant thereof, for instance as described in
WO2007/020290 and WO2008/020075. In another embodiment, the TGase
is from S. ladakanum or a variant thereof, for instance as
described in WO2008/020075.
[0400] The conjugation of GH to A-W--B according to the present
invention may be achieved by TGase-mediated modification leading to
selective alteration at specific lysine (Lys) or glutamine (Gln)
positions in the sequence of the GH compound depending on the
substrate used. Use of amines as substrates will leade to
modification of Glutamines whereas the use of primary amides will
lead to modification of Lysines. hGH (SEQ ID NO: 1) has 9 lysine
residues at positions 38, 41, 70, 115, 140, 145, 158, 168 and 172
and 13 glutamine residues at positions 22, 29, 40, 46, 49, 68, 69,
84, 91, 122, 137, 141 and 181, although not all of these are
readily available for modification nor suitably for modifications
as this will lead to diminished binding potency to the growth
hormone binding proteine hence leading to reduced biological
activity. The x-ray protein crystal structure between hGH and its
binding proteine (pdb: 3HHR) reveals that at least 4 lysines (38,
41, 168 and 172) takes part in binding to the binding proteine and
potentially only one of the glutamines (Gln 46). This renders the
glutamines more attractive as target for selective introduction of
an albumin binder linker.
[0401] These structural considerations are further supported by
findings summarised by N. Chene et al in Reprod. Nutr. Develop. 29,
1-25 (1989) where it's concluded that chemical modifications
affecting lysines have been found to have a negative effect on the
in vivo biological activity and on the binding capacity to the
liver receptors of GH.
Chemistry I
[0402] In an aspect the present invention relates to preparation of
a growth hormone conjugate of formula (I) wherein a GH compound is
treated with a property-modifying group using TGase catalyzed
chemistry. Initially, an aldehyde or a ketone functionality is
installed by a two step reaction using amino alcohols that
subsequently are treated with periodate to generate an aldehyde or
keto functionality by oxidative cleavage. Non limited examples of
amino alcohols for illustration only includes
1,3-diamino-2-propanol and 1-amino-2,3-dihydroxypropane.
[0403] In a further aspect the present invention relates to
preparation of a growth hormone conjugate of formula (I) comprising
treatment of an aldehyde or ketone derived from the GH compound
with a property-modifying group-derived aniline or heteroaryl amine
to yield an amine (III.fwdarw.IV).
[0404] In an embodiment, aldehyde derived from the GH compound is
treated with property-modifying group-derived aniline or
heteroarylamine.
[0405] The term "GH compound derived aldehyde (or ketone)" or "an
aldehyde or ketone derived from the GH compound" is intented to
indicate a GH compound to which an aldehyde or ketone functional
group has been covalently attached, or a GH compound on which an
aldehyde or ketone functional group has been generated. The
preparation of GH compound-derived aldehydes, such as compound
(III) illustrated below is well known to those skilled in the art,
and any of these known procedures may be used to prepare the GH
compound-derived aldehyde (III) required for the realization of the
invention disclosed herein.
[0406] In one embodiment, the conjugate A-W--B-GH (IV) is prepared
as illustrated below:
##STR00049## ##STR00050##
[0407] The TGase-mediated enzymatic reaction with GH (I) results in
the modification of Gin at position 141 and/or 40 affording (II).
The modified GH (II) is treated with periodate to cleave the
aminoalcohol to provide a GH derived aldehyde (III). Conjugation of
GH aldehyde (III) with A-W--B1-NH.sub.2 occurs via reductive
alkylation (III.fwdarw.IV). Reductive alkylation as exemplified
herein is well-recognized in the art and results in GH compounds
(IV) modified in position Gln(141) and/or 40.
Chemistry II
[0408] In one embodiment, the conjugate A-W--B-GH is prepared using
reductive amination in GH's N-terminal as illustrated below:
##STR00051##
[0409] Conjugation of GH to A-W--B1-CHO occurs via reductive
alkylation (GH.fwdarw.V). Reductive alkylation as exemplified above
is well-recognized in the art for modifying the N-terminal of
GH.
Chemistry III
[0410] In one embodiment, the conjugate A-W--B-GH is prepared as
illustrated below:
##STR00052##
Wherein the cysteine residue optionally is protected as a mixed
disulfide (VI) (GH--S--S--R) with R being a small organic moeity.
Non limited examples of mixed disulfides may include disulfides
between cystamine (R.dbd.--CH.sub.2CH.sub.2NH.sub.2); cysteine
(R.dbd.--CH.sub.2CH(C(O)OH)NH.sub.2); homocysteine
(R.dbd.--CH.sub.2CH.sub.2CH(C(O)OH)NH.sub.2); and gluthatione
(R.dbd.--CH.sub.2CH(C(O)NHCH.sub.2C(O)OH)NH--C(O)CH.sub.2CH.sub.2CH(C(O)O-
H)NH.sub.2).
[0411] The derivatization process utilise an albumin binding linker
A-W--B1-LG wherein LG represent an inorganic leaving group such as
--Cl, --Br, --I or an organic leaving group such as mesylate or
tosylate. Conjugation of GH with A-W--B1-LG occurs via nucleophilic
substitution (VII.fwdarw.IX).
Chemistry IV
[0412] In one embodiment, the conjugate A-W--B-GH is prepared as
illustrated below:
##STR00053##
[0413] Deprotected Cys GH compound (VII) as obtained from (VI)
above can be reacted with a malimide substituted albumin binder
linker (X) affording GH conjugate
A-W--B1-NHC(O)CH.sub.2CH.sub.2-pyrrolidin-2,5-dione-3-GH (XI).
[0414] wherein the hydrophilic spacer B1 has the formula
--X.sub.1--X.sub.2--X.sub.3--X.sub.4--
wherein [0415] X.sub.1 is
--W.sub.1--[(CHR.sup.1).sub.l1--W.sub.2].sub.m1--{[(CH.sub.2).sub.n1E1].s-
ub.m2--[(CHR.sup.2).sub.l2--W.sub.3].sub.m3}.sub.n2--, [0416]
X.sub.2 is
--[(CHR.sup.3).sub.l3--W.sub.4].sub.m4--{[(CH.sub.2).sub.n3E2].sub.m5--[(-
CHR.sup.4).sub.l4--W.sub.5].sub.m6}.sub.n4--, [0417] X.sub.3 is
--[(CHR.sup.5).sub.l5].sub.m7--, [0418] X.sub.4 is a valence bond,
[0419] l1, l2, l3, l4, and l5 independently are selected from 0-16,
[0420] m1, m3, m4, m6 and m7 independently are selected from 0-10,
[0421] m2 and m5 independently are selected from 0-25, [0422] n1,
n2, n3 and n4 independently are selected from 0-16, [0423] R.sup.1,
R.sup.2, R.sup.3, R.sup.4 and R.sup.5 independently are selected
from hydrogen, --C(O)OH, --C(O)NH.sub.2, --S(O)OH, --S(O).sub.2OH,
--NH--C(.dbd.NH)--NH.sub.2, C.sub.1-6-alkyl, aryl or hetaryl;
wherein the alkyl, aryl and hetaryl groups optionally are
substituted with halogen, --C(O)OH, --C(O)NH.sub.2, --S(O)OH,
--S(O).sub.2OH, --CN or --OH, [0424] E1 and E2 independently are
selected from --O--, --NR.sup.6--, --N(COR.sup.7)-- or a valence
bond; wherein R.sup.6 and R.sup.7 independently represent hydrogen
or C.sub.1-6-alkyl, [0425] W.sub.1 to W.sub.5 independently are
selected from --C(O)NH--, --NHC(O)--, --C(O)NHCH.sub.2--,
--CH.sub.2NHC(O)--, --C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--,
--OC(O)NH--, --NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--,
--C(O)CH.dbd.CH--, --CH.dbd.CHC(O)--, --(CH.sub.2).sub.s2--,
--C(O)--, --C(O)O--, --OC(O)--, or a valence bond; wherein s2 is 0
or 1.
Chemistry V
[0426] In one embodiment, the conjugate A-W--B-GH is prepared as
illustrated below:
##STR00054##
[0427] Albumine binders may be attached to single cys GH
derivatives using S-nitrosyl chemistry as described in
WO2009/024791.
[0428] Deprotected Cys GH compound (VII) is subjected to
S-nitrosylation by addition of a NO donor such as DEA NOnate (Sigma
Aldrich). Nitrosylated single cys GH (XII) is then reacted with an
allyl amine substituted albumine binder (XIII) affording oxime
(XIV) which after hydrolysis affords GH conjugate
A-W--B1-C(O)NHCH.sub.2C(O)CH.sub.2--Cys GH (XV) wherein the
hydrophilic spacer B1 has the formula
--X.sub.1--X.sub.2--X.sub.3--X.sub.4--
wherein [0429] X.sub.1 is
--W.sub.1--[(CHR.sup.1).sub.l1--W.sub.2].sub.m1--{[(CH.sub.2).sub.n1E1].s-
ub.m2--[(CHR.sup.2).sub.l2--W.sub.3].sub.m3}.sub.n2--, [0430]
X.sub.2 is
--[(CHR.sup.3).sub.l3--W.sub.4].sub.m4--{[(CH.sub.2).sub.n3E2].sub.m5--[(-
CHR.sup.4).sub.l4--W.sub.5].sub.m6}.sub.n4--, [0431] X.sub.3 is
--[(CHR.sup.5).sub.l5].sub.m7--, [0432] X.sub.4 is a valence bond,
[0433] l1, l2, l3, l4, and l5 independently are selected from 0-16,
[0434] m1, m3, m4, m6 and m7 independently are selected from 0-10,
[0435] m2 and m5 independently are selected from 0-25, [0436] n1,
n2, n3 and n4 independently are selected from 0-16, [0437] R.sup.1,
R.sup.2, R.sup.3, R.sup.4 and R.sup.5 independently are selected
from hydrogen, --C(O)OH, --C(O)NH.sub.2, --S(O)OH, --S(O).sub.2OH,
--NH--C(.dbd.NH)--NH.sub.2, C.sub.1-6-alkyl, aryl or hetaryl;
wherein the alkyl, aryl and hetaryl groups optionally are
substituted with halogen, --C(O)OH, --C(O)NH.sub.2, --S(O)OH,
--S(O).sub.2OH, --CN or --OH, [0438] E1 and E2 independently are
selected from --O--, --N(R.sup.6)--, --N(C(O)R.sup.7)-- or a
valence bond; wherein R.sup.6 and R.sup.7 independently represent
hydrogen or C.sub.1-6-alkyl, [0439] W.sub.1 to W.sub.5
independently are selected from --C(O)NH--, --NHC(O)--,
--C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--, --C(O)NHS(O).sub.2--,
--S(O).sub.2NHC(O)--, --OC(O)NH--, --NHC(O)O--, --C(O)CH.sub.2--,
--CH.sub.2C(O)--, --C(O)CH.dbd.CH--, --CH.dbd.CHC(O)--,
--(CH.sub.2).sub.s2--, --C(O)--, --C(O)O--, --OC(O)--, or a valence
bond; wherein s2 is 0 or 1.
[0440] A close relationship to the natural peptide is generally
regarded as an advantage with therapeutic interventions comprising
administration of variants or analogues of this natural peptide as
it minimizes the risk of e.g. any unwanted antibody generation.
[0441] GH may be modified in their C-terminal by use of
carboxypeptidase Y (EC. 3.4.16.5), and suitable modified substrates
as described in WO2007/093594. A two step procedure as described by
B. Peschke et al. "C-Terminally PEGylated GH derivatives" Bioorg.
Med. Chem. 15, 4382-4395, (2007), where C terminal alanine is
enzymatically exchanged with NE-(4-acetylbenzoyl)lysine, followed
by reaction with albumin binder derivatives according to the
invention.
[0442] As apparent from the above the invention further relates to
the intermediate linker applied in preparation of the conjugate
A-W--B-GH. Said linker compound may be described by us of formula
(III)
A-W--B1-U (III)
wherein A represent an albumin binding residue, B1 represents a
hydrophilic spacer, W is a chemical group linking A and B1, and U
represent a conjugating moiety.
[0443] Based on the above the conjugating moiety will vary
depending on the method of conjugation applied which may in the end
also be visible in the final hGH compound (A-W--B-GH).
[0444] In further embodiments of the compound, A-W--B1-U, A and W
are as defined in any of the above embodiments.
[0445] When the method described herein above as Chemistry IV, is
applied the compound A-W--B1-U, may further be defined, as an
embodiment, wherein U comprises or consists of an aryl, an
heteraryl, a substituted malimide or a pyrrolidine-2,5-dione such
as --NHC(O)CH.sub.2CH.sub.2-pyrrolidin-2.5-dione.
[0446] In alternative embodiments of compound A-W--B1-U, U
comprises D1-(CH.sub.2).sub.l6-D2, wherein D1 and D2 are
independently selected from --O--, --N(R6)-, --NC(O)R7- or a
valence bond; wherein R6 and R7 independently represent hydrogen or
C.sub.1-6-alkyl.
[0447] Likewise application of Chemistry III as described herein
above will apply linker compounds wherein U comprises or consists
of a leaving group, such as Cl, Br, I, --OH, --OS(O).sub.2Me,
--OS(O).sub.2CF.sub.3 or --OTs, or preferably compounds according
to formula (III), wherein the leaving group is a halogen compound
selected from CI, Br and I, preferably Br.
[0448] Further embodiments of the linker compounds (which are
applied in Chemistry V) are according to the invention defined by
formula (III), wherein U comprises or consists of an allyl amine
(H.sub.2C.dbd.CH--CH.sub.2--NH.sub.2), such as
--C(O)NHCH.sub.2--CH.dbd.CH.sub.2.
[0449] When the method described herein above as Chemistry I, is
applied the compound A-W--B1-U, may further be defined, as an
embodiment, wherein U comprises or consists of an amine
(--NH.sub.2).
[0450] In alternative embodiments, U may comprises or consists of
an aldehyde, such as --CHO.
[0451] The compounds may following be conjugated to any sort of
therapeutic compound which include an acceptor group to which "U"
may enable conjugation. In a preferred embodiment the therapeutic
compound is a polypeptide. Peptides may naturally include AA
residues which may function as an acceptor group, such as Gin
residues, Phe residues and Cys residues.
[0452] Alternatively such amino acid residues may be introduced in
an appropriate position in the polypeptide.
Pharmaceutical Compositions
[0453] Another purpose is to provide a pharmaceutical composition
comprising a growth hormone conjugate of the present invention,
such as a growth hormone conjugate of formula (I) or (II), which is
present in a concentration from 10.sup.-15 mg/mL to 200 mg/mL, such
as e.g. 10.sup.-10 mg/mL to 5 mg/mL and wherein said composition
has a pH from 2.0 to 10.0. The composition may further comprise
pharmaceutical exhibients, such as a buffer system,
preservative(s), tonicity agent(s), chelating agent(s), stabilizers
and surfactants. In one embodiment of the invention the
pharmaceutical composition is an aqueous composition, ie.
composition comprising water. Such composition is typically a
solution or a suspension. In a further embodiment of the invention
the pharmaceutical composition is an aqueous solution. The term
"aqueous composition" is defined as a composition comprising at
least 50% w/w water. Likewise, the term "aqueous solution" is
defined as a solution comprising at least 50% w/w water, and the
term "aqueous suspension" is defined as a suspension comprising at
least 50% w/w water.
[0454] In another embodiment the pharmaceutical composition is a
freeze-dried composition, whereto the physician or the patient adds
solvents and/or diluents prior to use.
[0455] In another embodiment the pharmaceutical composition is a
dried composition (e.g. freeze-dried or spray-dried) ready for use
without any prior dissolution.
[0456] In a further aspect the invention relates to a
pharmaceutical composition comprising an aqueous solution of a
growth hormone conjugate, such as a growth hormone conjugate of
formula (I) or (II), and a buffer, wherein said GH conjugate is
present in a concentration from 0.1-100 mg/mL or above, and wherein
said composition has a pH from about 2.0 to about 10.0.
[0457] In a another embodiment of the invention the pH of the
composition is selected from the list consisting of 2.0, 2.1, 2.2,
2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,
3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8,
4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1,
6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4,
7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and
10.0.
[0458] In a further embodiment of the invention the buffer is
selected from the group consisting of sodium acetate, sodium
carbonate, citrate, glycylglycine, histidine, glycine, lysine,
arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate,
sodium phosphate, and tris(hydroxymethyl)-aminomethan, bicine,
tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric
acid, aspartic acid or mixtures thereof. Each one of these specific
buffers constitutes an alternative embodiment of the invention.
[0459] In a further embodiment of the invention the composition
further comprises a pharmaceutically acceptable preservative. In a
further embodiment of the invention the preservative is 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-(p-chlorphenoxy)propane-1,2-diol) or mixtures thereof. In a
further embodiment of the invention the preservative is present in
a concentration from 0.1 mg/mL to 20 mg/mL. In a further embodiment
of the invention the preservative is present in a concentration
from 0.1 mg/mL to 5 mg/mL. In a further embodiment of the invention
the preservative is present in a concentration from 5 mg/mL to 10
mg/mL. In a further embodiment of the invention the preservative is
present in a concentration from 10 mg/mL to 20 mg/mL. Each one of
these specific preservatives constitutes an alternative embodiment
of the invention. The use of a preservative in pharmaceutical
compositions is well-known to the skilled person. For convenience
reference is made to Remington: The Science and Practice of
Pharmacy, 20.sup.th edition, 2000.
[0460] In a further embodiment of the invention the composition
further comprises an isotonic agent. In a further embodiment of the
invention the isotonic agent is selected from the group consisting
of a salt (e.g. sodium chloride), a sugar or sugar alcohol, an
amino acid (e.g. L-glycine, L-histidine, arginine, lysine,
isoleucine, aspartic acid, tryptophan, threonine), an alditol (e.g.
glycerol (glycerine), 1,2-propanediol (propyleneglycol),
1,3-propanediol, 1,3-butanediol) polyethyleneglycol (e.g. PEG 400),
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, for example, 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 obtained using the methods of the
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 the invention the isotonic agent is present
in a concentration from 1 mg/mL to 50 mg/mL. In a further
embodiment of the invention the isotonic agent is present in a
concentration from 1 mg/mL to 7 mg/mL. In a further embodiment of
the invention the isotonic agent is present in a concentration from
8 mg/mL to 24 mg/mL. In a further embodiment of the invention the
isotonic agent is present in a concentration from 25 mg/mL to 50
mg/mL. Each one of these specific isotonic agents constitutes an
alternative embodiment of the invention. The use of an isotonic
agent in pharmaceutical compositions is well-known to the skilled
person. For convenience reference is made to Remington: The Science
and Practice of Pharmacy, 20.sup.th edition, 2000.
[0461] In a further embodiment of the invention the composition
further comprises a chelating agent. In a further embodiment of the
invention the chelating agent is selected from salts of
ethylenediaminetetraacetic acid (EDTA), citric acid, and aspartic
acid, and mixtures thereof. In a further embodiment of the
invention the chelating agent is present in a concentration from
0.1 mg/mL to 5 mg/mL. In a further embodiment of the invention the
chelating agent is present in a concentration from 0.1 mg/mL to 2
mg/mL. In a further embodiment of the invention the chelating agent
is present in a concentration from 2 mg/mL to 5 mg/mL. Each one of
these specific chelating agents constitutes an alternative
embodiment of the invention. The use of a chelating agent in
pharmaceutical compositions is well-known to the skilled person.
For convenience reference is made to Remington: The Science and
Practice of Pharmacy, 20.sup.th edition, 2000.
[0462] In a further embodiment of the invention the composition
further comprises a stabilizer. The use of a stabilizer in
pharmaceutical compositions is well-known to the skilled person.
For convenience reference is made to Remington: The Science and
Practice of Pharmacy, 20.sup.th edition, 2000.
[0463] More particularly, compositions of the invention are
stabilized liquid pharmaceutical compositions whose therapeutically
active components include a protein that possibly exhibits
aggregate formation during storage in liquid pharmaceutical
compositions. By "aggregate formation" is intended a physical
interaction between the protein molecules that results in formation
of oligomers, which may remain soluble, or large visible aggregates
that precipitate from the solution. By "during storage" is intended
a liquid pharmaceutical composition or composition once prepared,
is not immediately administered to a subject. Rather, following
preparation, it is packaged for storage, either in a liquid form,
in a frozen state, or in a dried form for later reconstitution into
a liquid form or other form suitable for administration to a
subject. By "dried form" is intended the liquid pharmaceutical
composition or composition is dried either by freeze drying (i.e.,
lyophilization; see, for example, Williams and Polli, J. Parenteral
Sci. Technol., 38, 48-59, (1984)), spray drying (see Masters (1991)
in Spray-Drying Handbook (5.sup.th ed; Longman Scientific and
Technical, Essez, U.K.), pp. 491-676; Broadhead et al. Drug Devel.
Ind. Pharm. 18, 1169-1206, (1992); and Mumenthaler et al., Pharm.
Res., 11, 12-20, (1994)), or air drying (Carpenter and Crowe,
Cryobiology 25, 459-470, (1988); and Roser, Biopharm. 4, 47-53,
(1991)). Aggregate formation by a protein during storage of a
liquid pharmaceutical composition can adversely affect biological
activity of that protein, resulting in loss of therapeutic efficacy
of the pharmaceutical composition. Furthermore, aggregate formation
may cause other problems such as blockage of tubing, membranes, or
pumps when the protein-containing pharmaceutical composition is
administered using an infusion system.
[0464] The pharmaceutical compositions of the invention may further
comprise an amount of an amino acid base sufficient to decrease
aggregate formation by the protein during storage of the
composition. By "amino acid base" is intended an amino acid or a
combination of amino acids, where any given amino acid is present
either in its free base form or in its salt form. Where a
combination of amino acids is used, all of the amino acids may be
present in their free base forms, all may be present in their salt
forms, or some may be present in their free base forms while others
are present in their salt forms. In one embodiment, amino acids to
use in preparing the compositions of the invention are those
carrying a charged side chain, such as arginine, lysine, aspartic
acid, and glutamic acid. Any stereoisomer (i.e., L or D isomer, or
mixtures thereof) of a particular amino acid (methionine,
histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan,
threonine and mixtures thereof) or combinations of these
stereoisomers or glycine or an organic base such as but not limited
to imidazole, may be present in the pharmaceutical compositions of
the invention so long as the particular amino acid or organic base
is present either in its free base form or its salt form. In one
embodiment the L-stereoisomer of an amino acid is used. In one
embodiment the L-stereo-isomer is used. Compositions of the
invention may also be formulated with analogues of these amino
acids. By "amino acid analogue" is intended a derivative of the
naturally occurring amino acid that brings about the desired effect
of decreasing aggregate formation by the protein during storage of
the liquid pharmaceutical compositions of the invention. Suitable
arginine analogues include, for example, aminoguanidine, ornithine
and N-monoethyl L-arginine, suitable methionine analogues include
ethionine and buthionine and suitable cysteine analogues include
S-methyl-L cysteine. As with the other amino acids, the amino acid
analogues are incorporated into the compositions in either their
free base form or their salt form. In a further embodiment of the
invention the amino acids or amino acid analogues are used in a
concentration, which is sufficient to prevent or delay aggregation
of the protein.
[0465] In a further embodiment of the invention methionine (or
other sulphuric amino acids or amino acid analogous) may be added
to inhibit oxidation of methionine residues to methionine sulfoxide
when the protein acting as the therapeutic agent is a protein
comprising at least one methionine residue susceptible to such
oxidation. By "inhibit" is intended minimal accumulation of
methionine oxidized species over time. Inhibiting methionine
oxidation results in greater retention of the protein in its proper
molecular form. Any stereoisomer of methionine (L or D isomer) or
any combinations thereof can be used. The amount to be added should
be an amount sufficient to inhibit oxidation of the methionine
residues such that the amount of methionine sulfoxide is acceptable
to regulatory agencies. Typically, this means that the composition
contains no more than about 10% to about 30% methionine sulfoxide.
Generally, this can be obtained by adding methionine such that the
ratio of methionine added to methionine residues ranges from about
1:1 to about 1000:1, such as 10:1 to about 100:1.
[0466] In a further embodiment of the invention the composition
further comprises a stabilizer selected from the group of high
molecular weight polymers or low molecular compounds. In a further
embodiment of the invention the stabilizer is selected from
polyethylene glycol (e.g. PEG 3350), polyvinyl alcohol (PVA),
polyvinylpyrrolidone, carboxy/hydroxycellulose or derivates thereof
(e.g. HPC, HPC-SL, HPC-L and HPMC), cyclodextrins,
sulphur-containing substances as monothioglycerol, thioglycolic
acid and 2-methylthioethanol, and different salts (e.g. sodium
chloride). Each one of these specific stabilizers constitutes an
alternative embodiment of the invention.
[0467] The pharmaceutical compositions may also comprise additional
stabilizing agents, which further enhance stability of a
therapeutically active protein therein. Stabilizing agents of
particular interest to the present invention include, but are not
limited to, methionine and EDTA, which protect the protein against
methionine oxidation, and a nonionic surfactant, which protects the
protein against aggregation associated with freeze-thawing or
mechanical shearing.
[0468] In a further embodiment of the invention the composition
further comprises a surfactant. In a further embodiment of the
invention the surfactant is selected from a detergent, ethoxylated
castor oil, polyglycolyzed glycerides, acetylated monoglycerides,
sorbitan fatty acid esters, polyoxypropylene-polyoxyethylene block
polymers (eg. poloxamers such as Pluronic.RTM. F68, poloxamer 188
and 407, Triton X-100), polyoxyethylene sorbitan fatty acid esters,
polyoxyethylene and polyethylene derivatives such as alkylated and
alkoxylated derivatives (tweens, e.g. Tween-20, Tween-40, Tween-80
and Brij-35), monoglycerides or ethoxylated derivatives thereof,
diglycerides or polyoxyethylene derivatives thereof, alcohols,
glycerol, lectins and phospholipids (eg. phosphatidyl serine,
phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl
inositol, diphosphatidyl glycerol and sphingomyelin), derivates of
phospholipids (eg. dipalmitoyl phosphatidic acid) and
lysophospholipids (eg. palmitoyl lysophosphatidyl-L-serine and
1-acyl-sn-glycero-3-phosphate esters of ethanolamine, choline,
serine or threonine) and alkyl, alkoxyl (alkyl ester), alkoxy
(alkyl ether)-derivatives of lysophosphatidyl and
phosphatidylcholines, e.g. lauroyl and myristoyl derivatives of
lysophosphatidylcholine, dipalmitoylphosphatidylcholine, and
modifications of the polar head group, that is cholines,
ethanolamines, phosphatidic acid, serines, threonines, glycerol,
inositol, and the positively charged DODAC, DOTMA, DCP, BISHOP,
lysophosphatidylserine and lysophosphatidylthreonine, and
glycerophospholipids (eg. cephalins), glyceroglycolipids (eg.
galactopyransoide), sphingoglycolipids (eg. ceramides,
gangliosides), dodecylphosphocholine, hen egg lysolecithin, fusidic
acid derivatives-(e.g. sodium tauro-dihydrofusidate etc.),
long-chain fatty acids and salts thereof C.sub.6-C.sub.12 (eg.
oleic acid and caprylic acid), acylcarnitines and derivatives,
N.sup..alpha.-acylated derivatives of lysine, arginine or
histidine, or side-chain acylated derivatives of lysine or
arginine, N.sup..alpha.-acylated derivatives of dipeptides
comprising any combination of lysine, arginine or histidine and a
neutral or acidic amino acid, N.sup..alpha.-acylated derivative of
a tripeptide comprising any combination of a neutral amino acid and
two charged amino acids, DSS (docusate sodium, CAS registry no
[577-11-7]), docusate calcium, CAS registry no [128-49-4]),
docusate potassium, CAS registry no [7491-09-0]), SDS (sodium
dodecyl sulphate or sodium lauryl sulphate), sodium caprylate,
cholic acid or derivatives thereof, bile acids and salts thereof
and glycine or taurine conjugates, ursodeoxycholic acid, sodium
cholate, sodium deoxycholate, sodium taurocholate, sodium
glycocholate,
N-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, anionic
(alkyl-aryl-sulphonates) monovalent surfactants, zwitterionic
surfactants (e.g. N-alkyl-N,N-dimethylammonio-1-propanesulfonates,
3-cholamido-1-propyldimethylammonio-1-propanesulfonate, cationic
surfactants (quaternary ammonium bases) (e.g.
cetyl-trimethylammonium bromide, cetylpyridinium chloride),
nonionic surfactants (eg. dodecyl 13-D-glucopyranoside),
poloxamines (eg. Tetronic's), which are tetrafunctional block
copolymers derived from sequential addition of propylene oxide and
ethylene oxide to ethylenediamine, or the surfactant may be
selected from the group of imidazoline derivatives, or mixtures
thereof. Each one of these specific surfactants constitutes an
alternative embodiment of the invention.
[0469] The use of a surfactant in pharmaceutical compositions is
well-known to the skilled person. For convenience reference is made
to Remington: The Science and Practice of Pharmacy, 20.sup.th
edition, 2000.
[0470] It is possible that other ingredients may be present in the
pharmaceutical composition of the present invention. Such
additional ingredients may include wetting agents, emulsifiers,
antioxidants, bulking agents, tonicity modifiers, chelating agents,
metal ions, oleaginous vehicles, proteins (e.g., human serum
albumin, gelatine or proteins) and a zwitterion (e.g., an amino
acid such as betaine, taurine, arginine, glycine, lysine and
histidine). Such additional ingredients, of course, should not
adversely affect the overall stability of the pharmaceutical
composition of the present invention.
[0471] Pharmaceutical compositions containing a growth hormone
conjugate according to the present invention may be administered to
a patient in need of such treatment at several sites, for example,
at topical sites, for example, skin and mucosal sites, at sites
which bypass absorption, for example, administration in an artery,
in a vein, in the heart, and at sites which involve absorption, for
example, administration in the skin, under the skin, in a muscle or
in the abdomen.
[0472] Administration of pharmaceutical compositions according to
the invention may be through several routes of administration, for
example, lingual, sublingual, buccal, in the mouth, oral, in the
stomach and intestine, nasal, pulmonary, for example, through the
bronchioles and alveoli or a combination thereof, epidermal,
dermal, transdermal, vaginal, rectal, ocular, for examples through
the conjunctiva, uretal, and parenteral to patients in need of such
a treatment.
[0473] Compositions of the current invention may be administered in
several dosage forms, for example, as solutions, suspensions,
emulsions, microemulsions, multiple emulsion, foams, salves,
pastes, plasters, ointments, tablets, coated tablets, rinses,
capsules, for example, hard gelatine capsules and soft gelatine
capsules, suppositories, rectal capsules, drops, gels, sprays,
powder, aerosols, inhalants, eye drops, ophthalmic ointments,
ophthalmic rinses, vaginal pessaries, vaginal rings, vaginal
ointments, injection solution, in situ transforming solutions, for
example in situ gelling, in situ setting, in situ precipitating, in
situ crystallization, infusion solution, and implants.
[0474] Compositions of the invention may further be compounded in,
or attached to, for example through covalent, hydrophobic and
electrostatic interactions, a drug carrier, drug delivery system
and advanced drug delivery system in order to further enhance
stability of the growth hormone conjugate, increase
bioavailability, increase solubility, decrease adverse effects,
achieve chronotherapy well known to those skilled in the art, and
increase patient compliance or any combination thereof. Examples of
carriers, drug delivery systems and advanced drug delivery systems
include, but are not limited to, polymers, for example cellulose
and derivatives, polysaccharides, for example dextran and
derivatives, starch and derivatives, poly(vinyl alcohol), acrylate
and methacrylate polymers, polylactic and polyglycolic acid and
block co-polymers thereof, polyethylene glycols, carrier proteins,
for example albumin, gels, for example, thermogelling systems, for
example block co-polymeric systems well known to those skilled in
the art, micelles, liposomes, microspheres, nanoparticulates,
liquid crystals and dispersions thereof, L2 phase and dispersions
there of, well known to those skilled in the art of phase behaviour
in lipid-water systems, polymeric micelles, multiple emulsions,
self-emulsifying, self-microemulsifying, cyclodextrins and
derivatives thereof, and dendrimers.
[0475] Compositions of the current invention are useful in the
composition of solids, semisolids, powder and solutions for
pulmonary administration of growth hormone conjugate, using, for
example a metered dose inhaler, dry powder inhaler and a nebulizer,
all being devices well known to those skilled in the art.
[0476] Compositions of the current invention are specifically
useful in the composition of controlled, sustained, protracting,
retarded, and slow release drug delivery systems. More
specifically, but not limited to, compositions are useful in
composition of parenteral controlled release and sustained release
systems (both systems leading to a many-fold reduction in number of
administrations), well known to those skilled in the art. Even more
preferably, are controlled release and sustained release systems
administered subcutaneous. Without limiting the scope of the
invention, examples of useful controlled release system and
compositions are hydrogels, oleaginous gels, liquid crystals,
polymeric micelles, microspheres, nanoparticles,
[0477] Methods to produce controlled release systems useful for
compositions of the current invention include, but are not limited
to, crystallization, condensation, co-crystallization,
precipitation, co-precipitation, emulsification, dispersion, high
pressure homogenisation, encapsulation, spray drying,
microencapsulating, coacervation, phase separation, solvent
evaporation to produce microspheres, extrusion and supercritical
fluid processes. General reference is made to Handbook of
Pharmaceutical Controlled Release (Wise, D. L., ed. Marcel Dekker,
New York, 2000) and Drug and the Pharmaceutical Sciences vol. 99:
Protein Composition and Delivery (MacNally, E. J., ed. Marcel
Dekker, New York, 2000).
[0478] Parenteral administration may be performed by subcutaneous,
intramuscular, intraperitoneal or intravenous injection by means of
a syringe, optionally a pen-like syringe. Alternatively, parenteral
administration can be performed by means of an infusion pump. A
further option is a composition which may be a solution or
suspension for the administration of the growth hormone conjugate
in the form of a nasal or pulmonal spray. As a still further
option, the pharmaceutical compositions containing the growth
hormone conjugate of the invention can also be adapted to
transdermal administration, e.g. by needle-free injection or from a
patch, optionally an iontophoretic patch, or transmucosal, e.g.
buccal, administration.
[0479] The term "stabilized composition" refers to a composition
with increased physical stability, increased chemical stability or
increased physical and chemical stability.
[0480] The term "physical stability" of the protein composition as
used herein refers to the tendency of the protein to form
biologically inactive and/or insoluble aggregates of the protein as
a result of exposure of the protein to thermo-mechanical stresses
and/or interaction with interfaces and surfaces that are
destabilizing, such as hydrophobic surfaces and interfaces.
Physical stability of the aqueous protein compositions is evaluated
by means of visual inspection and/or turbidity measurements after
exposing the composition filled in suitable containers (e.g.
cartridges or vials) to mechanical/physical stress (e.g. agitation)
at different temperatures for various time periods. Visual
inspection of the compositions is performed in a sharp focused
light with a dark background. The turbidity of the composition is
characterized by a visual score ranking the degree of turbidity for
instance on a scale from 0 to 3 (a composition showing no turbidity
corresponds to a visual score 0, and a composition showing visual
turbidity in daylight corresponds to visual score 3). A composition
is classified physical unstable with respect to protein
aggregation, when it shows visual turbidity in daylight.
Alternatively, the turbidity of the composition can be evaluated by
simple turbidity measurements well-known to the skilled person.
Physical stability of the aqueous protein compositions can also be
evaluated by using a spectroscopic agent or probe of the
conformational status of the protein. The probe is preferably a
small molecule that preferentially binds to a non-native conformer
of the protein. One example of a small molecular spectroscopic
probe of protein structure is Thioflavin T. Thioflavin T is a
fluorescent dye that has been widely used for the detection of
amyloid fibrils. In the presence of fibrils, and perhaps other
protein configurations as well, Thioflavin T gives rise to a new
excitation maximum at about 450 nm and enhanced emission at about
482 nm when bound to a fibril protein form. Unbound Thioflavin T is
essentially non-fluorescent at the wavelengths.
[0481] Other small molecules can be used as probes of the changes
in protein structure from native to non-native states. For instance
the "hydrophobic patch" probes that bind preferentially to exposed
hydrophobic patches of a protein. The hydrophobic patches are
generally buried within the tertiary structure of a protein in its
native state, but become exposed as a protein begins to unfold or
denature. Examples of these small molecular, spectroscopic probes
are aromatic, hydrophobic dyes, such as antrhacene, acridine,
phenanthroline or the like. Other spectroscopic probes are
metal-amino acid complexes, such as cobalt metal complexes of
hydrophobic amino acids, such as phenylalanine, leucine,
isoleucine, methionine, and valine, or the like.
[0482] The term "chemical stability" of the protein composition as
used herein refers to chemical covalent changes in the protein
structure leading to formation of chemical degradation products
with potential less biological potency and/or potential increased
immunogenic properties compared to the native protein structure.
Various chemical degradation products can be formed depending on
the type and nature of the native protein and the environment to
which the protein is exposed. Elimination of chemical degradation
can most probably not be completely avoided and increasing amounts
of chemical degradation products is often seen during storage and
use of the protein composition as well-known by the person skilled
in the art. Most proteins are prone to deamidation, a process in
which the side chain amide group in glutaminyl or asparaginyl
residues is hydrolysed to form a free carboxylic acid. Other
degradations pathways involves formation of high molecular weight
transformation products where two or more protein molecules are
covalently bound to each other through transamidation and/or
disulfide interactions leading to formation of covalently bound
dimer, oligomer and polymer degradation products (Stability of
Protein Pharmaceuticals, Ahern. T. J. & Manning M. C., Plenum
Press, New York 1992). Oxidation (of for instance methionine
residues) can be mentioned as another variant of chemical
degradation. The chemical stability of the protein composition can
be evaluated by measuring the amount of the chemical degradation
products at various time-points after exposure to different
environmental conditions (the formation of degradation products can
often be accelerated by for instance increasing temperature). The
amount of each individual degradation product is often determined
by separation of the degradation products depending on molecule
size and/or charge using various chromatography techniques (e.g.
SEC-HPLC and/or RP-HPLC).
[0483] Hence, as outlined above, a "stabilized composition" refers
to a composition with increased physical stability, increased
chemical stability or increased physical and chemical stability. In
general, a composition must be stable during use and storage (in
compliance with recommended use and storage conditions) until the
expiration date is reached.
[0484] In one embodiment of the invention the pharmaceutical
composition comprising the growth hormone conjugate of formula (I)
or (II) is stable for more than 6 weeks of usage and for more than
3 years of storage.
[0485] In another embodiment of the invention the pharmaceutical
composition comprising the growth hormone conjugate of formula (I)
or (II) is stable for more than 4 weeks of usage and for more than
3 years of storage.
[0486] In a further embodiment of the invention the pharmaceutical
composition comprising the growth hormone conjugate of formula (I)
or (II) is stable for more than 4 weeks of usage and for more than
two years of storage.
[0487] In an even further embodiment of the invention the
pharmaceutical composition comprising the growth hormone conjugate
of formula (I) or (II) is stable for more than 2 weeks of usage and
for more than two years of storage.
[0488] All references, including publications, patent applications
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference was individually and
specifically indicated to be incorporated by reference and was set
forth in its entirety herein.
[0489] All headings and sub-headings are used herein for
convenience only and should not be construed as limiting the
invention in any way,
[0490] Any combination of the above-described elements in all
possible variations thereof is encompassed by the invention unless
otherwise indicated herein or otherwise clearly contradicted by
context.
[0491] The terms "a" and "an" and "the" and similar referents as
used in the context of describing the invention are to be construed
to cover both the singular and the plural (i.e. one or more),
unless otherwise indicated herein or clearly contradicted by
context.
[0492] Recitation of ranges of values herein are merely intended to
serve as a shorthand method of referring individually to each
separate value falling within the range, unless otherwise indicated
herein, and each separate value is incorporated into the
specification as if it were individually recited herein. Unless
otherwise stated, all exact values provided herein are
representative of corresponding approximate values (e.g., all exact
exemplary values provided with respect to a particular factor or
measurement can be considered to also pro-vide a corresponding
approximate measurement, modified by "about," where
appropriate).
[0493] All methods described herein can be performed in any
suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context.
[0494] The use of any and all examples, or exemplary language
(e.g., "such as") provided herein, is intended merely to better
illuminate the invention and does not pose a limitation on the
scope of the invention unless otherwise indicated. No language in
the specification should be construed as indicating any element is
essential to the practice of the invention unless as much is
explicitly stated.
[0495] 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.
[0496] A non-eshausive list of embodiment describing the invention
is provided here below.
List of Embodiments
Embodiment 1
[0497] A growth hormone conjugate which comprises a growth hormone
compound (GH) having
a) a single Cys mutation, b) an additional disulfide bridge, or c)
a single Cys mutation and an additional disulfide bridge, wherein
an albumin binding residue via a hydrophilic spacer is linked to
said GH, or a pharmaceutically acceptable salt thereof. 2. The
conjugate of embodiment 1, wherein GH represents a growth hormone
compound comprising an amino acid sequence having at least 80%
identity to the amino acid sequence of human growth hormone (hGH)
(SEQ ID NO: 1), such as at least 80%, at least 85%, at least 90%,
or at least 95% identity with hGH, or GH is hGH (SEQ ID NO: 1). 3.
The conjugate of embodiment 1, wherein GH or the GH conjugate has
at least 80% of the growth hormone activity of hGH. 4. The
conjugate of any one of embodiments 1-3 wherein the albumin binding
residue via a hydrophilic spacer is linked to a GH having a single
Cys mutation. 5. The conjugate of embodiment 4, wherein the single
Cys mutation is positioned in any one of the regions selected from
the N-terminal, H1, H2, L2 or H3 of GH. 6. The conjugate of
embodiment 5, wherein the GH has a single Cys mutation selected
from anyone of T3C, P5C, S7C, D11C, H18C, Q29C, E30C, E33C, A34C,
Y35C, E88C, Q91C, S95C, A98C, N99C, S100C, L101C, V102C, Y103C,
D107C, S108C, D112C, Q122C and G126C. 7. The conjugate of any one
of embodiments 1-3 wherein the albumin binding residue via a
hydrophilic spacer is linked to a GH having an additional disulfide
bridge. 8. The conjugate of embodiment 7, wherein the additional
disulfide bond is between a loop segment and a helical segment or
within loop segment or between loop segments or between helical
segments. 9. The conjugate of any one of embodiments 7-8, wherein
the GH comprises an additional disulfide bond wherein at least one
of the cysteines is present in a loop segment, such from amino acid
residues 128-154 (L3). 10. The conjugate of any one of embodiments
7-9, wherein the GH comprises an additional disulfide bond wherein
the additional disulfide bond which connects a loop segment with a
helical segment, such as helix B or H2. 11. The conjugate of any
one of embodiments 7-10, wherein the additional disulfide bond
connects L3 with H2. 12. The conjugate of any one of embodiments
7-11, wherein the additional disulfide bridge is between at least
one of the amino acid pairs in the positions corresponding to
R16C/L117C, A17C/E174C, H21C/M170C, D26C/V102C, D26C/Y103C,
N47C/T50C, Q49C/G161C, F54C/Y143C, F54C/S144C, F54C/F146C,
S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C,
P59C/Q137C, P61C/E66C, P61C/T67C, S71C/S132C, L73C/S132C,
L73C/F139C, R77C/I138C, R77C/F139C, L81C/Q141C, L81C/Y143C,
Q84C/Y143C, Q84C/S144C, S85C/Y143C, S85C/S144C, P89C/F146C,
F92C/F146C, F92C/T148C, R94C/D107C, V102C/A105C, L156C/F146C,
L156C/T148C and/or V185C/S188C in hGH (SEQ ID NO: 1), such as
Q84C/Y143C. 13. The conjugate of any one of embodiments 1-3 wherein
the albumin binding residue via a hydrophilic spacer is linked to a
GH having a single Cys mutation and an additional disulfide bridge.
14. The conjugate of embodiment 13 wherein the GH has a single Cys
mutation selected from any one of T3C, P5C, S7C, D11C, H18C, Q29C,
E30C, E33C, A34C, Y35C, Y42C, S55C, S57C, S62C, Q69C, E88C, Q91C,
S95C, A98C, N99C, S100C, L101C, V102C, Y103C, D107C, S108C, D112C,
Q122C and G126C. 15. The conjugate of any one of embodiments 13-14,
wherein the additional disulfide bond is between a loop segment and
a helical segment or within loop segment or between loop segments
or between helical segments. 16. The conjugate of any one of
embodiments 13-15, wherein the GH comprises an additional disulfide
bond wherein at least one of the cysteines is present in a loop
segment, such from amino acid residues 128-154 (L3). 17. The
conjugate of any one of embodiments 13-16, wherein the GH comprises
an additional disulfide bond wherein the additional disulfide bond
which connects a loop segment, with a helical segment, such as
helix B or H2. 18. The conjugate of any one of embodiments 13-17,
wherein the additional disulfide bond connects a loop segment, such
from amino acid residues 128-154 (L3), with helix B or H2. 19. The
conjugate of any one of embodiments 13-18, wherein the additional
disulfide bridge is between at least one of the amino acid pairs in
the positions corresponding to R16C/L117C, A17C/E174C, H21C/M170C,
D26C/V102C, D26C/Y103C, N47C/T50C, Q49C/G161C, F54C/Y143C,
F54C/S144C, F54C/F146C, S55C/Y143C, S57C/Y143C, I58C/Q141C,
I58C/Y143C, I58C/S144C, P59C/Q137C, P61C/E66C, P61C/T67C,
S71C/S132C, L73C/S132C, L73C/F139C, R77C/I138C, R77C/F139C,
L81C/Q141C, L81 C/Y143C, Q84C/Y143C, Q84C/S144C, S85C/Y143C,
S85C/S144C, P89C/F146C, F92C/F146C, F92C/T148C, R94C/D107C,
V102C/A105C, L156C/F146C, L156C/T148C and/or V185C/S188C in hGH
(SEQ ID NO: 1), such as Q84C/Y143C. 20. The conjugate of any one of
embodiments 1-19, wherein the albumin binding residue is selected
from
##STR00055##
[0498] wherein * denotes the attachment to the hydrophilic spacer
through a chemical group linking the albumin binding residue and
the hydrophilic spacer.
21. The conjugate of any one of embodiments 1-20, wherein the
chemical group linking the albumin binding residue and the
hydrophilic spacer has the formula
--W.sub.7--Y--,
[0499] wherein [0500] Y is
--(CH.sub.2).sub.l7--C.sub.3-10-cycloalkyl-W.sub.8-- or a valence
bond, [0501] l7 is 0-6, [0502] W.sub.7 is selected from --C(O)NH--,
--NHC(O)--, --C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--, --OC(O)NH--,
--NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--, --C(O)CH.dbd.CH--,
--CH.dbd.CHC(O)--, --(CH.sub.2).sub.s3--, --C(O)--, --C(O)O--,
--OC(O)--, or a valence bond; wherein s3 is 0 or 1, [0503] W.sub.8
is selected from --C(O)NH--, --NHC(O)--, --C(O)NHCH.sub.2--,
--CH.sub.2NHC(O)--, --C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--,
--OC(O)NH--, --NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--,
--C(O)CH.dbd.CH--, --CH.dbd.CHC(O)--, --(CH.sub.2).sub.s4--,
--C(O)--, --C(O)O--, --OC(O)--, or a valence bond; wherein s4 is 0
or 1. 22. The conjugate of any one of embodiments 1-21 wherein the
hydrophilic spacer has the formula
[0503] --X.sub.1--X.sub.2--X.sub.3--X.sub.4--
wherein [0504] X.sub.1 is
--W.sub.1--[(CHR.sup.1).sub.l1--W.sub.2].sub.m1--{[(CH.sub.2).sub.n1E1].s-
ub.m2--[(CHR.sup.2).sub.l2--W.sub.3].sub.m3}.sub.n2--, [0505]
X.sub.2 is
--[(CHR.sup.3).sub.l3--W.sub.4].sub.m4--{[(CH.sub.2).sub.n3E2].sub.m5--[(-
CHR.sup.4).sub.l4--W.sub.5].sub.m6}.sub.n4--, [0506] X.sub.3 is
--[(CHR.sup.5).sub.l5--W.sub.6].sub.m7--, [0507] X.sub.4 is
F-D1-(CH.sub.2).sub.l6-D2-, [0508] l1, l2, l3, l4, l5 and l6
independently are selected from 0-16, [0509] m1, m3, m4, m6 and m7
independently are selected from 0-10, [0510] m2 and m5
independently are selected from 0-25, [0511] n1, n2, n3 and n4
independently are selected from 0-16, [0512] F is aryl, hetaryl,
pyrrolidine-2,5-dione or a valence bond, wherein the aryl and
hetaryl groups are optionally substituted with halogen, --CN, --OH,
--C(O)OH, --C(O)NH.sub.2, --S(O).sub.2OH or C.sub.1-6-alkyl, [0513]
R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 independently are
selected from hydrogen, --C(O)OH, --C(O)NH.sub.2, --S(O)OH,
--S(O).sub.2OH, --NH--C(.dbd.NH)--NH.sub.2, C.sub.1-6-alkyl, aryl
or hetaryl; wherein the alkyl, aryl and hetaryl groups optionally
are substituted with halogen, --C(O)OH, --C(O)NH.sub.2, --S(O)OH,
--S(O).sub.2OH, --CN or --OH, [0514] D1, D2, E1 and E2
independently are selected from --O--, --N(R.sup.6)--,
--N(C(O)R.sup.7)-- or a valence bond; wherein R.sup.6 and R.sup.7
independently represent hydrogen or C.sub.1-6-alkyl, [0515] W.sub.1
to W.sub.5 independently are selected from --C(O)NH--, --NHC(O)--,
--C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--, --C(O)NHS(O).sub.2--,
--S(O).sub.2NHC(O)--, --OC(O)NH--, --NHC(O)O--, --C(O)CH.sub.2--,
--CH.sub.2C(O)--, --C(O)CH.dbd.CH--, --CH.dbd.CHC(O)--,
--(CH.sub.2).sub.s2--, --C(O)--, --C(O)O--, --OC(O)--, or a valence
bond; wherein s2 is 0 or 1, [0516] W.sub.6 is selected from
--C(O)NH--, --NHC(O)--, --C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--, --OC(O)NH--,
--NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--, --C(O)CH.dbd.CH--,
--CH.dbd.CHC(O)--, --(CH.sub.2).sub.s1--, --C(O)--, --C(O)O--,
--OC(O)--, --NHC(O)C.sub.1-6-alkyl, --C(O)NHC.sub.1-6-alkyl or a
valence bond; wherein s1 is 0 or 1 and the C.sub.1-6-alkyl group is
optionally substituted with oxo, pyrrolidine-2,5-dione,
--NHC(O)CH*CH.sub.2COOH or --NHC(O)CH.sub.2CH*COOH; wherein (*)
indicates the attachment point from the carbon atom of CH to
X.sub.4. 23. The conjugate of any one of embodiments 1-22, wherein
X.sub.4 is a valence bond and W.sub.6 is selected from either
pyrrolidine-2,5-dione, --NHC(O)CH*CH.sub.2COOH or
--NHC(O)CH.sub.2CH*COOH wherein (*) indicates the attachment point
from the carbon atom of CH to GH. 24. The conjugate of embodiment
22 wherein the hydrophilic spacer is selected from
##STR00056## ##STR00057##
[0516] 25. A growth hormone conjugate wherein the growth hormone
conjugate has the formula (I):
A-W--B-GH (I)
Wherein
[0517] GH represents a growth hormone compound having a single Cys
mutation, B represents a hydrophilic spacer linked to the sulphur
residue of the Cys mutation, W is a chemical group linking A and B,
and A represent an albumin binding residue; and pharmaceutically
acceptable salts thereof. 26. The conjugate of embodiment 25,
wherein GH represents a growth hormone compound comprising an amino
acid sequence having at least 80% identity to the amino acid
sequence of human growth hormone (hGH) (SEQ ID NO: 1), such as at
least 80%, at least 85%, at least 90%, or at least 95% identity
with hGH, or GH is hGH (SEQ ID NO: 1). 27. The conjugate of
embodiment 25, wherein GH or the GH conjugate has at least 80% of
the growth hormone activity of hGH. 28. The conjugate of any one of
embodiments 25-27, wherein the single Cys mutation is positioned in
any one of the regions selected from the N-terminal, H1, H2, L2 or
H3 of GH. 29. The conjugate of any one of embodiments 25-28 wherein
the GH has a single Cys mutation selected from any one of: T3C,
P5C, S7C, D11C, H18C, Q29C, E30C, E33C, A34C, Y35C, E88C, Q91C,
S95C, A98C, N99C, S100C, L101C, V102C, Y103C, D107C, S108C, D112C,
Q122C and G126C. 30. The conjugate of any one of embodiments 25-29,
wherein A is selected from
##STR00058## ##STR00059##
[0518] wherein * denotes the attachment to B through W.
31. The conjugate of any one of embodiments 25-30, wherein W has
the formula
--W.sub.7--Y--,
wherein [0519] Y is
--(CH.sub.2).sub.l7--C.sub.3-10-cycloalkyl-W.sub.8-- or a valence
bond, [0520] l7 is 0-6, [0521] W.sub.7 is selected from --C(O)NH--,
--NHC(O)--, --C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--, --OC(O)NH--,
--NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--, --C(O)CH.dbd.CH--,
--CH.dbd.CHC(O)--, --(CH.sub.2).sub.s3--, --C(O)--, --C(O)O--,
--OC(O)--, or a valence bond; wherein s3 is 0 or 1, [0522] W.sub.8
is selected from --C(O)NH--, --NHC(O)--, --C(O)NHCH.sub.2--,
--CH.sub.2NHC(O)--, --C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--,
--OC(O)NH--, --NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--,
--C(O)CH.dbd.CH--, --CH.dbd.CHC(O)--, --(CH.sub.2).sub.s4--,
--C(O)--, --C(O)O--, --OC(O)--, or a valence bond; wherein s4 is 0
or 1. 32. The conjugate of any one of embodiments 25-31 wherein B
has the formula
[0522] --X.sub.1--X.sub.2--X.sub.3--X.sub.4--
wherein [0523] X.sub.1 is
--W.sub.1--[(CHR.sup.1).sub.l1--W.sub.2].sub.m1--{[(CH.sub.2).sub.n1E1].s-
ub.m2--[(CHR.sup.2).sub.l2--W.sub.3].sub.m3}.sub.n2--, [0524]
X.sub.2 is
--[(CHR.sup.3).sub.l3--W.sub.4].sub.m4--{[(CH.sub.2).sub.n3E2].sub.m5--[(-
CHR.sup.4).sub.l4--W.sub.5].sub.m6}.sub.n4--, [0525] X.sub.3 is
--[(CHR.sup.5).sub.l5--W.sub.6].sub.m7--, [0526] X.sub.4 is
F-D1-(CH.sub.2).sub.l6-D2-, [0527] l1, l2, l3, l4, l5 and l6
independently are selected from 0-16, [0528] m1, m3, m4, m6 and m7
independently are selected from 0-10, [0529] m2 and m5
independently are selected from 0-25, [0530] n1, n2, n3 and n4
independently are selected from 0-16, [0531] F is aryl, hetaryl,
pyrrolidine-2,5-dione or a valence bond, wherein the aryl and
hetaryl groups are optionally substituted with halogen, --CN, --OH,
--C(O)OH, --C(O)NH.sub.2, --S(O).sub.2OH or C.sub.1-6-alkyl, [0532]
R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 independently are
selected from hydrogen, --C(O)OH, --C(O)NH.sub.2, --S(O)OH,
--S(O).sub.2OH, --NH--C(.dbd.NH)--NH.sub.2, C.sub.1-6-alkyl, aryl
or hetaryl; wherein the alkyl, aryl and hetaryl groups optionally
are substituted with halogen, --C(O)OH, --C(O)NH.sub.2, --S(O)OH,
--S(O).sub.2OH, --CN or --OH, [0533] D1, D2, E1 and E2
independently are selected from --O--, --N(R.sup.6)--,
--N(C(O)R.sup.7)-- or a valence bond; wherein R.sup.6 and R.sup.7
independently represent hydrogen or C.sub.1-6-alkyl, [0534] W.sub.1
to W.sub.5 independently are selected from --C(O)NH--, --NHC(O)--,
--C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--, --C(O)NHS(O).sub.2--,
--S(O).sub.2NHC(O)--, --OC(O)NH--, --NHC(O)O--, --C(O)CH.sub.2--,
--CH.sub.2C(O)--, --C(O)CH.dbd.CH--, --CH.dbd.CHC(O)--,
--(CH.sub.2).sub.s2--, --C(O)--, --C(O)O--, --OC(O)--, or a valence
bond; wherein s2 is 0 or 1, [0535] W.sub.6 is selected from
--C(O)NH--, --NHC(O)--, --C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--, --OC(O)NH--,
--NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--, --C(O)CH.dbd.CH--,
--CH.dbd.CHC(O)--, --(CH.sub.2).sub.s1--, --C(O)--, --C(O)O--,
--OC(O)--, --NHC(O)C.sub.1-6-alkyl, --C(O)NHC.sub.1-6-alkyl or a
valence bond; wherein s1 is 0 or 1 and the C.sub.1-6-alkyl group is
optionally substituted with oxo, pyrrolidine-2,5-dione,
--NHC(O)CH*CH.sub.2COOH or --NHC(O)CH.sub.2CH*COOH; wherein (*)
indicates the attachment point from the carbon atom of CH to
X.sub.4. 33. The conjugate of any one of embodiments 25-32, wherein
l1, l2, l3, l4, l5 and l6 independently are 0-6, m1, m3, m4, m6 and
m7 independently are 0-6, m2 and m5 independently are 0-10, and n1,
n2, n3 and n4 independently are 0-10. 34. The conjugate of any one
of embodiments 25-33, wherein D1 and D2 are independently selected
from --O-- or --N(R.sup.6)-- or a valence bond. 35. The conjugate
of any one of embodiments 25-34, wherein E1 and E2 are
independently selected from --O-- or --N(R.sup.6)-- or a valence
bond. 36. The conjugate of any one of embodiments 25-35, wherein
W.sub.1 through W.sub.8 independently are selected from the group
consisting of --C(O)NH--, --NHC(O)--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--,
--NHC(O)C.sub.1-6-alkyl, --C(O)NHC.sub.1-6-alkyl or a valence bond;
wherein the alkyl group is optionally substituted with oxo,
pyrrolidine-2,5-dione, --NHC(O)CH*CH.sub.2COOH or
--NHC(O)CH.sub.2CH*COOH; wherein (*) indicates the attachment point
from the carbon atom of CH to X.sub.4. 37. The conjugate of any one
of embodiments 25-36, wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4
and R.sup.5 independently are selected from hydrogen, --C(O)OH,
--C(O)NH.sub.2, --S(O).sub.2OH or C.sub.1-6-alkyl; wherein the
alkyl group optionally is substituted with --C(O)OH, --C(O)NH.sub.2
or --S(O).sub.2OH. 38. The conjugate of any one of embodiments
25-37, wherein
--{[(CH.sub.2).sub.n1E1].sub.m2--[(CHR.sup.2).sub.l2--W.sub.3].sub.m3}.su-
b.n2-- and
--{[(CH.sub.2).sub.n3E2].sub.m5--[(CHR.sup.4).sub.l4--W].sub.m6-
}.sub.n4--, wherein E1 and E2 are --O--, are selected from
##STR00060##
[0536] wherein * is intended to denote a point of attachment, ie,
an open bond.
39. The conjugate of any one of embodiments 25-38, wherein X.sub.4
is a valence bond and W.sub.6 is selected from either
pyrrolidine-2,5-dione, --NHC(O)CH*CH.sub.2COOH or
--NHC(O)CH.sub.2CH*COOH wherein (*) indicates the attachment point
from the carbon atom of CH to GH. 40. The conjugate of any one of
embodiments 25-39 wherein B is selected from
##STR00061## ##STR00062##
41. The conjugate of any one of embodiments 25-40, wherein said
compound is selected from
##STR00063## ##STR00064## ##STR00065## ##STR00066##
##STR00067##
42. A growth hormone conjugate wherein the growth hormone conjugate
has the formula (I).
A-W--B-GH (I)
Wherein
[0537] GH represents a growth hormone compound having an additional
disulfide bridge, B represents a hydrophilic spacer, W is a
chemical group linking A and B, and A represent an albumin binding
residue; and pharmaceutically acceptable salts thereof. 43. The
conjugate of embodiment 42, wherein GH represents a growth hormone
compound comprising an amino acid sequence having at least 80%
identity to the amino acid sequence of human growth hormone (hGH)
(SEQ ID NO: 1), such as at least 80%, at least 85%, at least 90%,
or at least 95% identity with hGH, or GH is hGH (SEQ ID NO: 1). 44.
The conjugate of embodiment 42, wherein GH or the GH conjugates has
at lease 80% of the growth hormone activity of hGH. 45. The
conjugate of embodiment 44, wherein the activity is measured in an
in viro BAF assay (assay I) 46. The conjugate of any one of
embodiments 42-45, wherein the GH comprises additional disulfide
bonds between a loop segment and a helical segment or within loop
segment or between loop segments or between helical segments. 47.
The conjugate of any one of embodiments 42-46, wherein the GH
comprises an additional disulfide bond wherein at least one of the
cysteines is present in a loop segment, such from amino acid
residues 128-154 (L3). 48. The conjugate of any one of embodiments
42-47, wherein the GH comprises an additional disulfide bond
wherein the additional disulfide bond which connects a loop
segment, with a helical segment, such as helix B or helix 2. 49.
The conjugate of any one of embodiments 42-48, wherein the GH
comprises an additional disulfide bond wherein the additional
disulfide bond connects L3 (128-154), with helix B or helix 2. 50.
The conjugate of any one of embodiments 42-49, wherein the
additional disulfide bridge is between at least one of the amino
acid pairs in the positions corresponding to R16C/L117C,
A17C/E174C, H21C/M170C, D26C/V102C, D26C/Y103C, N47C/T50C,
Q49C/G161C, F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C,
S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C,
P61C/E66C, P61C/T67C, S71C/S132C, L73C/S132C, L73C/F139C,
R77C/I138C, R77C/F139C, L81C/Q141C, L81C/Y143C, Q84C/Y143C,
Q84C/S144C, S85C/Y143C, S85C/S144C, P89C/F146C, F92C/F146C,
F92C/T148C, R94C/D107C, V102C/A105C, L156C/F146C, L156C/T148C
and/or V185C/S188C in hGH (SEQ ID NO: 1), such as Q84C/Y143C. 51.
The conjugate of any one of embodiments 42-50, wherein A is
selected from
##STR00068##
wherein * denotes the attachment to B through W. 52. The conjugate
of any one of embodiments 42-51, wherein W has the formula
--W.sub.7--Y--,
wherein [0538] Y is
--(CH.sub.2).sub.l7--C.sub.3-10-cycloalkyl-W.sub.8-- or a valence
bond, [0539] l7 is 0-6, [0540] W.sub.7 is selected from --C(O)NH--,
--NHC(O)--, --C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--, --OC(O)NH--,
--NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--, --C(O)CH.dbd.CH--,
--CH.dbd.CHC(O)--, --(CH.sub.2).sub.s3--, --C(O)--, --C(O)O--,
--OC(O)--, or a valence bond; wherein s3 is 0 or 1, [0541] W.sub.8
is selected from --C(O)NH--, --NHC(O)--, --C(O)NHCH.sub.2--,
--CH.sub.2NHC(O)--, --C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--,
--OC(O)NH--, --NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--,
--C(O)CH.dbd.CH--, --CH.dbd.CHC(O)--, --(CH.sub.2).sub.s4--,
--C(O)--, --C(O)O--, --OC(O)--, or a valence bond; wherein s4 is 0
or 1. 53. The conjugate of any one of embodiments 42-52 wherein B
has the formula
[0541] --X.sub.1--X.sub.2--X.sub.3--X.sub.4--
wherein [0542] X.sub.1 is
--W.sub.1--[(CHR.sup.1).sub.l1--W.sub.2].sub.m1--{[(CH.sub.2).sub.n1E1].s-
ub.m2--[(CHR.sup.2).sub.l2--W.sub.3].sub.m3}.sub.n2--, [0543]
X.sub.2 is
--[(CHR.sup.3).sub.l3--W.sub.4].sub.m4--{[(CH.sub.2).sub.n3E2].sub.m5--[(-
CHR.sup.4).sub.l4--W.sub.5].sub.m6}.sub.n4--, [0544] X.sub.3 is
--[(CHR.sup.5).sub.l5--W.sub.6].sub.m7--, [0545] X.sub.4 is
F-D1-(CH.sub.2).sub.l6-D2-, [0546] l1, l2, l3, l4, l5 and l6
independently are selected from 0-16, [0547] m1, m3, m4, m6 and m7
independently are selected from 0-10, [0548] m2 and m5
independently are selected from 0-25, [0549] n1, n2, n3 and n4
independently are selected from 0-16, [0550] F is aryl, hetaryl,
pyrrolidine-2,5-dione or a valence bond, wherein the aryl and
hetaryl groups are optionally substituted with halogen, --CN, --OH,
--C(O)OH, --C(O)NH.sub.2, --S(O).sub.2OH or C.sub.1-6-alkyl, [0551]
R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 independently are
selected from hydrogen, --C(O)OH, --C(O)NH.sub.2, --S(O)OH,
--S(O).sub.2OH, --NH--C(.dbd.NH)--NH.sub.2, C.sub.1-6-alkyl, aryl
or hetaryl; wherein the alkyl, aryl and hetaryl groups optionally
are substituted with halogen, --C(O)OH, --C(O)NH.sub.2, --S(O)OH,
--S(O).sub.2OH, --CN or --OH, [0552] D1, D2, E1 and E2
independently are selected from --O--, --N(R.sup.6)--,
--N(C(O)R.sup.7)-- or a valence bond; wherein R.sup.6 and R.sup.7
independently represent hydrogen or C.sub.1-6-alkyl, [0553] W.sub.1
to W.sub.5 independently are selected from --C(O)NH--, --NHC(O)--,
--C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--, --C(O)NHS(O).sub.2--,
--S(O).sub.2NHC(O)--, --OC(O)NH--, --NHC(O)O--, --C(O)CH.sub.2--,
--CH.sub.2C(O)--, --C(O)CH.dbd.CH--, --CH.dbd.CHC(O)--,
--(CH.sub.2).sub.s2--, --C(O)--, --C(O)O--, --OC(O)--, or a valence
bond; wherein s2 is 0 or 1, [0554] W.sub.6 is selected from
--C(O)NH--, --NHC(O)--, --C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--, --OC(O)NH--,
--NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--, --C(O)CH.dbd.CH--,
--CH.dbd.CHC(O)--, --(CH.sub.2).sub.s1--, --C(O)--, --C(O)O--,
--OC(O)--, --NHC(O)C.sub.1-6-alkyl, --C(O)NHC.sub.1-6-alkyl or a
valence bond; wherein s1 is 0 or 1 and the C.sub.1-6-alkyl group is
optionally substituted with oxo, pyrrolidine-2,5-dione,
--NHC(O)CH*CH.sub.2COOH or --NHC(O)CH.sub.2CH*COOH; wherein (*)
indicates the attachment point from the carbon atom of CH to
X.sub.4. 54. The conjugate of any one of embodiments 42-53, wherein
l1, l2, l3, l4, l5 and l6 independently are 0-6, m1, m3, m4, m6 and
m7 independently are 0-6, m2 and m5 independently are 0-10, and n1,
n2, n3 and n4 independently are 0-10. 55. The conjugate of any one
of embodiments 42-54, wherein D1 and D2 are independently selected
from --O-- or --N(R.sup.6)-- or a valence bond. 56. The conjugate
of any one of embodiments 42-55, wherein E1 and E2 are
independently selected from --O-- or --N(R.sup.6)-- or a valence
bond. 57. The conjugate of any one of embodiments 42-56, wherein
W.sub.1 through W.sub.8 independently are selected from the group
consisting of --C(O)NH--, --NHC(O)--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--, --NHC(O)C.sub.1-6-alkyl
or --C(O)NHC.sub.1-6-alkyl or a valence bond; wherein the alkyl
group is optionally substituted with oxo, pyrrolidine-2,5-dione,
--NHC(O)CH*CH.sub.2COOH or --NHC(O)CH.sub.2CH*COOH; wherein (*)
indicates the attachment point from the carbon atom of CH to
X.sub.4. 58. The conjugate of any one of embodiments 42-57, wherein
R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 independently are
selected from hydrogen, --C(O)OH, --C(O)NH.sub.2, --S(O).sub.2OH or
C.sub.1-6-alkyl; wherein the alkyl group optionally is substituted
with --C(O)OH, --C(O)NH.sub.2 or --S(O).sub.2OH. 59. The conjugate
of any one of embodiments 42-58, wherein
--{[(CH.sub.2).sub.n1E1].sub.m2--[(CHR.sup.2).sub.l2--W.sub.3].sub.m3}.su-
b.n2-- and
--{[(CH.sub.2).sub.n3E2].sub.m5--[(CHR.sup.4).sub.l4--W].sub.m6-
}.sub.n4--, wherein E1 and E2 are --O--, are selected from
##STR00069##
[0555] wherein * is intended to denote a point of attachment, ie,
an open bond.
60. The conjugate of any one of embodiments 42-59 wherein B is
selected from
##STR00070## ##STR00071##
61. The conjugate of any one of embodiments 42-60, wherein A via B
is attached to the glutamine residue in the position corresponding
to position 40, position 141 in hGH SEQ ID NO: 1, or the N-terminal
residue of the growth hormone compound. 62. The conjugate of any
one of embodiments 42-61, wherein said compound is selected
from
##STR00072##
63. A growth hormone conjugate wherein the growth hormone conjugate
has the formula (I):
A-W--B-GH (I)
wherein GH represents a growth hormone compound having a single Cys
mutation and an additional disulfide bridge, B represents a
hydrophilic spacer linked to the sulphur residue of the Cys
mutation, W is a chemical group linking A and B, and A represent an
albumin binding residue; and pharmaceutically acceptable salts
thereof. 64. The conjugate of embodiment 63, wherein GH represents
a growth hormone compound comprising an amino acid sequence having
at least 80% identity to the amino acid sequence of human growth
hormone (hGH) (SEQ ID NO: 1), such as at least 80%, at least 85%,
at least 90%, or at least 95% identity with hGH, or GH is hGH (SEQ
ID NO: 1). 65. The conjugate of embodiment 64, wherein GH or the GH
conjugate has at least 80% of the growth hormone activity of hGH.
66. The conjugate of any of the embodiments 63-65, wherein the
single Cys mutation is positioned in any one of the regions
selected from the N-terminal, H1, H2, L2 or H3 of GH. 67. The
conjugate of any one of embodiments 63-66 wherein the GH has a
single Cys mutation selected from any one of; T3C, P5C, S7C, D11C,
H18C, Q29C, E30C, E33C, A34C, Y35C, K38C, E39C, Y42C, S43C, D47C,
P48C, S55C, S57C, P59C, S62, E65C, Q69C, E88C, Q91C, S95C, A98C,
N99C, S100C, L101C, V102C, Y103C, D107C, S108C, D112C, Q122C,
G126C, E129C, D130C, G131C, P133C, T135C, G136C, T142C, D147C,
N149C, D154C, A155C, L156C, R178C, E186C, G187C and G190C, such as
any one of; T3C, P5C, S7C, D11C, H18C, Q29C, E30C, E33C, A34C,
Y35C, E88C, Q91C, S95C, A98C, N99C, S100C, L101C, V102C, Y103C,
D107C, S108C, D112C, Q122C and G126C. 68. The conjugate of any one
of embodiments 63-67, wherein the additional disulfide bond is
between a loop segment and a helical segment or within loop segment
or between loop segments or between helical segments. 69. The
conjugate of any one of embodiments 63-68, wherein the GH comprises
an additional disulfide bond wherein at least one of the cysteines
is present in a loop segment, such from amino acid residues 128-154
(L3). 70. The conjugate of any one of embodiments 63-69, wherein
the additional disulfide bond connects a loop segment, with a
helical segment, such as H2. 71. The conjugate of any one of
embodiments 63-70, wherein the additional disulfide bond connects
L3, with helix H2. 72. The conjugate of any one of embodiments
63-71, wherein the additional disulfide bridge is between at least
one of the amino acid pairs in the positions corresponding to
R16C/L117C, A17C/E174C, H21C/M170C, D26C/V102C, D26C/Y103C,
N47C/T50C, Q49C/G161C, F54C/Y143C, F54C/S144C, F54C/F146C,
S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C,
P59C/Q137C, P61C/E66C, P61C/T67C, S71C/S132C, L73C/S132C,
L73C/F139C, R77C/I138C, R77C/F139C, L81C/Q141C, L81C/Y143C,
Q84C/Y143C, Q84C/S144C, S85C/Y143C, S85C/S144C, P89C/F146C,
F92C/F146C, F92C/T148C, R94C/D107C, V102C/A105C, L156C/F146C,
L156C/T148C and/or V185C/S188C in hGH (SEQ ID NO: 1), such as
Q84C/Y143C. 73. The conjugate of any one of embodiments 63-72,
wherein A is selected from
##STR00073## ##STR00074##
[0556] wherein * denotes the attachment to B through W.
74. The conjugate of any one of embodiments 63-73, wherein W has
the formula
--W.sub.7--Y--,
wherein [0557] Y is
--(CH.sub.2).sub.l7--C.sub.3-10-cycloalkyl-W.sub.8-- or a valence
bond, [0558] l7 is 0-6, [0559] W.sub.7 is selected from --C(O)NH--,
--NHC(O)--, --C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--, --OC(O)NH--,
--NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--, --C(O)CH.dbd.CH--,
--CH.dbd.CHC(O)--, --(CH.sub.2).sub.s3--, --C(O)--, --C(O)O--,
--OC(O)--, or a valence bond; wherein s3 is 0 or 1, [0560] W.sub.8
is selected from --C(O)NH--, --NHC(O)--, --C(O)NHCH.sub.2--,
--CH.sub.2NHC(O)--, --C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--,
--OC(O)NH--, --NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--,
--C(O)CH.dbd.CH--, --CH.dbd.CHC(O)--, --(CH.sub.2).sub.s4--,
--C(O)--, --C(O)O--, --OC(O)--, or a valence bond; wherein s4 is 0
or 1. 75. The conjugate of any one of embodiments 63-74 wherein B
has the formula
[0560] --X.sub.1--X.sub.2--X.sub.3--X.sub.4--
wherein [0561] X.sub.1 is
--W.sub.1--[(CHR.sup.1).sub.l1--W.sub.2].sub.m1--{[(CH.sub.2).sub.n1E1].s-
ub.m2--[(CHR.sup.2).sub.l2--W.sub.3].sub.m3}.sub.n2--, [0562]
X.sub.2 is
--[(CHR.sup.3).sub.l3--W.sub.4].sub.m4--{[(CH.sub.2).sub.n3E2].sub.m5--[(-
CHR.sup.4).sub.l4--W.sub.5].sub.m6}.sub.n4--, [0563] X.sub.3 is
--[(CHR.sup.5).sub.l5--W.sub.6].sub.m7--, [0564] X.sub.4 is
F-D1-(CH.sub.2).sub.l6-D2-, [0565] l1, l2, l3, l4, l5 and l6
independently are selected from 0-16, [0566] m1, m3, m4, m6 and m7
independently are selected from 0-10, [0567] m2 and m5
independently are selected from 0-25, [0568] n1, n2, n3 and n4
independently are selected from 0-16, [0569] F is aryl, hetaryl,
pyrrolidine-2,5-dione or a valence bond, wherein the aryl and
hetaryl groups are optionally substituted with halogen, --CN, --OH,
--C(O)OH, --C(O)NH.sub.2, --S(O).sub.2OH or C.sub.1-6-alkyl, [0570]
R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 independently are
selected from hydrogen, --C(O)OH, --C(O)NH.sub.2, --S(O)OH,
--S(O).sub.2OH, --NH--C(.dbd.NH)--NH.sub.2, C.sub.1-6-alkyl, aryl
or hetaryl; wherein the alkyl, aryl and hetaryl groups optionally
are substituted with halogen, --C(O)OH, --C(O)NH.sub.2, --S(O)OH,
--S(O).sub.2OH, --CN or --OH, [0571] D1, D2, E1 and E2
independently are selected from --O--, --N(R.sup.6)--,
--N(C(O)R.sup.7)-- or a valence bond; wherein R.sup.6 and R.sup.7
independently represent hydrogen or C.sub.1-6-alkyl, [0572] W.sub.1
to W.sub.5 independently are selected from --C(O)NH--, --NHC(O)--,
--C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--, --C(O)NHS(O).sub.2--,
--S(O).sub.2NHC(O)--, --OC(O)NH--, --NHC(O)O--, --C(O)CH.sub.2--,
--CH.sub.2C(O)--, --C(O)CH.dbd.CH--, --CH.dbd.CHC(O)--,
--(CH.sub.2).sub.s2--, --C(O)--, --C(O)O--, --OC(O)--, or a valence
bond; wherein s2 is 0 or 1, [0573] W.sub.6 is selected from
--C(O)NH--, --NHC(O)--, --C(O)NHCH.sub.2--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--, --OC(O)NH--,
--NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--, --C(O)CH.dbd.CH--,
--CH.dbd.CHC(O)--, --(CH.sub.2).sub.s1--, --C(O)--, --C(O)O--,
--OC(O)--, --NHC(O)C.sub.1-6-alkyl, --C(O)NHC.sub.1-6-alkyl or a
valence bond; wherein s1 is 0 or 1 and the C.sub.1-6-alkyl group is
optionally substituted with oxo, pyrrolidine-2,5-dione,
--NHC(O)CH*CH.sub.2COOH or --NHC(O)CH.sub.2CH*COOH; wherein (*)
indicates the attachment point from the carbon atom of CH to
X.sub.4. 76. The conjugate of any one of embodiments 63-75, wherein
l1, l2, l3, l4, l5 and l6 independently are 0-6, m1, m3, m4, m6 and
m7 independently are 0-6, m2 and m5 independently are 0-10, and n1,
n2, n3 and n4 independently are 0-10. 77. The conjugate of any one
of embodiments 63-75, wherein D1 and D2 are independently selected
from --O-- or --N(R.sup.6)-- or a valence bond. 78. The conjugate
of any one of embodiments 63-77, wherein E1 and E2 are
independently selected from --O-- or --N(R.sup.6)-- or a valence
bond. 79. The conjugate of any one of embodiments 63-78, wherein
W.sub.1 through W.sub.8 independently are selected from the group
consisting of --C(O)NH--, --NHC(O)--, --CH.sub.2NHC(O)--,
--C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--, --NHC(O)C.sub.1-6-alkyl
or --C(O)NHC.sub.1-6-alkyl or a valence bond; wherein the alkyl
group is optionally substituted with oxo, pyrrolidine-2,5-dione,
--NHC(O)CH*CH.sub.2COOH or --NHC(O)CH.sub.2CH*COOH; wherein (*)
indicates the attachment point from the carbon atom of CH to
X.sub.4. 80. The conjugate of any one of embodiments 63-79, wherein
R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 independently are
selected from hydrogen, --C(O)OH, --C(O)NH.sub.2, --S(O).sub.2OH or
C.sub.1-6-alkyl; wherein the alkyl group optionally is substituted
with --C(O)OH, --C(O)NH.sub.2 or --S(O).sub.2OH. 81. The conjugate
of any one of embodiments 63-80, wherein
--{[(CH.sub.2).sub.n1E1].sub.m2--[(CHR.sup.2).sub.l2--W.sub.3].sub.m3}.su-
b.n2-- and
--{[(CH.sub.2).sub.n3E2].sub.m5--[(CHR.sup.4).sub.l4--W].sub.m6-
}.sub.n4--, wherein E1 and E2 are --O--, are selected from
##STR00075##
[0574] wherein * is intended to denote a point of attachment, ie,
an open bond.
82. The conjugate of any one of embodiments 63-81, wherein X.sub.4
is a valence bond and W.sub.6 is selected from either
pyrrolidine-2,5-dione, --NHC(O)CH*CH.sub.2COOH or
--NHC(O)CH.sub.2CH*COOH wherein (*) indicates the attachment point
from the carbon atom of CH to GH. 83. The conjugate of any one of
embodiments 63-82 wherein B is selected from
##STR00076## ##STR00077##
84. The conjugate of any one of embodiments 1-83, wherein one
albumin binding residue (A) via a hydrophilic spacer (B) is linked
to said GH. 85. The conjugate of any one of embodiments 63-84,
wherein said compound is selected from
##STR00078## ##STR00079## ##STR00080## ##STR00081##
##STR00082##
86. The conjugate of any one of embodiments 1-85 wherein the
hydrophilic spacer has m Log P<0. 87. The conjugate of any one
of embodiments 1-86, wherein the molar weight of said hydrophilic
spacer is in the range from 80 D to 1500 D or in the range from 300
D to 1100 D. 88. The conjugate of any one of embodiments 1-87,
wherein said albumin binding residue is a lipophilic residue. 89.
The conjugate of any one of embodiments 1-88, wherein said albumin
binding residue binds non-covalently to albumin. 90. The conjugate
of any one of embodiments 1-89, wherein said albumin binding
residue is negatively charged at physiological pH. 91. The
conjugate of any one of embodiments 1-90, wherein said albumin
binding residue has a binding affinity towards human serum albumin
that is below about 10 .mu.M or below about 1 .mu.M. 92. The
conjugate of any one of embodiments 1-91, wherein said albumin
binding residue is selected from a straight chain alkyl group, a
branched alkyl group, a group which has an .omega.-carboxylic acid
group or an .omega.-carboxylic acid isoster. 93. The conjugate of
any one of embodiments 1-92, wherein said albumin binding residue
has from 6 to 40 carbon atoms, from 8 to 26 carbon atoms or from 8
to 20 carbon atoms. 94. The conjugate of any one of embodiments
1-93, wherein said albumin binding residue is a peptide, such as a
peptide comprising less than 40 amino acid residues. 95. The
conjugate of any one of embodiments 1-94, wherein two albumin
binding residues (A) via a hydrophilic spacer (B) is linked to said
GH. 96. The conjugate of any one of embodiments 1-95 for use in
therapy. 97. A pharmaceutical composition comprising a conjugate of
any one of embodiments 1-95, optionally in combination with a
pharmaceutical acceptable excipient. 98. A pharmaceutical
composition of embodiment 97, wherein said composition can be
administered through lingual, sublingual, buccal, in the mouth,
oral, in the stomach and intestine, nasal, pulmonary, epidermal,
dermal, transdermal, and parenteral to patients. 99. A method of
treating growth hormone deficiency (GHD), the method comprising
administrating to a patient in need thereof an effective amount of
a therapeutivcally effective amount of a conjugate of any one of
embodiments 1-95. 100. A method of treating Turner Syndrome;
Prader-Willi syndrome (PWS); Noonan syndrome; Down syndrome;
chronic renal disease, juvenile rheumatoid arthritis; cystic
fibrosis, HIV-infection in children receiving HAART treatment
(HIV/HALS children); short children born short for gestational age
(SGA); short stature in children born with very low birth weight
(VLBW) but SGA; skeletal dysplasia; hypochondroplasia;
achondroplasia; idiopathic short stature (ISS); GHD in adults;
fractures in or of long bones, such as tibia, fibula, femur,
humerus, radius, ulna, clavicula, matacarpea, matatarsea, and
digit; fractures in or of spongious bones, such as the scull, base
of hand, and base of food; patients after tendon or ligament
surgery in e.g. hand, knee, or shoulder; patients having or going
through distraction oteogenesis; patients after hip or discus
replacement, meniscus repair, spinal fusions or prosthesis
fixation, such as in the knee, hip, shoulder, elbow, wrist or jaw;
patients into which osteosynthesis material, such as nails, screws
and plates, have been fixed; patients with non-union or mal-union
of fractures; patients after osteatomia, e.g. from tibia or
1.sup.st toe; patients after graft implantation; articular
cartilage degeneration in knee caused by trauma or arthritis;
osteoporosis in patients with Turner syndrome; osteoporosis in men;
adult patients in chronic dialysis (APCD); malnutritional
associated cardiovascular disease in APCD; reversal of cachexia in
APCD; cancer in APCD; chronic abstractive pulmonal disease in APCD;
HIV in APCD; elderly with APCD; chronic liver disease in APCD,
fatigue syndrome in APCD; Crohn's disease; impaired liver function;
males with HIV infections; short bowel syndrome; central obesity;
HIV-associated lipodystrophy syndrome (HALS); male infertility;
patients after major elective surgery, alcohol/drug detoxification
or neurological trauma; aging; frail elderly; osteo-arthritis;
traumatically damaged cartilage; erectile dysfunction;
fibromyalgia; memory disorders; depression; traumatic brain injury;
subarachnoid haemorrhage; very low birth weight; metabolic
syndrome; glucocorticoid myopathy; short stature due to
glucucorticoid treatment in children, the acceleration of the
healing of muscle tissue, nervous tissue or wounds; the
acceleration or improvement of blood flow to damaged tissue; or the
decrease of infection rate in damaged tissue, the method comprising
administrating to a patient in need thereof an effective amount of
a therapeutivcally effective amount of a conjugate of any one of
embodiments 1-95. 101. The use of a conjugate of any one of
embodiments 1-95 in the manufacture of a medicament for the
treatment of growth hormone deficiency (GHD). 102. The use of a
conjugate of any one of embodiments 1-95 in the manufacture of a
medicament for the treatment of Turner Syndrome; Prader-Willi
syndrome (PWS); Noonan syndrome; Down syndrome; chronic renal
disease, juvenile rheumatoid arthritis; cystic fibrosis,
HIV-infection in children receiving HAART treatment (HIV/HALS
children); short children born short for gestational age (SGA);
short stature in children born with very low birth weight (VLBW)
but SGA; skeletal dysplasia; hypochondroplasia; achondroplasia;
idiopathic short stature (ISS); GHD in adults; fractures in or of
long bones, such as tibia, fibula, femur, humerus, radius, ulna,
clavicula, matacarpea, matatarsea, and digit; fractures in or of
spongious bones, such as the scull, base of hand, and base of food;
patients after tendon or ligament surgery in, e.g., hand, knee, or
shoulder; patients having or going through distraction oteogenesis;
patients after hip or discus replacement, meniscus repair, spinal
fusions or prosthesis fixation, such as in the knee, hip, shoulder,
elbow, wrist or jaw; patients into which osteosynthesis material,
such as nails, screws and plates, have been fixed; patients with
non-union or mal-union of fractures; patients after osteatomia,
e.g., from tibia or 1.sup.st toe; patients after graft
implantation; articular cartilage degeneration in knee caused by
trauma or arthritis; osteoporosis in patients with Turner syndrome;
osteoporosis in men; adult patients in chronic dialysis (APCD);
malnutritional associated cardiovascular disease in APCD; reversal
of cachexia in APCD; cancer in APCD; chronic abstractive pulmonal
disease in APCD; HIV in APCD; elderly with APCD; chronic liver
disease in APCD, fatigue syndrome in APCD; Crohn's disease;
impaired liver function; males with HIV infections; short bowel
syndrome; central obesity; HIV-associated lipodystrophy syndrome
(HALS); male infertility; patients after major elective surgery,
alcohol/drug detoxification or neurological trauma; aging; frail
elderly; osteo-arthritis; traumatically damaged cartilage; erectile
dysfunction; fibromyalgia; memory disorders; depression; traumatic
brain injury; subarachnoid haemorrhage; very low birth weight;
metabolic syndrome; glucocorticoid myopathy; short stature due to
glucucorticoid treatment in children, the acceleration of the
healing of muscle tissue, nervous tissue or wounds; the
acceleration or improvement of blood flow to damaged tissue; or the
decrease of infection rate in damaged tissue. 103. A compound of
formula (III)
A-W--B1-U (III)
wherein A represent an albumin binding residue, B1 represents a
hydrophilic spacer, W is a chemical group linking A and B1, and U
represent a conjugating moiety. 104. The compound according to
embodiment 103, wherein A and W are as defined in any of the above
embodiments. 105. The compound according to embodiment 103 or
embodiment 104, wherein U comprises or consists of an aryl, an
heteraryl, a substituted malimide or a pyrrolidine-2,5-dione such
as --NHC(O)CH.sub.2CH.sub.2-pyrrolidin-2.5-dione. 106. The compound
according to embodiment 103 or embodiment 104, wherein U comprises
D1-(CH.sub.2).sub.l6-D2, wherein D1 and D2 are independently
selected from --O--, --N(R6)-, --NC(O)R7- or a valence bond;
wherein R6 and R7 independently represent hydrogen or
C.sub.1-6-alkyl. 107. The compound according to embodiment 103 or
embodiment 104, wherein U comprises or consists of a leaving group,
such as Cl, Br, I, --OH, --OS(O).sub.2Me, --OS(O).sub.2CF.sub.3,
--Ots. 108. The compound according to embodiments 107, wherein the
leaving group is a halogen compound selected from Cl, Br and I,
preferably Br. 109. The compound according to embodiment 103 or
embodiment 104, wherein U comprises or consists of an allyl amine
(H.sub.2C.dbd.CH--CH.sub.2--NH.sub.2), such as
--C(O)NHCH.sub.2--CH.dbd.CH.sub.2. 110. The compound according to
embodiment 103 or embodiment 104, wherein U comprises or consists
of an amine, such as --NH.sub.2. 111. The compound according to any
of the embodiments 103-110, wherein the therapeutic compound is a
polypeptide. 112. The compound according to any of the embodiments
103-110, wherein the therapeutic compound is a polypeptide with a
single free cystine. 113. The compound according to embodiment 103
or embodiment 104, wherein U comprises or consists of an aldehyde,
such as --CHO. 114. The compound according to any of the embodiment
103-113, wherein the hydrophilic spacer B1 has the formula
--X.sub.1--X.sub.2--X.sub.3--X.sub.4--
wherein [0575] X.sub.1 is
--W.sub.1--[(CHR.sup.1).sub.l1--W.sub.2].sub.m1--{[(CH.sub.2).sub.n1E1].s-
ub.m2--[(CHR.sup.2).sub.l2--W.sub.3].sub.m3}.sub.n2--, [0576]
X.sub.2 is
--[(CHR.sup.3).sub.l3--W.sub.4].sub.m4--{[(CH.sub.2).sub.n3E2].sub.m5--[(-
CHR.sup.4).sub.l4--W.sub.5].sub.m6}.sub.n4--, [0577] X.sub.3 is
--[(CHR.sup.5).sub.l5].sub.m7--, [0578] X.sub.4 is a valence bond,
[0579] l1, l2, l3, l4, and l5 independently are selected from 0-16,
[0580] m1, m3, m4, m6 and m7 independently are selected from 0-10,
[0581] m2 and m5 independently are selected from 0-25, [0582] n1,
n2, n3 and n4 independently are selected from 0-16, [0583] R.sup.1,
R.sup.2, R.sup.3, R.sup.4 and R.sup.5 independently are selected
from hydrogen, --C(O)OH, --C(O)NH.sub.2, --S(O)OH, --S(O).sub.2OH,
--NH--C(.dbd.NH)--NH.sub.2, C.sub.1-6-alkyl, aryl or hetaryl;
wherein the alkyl, aryl and hetaryl groups optionally are
substituted with halogen, --C(O)OH, --C(O)NH.sub.2, --S(O)OH,
--S(O).sub.2OH, --CN or --OH, E1 and E2 independently are selected
from --O--, --N(R.sup.6)--, --N(C(O)R.sup.7)-- or a valence bond;
wherein R.sup.6 and R.sup.7 independently represent hydrogen or
C.sub.1-6-alkyl, [0584] W.sub.1 to W.sub.5 independently are
selected from --C(O)NH--, --NHC(O)--, --C(O)NHCH.sub.2--,
--CH.sub.2NHC(O)--, --C(O)NHS(O).sub.2--, --S(O).sub.2NHC(O)--,
--OC(O)NH--, --NHC(O)O--, --C(O)CH.sub.2--, --CH.sub.2C(O)--,
--C(O)CH.dbd.CH--, --CH.dbd.CHC(O)--, --(CH.sub.2).sub.s2--,
--C(O)--, --C(O)O--, --OC(O)--, or a valence bond; wherein s2 is 0
or 1.
[0585] The description herein of any aspect or embodiment of the
invention using terms such as "comprising", "having", "including"
or "containing" with reference to an element or elements is
intended to provide support for a similar aspect or embodiment of
the invention that "consists of", "consists essentially of", or
"substantially comprises" that particular element or elements,
unless otherwise stated or clearly contradicted by context (e.g., a
composition described herein as comprising a particular element
should be understood as also describing a composition consisting of
that element, unless otherwise stated or clearly contradicted by
context).
[0586] This invention includes all modifications and equivalents of
the subject matter recited in the aspects or claims presented
herein to the maximum extent permitted by applicable law.
[0587] The present invention is further illustrated by the
following examples which, however, are not to be construed as
limiting the scope of protection. The features disclosed in the
foregoing description and in the following examples may, both
separately and in any combination thereof, be material for
realising the invention in diverse forms thereof.
EXAMPLES
Abbreviations
[0588] amu=atomic mass units CV=column volumes hr(s)=hour(s)
Hz=hertz L=liter(s) M=molar mbar=millibar mg=milligram(s)
min.=minute(s) mL=milliliter(s) mM=millimolar mm=milimeter(s)
mmol=millimole(s) nmol=nanomole(s) mol=mole(s) .mu.L=microliters
N=normal nm=nanometer(s) sec=second(s) ppm=parts per million
ESI=electrospray ionization i.v.=intravenous m/z=mass to charge
ratio MS=mass spectrometry HPLC=high pressure liquid chromatography
RP=reverse phase HPLC-MS=high pressure liquid chromatography-mass
spectrometry NMR.dbd.nuclear magnetic resonance spectroscopy
p.o.=per oral rt or RT=room temperature s.c.=subcutaneous
tr=retention time Boc=tert butyloxycarbonyl O-t-Bu=tert butyl ester
t-Bu=tert butyl Boc-4-ABZ-OH=4-tert-Butoxycarbonylamino-benzoic
acid DCM=dichloromethane, CH.sub.2Cl.sub.2, methylenechloride
DIC=diisopropylcarbdiimide
DIPEA=N,N-diisopropylethylamine
DMF=N,N-dimethylformamide
[0589] DMSO=dimethylsulfoxide DTT=dithiothreitol
EDAC=1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
Et.sub.2O=diethyl ether EtOAc=ethyl acetate
Fmoc=9H-fluoren-9-ylmethoxycarbonyl Fmoc-Glu-O-t-Bu=N-Fmoc-glutamic
acid-1-t-butyl ester
Fmoc-Lys(Mtt)-OH.dbd.(S)-6-[(Diphenyl-p-tolyl-methyl)-amino]-2-(9H-fluore-
n-9-ylmethoxycarbonylamino)-hexanoic acid
Fmoc-OEG-OH=(2[2-(Fmoc-amino)ethoxy]ethoxy)acetic acid
OEG=(2[2-(amino)ethoxy]ethoxy)acetyl
[0590]
Fmoc-Thx-OH.dbd.N-Fmoc-trans-4-aminomethylcyclohexancarboxylic acid
H.sub.2O=water HOBt=1-hydroxybenzotriazole MeCN=acetonitrile
MeOH=methanol MTP=3-methyl-thio-1-propanol NaCl=sodium chloride
NaOH=sodium hydroxide
NMP=N-methylpyrrolidin-2-one
[0591] OEG=(2[2-(amino)ethoxy]ethoxy)acetic acid
TFA=trifuloroacetic acid THF=tetrahydrofuran TIS=triisopropylsilane
CDCl.sub.3=deuterio chloroform CD.sub.3OD=tetradeuterio methanol
DMSO-d.sub.6=hexadeuterio dimethylsulfoxide
TNBS=trinitrobenzensulfonic acid
TSTU=O--(N-Succinimidyl)-1,1,3,3-tetramethyl uranium
tetrafluoroborate
[0592] The examples also make use of the following general
methods:
General Method for Preparing a hGH Compounds.
[0593] The gene coding for the growth hormone compound was inserted
recombinantly into a plasmid vector. A suitable E. coli strain was
subsequently transformed using the plasmid vector. hGH or GH
variants may be expressed with an N-terminal methionine or as a
MEAE fusion from which the MEAE sequence is subsequently cleaved
off.
[0594] Cell stock was prepared in 25% glycerol and stored at
-80.degree. C. Glycerol stock strain was inoculated into LB plates
and subsequently incubated at 37.degree. C. overnight. The content
of each plate was washed with LB medium and diluted into 500 mL LB
medium for expression. The cultures were incubated at 37.degree. C.
with shaking at 220 rpm until OD.sub.600 0.6 was reached.
Succeeding induction was performed using 0.2 mM IPTG at 25.degree.
C. for 6 hours. Cells were finally harvested by centrifugation.
[0595] Cells were subsequently suspended in 10 mM Tris-HCl, pH=9.0
containing 0.05% Tween 20, 2.5 mM EDTA, 10 mM cysteamine and 4M
urea, and disrupted using a cell disrupter at 30 kPSI. The
supernatant was collected by centrifugation and subsequently
subjected to chromatographic purification.
[0596] The purification was performed using ion-exchange
chromatography and hydrophibic interaction, followed by removal of
the peptide tag using human dipeptidyl peptidase I (hDPPI)
expressed from CHO cell. Final purification was achieved by
isoprecipitation and ion-exchange chromatography. The purification
could also be achieved by using but not limited to ion-exchange
chromatography, hydrophobic interaction chromatography, affinity
chromatography, size exclusion chromatography and membrane based
separation techniques known to a person skilled in the art.
Protein Chemical Characterization of Purified Growth Hormone
Compounds.
[0597] The intact purified protein was analysed using MALDI-MS. The
observed mass corresponded to the theoretical mass deduced from the
amino acid sequence. The expected linkage disulfide bonds may be
demonstrated by peptide mapping using trypsin and AspN digestion
followed by MALDI-MS analysis of the digest before and after
reduction of the disulfide bonds with DTT.
Assay for Measuring Rate of Protease Degradation of GH and hGH
Compound Conjugates
[0598] The compound of interest is digested by a relevant protease
(Trypsin, Chymotrypsin, Pepsin, Elastase, Factor VIIa, Factor Xa,
Proteinase K, Carboxy peptidase, DPPIV, Neutral Endopeptidase,
Granzyme B, Proline-endopeptidase, Staphylococcal peptidase I,
Thermolysin, Thrombin, Arg-C proteinase, Asp-N endopeptidase,
Caspase 1-10, Clostripain, Enterokinase, Glutamyl endopeptidase,
Granzyme B, LysC, LysN, Proline-endopeptidase and Staphylococcal
peptidase I or tissue extracts.) in an appropriate buffer (e.g. PBS
or ammonium bicarbonate) at 37.degree. C. for up till 24 hrs.
Proteolytic degradation is assessed by a HPLC assay.
Proteolytic Digestion:
[0599] 100 .mu.L of test compound solution at 1 mg/mL in ammonium
bicarbonate buffer is degraded by enzyme for up till 24 hrs at
37.degree. C. Sub-samples are taken to various time points and the
proteolytic reaction is stopped by acidifying the sample by 10
times dilution into 1% TFA. These diluted samples are analysed by
reversed phase HPLC to estimate the degree of proteolytic
digestion.
HPLC Method:
[0600] 10 .mu.L of the above solution is injected on a reversed
phase Vydac C4 2.times.150 mm column eluted with a linear gradient
from 0.1% TFA in water to 100% acetonitrile containing 0.1% TFA
over a period of 30 min at a flow rate of 0.2 ml/min. Detection of
peaks is performed at 214 nm UV absorption. Percentage (%) intact
compound at time point t=T is calculated from the peak area at time
point t=T (A.sub.T) and the peak area at t=0 (A.sub.0) as
(A.sub.T/A.sub.0).times.100%. Percentage (%) intact compound is
plotted against time using GraphPad Prims software ver. 5.01. Half
life (T.sub.1/2) is calculated as one phase decay also by GraphPad
Prism software. Examples of enzymes that may be used are elastase
(Sigma from porcine pancrease) and chymotrypsin (Roche sequencing
grade). Example of buffer is 50 mM ammonium bicarbonate,
pH=8.5.
Capillary Electrophoresis:
[0601] Capillary electrophoresis was carried out using an Agilent
Technologies 3DCE system (Agilent Technologies). Data acquisition
and signal processing were performed using Agilent Technologies
3DCE ChemStation. The capillary was a 64.5 cm (56.0 cm efficient
length) 50 .mu.m i.d. "Extended Light Path Capillary" from Agilent.
UV detection was performed at 200 nm (16 nm Bw, Reference 380 nm
and 50 nm Bw). The running electrolyte was phosphate buffer 50 mM
pH 7 (method A). The capillary was conditioned with 0.1M NaOH for 3
min, then with Milli-Q water for 2 min and with the electrolyte for
3 min. After each run, the capillary was flushed with milli-Q water
for 2 min, then with phosphoric acid for 2 min, and with milli-Q
water for 2 min. The hydrodynamic injection was done at 50 mbar for
4.0 sec. The voltage was +25 kV. The capillary temperature was
30.degree. C. and the runtime was 10.5 min.
Maldi-Tof Mass Spectrometry:
[0602] Molecular weights were determined using the Autoflex
Maldi-Tof instrument (Bruker). Samples were prepared using
alfa-cyano-4-hydroxy-cinnamic acid as matrix.
RP-HPLC:
[0603] RP-HPLC analysis was performed on a Agilent 1100 system
using a Vydac 218TP54 4.6 mm.times.250 mm 5 .mu.m C-18 silica
column (The Separations Group, Hesperia). Detection was by UV at
214 nm, 254 nm, 280 nm and 301 nm. The column was equilibrated with
0.1% trifluoracetic acid/H.sub.2O and the sample was eluted by a
suitable gradient of 0 to 90% acetonitrile against 0.1%
trifluoracetic acid/H.sub.2O.
LC-MS:
[0604] LC-MS analysis was performed on a PE-Sciex API 100 or 150
mass spectrometer equipped with two Perkin Elmer Series 200
Micropumps, a Perkin Elmer Series 200 autosampler, an Applied
Biosystems 785A UV detector and a Sedex 75 Evaporative Light
scattering detector. A Waters Xterra 3.0 mm.times.50 mm 5.mu. C-18
silica column was eluted at 1.5 ml/min at room temperature. It was
equilibrated with 5% MeCN/0.1% TFA/H.sub.2O and eluted for 1.0 min
with 5% MeCN/0.1% TFA/H.sub.2O and then with a linear gradient to
90% MeCN/0.1% TFA/H.sub.2O over 7 min. Detection was by UV
detection at 214 nm and Evaporative light Scattering. A fraction of
the column eluate was introduced into the ionspray interface of a
PE-Sciex API 100 mass spectrometer. The mass range 300-2000 amu was
scanned every 2 seconds during the run.
Quantification of Protein:
[0605] Protein concentrations were estimated by measuring
absorbance at 280 nm using a NanoDrop ND-1000
UV-spectrofotometer.
Enzymatic Peptide Mapping for Determination of Site(s) of
Derivatization:
[0606] Peptide mapping was performed using Asp-N digestion of the
reduced and alkylated protein. First the protein was treated with
DTT and iodoacetamide according to standard procedures. The
alkylated product was purified using HPLC. Subsequently the
alkylated purified product was digested overnight with endoprotease
Asp-N(Boehringer) at an enzyme:substrate ratio of 1:100. The digest
was HPLC separated using a C-18 column and standard TFA/MeCN buffer
system. The resulting peptide map was compared to that of
underivatized hGH and fractions with different retention times were
collected and further analyzed using Maldi-tof mass
spectrometry.
Sds Page:
[0607] SDS poly-acrylamide gel electrophoresis was performed using
NuPAGE 4%-12% Bis-Tris gels (Invitrogen NP0321BOX). The gels were
silver stained (Invitrogen LC6100) or Coomassie stained (Invitrogen
LC6065) and where relevant also stained for PEG with barium iodide
as described by M. M. Kurfurst in Anal. Biochem. 200(2), 244-248,
(1992).
Protein Chromatography:
[0608] Protein chromatography was performed on an Akta Explorer
chromatographic system and columns from GE Health Care. Anion
exchange was done using a Q-Sepharose HP 26/10 column. Starting
buffer was 20 mM triethanolamine buffer pH 8.5 and eluting buffer
was starting buffer+0.2 M NaCl. The compounds were typically eluted
with a gradient of 0-75% eluting buffer over 15 column volumes.
De-salting and buffer exchange was performed using a HiPrep 26/10
column.
TNBS Test
[0609] A solution of 10% DIPEA in DMF (solution 1) and a solution
of 1 M aqueous TNBS (solution 2) was prepared. A few resin beads
were placed in a small test tube and 1-3 drops of each solution (1
and 2) were added. After a short mixing the mixture was left at
room temperature for 10 min. and the beads inspected. Intensely
orange or red beads indicate positive results (i.e presence of free
amines); yellow or slightly orange beads indicate slightly positive
and colorless beads are negative.
Log P Calculation
[0610] Log P values can be calculated as m Log P and/or c Log P for
the albumin binder part and/or the hydrophilic spacer part using
published algorithms (J. Am. Chem. Soc., 86, 5175-5180, (1964) "A
New Substituent Constant, v, Derived from Partition Coefficients",
C. A. Lipinski et al. Advanced Drug Delivery Reviews, 23, 3-25
(1997), "Experimental and Computational Approaches to Estimate
Solubility and Permeability in Drug Discovery and Development
Settings" and I. Moriguchi, S. Hirono, I. Nakagome, H. Hirano,
Chem. and Pharm. Bull., 42, 976-978, (1994) "Comparison of
Reliability of log P Values for Drugs Calculated by Several
Methods". Herein clog P--Pomona College log P (octanol/water
partition coefficient) is calculated with Sybyl 7.0 from Tripos
(http://www.tripos.com) version 4.2 of the clog P algorithm and
version 22 of its associated fragment database as provided by
BioByte Corp (http://www.biobyte.com/).
Preparation of Albumin Binders
Example 1
4-(1H-Tetrazol-16-yl-hexadecanoylsulfamoyl)butanoyl-OEG-yGlu-yGlu-OEG-N.su-
b.6(C(O)CH.sub.2Br)Lys-OH (I)
##STR00083##
[0612] The compound (I) was synthesised on solid support according
to scheme 1, in 1 mM scale using standard Fmoc-peptide chemistry on
an ABI433 synthetizer. Peptide was assembled on a
Fmoc-Lys(MTT)-Wang resin using Fmoc-OEG-OH and Fmoc-Glu-OtBu
protected amino acids.
4-(16-1H-Tetrazol-5-yl-hexadecanoylsulfamoyl)butyric acid was
manual coupled using DIC/NHS in DCM/NMP, 2 eq. over night, TNBS
test showed the reaction to be completed. The resin was then
treated with 50 mL DCM/TFA/TIS/water (94:2:2:2) in a flowthrough
arrangement until the yellow colour disappeared, .about.20 min.
followed by washing and neutralizing with DIPEA/DMF. Bromo acetic
acid (4 mM) in DCM/NMP (1:1) was activated with a 1 mM mixture of
NHS and DIC, filtered and added to the resin with addition of
further 1 mM of DIPEA. After 1 hr the reaction was completed. The
resin was treated with 80 mL TFA/TIS/water (95:2.5:2.5) for 1 hr.
Evaporated with a stream of N.sub.2, precipitated by addition of
Et.sub.2O and washed with Et.sub.2O and dried. Crude product was
purified on preparative HPLC (2 runs), with a gradient from 30-80%
0.1 TFA/MeCN against 0.1% TFA in water. Fractions were collected
and lyophilized with .about.50% MeCN affording compound (I).
[0613] TOF-MS: mass 1272.52 (M+1)
##STR00084##
Example 2
[0614] In a similar way as described in Example 1 above the
following compound was prepared using Fmoc-Lys(Mtt)-OH and Wang
Resin.
##STR00085##
[0615] TOF-MS: mass 536.52 (M+1)
Example 3
[0616] In a similar way as described in Example 1 above the
following compound was prepared using Fmoc-Lys(Mtt)-OH and Wang
Resin.
##STR00086##
[0617] TOF-MS: mass 810.80 (M+1)
Example 4
[0618] In a similar way as described in Example 1 above the
following compound was prepared using Fmoc-Lys(Mtt)-OH and Wang
Resin.
##STR00087##
[0619] TOF-MS: mass 844.84 (M+1)
Example 5
[0620] In a similar way as described in Example 1 above the
following compound was prepared using Fmoc-Lys(Mtt)-OH and Wang
Resin.
##STR00088##
[0621] TOF-MS: mass 1151.27 (M+1)
Example 6
[0622] In a similar way as described in Example 1 above the
following compound was prepared using Fmoc-Lys(Mtt)-OH and Wang
Resin.
##STR00089##
[0623] TOF-MS: mass 1272.30 (M+1)
Example 7
[0624] In a similar way as described in Example 1 above the
following compound was prepared using Fmoc-Lys(Mtt)-OH and Wang
Resin.
##STR00090##
[0625] TOF-MS: mass 984.01 (M+1)
Example 8
[0626] In a similar way as described in Example 1 above the
following compound was prepared using Fmoc-Lys(Mtt)-OH and Wang
Resin.
##STR00091##
[0627] TOF-MS: mass 882.95 (M+1)
Example 9
[0628] In a similar way as described in Example 1 above the
following compound was prepared using Fmoc-Lys(Mtt)-OH and Wang
Resin.
##STR00092##
[0629] TOF-MS: mass 782.74 (M+1)
Example 10
[0630] In a similar way as described in Example 1 above the
following compound was prepared using Fmoc-Lys(Mtt)-OH and Wang
Resin.
##STR00093##
[0631] TOF-MS: mass 1127.14 (M+1)
Example 11
[0632] In a similar way as described in Example 1 above the
following compound was prepared using Fmoc-Lys(Mtt)-OH, iodoacetic
acid and Wang Resin.
##STR00094##
[0633] TOF-MS: mass 1174.14 (M+1)
Example 12
[0634] In a similar way as described in Example 1 above the
following compound was prepared using Fmoc-Lys(Mtt)-OH,
chloroacetic acid and Wang Resin.
##STR00095##
[0635] TOF-MS: mass 1061.89 (M+1)
Example 13
(19-carboxynonadecanoyl)-Thx-.gamma.Glu-Glu-N-{3-[2-(2-{3-[3-malimidopropi-
onylamino]propoxy}ethoxy)ethoxy]propyl} amide (II)
##STR00096##
[0637] 2-Chlorotrityl resin (2.0 g, 2.6 mmol) was svelled in DCM
for 0.5 hr. A solution of 4,7,10-trioxa-1,13-diamine in DCM (30 mL)
was added. Resin was stirred at rt for 1 hr. The resin was washed
once with DCM, then added a solution of DIPEA:MeOH:DCM (15 mL:15
mL:20 mL). The resin was shaken for 0.5 hr, then washed trice with
DCM. Fmoc-Glu(OtBu)OH, Fmoc-Glu-OtBu and FmocThexOH were then
coupled sequentially by standard peptide chemistry as follows: A
0.5 M solution each of Fmoc-AA-OH/DIC/HOBt in NMP (11.7 mL) was
mixed and after 2 min. added to the resin. The resin was shaken for
45 min. at rt. and then washed with 5.times.NMP and 5.times.DCM. A
solution of Ac.sub.2O/DIPEA/NMP (1:1:5) was added and the resin was
stirred at rt for 10 min. The resin was washed (5.times.NMP and
5.times.DCM). The resin was then treated with 30% piperidin in NMP
for 2.times.10 min. and finally washed with 5.times.NMP and
5.times.DCM. The peptide was then added a 0.25 M solution of
eicosanediacid (6 eq) containing 0.125 M HOAt (3 eq), 0.125 M DIC
(3 eq) and 0.125 M lutidine (3 eq). The resin was shaken at rt for
2 hrs following by washing with 5.times.NMP and 8.times.DCM.
Product was cleaved from the resin using 10% TFA-DCM for 20 min.
The resin was filtratered off and treated one more time with 10%
TFA-DCM for an additional 20 min. The combined filtrates were
collected, and evaporated to dryness.
[0638] The dry product from above was dissolved in DMF (6 mL), and
added TSTU-activated 3 maleimidopropionic acid (premade by reacting
TSTU with was 3 maleimidopropionic acid in DMF (2 mL) for 45 min.)
and DIPEA (200 .mu.L). The mixture was stirred at rt. for 1 hr. The
reaction mixture was evaporated to dryness and the residue
dissolved in 95% TFA-MilliQ water and stirred at rt. for 20 min.
The mixture was evaporated to dryness. To the residue was added a
minimum of water to precipitate solid. The precipitate was filtered
off and recrystallized from MeCN. The crystals were collected and
washed extensively with Et.sub.2O affording compound (II) as a
white solid.
[0639] TOF-MS: mass 1094.39 (M+1).
Example 14
[0640] In a similar way as described in Example 1 above and
depicted in scheme 2 below the following compound was prepared
using Boc-Gly-PAM resin as starting material and
mono-tert-butyl-eicosanoic acid, 4-Boc-aminobenzoic acid,
Fmoc-Thx-OH, Fmoc-OEG-OH, Fmoc-Glu(O-t-Bu)-OH, Fmoc-Glu(OH)-t-Bu
protected amino acids. After mild deprotection, peptide product was
cleaved from the resin using 2,2-dimethoxyethylamine followed by
deacetalisation using TFA which afforded albumin binder (IV).
##STR00097##
[0641] TOF-MS: mass 1128.38 (M+1)
##STR00098##
Example 15
[0642] In a similar way as described in Example 14 above the
following compound was prepared using Boc-Gly-PAM resin as starting
material and mono-tert-butyl-eicosanoic acid, 4-Boc-aminobenzoic
acid, Fmoc-Thx-OH, Fmoc-OEG-OH, Fmoc-Glu(O-t-Bu)-OH,
Fmoc-Glu(OH)-t-Bu protected amino acids. After mild deprotection,
peptide product was cleaved from the resin using
2,2-dimethoxyethylamine followed by deacetalisation using TFA which
afforded albumin binder (V).
##STR00099##
[0643] TOF-MS: Rt=15.2 min, mass=967.11 (M+1)
Example 16
[0644] In a similar way as described in Example 14 above compound
(VI) was prepared using Fmoc-Lys(Mtt)-Wang resin as starting
material and mono-tert-butyl-octadecanedioic acid,
Boc-Ser(t-Bu)-OH, Fmoc-OEG-OH, Fmoc-Glu(O-t-Bu)-OH, and oxidised
Fmoc-Cys-OH protected amino acids. The peptide product was cleaved
from the resin using 2.5% TIS, 2.5% H.sub.2O in THF for 3 hrs and
purified by prep-HPLC:
[0645] Column: 2 cm C18
[0646] Eluent A: 0.1% TFA i Milli-Q water
[0647] Eluent B: 0.1% TFA i MeCN
[0648] Start % B: 40%
[0649] End % B: 75%
[0650] Gradient: 5 min. with 10% MeCN, 5-10 min. up to start % B
over 51 min., 5 min. with end % B+10% MeCN approx. 1 hr
[0651] Fractions were analysed by LC-MS-TOF.
[0652] Desired fractions were collected, pooled and lyophilised
affording compound (VI)
##STR00100##
[0653] TOF-MS: Rt=6.3 min, mass=955.1 (M+1)
Oxidation of (VI):
MTP-Solution:
[0654] 3-Methyl-thio-1-propanol (290 mg) dissolved in 4 mL 25 mM
HEPES, pH=7.00
Periodate-Solution:
[0655] 96 mg NaIO.sub.4 dissolved in 2 mL Milli-Q water
[0656] To a solution of compound (VI) in Milli-Q water (1 mL) was
added MTP-solution (3.6 mL)+periodate-solution (560 .mu.L) and the
pH was adjusted to 9.5 with one drop of 1N NaOH. The reaction flask
was covered with tin-foil and stirred for 1 hr at RT. An additional
portion of periodate-solution (560 .mu.L) was added and the
reaction mixture was left for 4.5 hours at ambient temperature. The
resulting mixture was run through two NAP columns, to get rid of
the NaIO.sub.4. The columns were prewashed with 25 mM HEPES
(5.times.2.5 mL) pH=7.0. Sample (2.5 mL) applicated on each column
and eluated with 3.5 mL 25 mM HEPES, pH=7.00. 2.times.3.5 mL were
pooled in total--10.5 mL containing keto-aldehyde (VII) which were
used directly for conjugation with an GH analogue.
##STR00101##
[0657] TOF-MS: mass=924.08 (M+1)
Example 17
4-(1H-Tetrazol-16-yl-hexadecanoylsufamoyl)butanoyl-OEG-.gamma.Glu-.gamma.G-
lu-OEG-N.sup..epsilon.(4-aminobenzoyl)Lys-NH.sub.2 (VIII)
##STR00102##
[0659] The compound (VIII) was synthesised on solid support
according to scheme 3. Fmoc protected Rink-Amide-Resin (2.2 g, 0.6
mMol/g) was weighed into a flask. The resin was swelled with NMP
(3.times.30 mL) for 2 hrs. The resin was shaken with 25% piperidine
in NMP (30 mL) for 10 min. The resin was drained and treated with
25% piperidine in NMP (30 mL) for 1 hr followed by draining and
wash with NMP (6.times.30 mL). Fmoc-Lys(Mtt)-OH and HOBt were
weighed into a flask, dissolved in bromo phenol blue in NMP (30 mL,
0.5 mM). This solution was added to the drained resin above
followed by addition of DIC. The reaction was shaken at ambient
temperature for 21 hrs. The resin was drained and washed with NMP
(6.times.30 mL) followed by washing with DCM (3.times.30 mL). The
resin was treated with hexafluorisopropanol (20 mL) for 10 min.
Shaken for 10 min. The resin was drained and washed with DCM
(3.times.30 mL). The resin was treated with hexafluorisopropanol
(20 mL) for 10 min again and shaken for 10 min. The resin was
drained and washed with DCM (3.times.30 mL) followed by drained and
washed with NMP (3.times.30 mL). 4-(Boc-amino)benzoic acid and HOBt
were weighed into a flask, dissolved in bromo phenol blue in NMP
(30 mL, 0.5 mM). This solution was added to the drained resin above
followed by addition of DIC. The reaction was shaken at ambient
temperature. The resin was drained and washed with NMP (6.times.30
mL). The resin was shaken with 25% piperidine in NMP (10 mL) for 10
min. The resin was drained and treated with 25% piperidine in NMP
(10 mL) for 1 hr followed by draining and wash with NMP (6.times.15
mL). Fmoc-OEG-OH and HOBt were weighed into a flask, dissolved in
brom phenol blue in NMP (15 mL, 0.5 mM). This solution was added to
the drained resin followed by addition of DIC. The reaction was
shaken at ambient temperature for 23 hrs. The resin was drained and
washed with NMP (6.times.15 mL). The resin was shaken with 25%
piperidine in NMP (10 mL) for 10 min. The resin was drained and
treated with 25% piperidine in NMP (10 mL) for 1 hr followed by
draining and wash with NMP (6.times.15 mL). Fmoc-Glu-O-t-Bu and
HOBt were weighed into a flask, dissolved in bromo phenol blue in
NMP (15 mL, 0.5 mM). This solution was added to the drained resin
followed by addition of DIC. The reaction was shaken at ambient
temperature for 18 hrs. The resin was drained and washed with NMP
(6.times.15 mL). The resin was shaken with 25% piperidine in NMP
(10 mL) for 10 min. The resin was drained and treated with 25%
piperidine in NMP (10 mL) for 1 hour followed by draining and wash
with NMP (6.times.15 mL). Fmoc-Glu-O-t-Bu and HOBt were weighed
into a flask, dissolved in 15 ml 0.5 mM bromo phenol blue in NMP.
This solution was added to the drained resin followed by addition
of DIC. The reaction was shaken at ambient temperature for 18 hrs.
The resin was drained and washed with NMP (6.times.15 mL). The
resin was shaken with 25% piperidine in NMP (10 mL) for 10 min. The
resin was drained and treated with 25% piperidine in NMP (10 mL)
for 1 hr followed by draining and washing with NMP (6.times.15 mL).
Fmoc-OEG-OH and HOBt were weighed into a flask, dissolved in bromo
phenol blue in NMP (15 mL, 0.5 mM). This solution was added to the
drained resin followed by addition of DIC. The reaction was shaken
at ambient temperature. The resin was drained and washed with NMP
(6.times.15 mL). The resin was shaken with 25% piperidine in NMP
(10 mL) for 10 min. The resin was drained and treated with 25%
piperidine in NMP (10 mL) for 1 hr followed by draining and washing
with NMP (6.times.15 mL).
[0660] 4-(16-1H-Tetrazol-5-yl-hexadecanoylsulfamoyl)butyric acid
and HOBt were weighed into a flask, dissolved in bromo phenol blue
in NMP (15 mL, 0.5 mM). This solution was added to the drained
resin followed by the addition of DIC. The reaction was shaken at
ambient temperature for 21 hrs. The resin was drained and washed
with NMP (6.times.15 mL) followed by draining and wash with DCM
(6.times.15 mL). The resin was cleaved with a mixture of 95% TFA in
water (10 mL)+DCM (0.25 mL) and TIS (0.25 mL). The resin was shaken
for 2 hours at ambient temperature and filtered into ice cold
Et.sub.2O (75 mL). The resulting precipitate was isolated by
centrifugation followed by washing with Et.sub.2O (3.times.) and
dried in vacuum for 48 hours affording crude 300 mg of compound
(VIII).
[0661] Crude compound (VIII) was purified on prep-HPLC (GILSON),
30->80% MeCN. Pooled fractions were evaporated to dryness and
the residue dissolved in H.sub.2O/MeCN 1:1 and freezedried over
night affording 170 mg of the compound (VIII).
[0662] TOF-MS: Rt=4.7 min, mass 1268.71 (M+1)
##STR00103## ##STR00104##
Example 18
[0663] In a similar way as described in Example 17 above the
following compound was prepared using Fmoc-Lys(Mtt)-OH and Wang
Resin.
##STR00105##
Example 19
[0664] In a similar way as described in Example 17 above the
following compound was prepared using FMOC-Lys(Mtt)-OH and Rink
amid resin.
##STR00106##
[0665] TOF-MS: mass 1333.64 (M+1)
Example 20
[0666] In a similar way as described in Example 17 above the
following compound was prepared using FMOC-Lys(Mtt)-OH and Rink
amid resin.
##STR00107##
[0667] TOF-MS: mass 1320.67 (M+1)
Example 21
[0668] In a similar way as described in Example 17 above the
following compound was prepared using FMOC-Lys(Mtt)-OH and Rink
amid resin.
##STR00108##
[0669] TOF-MS: mass 2114.64 (M+1)
Example 22
[0670] In a similar way as described in Example 17 above the
following compound was prepared using FMOC-Lys(Mtt)-OH and Rink
amid resin.
##STR00109##
[0671] TOF-MS: mass 1534.82 (M+1)
Example 23
[0672] In a similar way as described in Example 17 above the
following compound was prepared using FMOC-Lys(Mtt)-OH and Rink
amid resin.
##STR00110##
[0673] TOF-MS: mass 823.05 (M+1)
Example 24
[0674] In a similar way as described in Example 17 above the
following compound was prepared using FMOC-Lys(Mtt)-OH and Wang
Resin.
##STR00111##
[0675] TOF-MS: mass 980.22 (M+1)
Example 25
[0676] In a similar way as described in Example 17 above the
following compound was prepared using FMOC-Lys(Mtt)-OH and Rink
amid resin.
##STR00112##
[0677] TOF-MS: mass 851.10 (M+1)
Example 26
[0678] In a similar way as described in Example 17 above the
following compound was prepared using FMOC-Lys(Mtt)-OH and Rink
amid resin.
##STR00113##
[0679] TOF-MS: mass 1258.51 (M+1)
Example 27
[0680] In a similar way as described in Example 17 above the
following compound was prepared using FMOC-Lys(Mtt)-OH and Wang
Resin.
##STR00114##
[0681] TOF-MS: mass 1269.49 (M+1)
Example 28
[0682] In a similar way as described in Example 17 above the
following compound was prepared using FMOC-Lys(Mtt)-OH and Rink
amid resin.
##STR00115##
[0683] TOF-MS: mass 841.04 (M+1)
Example 29
[0684] In a similar way as described in Example 17 above the
following compound was prepared using FMOC-Lys(Mtt)-OH and Rink
amid resin.
##STR00116##
[0685] TOF-MS: mass 863.07 (M+1)
Example 30
[0686] In a similar way as described in Example 17 above the
following compound was prepared using FMOC-Lys(Mtt)-OH and Rink
amid resin.
##STR00117##
[0687] TOF-MS: mass 855.07 (M+1)
Example 31
[0688] In a similar way as described in Example 17 above the
following compound was prepared using FMOC-Lys(Mtt)-OH and Rink
amid resin.
##STR00118##
[0689] TOF-MS: mass 883.12 (M+1)
Example 32
[0690] In a similar way as described in Example 17 above the
following compound was prepared using FMOC-Lys(Mtt)-OH and Rink
amid resin.
##STR00119##
[0691] TOF-MS: mass 1123.35 (M+1)
Example 33
[0692] In a similar way as described in Example 17 above the
following compound was prepared using FMOC-Lys(Mtt)-OH and Rink
amid resin.
##STR00120##
[0693] TOF-MS: Rt=4.7 min, mass 1267.45 (M+1)
Example 34
[0694] In a similar way as described in Example 17 above the
following compound was prepared using FMOC-Lys(Mtt)-OH and Rink
amid resin.
##STR00121##
[0695] TOF-MS: mass 1310.67 (M+1)
Example 35
[0696] In a similar way as described in Example 17 above the
following compound was prepared using FMOC-Lys(Mtt)-OH and Rink
amid resin.
##STR00122##
[0697] TOF-MS: mass 1308.58 (M+1)
Example 36
[0698] In a similar way as described in Example 17 above the
following compound was prepared using FMOC-Glu(ODmab)-OH and
2-chlorotrityl chloride resine.
##STR00123##
[0699] TOF-MS: mass 1235.56 (M+1)
Example 37
[0700] In a similar way as described in Example 17 above the
following compound was prepared using FMOC-Glu(ODmab)-OH and
2-chlorotrityl chloride resine.
##STR00124##
[0701] TOF-MS: mass 1173.40 (M+1)
Example 38
[0702] In a similar way as described in Example 17 above the
following compound was prepared using FMOC-Lys(Mtt)-OH and Rink
amid resin.
##STR00125##
[0703] TOF-MS: mass 703.93 (M+1)
Example 39
[0704] In a similar way as described in Example 17 above the
following compound was prepared using FMOC-Lys(Mtt)-OH and Rink
amid resin.
##STR00126##
[0705] TOF-MS: mass 689.90 (M+1)
Example 40
[0706] In a similar way as described in Example 17 above the
following compound was prepared using FMOC-Glu(ODmab)-OH and
2-chlorotrityl chloride resine.
##STR00127##
[0707] TOF-MS: mass 1182.34 (M+1)
Example 41
17-[(S)-3-(2-{2-[(2-{2-[(5-Aminopentylcarbamoyl)methoxy]ethoxy}ethylcarbam-
oyl)-methoxy]ethoxy}ethylcarbamoyl)-1-carboxy-propylcarbamoyl]-heptadecano-
ic acid
##STR00128##
[0709] N-tert-butoxycarbonyl cadaverine (24.3 mg; 0.12 mmol) was
added to a solution of
17-((S)-1-Carboxy-3-{2-[2-({2-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonylmet-
hoxy)-ethoxy]ethylcarbamoyl}methoxy)ethoxy]ethylcarbamoyl}propylcarbamoyl)-
-heptadecanoic acid (100 mg; 0.12 mmol) and DIPEA (46.68 mg; 0.36
mmol) in THF (2.0 ml). Reaction mixture was stirred over night at
room temperature, and then concentrated in vacuo. The residue was
dissolved in a mixture of water (5 ml) and THF (2 ml), and purified
by preparative HPLC (RP18 column). Fractions containing the
Boc-protected coupling product was pooled and taken to dryness. The
residual was dissolved in 50% TFA-DCM (4 ml) and stirred for 1h at
room temperature, then concentrated in vacuo to provide 54 mg (55%)
of the title material as its trifluoroacetic acid salt.
[0710] TOF-MS: mass 815.5 (M+1)
Preparation of GH Albumin Binder Compounds:
Example 42
[0711] The use of a transglutaminase to attach a handle to GH has
previously been described in WO2005/070468 and may be used in
accordance with the present invention for attachment of an albumine
binder. The TGase used is microbial transglutaminase from
Streptoverticillium mobaraense according to U.S. Pat. No.
5,156,956. A general method is described in the section Chemistry I
above.
1. Coupling of Transaminated and Oxidised GH Compound (I) with an
Albumine Binder (II)
[0712] The following solution was prepared:
[0713] Buffer A: Triethanolamine (119 mg, 0.8 mmol) was dissolved
in water (40 mL) and pH adjusted to 8.5.
[0714] (A) Transamination of hGH (III) with
1,3-diamino-2-propanol
##STR00129##
[0715] In the next step, transaminated GH (III) is added periodate.
The oxidation is typically done at low temperature, such as
4-10.degree. C. over 30 min. optionally in the dark. Periodate may
oxidize metheonine residues in GH to their corresponding metheonine
sulfoxide residues. To minimize this oxidation risk, small molecule
organic thioethers may be added during periodate oxidation. A
suitable organic thioether is 3-methylthiopropan-1-ol but the
skilled person will be able to suggest others.
Oxidation of Transaminated GH Compound (IV):
##STR00130##
[0717] Buffer change may be performed in order to obtain an acid
solution required for efficient sodium cyano borohydride reduction.
Typically, an excess of A-W--B1-NH2 amine is used, and sodium
cyanoborohydride may be added in smaller portions over time.
[0718] The following solutions were prepared:
[0719] Buffer A: Triethanolamine (119 mg, 0.8 mmol) was dissolved
in water (40 mL) and pH adjusted to 8.5.
[0720] Buffer B: 3-methylthiopropanol (725 mg, 7.1 mmol) was
dissolved in Buffer A (10 mL).
[0721] Buffer C: HEPES (5.96 g) was dissolved in water (1.0 L) and
pH adjusted to 7.0 Periodate: NaIO.sub.4 (48.1 mg, 0.225 mmol) was
dissolved in water (1.0 mL).
[0722] To a solution of (IV) (10 mg, 0.5 .mu.mol) was added Buffer
B (0.2 mL) followed by the periodate solution (0.03 mL). After 20
min. of cold incubation the mixture is dialyzed 4 times with buffer
C. The residue is concentrated to 1 mL.
[0723] (C) Reductive Amination of (I) with Albumin Binder (II)
##STR00131##
[0724] Albumin binder (II) as described in Example 17 through 40
may be used.
[0725] The final solution from (B) (1 mL, 10 mg, 0.45 .mu.mol (I))
was mixed with an albumine binder (II) solution (2 mL, 10 mg, 0.3
.mu.mol) in 25 mM HEPES buffer pH 7.0 and the resulting mixture was
slowly rotated at room temperature for 1 hr. After 1 hr
NaCNBH.sub.3 (100 .mu.L of a solution of NaCNBH.sub.3 (20 mg) in
water (0.5 mL)) was added portionwise. The mixture is kept at room
temperature in the dark for 18-24 hrs.
[0726] The later reaction may be performed as follows:
[0727] A solution of oxidized transaminated GH is added a solution
of albumin binder linker in a mixture of AcOH (1.5 mL) and 50 mM
MES (0.5 mL) at pH 6.00. The resulting reaction mixture is gently
shaken at RT for 30 min. at which time a NaCNBH.sub.3 solution (15
.mu.L, (22 mg NaCNBH.sub.3 dissolved in 500 .mu.L Milli-Q
water+AcOH (15 .mu.L))) is added. The sample is covered with tin
foil and stirrer over night at RT.
[0728] The conjugate can be isolated by anion exchange
chromatography as follows: Acetic acid is removed by buffer changed
with pure water (3.times.) using Amicon Ultral5 devices (Ultracel
10K tubes) by centrifugation at 4000 rpm/min. for 3.times.8 min.
The mixture is then buffer changed to 20 mM TEA, pH: 8.50 using
Amicon Filter devises and diluted to a final volume of 50 mL with
20 mM TEA, before loading it on a HiLoad Q Sepharose, 26/10 column.
The column is initially washed with 20 mM TEA, pH 8.50 (buffer A)
and then eluted with 20 mM TEA, 500 mM NaCl, pH 8.50 (buffer B)
using a 0-100% (B) gradient over 20 CV, with a flow rate of 2
mL/min. The pooled fractions were buffer changed 5 times to 10 mM
ammoniumbicarbonate buffer in pure water using Amicon Ultral5
devices (Ultracel 10K tubes) by centrifugation at 4000 rpm/min. for
3.times.8 min Using the albumin binder from Example 18 will result
in the following compound
42.1
##STR00132##
[0729] TOF-MS: mass 23301.63
[0730] The following compound was prepared using the albumin binder
from Example 41
42.2
##STR00133##
[0731] TOF-MS: mass 23727.6245
[0732] To a solution of hGH (1 mg, 45 nmol) and
17-[(S)-3-(2-{2-[(2-{2-[(5-aminopentylcarbamoyl)methoxy]ethoxy}ethylcarba-
moyl)
methoxy]ethoxy}ethylcarbamoyl)-1-carboxy-propylcarbamoyl]heptadecano-
ic acid (2.10 mg; 2250 nmol) in 20 mM triethanolamin (1000 ul; pH
8.5) was added TGase (0.12 nmol; Streptoverticillium mobaraense).
The reaction mixture was incubated at 25.degree. C. for 146 hrs,
where upon double derivatized hGH analogue of the above formula was
obtained.
Example 43
[0733] 1. Coupling of a GH Compound (I)N-Terminally with an
Albumine Binder (IV)
[0734] (A) Reductive Alkylation of (I) with an Albumin Binder
Aldehyde (IV)
##STR00134##
[0735] The derivatization process as shown above utilise an albumin
binding linker A-W--B wherein B has a terminal aldehyde
functionality. Conjugation of hGH with A-W--B-CHO occurs via
reductive alkylation (hGH.fwdarw.VI). Reductive alkylation is
exemplified herein and is well-recognized in the art and results in
hGH compounds modified at the N-terminal position.
[0736] Albumin binder (IV) was obtained as described in Example
14.
Synthesis of:
2-(C.sub.20diacid-Trx-.gamma.Glu-Glu-OEG-OEG-Gly-Glycin
amid)-ethyl-N.sup..alpha.1-hGH [Q84C, Y143C]
[0737] 43.0
##STR00135##
[0738] hGH[Q84C, Y143C] (23 mg) was dissolved in Hepes buffer (2.3
mL 0.25 mM pH 7.0).
C.sub.20diacid-Trx-.gamma.Glu-Glu-OEG-OEG-Gly-Gly-dimethylacetal (2
mg, see example 14 above) was treated with TFA (50 .mu.L) for 6
min. and evaporated to dryness in vacuum. The residue was stripped
with EtOH (200 .mu.L) and evaporated to dryness in vacuum. The
residue was dissolved in DMF (100 .mu.L) and added to the hGH
solution. A precipitate was formed and redissolved by addition of
DMF (1 mL). After 1 hr a solution of NaCNBH.sub.3 (20 mg, in 0.5 mL
MeCN (230 .mu.L)) was added portionwise and left for 20 hrs. The
reaction was quenched by addition of AcOH (2 mL) and diluted with
water to a total volume of 20 mL and purified on prep. HPLC on a
C18 column with a gradient of 0.1% TFA in MeCN from 40-80% against
0.1% TFA in water. The latest eluting peak was collected, diluted
from 70% MeCN to 10% with water and lyophilized affording 4.51 mg
of 2-(C.sub.20diacid-Trx-.gamma.Glu-Glu-OEG-OEG-Gly-Glycin
amid)-ethyl-N.sup..alpha.1-hGH [Q84C, Y143C].
[0739] TOF-MS: Rt=15.25 min, mass=23150
[0740] In a similar way as described above the following compound
was prepared using albumin binder from Example 16.
43.1
##STR00136##
[0741] TOF-MS: Rt=15.2 min, mass=23033
[0742] In a similar way as described above the following compound
was prepared using albumin binder from Example 15.
43.2
##STR00137##
[0743] TOF-MS: Rt=15 min, mass=22989.1
Example 44
[0744] 1. Coupling of a GH Compound (VII) Having an Internal Free
Single Cys with an Albumine Binder (VIII) [0745] 1) Liberation of
free Cys GH (VII) via reduction of disulfide (VI) with a suitable
selective reducing agent: [0746] 2) Alkylation of free Cys GH (VII)
with a halogen activated albumin binder (VIII) affording Cys
conjugated GH compound (IX)
##STR00138##
[0746] 44.1
##STR00139## [0747]
2-(C.sub.20diacid-Trx-.gamma.Glu-OEG-OEG-6Lys)-carbonylmethylene-S.sup.10-
1hG H[L101C] Preparation of hGH[L101C] (VII):
[0748] hGH[L101C] as obtained above had part of its free cystein
blocked with glutathione and cystamine. Deblocking was performed
enzymatically using glutaredoxin II (Grx2) in an equilibrium buffer
containing GSH and GSSG. Deblocked hGH[L101C] was separated from
low molecular weight GSH/GSSG by buffer exchanged on a Sephadex G25
column.
Conjugation of Albumin Binder (VIII) with hGH[L101C] (VII):
[0749] Albumin binder from example 5 (78 mg, 5 eq) was dissolved in
170 mL HEPES/EDTA buffer with 5% hydroxypropyl-.beta.-cyclodextrin
and added MTP (2.1 mL, 1%) and 0.5 M NaCl (6.34 g). To this mixture
was added concentrated hGH[L101C] (1 eq, 46 mL) and the mixture was
left over night at RT. The solution became cloudy over night. As
HPLC indicated unreacted starting material another 5 eq. albumin
binder from example 5 dissolved in a minimum of NMP was added. The
resulting mixture was stirred at RT for an additional 16 hrs.
Purification:
[0750] Buffers used:
[0751] Buffer A.
[0752] 20 mM Triethanolamine (TEA)+10% Ethylen glycol
[0753] 5.96 g triethanolamine
[0754] 200 mL ethylen glycol
[0755] MQ water added to 2 L.
[0756] pH adjusted to 8.5 with 1N HCl
[0757] Buffer B:
[0758] 20 mM Triethanolamine (TEA)+1.0 M NaCl+10% Ethylen
glycol
[0759] 5.96 g triethanolamine
[0760] 116.88 g NaCl
[0761] 200 mL ethylen glycol
[0762] MQ water added to 2 L.
[0763] pH adjusted to 8.5 with 1N HCl
[0764] The reaction buffer was changed to TEA-buffer A with ethylen
glycol on a Sephadex column over 3 runs:
[0765] Column: 50/30 Sephadex G25 fine
[0766] Buffer A:
[0767] Flow: 10 mL/min
[0768] Temp: RT (fractions collected at 12.degree. C.)
[0769] Fractions: 30 mL per fraction
[0770] Desired fractions were collected, pooled and then purified
on a Q Sepharose column:
[0771] Column: 26/10 Q Sepharose HP
[0772] Buffer A:
[0773] Buffer B:
[0774] Gradient 1: 0-10% Buffer B over 1 CV
[0775] Gradient 2: 10-40% buffer B over 20 CV
[0776] Gradient 3: 40-100% Buffer B over 1 CV
[0777] Flow: 8 mL/min
[0778] Temp: RT (fractions collected at RT)
[0779] Fractions: 5 mL per fraction
[0780] Desired fractions were collected, pooled and buffer
exchanged to 10 mM ammoniumbicarbonate
[0781] pH 8.0 on a Sephadex G25 column:
[0782] Column: 50/30 Sephadex G25 fine
[0783] Buffer A: 10 mM Ammoniumbicarbonate pH 8.0
[0784] Flow: 10 mL/min
[0785] Temp: RT (fractions collected at 12.degree. C.)
[0786] Fractions: 30 mL per fraction
[0787] Five fractions were pooled and freezedried.
[0788] The pool was analysed by MS and large amounts of dimer (MS
44491.7) was seen.
[0789] The freezedried vials were dissolved in buffer A and
purified again on a new Q Sepharose column:
[0790] Column: 26/10 Q Sepharose HP
[0791] Buffer A:
[0792] Buffer B:
[0793] Gradient 1: 0-10% Buffer B over 1 CV
[0794] Gradient 2: 10-40% buffer B over 20 CV
[0795] Gradient 3: 40-100% Buffer B over 1 CV
[0796] Flow: 8 mL/min
[0797] Temp: RT (fractions collected at RT)
[0798] Fractions: 5 mL per fraction
[0799] Fractions were pooled and desalted/buffer exchanged to 10 mM
ammoniumbicarbonate by ultrafiltration. The pool was concentrated
to 25 mL and quantified by RP-HPLC and MS-TOF:
[0800] TOF-MS: Rt=16.15 min, mass=23315.96
[0801] The following compounds were prepared using the same
method.
44.2
##STR00140##
[0802] TOF-MS: Rt=15.24 min, mass=22676.8
44.3
##STR00141##
[0803] TOF-MS: Rt=10.5 min, mass=22975.1
44.4
##STR00142##
[0804] OF-MS: Rt=15.5 min, mass=23009
44.5
##STR00143##
[0805] TOF-MS: Rt=14.0 min, mass=23305.5
44.6
##STR00144##
[0806] TOF-MS: Rt=15.27 min, mass=23148
44.7
##STR00145##
[0807] TOF-MS: Rt=16.40 min, mass=23048
44.8
##STR00146##
[0808] TOF-MS: Rt=15.3 min, mass=22884.4
44.9
##STR00147##
[0809] TOF-MS: Rt=14.6 min, mass=23291.4
44.10
##STR00148##
[0810] TOF-MS: Rt=15.05 min, mass=23097.76
44.11
##STR00149##
[0811] TOF-MS: Rt=14.2 min, mass=23420.83
44.12
##STR00150##
[0812] TOF-MS: Rt=15.7 min, mass=23289.6
44.13
##STR00151##
[0813] TOF-MS: Rt=17.0 min, mass=23324.55
44.15
##STR00152##
[0814] TOF-MS: Rt=12.85 min, mass=23337.5
44.16
##STR00153##
[0815] TOF-MS: Rt=15.24 min, mass=22676.8
Example 45
[0816] 1. Coupling of a GH Compound (VII) Having an Internal Free
Single Cys with an Albumine Binder (X) [0817] 1) Alkylation of free
Cys GH (VII) with a malimide substituted albumin binder (X)
affording Cys conjugated GH compound (XI)
##STR00154##
[0818] Deprotected Cys GH compound (VII) as obtained above in
Example 44 can be reacted with a malimide substituted albumin
binder linker (X) affording GH conjugate
A-W--B1-NHC(O)CH.sub.2CH.sub.2-pyrrolidin-2,5-dione-3-hGH (XI)
wherein B1 is defined as described in Chemistry IV above.
[0819] Conjugation of Maleimide Functionalised Albumin Binder (X)
to hGH L101C
Step (a) Deblocking of Cysteine Residue
[0820] Glutathione/cysteamine blocked Cys hGH (VI) was
enzymatically deblocked using glutaredoxin II (Grx2) in an
equilibrium buffer containing GSH and GSSG. Deblocked Cys hGH (VII)
was separated from low molecular GSH/GSSG by buffer exchanged on a
Sephadex G25 column.
Step (b) Coupling to Maleimide Functionalized Albumin Binder
(X)
[0821] Maleimide functionalized albumin binder (X) was dissolved in
buffer containing 5% hydroxypropyl-.beta.-cyclodextrin. The
solution was then added to de-blocked Cys hGH (VII) and allowed to
reacted over night at room temperature.
[0822] After conjugation the conjugated protein was purified on a Q
Sepharose HiLoad column in 20 mM triethanolamine buffer containing
10% ethylene glycol at pH 8.5 using a gradient of sodium chloride.
The collected fractions were pooled and transferred to 10 mM
ammonium bicarbonate using a G25 column and lyophilized.
45.1
##STR00155##
[0823] TOF-MS: Rt=16.0 min, mass=23352
45.2
##STR00156##
[0824] TOF-MS: Rt=15.98 min, mass=23338
45.3
##STR00157##
[0825] TOF-MS: Rt=16.62 min, mass=23324.6
45.4
##STR00158##
[0826] TOF-MS: Rt=16.20 min, mass=23339.7
45.5
##STR00159##
[0827] TOF-MS: Rt=15.72 min, mass=23316.35
45.6
##STR00160##
[0828] TOF-MS: Rt=17.2 min, mass=23365.9
45.7
##STR00161##
[0829] TOF-MS: Rt=17.2 min, mass=23366
45.8
##STR00162##
[0830] TOF-MS: Rt=16.5 min, mass=23366
45.9
##STR00163##
[0831] TOF-MS: Rt=16.8 min, mass=23323.8
45.10
##STR00164##
[0832] TOF-MS: Rt=17.1 min, mass=23382
45.11
##STR00165##
[0833] TOF-MS: Rt=17.2 min, mass=23338.8
45.12
##STR00166##
[0834] TOF-MS: Rt=17 min, mass=23353.9
45.13
##STR00167##
[0835] TOF-MS: Rt=15.65 min, mass=23323.7
45.14
##STR00168##
[0836] TOF-MS: Rt=16.5 min, mass=23365.8
45.15
##STR00169##
[0837] TOF-MS: Rt=17.2 min, mass=23365.9
Example 46
Assay (I) BAF-3GHR Assay to Determine Growth Hormone Activity
[0838] The BAF-3 cells (a murine pro-B lymphoid cell line derived
from the bone marrow) was originally IL-3 dependent for growth and
survival. IL-3 activates JAK-2 and STAT which are the same
mediators GH is activating upon stimulation. After transfection of
the human growth hormone receptor the cell line was turn into a
growth hormone-dependent cell line. This clone can be used to
evaluate the effect of different growth hormone samples on the
survival of the BAF-3GHR.
[0839] The BAF-3GHR cells are grown in starvation medium (culture
medium without growth hormone) for 24 hrs at 37.degree. C., 5%
CO.sub.2.
[0840] The cells are washed and re-suspended in starvation medium
and seeded in plates. 10 .mu.L of growth hormone compound or human
growth hormone in different concentrations or control is added to
the cells, and the plates are incubated for 68 hrs at 37.degree.
C., 5% CO.sub.2.
[0841] AlamarBlue.RTM. is added to each well and the cells are then
incubated for another 4 hrs. The AlamarBlue.RTM. is a redox
indicator, and is reduced by reactions innate to cellular
metabolism and, therefore, provides an indirect measure of viable
cell number.
[0842] Finally, the metabolic activity of the cells is measure in a
fluorescence plate reader. The absorbance in the samples is
expressed in % of cells not stimulated with growth hormone compound
or control and from the concentration-response curves the activity
(amount of a compound that stimulates the cells with 50%) can be
calculated.
[0843] In vitro potency of compound 45.4 in the BAF-3 hGH receptor
assay is shown in table 1 below.
[0844] Protease stability of compound 45.5 was determined as
described in the general method by incubating the compound for 4
hours with chymotrypsin or elastase. The percent of intact GH
compound was measured and the results are included in table 1.
TABLE-US-00002 TABLE 1 Data relating to compound 45.4 Chymo- Elas-
Ratio trypsin (% tase (% EC50 (EC.sub.50 cmp/ intact GH intact GH
Compund (nM) EC.sub.50 hGH) n compound) compound) hGH 0.026 .+-. 1
6 40 25 0.012 45.4 0.09 .+-. 3.5 6 75 65 0.043
Example 47
Pharmacokinetics
[0845] The pharmacokinetic of the compounds of the examples is
investigated in male Spraque Dawley rats after intravenous (i.v.)
and subcutaneous (s.c.) single dose administration.
[0846] Test compounds are diluted to a final concentration of 1
mg/mL in a dilution buffer consisting of: Glycine 20 mg/mL,
mannitol 2 mg/mL, NaHCO.sub.3 2.5 mg/mL, pH adjusted to 8.2.
[0847] The test compounds are studied in male Spraque Dawley rats
weighing 250 g. The test compounds are administered as a single
injection either i.v. in the tail vein or s.c. in the neck with a
25 G needle at a dose of 60 nmol/kg body weight.
[0848] For each test compound blood sampling is conducted according
to the following schedule presented in table 2.
TABLE-US-00003 TABLE 2 Blood sampling schedule for each test
compound. Animal Sampling time (h) no. RoA Predose 0.08 0.25 0.5 1
2 4 6 8 18 24 48 72 1 s.c. X X X X X X 2 X X X X X X 3 X X X X 4 X
X X X 5 X X 6 X X 7 i.v. X X X X X X X 8 X X X X X X X 9 X X X 10 X
X X
[0849] At each sampling time 0.25 ml blood is drawn from the tail
vein using a 25 G needle. The blood is sampled into a EDTA coated
test tube and stored on ice until centrifugation at 1200.times.G
for 10 min at 4.degree. C. Plasma is transferred to a Micronic tube
and stored at -20.degree. C. until analysis.
[0850] Test compound concentrations are determined by a sandwich
ELISA using a guinea pig anti-hGH polyclonal antibody as catcher,
and biotinylated hGH binding-protein (soluble part of human GH
receptor) as detector. The limit of detection of the assay was 0.2
nM.
[0851] A non-compartmental pharmacokinetic analysis is performed on
mean concentration-time profiles of each test compound using
WinNonlin Professional (Pharsight Inc., Mountain View, CA, USA).
The pharmacokinetic parameter estimates of terminal half-life
(t.sub.1/2) and mean residence time (MRT) are calculated.
TABLE-US-00004 TABLE 3 Half-life (t.sub.1/2) and mean residence
time (MRT) of GH compounds from the examples in Sprague Dawley rats
after single dose i.v. and s.c. administration. Compound T.sub.1/2
MRT (Example #) RoA (h) (h) 43.0 i.v. 7.2 9.8 43.2 i.v. 4.4 7.4
44.1 i.v. 5.6 7.2 44.3 i.v. 1.3 0.9 44.4 i.v. 2.5 2.7 44.5 i.v. 4.1
6.8 44.6 i.v. 3.2 4.0 44.7 i.v. 3.8 6.0 44.9 i.v. 4.2 6.5 44.10 i.v
4.1 7.1 45.4 i.v. 8.6 9.8 45.4 s.c. 19.8 31.2 45.12 i.v. 5.8
6.8
[0852] The bioavailablitiy of example 45.4 was estimated to 48.3%.
The time to maximum plasma concentration (t max) after subcutaneous
administration was 8.0 hrs. Cmax was 1670 and 151 nM after i.v. and
s.c. administration, respectively. The extrapolated plasma
concentration at time zero after i.v. administration concentration
was 1710 nM.
Example 48
[0853] The in vitro potentcy and half lives of a series of
compounds were determined as described above. The conjugates of the
compounds are identical but attached via an alternative cysteine
introduced by mutation as described in the table 4.
TABLE-US-00005 TABLE 4 In vitro potency and half lives (t.sub.1/2).
T.sub.1/2 In vitro (i.v. Rat) MRT Attachement site Compound potency
(hour) (hour) (variant) hGH 1.0 (def) 0.23 -- 45.1 2.6 3.8 T135C
45.2 2.8 8.7 D154C 45.3 2.1 3.1 Q69C 45.4 2.9 6.3/8.6 7.8/9.8 L101C
45.5 4.1 4.5 L18C 45.6 4.1 5.5 Y42C 45.7 0.72 6.5 S95C 45.8 0.59
2.0 S62C 45.9 1.8 4.1 E88C 45.10 2.6 3.4 4.3 A98C 45.11 3.1 5.8 6.8
N99C 45.12 2.5 1.9 3.0 V102C 45.13 16.5 1.9 2.6 E30C 45.14 4.4 1.5
2.0 S100C
Example 49
In Vivo Dose-Response Study in Hypophysectomised Sprague Dawley
Rats
[0854] The in vivo dose-response relationship is studied in
hypophysectomised male Sprague Dawley rats. The hypophysectomised
rat is a well known and recognised animal model of growth hormone
deficiency, where no production of growth hormone occurs after the
surgical removal of the pituitary gland. This also leads to low
circulating levels of insulin-like growth factor-1 (IGF-1) another
important clinical feature of growth hormone deficiency in
humans.
[0855] The hypophysectomy is performed on 4 week old male rats
weighing 90-100 g. The animals entering the study 3-4 weeks after
the surgery weighing 100-110 g. Animals with a body weight gain of
more than 10% during the 3-4 weeks after surgery are not allowed to
enter the study.
[0856] Dose response studies are performed using five dose levels
of compound 45.4 from 1-150 nmol/rat.
[0857] Baseline levels of plasma IGF-1 in hypophysectomised Sprague
Dawley rats were approximately 80-100 ng/mL in all dosing groups.
After a single dose IGF-1 levels rapidly increased to 800-1000
ng/mL on Day 1 almost independently of dose. The plasma IGF-1
levels declined again during the following days in a dose-dependent
manner with the fastest decline seen with the lowest dose and the
slowest decline with the highest dose. At the highest dose the
IGF-1 plasma level was maintained at 800-900 ng/mL for 3 days
before it started to decline more rapidly. IGF-1 plasma
concentration levels were elevated compared to the vehicle control
group for all dosing groups until Day 3. For dosing groups 10 nmol,
50 nmol and 150 nmol it was elevated during the entire study (7
days).
Example 50
Disappearance
[0858] It is hypothesized that absorption rate is related to the
ability of a molecule to pass the tight junctions of the
subcutaneous capillaries, a property related to molecular size. A
PEG-hGH with a 40 kDa PEG has an apparent molecular weight (mw) of
150 250 kDa. A hGH molecule with covalent bound albumin has mw=87
kDa, whereas a hGH molecule with a non-covalent bound albumin will
be dissociated from albumin part of the time and thus have mw=22
kDa. The time spend in the dissociated state will depend on the
affinity of the albumin binding moiety. Thus the absorption rate of
such compounds should be faster than for PEG-hGH and the rate
should increase with the use of albumin binding moieties having
lower affinity for albumin.
[0859] The test solutions were diluted in standard buffer
consisting of: Glycine 20 mg/mL, mannitol 2 mg/mL, NaHCO.sub.3 2.4
mg/mL, pH adjusted to 8.2.
[0860] Iodination with .sup.125I was performed by Chemistry &
Isotope Lab. Novo Nordisk A/S. The final radioactive formulation
had a specific radioactive activity of 3 .mu.Ci/mL and was supplied
in 3 mL Penfills.
[0861] The solutions were stored at 2-8.degree. C. until used.
[0862] Disapperence rate for selected compounds were meaussered in
five female pigs of crossbred LYD. The pigs are weighted, fasted
and issued a special "pig coat" in order to carry the gamma counter
and transmitter and placed in single pens before the start of the
study.
[0863] All pigs are fasted for 18 hrs prior the study.
[0864] The animals were dosed (60 nmol) subcutaneously on the left
and the right side of the neck respectively with a Novopen3.RTM.
and a NovoFine.RTM. 28G needle with fixed black needle stopper.
Injection depth was 5 mm.
[0865] The disappearance of the radioactive depots were measured by
portable equipment for about 24-48 hrs.
[0866] For each individual animal, the results were presented as
AUC (0-45 hrs) as shown in table 5.
[0867] The in vitro potentcy, half lives and further
characteristics of a series of compounds were determined as
described above and shown in the table 5.
TABLE-US-00006 TABLE 5 Compound charateristics. T.sub.1/2
Disapperance Duration of In vitro (i.v. Rat) AUC IGF-1 increase
Compound potency (hour) (0-45 hour) (hour) hGH 1.0 (def) 0.23 519
-- 44.9 8.2 4.2 2259 >48 44.8 1.8 2.6 1490 >24 44.1 4.7 5.6
1750 >48 44.3 1.5 1.3 2152 <24 44.6 4.7 3.2 1558 <24 44.7
4.5 3.8 2039 <24 44.5 5.8 4.1 1599 >48 44.4 3.9 2.5 1588
>48
Example 51
In Vivo Study in Pigs
[0868] To further confirm the functionality of hGH albumin
conjugates according to the invention three compounds were selected
for additional pharmacokinetic studies in pigs. Compounds equal to
compounds 44.1, 44.4 and 44.5 were prepared conjugating the albumin
binder to the hGH variant after removal of the MAEA purification
tag.
[0869] The test compounds were diluted to the final concentration
of 100 nmol/mL in standard buffer (20 mg/mL Glycine, 2 mg/mL
Mannitol, 2.4 mg/mL NaHCO.sub.3, pH adjusted to 8.2). Twenty four
male Gottingen minipigs 5 months of age and weighing 9-12 kg were
used in the study. Each test compound was dosed to eight animals
with four minipigs receiving intravenous bolus administration and
four animals receiving subcutaneous administration. The intravenous
injections were given through a 24 G Venflon in the ear. The dose
was given as a bolus over maximum 5 seconds followed by 2 mL of
0.9% NaCl. The subcutaneous injections were given on the right side
of the neck, approximately 5-7 cm from the ear and 7-9 cm from the
middle of the neck. Each animal received a single dose of test
compound of 10 nmol/kg. Blood samples were collected from each
animal at the following time points: Predose, 0.08, 0.25, 0.5, 1,
2, 4, 6, 8, 18, 24, 48, 72, 96, 120, 168, 240, and 336 hours post
injection. Plasma was isolated from each blood sample and stored at
-20.degree. C. before analysed for test compound. Plasma
concentration-time data were analysed by a non-compartmental
pharmacokinetic method.
TABLE-US-00007 TABLE 6 Pharmacokinetic parameter estimates after
single dose subcutaneous administration of 10 nmol/kg. ABW-Halo
AUC/dose Compound as in (h*kg/l) T.sub.1/2 (h) MRT (h) MAT (h) F
(%) 51.1 44.1 139 (37.5) 11 (1.2) 30.6 (4.1) 10.0 38.6 51.2 44.5
101 (22.5) 12 (2.7) 25.2 (3.9) 11.6 60.8 51.3 44.4 144 (34.7) 12.6
(3.5) 33.1 (1.7) 12.1 35.6 Mean .+-. SD in ( )
[0870] Table 6 is showing key pharmacokinetic parameters for the
three test compounds. The AUC/Dose is an estimate for the dose
corrected exposure of the test compounds. T, is the terminal
half-life of the test compounds after the absorption phase has been
completed. MRT is the mean residence time of the test compounds
corresponding to the time the average test compound molecule is in
the body. MAT is the corresponding mean absorption time and is an
estimate of the average time the molecule is in the absorption
phase. F is the absolute bioavailability of the test compounds
relative to intravenous administration.
Sequence CWU 1
1
11191PRTHomo sapiens 1Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp
Asn Ala Met Leu Arg1 5 10 15Ala His Arg Leu His Gln Leu Ala Phe Asp
Thr Tyr Gln Glu Phe Glu 20 25 30Glu Ala Tyr Ile Pro Lys Glu Gln Lys
Tyr Ser Phe Leu Gln Asn Pro 35 40 45Gln Thr Ser Leu Cys Phe Ser Glu
Ser Ile Pro Thr Pro Ser Asn Arg 50 55 60Glu Glu Thr Gln Gln Lys Ser
Asn Leu Glu Leu Leu Arg Ile Ser Leu65 70 75 80Leu Leu Ile Gln Ser
Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val 85 90 95Phe Ala Asn Ser
Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp 100 105 110Leu Leu
Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu 115 120
125Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser
130 135 140Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys
Asn Tyr145 150 155 160Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp
Lys Val Glu Thr Phe 165 170 175Leu Arg Ile Val Gln Cys Arg Ser Val
Glu Gly Ser Cys Gly Phe 180 185 190
* * * * *
References