U.S. patent application number 13/227910 was filed with the patent office on 2012-02-16 for conjugates of a polypeptide and an oligosaccharide.
This patent application is currently assigned to N.V. Organon. Invention is credited to Ebo Sybren Bos, Martin De Kort, Meertinus Jan Smit, Constant Adriaan Anton Van Boeckel.
Application Number | 20120039843 13/227910 |
Document ID | / |
Family ID | 35532856 |
Filed Date | 2012-02-16 |
United States Patent
Application |
20120039843 |
Kind Code |
A1 |
Bos; Ebo Sybren ; et
al. |
February 16, 2012 |
CONJUGATES OF A POLYPEPTIDE AND AN OLIGOSACCHARIDE
Abstract
The present invention relates to conjugates of a polypeptide and
an oligosaccharide, wherein the polypeptide is conjugated to at
least one oligosaccharide-spacer residue, the oligosaccharide being
a synthetic sulfated oligosaccharide comprising 4-18 monosaccharide
units and per se having affinity to antithrombin III and the spacer
being a bond or an essentially pharmacologically inactive flexible
linking residue, or a pharmaceutically acceptable salt thereof. The
conjugates of the invention have improved pharmacokinetic
properties when compared to the original polypeptides (i.e. the
corresponding non-conjugated polypeptides per se).
Inventors: |
Bos; Ebo Sybren; (Oss,
NL) ; De Kort; Martin; (Oss, NL) ; Smit;
Meertinus Jan; (Oss, NL) ; Van Boeckel; Constant
Adriaan Anton; (Oss, NL) |
Assignee: |
N.V. Organon
Oss
NL
|
Family ID: |
35532856 |
Appl. No.: |
13/227910 |
Filed: |
September 8, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11815131 |
Jan 25, 2008 |
|
|
|
PCT/EP2006/050551 |
Jan 31, 2006 |
|
|
|
13227910 |
|
|
|
|
Current U.S.
Class: |
424/85.2 ;
514/11.7; 514/11.9; 514/20.9; 530/307; 530/308; 530/322; 530/351;
536/17.4 |
Current CPC
Class: |
A61P 11/08 20180101;
A61K 47/62 20170801; A61K 47/549 20170801; A61P 3/04 20180101; A61P
19/10 20180101; A61P 43/00 20180101; A61P 31/18 20180101; A61P
19/08 20180101; A61P 3/10 20180101; A61P 9/10 20180101; A61P 1/00
20180101; A61P 7/02 20180101; A61K 47/61 20170801; A61P 25/00
20180101; A61P 35/00 20180101; A61P 3/00 20180101 |
Class at
Publication: |
424/85.2 ;
530/307; 536/17.4; 530/308; 530/322; 530/351; 514/11.7; 514/11.9;
514/20.9 |
International
Class: |
A61K 38/20 20060101
A61K038/20; C07K 14/575 20060101 C07K014/575; C07K 14/605 20060101
C07K014/605; C07K 9/00 20060101 C07K009/00; A61P 3/00 20060101
A61P003/00; A61K 38/26 20060101 A61K038/26; A61K 38/23 20060101
A61K038/23; A61K 38/14 20060101 A61K038/14; C07K 14/435 20060101
C07K014/435; C07K 14/585 20060101 C07K014/585; C07K 14/55 20060101
C07K014/55 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 1, 2005 |
EP |
05100688.0 |
Claims
1. A conjugate of a calcitonin, ganirelix, GLP-1,
[D-Ala.sup.8]-GLP-1(7-36), adrenomedullin, ADM(27-52),
kisspeptin-10, octreotide or interleukin-2 polypeptide and an
oligosaccharide, wherein the polypeptide is conjugated to at least
one oligosaccharide-spacer residue having the structure (II)
##STR00027## wherein R is independently or (1-8C)alkoxy, or a
pharmacologically inactive flexible linking residue, the charge of
OSO.sub.3.sup.- and COO.sup.- being compensated by positively
charged counter ions and wherein one pharmacologically inactive
flexible linking residue is present or a pharmaceutically
acceptable salt thereof.
2-3. (canceled)
4. The conjugate of claim 1, having a circulating plasma level
of.ltoreq.50 nM.
5. The conjugate of claim 1, wherein the oligosaccharide per se has
an anticoagulant activity which is of subtherapeutic level when
compared to the pharmacological activity of the polypeptide per
se.
6-7. (canceled)
8. The conjugate of claim 1, wherein the oligosaccharide residue
has the structure (III) ##STR00028## wherein R is independently
OSO.sub.3.sup.- or (1-8C)alkoxy, the charge of OSO.sub.3.sup.- and
COO.sup.- being compensated by positively charged counterions.
9. The conjugate of claim 8, wherein the oligosaccharide residue
has the structure (IV) ##STR00029## wherein R is independently
OCH.sub.3 or OSO.sub.3.sup.-.
10. The conjugate of claim 9, wherein both R groups in (IV) are
OSO.sub.3.sup.-.
11-21. (canceled)
22. The conjugate of claim 1, having the structure ##STR00030##
23. The conjugate of claim 1, having the structure ##STR00031##
24. A pharmaceutical composition comprising the conjugate of claim
1 and pharmaceutically suitable auxiliaries.
25. The conjugate of claim 1 for use in therapy.
26. (canceled)
27. A process for the preparation of a conjugate of a calcitonin
ganirelix, GLP-1, [D-Ala.sup.8]-GLP-1(7-36), adrenomedullin,
ADM(27-52), kisspeptin-10, octreotide or interleukin-2 polypeptide
and an oligosaccharide, wherein the polypeptide is conjugated to at
least one oligosaccharide-spacer residue having the structure (II)
##STR00032## wherein R is independently or (1-8C)alkoxy, or a
pharmacologically inactive flexible linking residue, the charge of
OSO.sub.3.sup.- and COO.sup.- being compensated by positively
charged counter ions and wherein one pharmacologically inactive
flexible linking residue is present, or a pharmaceutically
acceptable salt thereof, comprising (a) an optional step wherein
the polypeptide is adapted for conjugation, and (b) a coupling step
wherein the optionally adapted polypeptide is reacted with the
oligosaccharide-spacer residue.
28-29. (canceled)
30. A process for the preparation of a therapeutically active
conjugate comprising a calcitonin, ganirelix, GLP-1,
[D-Ala.sup.8]-GLP-1(7-36), adrenomedullin, ADM(27-52),
kisspeptin-10, octreotide or interleukin-2 polypeptide the
conjugate having negligible anticoagulant activity, wherein the
conjugate has a longer plasma half-life than the original
polypeptide while the biological activity is essentially retained,
comprising a step wherein an oligosaccharide having the structure
(II) ##STR00033## wherein R is independently or (1-8C)alkoxy, or a
pharmacologically inactive flexible linking residue, the charge of
OSO.sub.3.sup.- and COO.sup.- being compensated by positively
charged counter ions and wherein one pharmacologically inactive
flexible linking residue is present, is covalently attached to a
polypeptide .
31-32. (canceled)
Description
[0001] The present invention relates to new conjugates of
polypeptides and oligosaccharides, a process for their preparation,
pharmaceutical compositions containing the compounds as active
ingredients, as well as the use of said compounds for the
manufacture of medicaments.
[0002] Recent developments of recombinant DNA techniques and
advanced peptide synthetic methods have permitted the commercial
production of medically useful quantities of therapeutic
polypeptides. The short half-life of many therapeutic polypeptides,
however, has historically posed a challenge to the administration
of these compounds. There are several important polypeptide-based
drugs currently in use which would benefit from increased
half-life. Examples are erythropoietin, insulin, interferon
.alpha.-2b, interferon .beta., interferon .gamma., granulocyte
colony stimulating factor, human growth hormone, granulocyte
macrophage colony stimulating factor, relaxin, urokinase,
streptokinase, tissue plasminogen activator, calcitonin,
interleukin-2 and tumor necrosis factor with half-lives
significantly) less than a few hours. Insulin, for example, has a
half-life of only about 12 minutes in man. Other examples of
polypeptides that are being developed as potential therapeutic
agents but suffer from short half-lives are adrenomedullin,
glucagon like peptide (GLP-1) and kisspeptin (metastin). Extending
the half-life of therapeutic polypeptides can improve current
treatment by allowing dosing amounts and frequency of dosing to be
reduced (Curr. Opin. Drug Disc. Dev. 2005, 8, 590-600).
[0003] Many proteins have already been subjected to studies aimed
at extending the in vivo half-life employing e.g. adaptation by
PEGylation (i.e. conjugation with a .about.1-30 kDa polyethylene
glycol-moiety; Drug Discovery Today 2005, 10, 1451-1458). Currently
available are for example PEGylated analogs of insulin with an
extended half-life. Beside the reduced clearance rate, important
aspects of the latter insulin derivatives are the reduced
immunogenicity (e.g U.S. Pat. No. 4,179,337) and increased
solubility. Further, developments in PEGylation of insulin also led
to physically and proteolytically more stable conjugates than
native insulin (see for example WO 2004/091494, WO 2002/098232, US
2005/0152848).
PEGylated erythropoietin with a longer serum half-life is for
example described in WO 2004/022577. It has further been found that
by altered glycosylation of erythropoietin, the half-life
increases. In addition, hyperglycosylated analogs of erythropoietin
were reported to have higher in vivo activity (WO 2000/24893).
Other examples of PEGylated (poly)peptides with prolonged duration
of action are glucagon-like peptide-1 (GLP-1) (WO 2005/058954, WO
2004/093823; Bioconjugate Chem. 2005, 16, 377-382; Biomaterials,
2005, 26, 3597-3606), glucose-dependent insulinotropic polypeptide
(GIP) (Bioorg. Med. Chem. Lett., 2005, 15, 4114-4117), calcitonin
(Pharm. Dev. Technol. 1999, 4, 269-275) and octreotide (Pharm. Res.
2005, 22, 743-749). Still, the use of PEG has limitations. PEG is
obtained by chemical synthesis and, like all synthetic polymers, is
polydisperse. This means that a batch of PEG consists of molecules
having different numbers of monomers, resulting in a Gaussian
distribution of the molecular weights. When a polypeptide is
PEGylated, this leads to a collection of conjugates, which may have
different biological properties, in particular in half-lives and
immunogenicity. Reproducibility of the pharmacological activities
of PEGylated polypeptides may therefore be a serious drawback of
the technique. Also, it is known that PEGylation of proteins is
often accompanied by loss of biological activity. Further, the use
of PEG may cause problems relating to excretion from the body. At
high molecular weights PEGs can accumulate in the liver, leading to
macromolecular syndrome. Consequently, PEGylation of drugs should
be performed with great care. Similar results as with PEGylation
were obtained by derivatization of polypeptides with
polysaccharides, in particular with polysialic acid chains (e.g. WO
92/22331 and WO 2001/87922). In JP 02/231077, heparin--superoxide
dismutase (SOD) conjugates are described. Preferably, a number of
heparin molecules are attached to SOD resulting in conjugates
having a longer half-life than native SOD while retaining about 90%
of the enzymatic activity. Other conjugates of polypeptides with
increased half-life are exemplified by conjugated derivatives of
insulin (WO 2003/013573, WO 05/012346) or GLP-1 (Bioorg. Med. Chem.
Lett. 2004, 14, 4395-4398) which bind to circulating serum albumin.
The binding to serum albumin in those compounds is based in
particular on hydrophobic interactions of the binding moiety within
the conjugate with human serum albumin. The higher the
hydrophobicity of that moiety, the stronger the binding affinity to
human serum albumin. Although a wide range of binding moieties is
suitable, a drawback of such conjugates is the low affinity and
selectivity of the interaction of the conjugates with human serum
albumin with as a result a poor predictability of the
pharmacodynamic behavior. Alternatively, fusing the gene for human
insulin directly to that for human serum albumin results in a
long-acting form of insulin that is active in reducing blood
glucose levels for a prolonged period after subcutaneous
administration (Duttaroy et al. Diabetes 2005, 54, 251-258).
However, in this case the bioavailability of the fused polypeptide,
as well as the binding affinity for the target receptor, is
reduced. Further, in WO 2000/40253 conjugates of, for instance, a
peptide and, specifically, glycosaminoglycan chain(s) are
disclosed, which are considered as synthetic proteoglycans. In
those conjugates the pharmacological activity of the conjugated
glycosaminoglycan has a significant impact on the therapeutic
activity of the conjugates. Also, oligosaccharides are attached to
pharmaceutically active compounds in order to increase the
solubility thereof (WO 2004/03971).
[0004] The present invention relates to new conjugates of
polypeptides with increased half-lives, being conjugates of a
polypeptide and an oligosaccharide, wherein the polypeptide is
conjugated to at least one synthetic sulfated
oligosaccharide-spacer residue, the oligosaccharide comprising 4-18
monosaccharide units and per se having affinity to antithrombin III
and the spacer being a bond or an essentially pharmacologically
inactive flexible linking residue, or a pharmaceutically acceptable
salt thereof. Preferred oligosaccharides consist of 4-6
monosaccharide units and in particular preferred are
pentasaccharides. The conjugates of the invention have improved
pharmacokinetic properties--and thus improved pharmacological
properties--when compared to the original polypeptides (i.e. the
corresponding non-conjugated polypeptides per se).
[0005] The present invention further relates to a novel technology
based on a process for the preparation of a therapeutically active
conjugate comprising a polypeptide and having negligible
anti-thrombotic activity, comprising a step wherein a synthetic
sulfated oligosaccharide, in particular a pentasaccharide, which
per se has affinity to antithrombin III (ATIII), is covalently
attached to a polypeptide through a bond or an essentially
pharmacologically inactive flexible linking residue.
ATIII is a serine protease inhibitor, present in blood plasma,
which interrupts the coagulation cascade to provide a feed back
loop. The half-life of a sulfated pentasaccharide is essentially
based on its affinity to ATIII (see e.g. F. Paolucci et al. Clin.
Pharmacokinet. 2002; 41 Suppl. 2: 11-18). In the conjugates of this
invention the serum half-life is longer than the half-life of the
original polypeptide as a result of the half-life of the
pentasaccharide which largely accounts for the half-life of the
conjugate. Furthermore, the conjugates of the invention not only
have a longer half-life, but they also have tunable pharmacokinetic
properties based on the specific interaction between the
pentasaccharide part of the conjugate and ATIII (the latter
interaction is described e.g. in Westerduin et. al. Bioorg. Med.
Chem. 1994, 1267-1280; van Amsterdam et al., Arterioscler Thromb
Vasc Biol. 1995; 15:495-503). In an embodiment of the present
invention the oligosaccharide-polypeptide conjugate (the
oligosaccharide in particular consisting of 4-6 monosaccharide
units and most particularly being a pentasaccharide) has a
circulating plasma level of .ltoreq.50 nM. Up to this concentration
the ATIII-mediated anticoagulant activity of the oligosaccharide
(in particular pentasaccharide) is insignificant in particular with
respect to bleeding risks. (see for instance (1) F. Donat et al.,
Clin. Pharmacokinet. 2002; 41 Suppl. 2: 1-9; (2) S. J. Keam et al.
Drugs 2002; 62 (11):1673-1685 and (3) The Rembrandt Investigators
Circulation 2000; 102: 2726-2731). According to an embodiment of
this invention, the oligosaccharide (in particular consisting of
4-6 monosaccharide units and most particularly being a
pentasaccharide) used in the conjugates per se has an anticoagulant
activity which is of subtherapeutic level when compared to the
pharmacological activity of the polypeptide per se. Subtherapeutic
in this respect means: having a lower than therapeutic effect and
without side-effects, such as bleeding risks. For example, diabetes
type 1 patients require (long half-life) insulin injections to
complement (basal) therapeutic plasma levels of .about.[0.1-1.0]
nM, which is well in the subtherapeutic range of the
pentasaccharides used in the present conjugates. A person skilled
in the art will understand how to select conjugates with a proper
balance between the therapeutic levels of the polypeptide and the
pentasaccharide, respectively. The polypeptides in the conjugates
of the present invention retain their biological activity.
Furthermore, the linear pharmacokinetic behavior of ATM-bound
pentasaccharide in the conjugates of this invention accounts for a
highly predictable therapeutic effect of the conjugated
polypeptides, since the conjugates remain largely in the
intravascular compartment after i.v. or s.c. dosing.
[0006] The oligosaccharide residue in the conjugates of this
invention is a residue from a synthetic sulfated oligosaccharide
which per se has affinity to antithrombin III (ATIII). Sulfated
oligosaccharides, and in particular pentasaccharides, generally
have affinity to ATIII, however, a person skilled in the art can
easily check the affinity of an oligosaccharide to ATIII (van
Amsterdam et al., Arterioscler Thromb Vasc Biol. 1995; 15:495-503)
and select the desired affinity level. Suitable synthetic
oligosaccharide residues and in particular pentasaccharide residues
may be derived from the oligo- and pentasaccharides disclosed in EP
0,454,220, EP 0,529,715, WO 98/03554, WO 99/36428, J. Med. Chem.
2005; 48, 349-352, Angew. Chem. Intl. Ed. Engl. 1994, 32, 1671-1690
and the like.
The oligo- and pentasaccharide residues may be conjugated to the
polypeptide directly or via a linking residue attached to any
chemically suitable position within the pentasaccharide residue.
Therefore, in an embidoment of this invention the conjugates are
conjugates wherein the oligosaccharide-spacer residue has the
structure (I)
##STR00001##
wherein one essentially pharmacologically inactive flexible linking
residue is present and wherein R is independently OSO.sub.3.sup.-,
(1-8C)alkoxy or an essentially pharmacologically inactive flexible
linking residue, and Ra is independently OSO.sub.3.sup.-,
(1-8C)alkoxy, an essentially pharmacologically inactive flexible
linking residue or an oligosaccharide residue, comprising 1-13
monosaccharide units, and Rb is independently (1-8C)alkoxy, an
essentially pharmacologically inactive flexible linking residue or
an oligosaccharide residue, comprising 1-13 monosaccharide units,
the charge being compensated by positively charged counterions.
More preferred are conjugates wherein the oligosaccharide-spacer
residue is a pentasaccharide- spacer residue having the structure
(II)
##STR00002##
wherein one essentially pharmacologically inactive flexible linking
residue is present and wherein R is independently OSO.sub.3.sup.-
or (1-8C)alkoxy, or an essentially pharmacologically inactive
flexible linking residue, the charge being compensated by
positively charged counter ions. Further preferred are conjugates
wherein the pentasaccharide residue has the structure (III)
##STR00003##
wherein R is independently OSO.sub.3.sup.- or (1-8C)alkoxy, the
charge being compensated by positively charged counterions. Highly
preferred compounds according to the invention are compounds
wherein the pentasaccharide residue has the structure (IV)
##STR00004##
wherein R is independently OCH.sub.3 or OSO.sub.3.sup.-, and in
particular both R groups in (II) are OSO.sub.3.sup.-.
[0007] According to this invention, synthetic sulfated
oligosaccharide residues, in particular pentasaccharide residues,
with affinity to ATIII can be conjugated to any polypeptide. For
example, the polypeptide can be a bioactive peptide (e.g., 3 to 50
amino acids in length) or can be a longer polypeptide that may or
may not have catalytic activity. Non-limiting examples of bioactive
peptides include neurotransmitters such as conantokin G, dynorphin,
endorphin, enkephalin, or neurotensin; gastric activators such as
bombesin, motilin. or gastrin; calcium regulators such as
calcitonin or parathyroid hormone (PTH); bone resorption modulators
such as osteoprotegerin (OPG); stimulators of osteoblastic activity
such as adrenomedullin or truncated derivatives thereof such as
ADM(27-52); hormones such as vasoactive intestinal polypeptide,
corticotropin, secretin; hormone inhibitors such as somatostatin;
hormone stimulators such as melanocyte stimulating hormone,
luteinizing hormone releasing factor, or sermorelin; anti-diabetic
agents such as glucagons, amylin, glucagon-like peptide-1 (GLP-1)
or truncated derivatives thereof such as GLP-1(7-36), GLP-2,
glucose-dependent insulinotropic polypeptide (GIP) or insulin
("Humulin," Eli Lilly); anti-infectives such as lysostaphin;
appetite suppressing hormones such as obestatin; vasoconstrictors
such as angiotensin II; vasodilators such as bradykinin, substance
P or kallidin; natriuretic agents such as atrial natriuretic
polypeptide (ANP); antidiuretic hormones such as vasopressin or
desmopressin; and oxytocic agents such as oxytocin. Additional
examples of polypeptides that can be used include human growth
hormone ("Humantrope," Genentech); rLH; rG-CSF ("Neupogen," Amgen);
erythropoietin ("Epogen," Amgen); interferon .alpha.-2a, interferon
.alpha.-2b, interferon .beta., or interferon .gamma.; factor VIII
or other blood clotting factors such as protein C or factor VIIa;
follicle stimulating hormone (FSH); a cytokine such as an
interleukin (IL) (e.g., IL-1 -1, -2, -3, -4, -5, -6, -7, -8, -9,
-10, -11. -12, or -18); hemoglobin; superoxide dismutase; soluble
CD4 or CD4 receptor; platelet GpIIb/IIIa analogs and their
receptors ("ReoPro," Johnson & Johnson); glucocerebrosidase
("Ceredase" or "Cerezyme," Genzyme); ACTH; somatotropin;
parathyroid hormone, antidiuretic hormone; prolactin; rHGH, such as
pegvisomant ("Somavert", Pfizer); GnRH agonists, such as leuprolide
("Lupron", "Leprorelin", Takeda) or nafareline ("Synarel", Roche)
and GnRH antagonists, such as ganirelix ("Antagon", Organon); GHRH
agonists, such as sermorelin ("Geref", Serono); octreotide
("Sandostatin", Novartis); or thrombolytics such as streptokinase,
staphylokinase, urokinase, or tissue plasminogen activator
("Activase," Genentech); metastin (KISS1 or kisspeptin-54) or
truncated derivatives thereof such as kisspeptin-10.
Preferred polypeptides have a molecular weight of .about.0.3-50
kDa. Other preferred polypeptides have a molecular weight of
.about.0.3-20 kDa. Also preferred are polypeptides which have a
molecular weight of .about.0.3-7.5 kDa. Further preferred
polypeptides are insulin (t1/2=12 min; Mw=5.8 kDa), calcitonin (t
1/2=20 min; Mw=3.4 kDa), GLP-1(7-36) (t 1/2=6 min; Mw=3.4 kDa),
adrenomedullin (t 1/2=20 min; Mw=6.0 kDa), ADM(27-52) (Mw3.0 kDa),
octreotide (t 1/2=1.7 h, Mw=1.0 kDa), interleukin-2 (t 1/2=20 min;
Mw=15 kDa) and ganirelix (t 1/2=12 h; Mw=1.6 kDa). In particular
preferred are insulin and [D-Ala.sup.8]-GLP-1(7-36). A further
embodiment of this invention is a polypeptide conjugate
monosubstituted with a pentasaccharide-spacer residue.
[0008] The spacer is a bond or an essentially pharmacologically
inactive, flexible, linking residue. Preferably, the spacer is an
essentially pharmacologically inactive flexible linking residue, in
particular having 10-50 atoms counted along the "backbone" of the
spacer, the oxygen of the oligosaccharide residue not included. The
term "essentially pharmacologically inactive" as used herein means
that the spacer does not contain atoms or groups which show
pharmacologically activity per se at the doses at which the
compounds of the invention are therapeutically effective. Thus, at
doses at which the compounds of the present invention are used as
therapeutic drugs, the nature of the spacer does not lead to
demonstrable pharmacological side-effects.
[0009] The spacer may comprise (somewhat) rigid elements, such as
ring structures and unsaturated bonds. The spacer of the compounds
of the invention is preferably flexible. Suitable spacers may
easily be designed by a person skilled in the art. For synthetic
reasons longer spacers are considered less suitable, however,
longer spacers may still successfully be applied in the compounds
of the present invention. Preferred spacers comprise at least one
--(CH.sub.2CH.sub.2O)-- element.
[0010] Representative examples of the conjugates of the present
invention are conjugates of the following structures:
##STR00005##
wherein R1=R2=H,
##STR00006##
or wherein R1=R3=H,
##STR00007##
and wherein Y is selected from structures A, B, C and D
##STR00008## ##STR00009##
or other salts thereof, but also conjugates wherein the spacer is a
different one or is attached to the pentasaccharide at another
position. Preferred is the sodium salt. And preferably, Y is
selected from structures A and B.
[0011] Commonly used chemical abbreviations that are not explicitly
defined in this disclosure may be found in The American Chemical
Society Style Guide, Second Edition, American Chemical Society,
Washington, DC (1997), "2001 Guidelines for Authors" J. Org. Chem.
66(1), 24A (2001), "A Short Guide to Abbreviations and Their Use in
Polypeptide Science" J. Polypeptide. Sci. 5, 465-471 (1999).
The term polypeptide refers to a chain of at least three amino
acids, regardless of post-translational modifications. Polypeptides
can be naturally occurring, chemically synthesized, or
recombinantly produced polymers of amino acids. Polypeptides that
have three to 50 amino acids typically are classified as peptides.
The phrase "polypeptide with catalytic activity" means an enzyme.
The term insulin as used herein refers to the naturally occurring
hypoglycemic polypeptide found in mammals, including humans, rat,
guinea pig, and rabbits, as well as to recombinant insulin and
similar hypoglycemic polypeptides disclosed in U.S. Pat. Nos.
4,652,525, 4,431,740, 5,268,453, 5,506,202, 5,514,646, and
5,700,662. In the description of the conjugates of the invention
further the following definitions are used. The terms (1-4C)alkyl
and (1-8C)alkyl mean a branched or unbranched alkyl group having
1-4 and 1-8 carbon atoms, respectively, for example methyl, ethyl,
propyl, isopropyl, butyl, sec-butyl, tert-butyl, hexyl and octyl.
Methyl and ethyl are preferred alkyl groups. The term (1-8C)alkoxy
means an alkoxy group having 1-8 carbon atoms, the alkyl moiety
having the meaning as previously defined. Methoxy is a preferred
alkoxy group. The spacer length is the number of atoms of the
spacer, counted along the shortest chain between the
oligosaccharide residue and the polypeptide, not counting the
oxygen atom of the oligosaccharide residue which is connected to
the spacer.
[0012] An embodiment of this invention is further a process for the
preparation of a therapeutically active conjugate comprising a
polypeptide, the conjugate having negligible anti-thrombotic
activity, wherein the conjugate has a longer plasma half-life than
the original polypeptide while the biological activity is
essentially retained, comprising a step wherein a synthetic
sulfated oligosaccharide, in particular wherein the oligosaccharide
consists of 4-6 monosaccharide units, and most particularly being a
sulfated pentasaccharide, per se having affinity to antithrombin
III is attached to the polypeptide, optionally through an
essentially pharmacologically inactive flexible linking
residue.
General Synthetic and Analytic Aspects
Synthesis of Pentasaccharides
[0013] The ATIII-binding oligosaccharide, in particular
pentasaccharide, of the compounds of the present invention can be
prepared as described for instance in Angew. Chem. Intl. Ed. Engl.
1994, 32, 1671-1690. Different oligo- and pentasaccharides with
altered affinity for ATIII may be obtained by varying the
intermediate mono-, di- or tetrasaccharide building blocks, for
instance, by introduction of (permanent) alkyl groups or
application of different (temporary) protecting groups giving
access to differently sulfated oligo- and pentasaccharides in a
controlled fashion (e.g. Westerduin et. al. Bioorg. Med. Chem.
1994, 1267). The spacer may be introduced as described for instance
in WO 2001/42262. The oligo- and pentasaccharide-spacer molecule
may further be derivatised with linking residues such as the
gamma-maleimido butyryl (GMB) group, the N-hydroxysuccinimide (NHS)
group or optionally protected thiol group (e.g. Angew. Chem. Intl.
Ed. Engl. 1996, 35, 331-333) to allow direct coupling with an
optionally modified polypeptide.
Conjugation
[0014] In general, the conjugates of the invention are produced
according to a process comprising (a) an optional step wherein the
polypeptide is adapted for conjugation, and (b) a coupling step
wherein the optionally adapted polypeptide is reacted with an
oligo- or pentasaccharide-spacer molecule.
[0015] General synthetic methods for the production of
bioconjugates are described in "Bioconjugate Techniques" by Greg T.
Hermanson, 1996, Academic Press. In addition, for conjugation may
be considered a Staudinger ligation (such as described by K. L.
Kiick et al. Proc. Nat. Acad. Sci. 2002; 99:19-24) or a Huisgen's
1,3-dipolar cycloaddition using a pentasaccharide derivative and
polypeptide independently modified with an alkyne or azide
functional group. Alternatively, enzymatic reactions such as the
regioselective IgA protease mediated elongation of polypeptides at
the N-terminus (as described by M. Lewinska et al. in Bioconjugate
Chem. 2004, 15, 231-234) or the transglutaminase catalyzed
introduction of amino spacer containing oligosaccharides (as
described by M. Sato et al. in J. Am. Chem. Soc. 2004, 126,
14013-14022) can be adapted for conjugation of a pentasaccharide
spacer residue to an optionally modified polypeptide. Further,
PEGylation of for instance insulin, GLP-1 and octreotide is well
documented. In these proteins, a .about.5-30 kDa PEG-moiety can be
introduced without abolishing their biological activities; such
strategies may be followed for the (site-specific) introduction of
a pentasaccharide(spacer)-moiety. Furthermore, it is a prerequisite
that binding of the pentasaccharide-conjugate to ATIII (.about.50
kDa) has no substantial deleterious effect on the biological
activity of the polypeptide.
Insulin: The N-terminal B-1 and near C-terminal B-29 Lysine amino
functions are not essential for the bioactivity of insulin.
B1-PEGylated insulin has been prepared (S. W. Kim et al., Adv. Drug
Del. Rev. 2002, 54, 505-530) in 20% overall yield via reaction of a
N-hydroxysuccinimide (NHS) activated PEG derivative with
di-N-Boc-protected insulin. Similar reaction of a bifunctional
coupling reagent such as N-maleimidobutyryloxy succinimide ester
(GMBS) gives access to B1-modified insulin pentasaccharide
conjugates. Alternatively, the B29 Lys residue of unprotected
Zn.sup.2+-insulin can be selectively modified with an excess of NHS
ester at pH .about.10-11 in .about.60% yield. Other well
established methods for the regioselective conjugation to insulin
may be adapted from WO 98/02460, WO 2004/091494, WO 2005/012346, US
2005/0152848, Jensen et al. J. Pept. Sci. 2005, 11, 339-346, Lee et
al. Bioconj. Chem. 2005, 16, 615-620, Jain et al. Biochim. Biophys.
Act. 2003, 1622, 42-49, Tessmar et al. Tissue Engin. 2004, 10, 3,
441-453). Ganirelix: NHS ester derivatives of a pentasaccharide can
be conjugated to the free N-terminal amino group of de-N-Ac
ganirelix or an amino spacer containing pentasaccharide derivative
can be conjugated, optionally via an additional spacer, to the free
terminal carboxylic acid group of desamido ganirelix which can in
turn be obtained by advanced (solid phase) peptide synthesis as
described for instance in J. Med. Chem. 1992, 35, 3942-3948.
Octreotide: The N-terminal D-Phe amino acid residue of octreotide,
a commercially available peptide, can be modified with up to 5 kDa
PEG without abolishing the bioactivity (D. Hee et al. Pharm. Res.
2005, 22, 743-749). Regiospecific functionalization of the
N-terminal amino group can be achieved with an excess of
bifunctional NHS ester linking reagent at pH .about.6, upon which
further conjugation to a carrier pentasaccharide can be effected
according to general synthetic methods for the production of
bioconjugates as described above (e.g. by conjugation of a
pentasaccharide spacer residue containing a thiol group to a
maleimide derivative of octreotide). ADM(27-52): The N-terminal
half of full length adrenomedullin (ADM) is not essential for its
osteogenic activity and inhibitory effect on vascular
calcification. Regiospecific conjugation of a
pentasaccharide-spacer residue to the N-terminal Ala residue of
ADM(27-52) can be achieved by synthesizing optionally N-terminally
modified ADM(27-52) using well established methods employing solid
phase peptide synthesis and general synthetic methods for the
production of bioconjugates as described above.
[D-Ala.sup.8]-GLP-1(7-36): The C-terminal portion of GLP-1(7-36)
and it's derivatives such as Exendin-4(1-39) form an
.alpha.-helical structure in which amino acid residues are exposed
that are important for receptor binding. Extension of this amino
acid sequence with an additional lysine residue that is modified at
the N.sup.c-position with a maleimide function, using an adapted
solid phase peptide synthesis as described for instance in WO
2005/058954, still exhibits receptor binding and in vivo functional
activity after covalent binding to the Cys.sup.34 amino acid of
human serum albumin, while the proteolytic stability may (further)
be improved by incorporating a D-Ala residue at position 2 (Bioorg.
Med. Chem. Lett. 2004, 14, 4395-4398). In a similar manner
GLP-1(7-36) or analogues thereof can be conjugated to a suitably
functionalized pentasaccharide-spacer moiety (e.g containing a
thiol group). Alternatively, a Cys amino acid can be incorporated
in the peptide sequence, preferably at position(s) 11, 12, 16, 22,
23, 24, 25, 26, 27, 30, 34, 35 or 36 or added at position 37, and
which may be coupled to a suitably functionalized
pentasaccharide-spacer moiety (e.g. containing a maleimide group),
using methods similar as described for the PEGylation of GLP1
derivatives (WO 2004/093823). Furthermore, conjugates of GLP-1 may
be obtained by direct coupling to a bifunctional NHS ester linking
reagent to GLP-1, followed by separation of the positional isomers
(as described for instance for the direct PEGylation of GLP-1 by
Lee et al. Bioconjugate Chem. 2005, 16, 377-382) and coupling to a
suitably functionalized pentasaccharide-spacer moiety.
Interleukin-2 (IL-2): the free Cys.sup.125 amino acid of
commercially available native recH-IL2, or free (additional) Cys
amino acids of IL2 muteins that are still biologically active, can
be reacted with a pentasaccharide-spacer moiety containing a
maleimide group according to a similar protocol as described for
the PEGylation of IL2 (U.S. Pat. No. 5,206,344) with
PEG-maleimide.
[0016] The peptide coupling, a possible procedural step in the
above described method to prepare the compounds of the invention,
can be carried out by methods commonly known in the art for the
coupling--or condensation--of peptide fragments such as by the
azide method, mixed anhydride method, activated ester method, the
carbodiimide method, or, preferably, under the influence of
ammonium/uronium salts like TBTU, especially with the addition of
catalytic and racemisation suppressing compounds like
N-hydroxysuccinimide, N-hydroxybenzotriazole and
7-aza-N-hydroxybenzotriazole. Overviews are given in The Peptides,
Analysis, Synthesis, Biology, Vol. 3, E. Gross and. J. Meienhofer,
eds. (Academic Press, New York, 1981) and Peptides: Chemistry and
Biology, N. Sewald and H.-D. Jakubke (Wiley-VCH, Weinheim,
2002).
[0017] Amine functions present in the compounds may be protected
during the synthetic procedure by an N-protecting group, which
means a group commonly used in peptide chemistry for the protection
of an .alpha.-amino group, like the tert-butyloxycarbonyl (Boc)
group, the benzyloxycarbonyl (Z) group, the
9-fluorenylmethyloxycarbonyl (Fmoc) group or the phthaloyl (Phth)
group, or may be introduced by demasking of an azide moiety.
Overviews of amino protecting groups and methods for their removal
is given in the above mentioned The Peptides, Analysis, Synthesis,
Biology, Vol. 3 and Peptides: Chemistry and Biology.
[0018] The compounds of the invention, which may occur in the form
of a free base, may be isolated from the reaction mixture in the
form of a pharmaceutically acceptable salt. The pharmaceutically
acceptable salts may also be obtained by treating the free base of
formula (I) with an organic or inorganic acid such as hydrogen
chloride, hydrogen bromide, hydrogen iodide, sulfuric acid,
phosphoric acid, acetic acid, propionic acid, glycolic acid, maleic
acid, malonic acid, methanesulphonic acid, fumaric acid, succinic
acid, tartaric acid, citric acid, benzoic acid, ascorbic acid and
the like.
[0019] The compounds of this invention or intermediates thereof may
possess chiral carbon atoms, and may therefore be obtained as a
pure enantiomer, or as a mixture of enantiomers, or as a mixture
containing diastereomers. Methods for obtaining the pure
enantiomers are well known in the art, e.g. crystallization of
salts which are obtained from optically active acids and the
racemic mixture, or chromatography using chiral columns. For
diastereomers straight phase or reversed phase columns may be
used.
Physico- and Biochemical Analysis
[0020] Several techniques to monitor the effect of the reaction
with bifunctional linkers and/or reactive pentasaccharide-moieties
on the bioactivity of the protein are available. In this respect,
biomolecular interaction analysis (BIA), using soluble receptor
molecules as complementary binding agents (insulin) and
determination of enzyme activity are valuable tools. Binding
studies of the pentasaccharide-conjugate to ATIII can be included
in these assays as well. Ion exchange, size exclusion
chromatography and ATIII affinity chromatography are available
methods for subfractionation of the pentasaccharide conjugates,
while electrophoresis techniques are suitable for orthogonal,
qualitative and quantitative characterization (e.g. SDS-PAGE, CZE).
Conjugation sites can be identified by MALDI-TOF MS analysis and
N-terminal sequencing of the conjugates.
Pharmacokinetic (PK) Studies
[0021] PK studies to determine the in vivo half-life of the
unmodified polypeptide and the corresponding pentasaccharide
conjugates can be carried out in rats. Several options are
available, e.g. radiolabeling with .sup.125I employing Iodogen or
lactoperoxidase ionisation to induce electrophilic substitution or
using Bolton-Hunter reagent as labeling moiety and determining
gamma-radiation in plasma samples. Other methods known in the art
are based on injection of unlabeled conjugates followed by
immunochemical analysis through ELISA or Luminex technology.
Pharmacological Evaluation
[0022] The pharmacological effects of conjugation of polypeptides
of the invention to an ATIII binding pentasaccharide can be studied
in in vitro assays and in vivo animal models as described
below.
[0023] Insulin is a 5.8 kDa protein consisting of two peptide
chains which are held together by two disulfide bridges.
Site-specific chemical modification at one of the Lys
.epsilon.-amino or N-terminal .alpha.-amino groups is well
documented. The effect of conjugation of a pentasaccharide to
insulin can be studied by analyzing serum samples for glucose,
insulin and C-peptide content (a biomarker to correct for
endogenous insulin secretion). A glucometer and human insulin and
C-peptide radioimmunoassays are commercially available. In vivo
effects of insulin on the blood glucose levels can be measured in
rats or Beagle dogs.
[0024] The pharmacological in vitro and in vivo effects of
pentasaccharide conjugation of the decapeptide mimetic GnRH
antagonist ganirelix can be studied in established assays and
animal models. Advanced polypeptide synthesis will deliver a
well-defined molecule of which the effects can be compared to
ganirelix blocking oocyte maturation and ovulation in mouse and
rat. Comparison of biological half-life of ganirelix and its
pentasaccharide-conjugated counterpart may for instance be studied
by determining at which time after administration the natural
process of maturation and ovulation has restored.
[0025] GLP-1(7-36) is a well known and well studied insulinotropic
endocrine hormone inducing numerous biological effects such as
stimulating insulin secretion, inhibiting glucagon secretion,
gastric or intestinal motility, enhancing glucose utilization and
inducing weight loss. It is rapidly degraded by dipeptidyl
peptidase IV (DPPIV). The latter premature degradation may for
instance be circumvented by replacement of the amino acid residue
at position 8 (e.g. by D-alanine). Other approaches such as
modification with large fatty acid chains or PEG have resulted in
biologically active GLP-1 or analogs thereof (as defined in e.g. WO
2004/093823). The (additional) stabilizing effect of conjugating an
optionally modified GLP-1(7-36) derivative to a carrier
pentasaccharide can be studied by measuring the stability of the
conjugate in vitro in the presence of DPPIV and in vivo by
measuring the plasma half life using immunochemical analysis. The
functional activity of the GLP-1 pentasaccharide conjugate can be
determined in vitro by measuring the ability to bind to and
activate the GLP-1 receptor and in vivo pharmacodynamic effects can
be studied by analyzing serum samples for glucose and insulin.
[0026] Adrenomedullin is a 52-amino acid polypeptide with numerous
biological functions such as vasodilation, bronchodilation,
neurotransmission, growth regulation and regulation of bone
formation. The truncated fragment ADM(27-52) lacks the structural
requirements for vasodilator activity but is still able to
stimulate the growth of cultured rat osteoblasts in a
dose-dependent manner (Regulatory Peptides 2003, 112, 79-86). In
addition, it was recently established that ADM(27-52) inhibits
vascular calcification in rats (Regulatory Peptides 2005, 129,
125-132) and may thus have potential therapeutic application in the
prevention of artery calcification. The effect of conjugating
ADM(27-52) to a carrier pentasaccharide may be assessed by well
established assays, measuring the in vitro osteogenic activity in
cultures of actively growing fetal rat osteoblasts or the in vivo
increase in index of bone formation (without affecting bone
resorption).
[0027] Octreotide is a synthetic octapeptide analogue of
somatostatin and is clinically used for the treatment of acromegaly
and certain endocrine tumors. It has been shown that long acting
depot formulations (e.g. Sanostatin LAR depot, Novartis Pharma,
Basel, Switzerland) are at least as effective in the lowering of
plasma growth hormone and insulin-like growth factor (IGF-I) levels
compared with three-daily subcutaneous injections. The effect of
conjugation of octreotide to a pentasaccharide on its
pharmacokinetic and pharmacodynamic properties can be studied in
male rats by using established radio immunoassays to determine
levels of conjugated octreotide and altered levels of IGF-I.
[0028] Interleukin-2 (IL-2) is a protein produced naturally in the
body by white blood cells (T-lymphocytes) and is an important
protein of the immune system. It is commercially available
(Aldesleukin, Proleukin.RTM., Chiron, U.S.) as a drug and is used
in the treatment for some types of cancer (hairy cell leukemia) and
is used in conjunction with anti-HIV therapy to induce increases in
CD4 cell counts. The specific bioactivity of pentasaccharide-IL-2
conjugates can be determined in vitro using the IL-2 cell
proliferation bioassay described by Gillis et al. (J. Immunol.
1978, 120, 2027-2032).
Pharmaceutical Formulations
[0029] The conjugates of the invention may be administered
enterally or parenterally. The exact dose and regimen of these
compounds and compositions thereof will necessarily be dependent
upon the biological activity of the polypeptide per se, the needs
of the individual subject to whom the medicament is being
administered, the degree of affliction or need and the judgment of
the medical practitioner. In general, parenteral administration
requires lower dosages than other methods of administration which
are more dependent upon absorption. However, the daily dosages are
for humans preferably 0.0001-1 mg per kg body weight, more
preferably 0.001-0.1 mg per kg body weight.
The medicament manufactured with the compounds of this invention
may also be used as adjuvant in therapy. In such a case, the
medicament is administered with other compounds useful in treating
such disease states. Mixed with pharmaceutically suitable
auxiliaries, e.g. as described in the standard reference, Gennaro
et al., Remington's Pharmaceutical Sciences, (18th ed., Mack
Publishing Company, 1990, see especially Part 8: Pharmaceutical
Preparations and Their Manufacture) the compounds may be compressed
into solid dosage units, such as pills, tablets, or be processed
into capsules or suppositories. By means of pharmaceutically
suitable liquids the compounds can also be applied in the form of a
solution, suspension, emulsion, e.g. for use as an injection
preparation, or as a spray. For making dosage units, e.g. tablets,
the use of conventional additives such as fillers, colorants,
polymeric binders and the like is contemplated. In general any
pharmaceutically acceptable additive which does not interfere with
the function of the active compounds can be used. Suitable carriers
with which the compositions can be administered include lactose,
starch, cellulose derivatives and the like, or mixtures thereof,
used in suitable amounts. When administration is intravenous,
pharmaceutical compositions may be given as a bolus, as two or more
doses separated in time, or as a constant or non-linear flow
infusion. Thus, compositions of the invention can be formulated for
any route of administration. Typically, compositions for
intravenous administration are solutions in sterile isotonic
aqueous buffer. Where necessary, the composition may also include a
solubilizing agent, a stabilizing agent, and a local anesthetic
such as lidocaine to ease pain at the site of the injection.
Generally, the ingredients will be supplied either separately, e.g.
in a kit, or mixed together in a unit dosage form, for example, as
a dry lyophilized powder or water free concentrate. The composition
may be stored in a hermetically sealed container such as an ampule
or sachette indicating the quantity of active agent in activity
units. Where the composition is administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade "water for injection," saline, or other suitable intravenous
fluids. Where the composition is to be administered by injection,
an ampule of sterile water for injection or saline may be provided
so that the ingredients may be mixed prior to administration.
Pharmaceutical compositions of this invention comprise the
compounds of the present invention and pharmaceutically acceptable
salts thereof, with any pharmaceutically acceptable ingredient,
excipient, carrier, adjuvant or vehicle.
[0030] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, suitable methods and materials are described in
this document. All publications, patent applications, patents, and
other references mentioned herein are incorporated by reference in
their entirety. In case of conflict, the present specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and not intended to be
limiting.
[0031] The invention is further illustrated by, but not limited to,
the following examples. It will be understood that various
modifications may be made with different pentasaccharides, spacers
and polypeptides without departing from the spirit and scope of
this invention.
LEGENDS TO THE FIGURES
[0032] FIG. 1. Recognition of pentasaccharide-insulin conjugate 6
(Insulin-penta) by insulin-specific ELISA.
[0033] FIG. 2A. Biomolecular interaction analysis of
pentasaccharide(PS)-insulin conjugate 6.
Reaction of immobilized anti-insulin antibody with insulin
conjugate with subsequent binding to human insulin receptor.
[0034] FIG. 2B. Biacore analysis of pentasaccharide(PS)-insulin
conjugate 6.
Reaction of immobilized anti-insulin antibody with insulin
conjugate with subsequent binding of human ATIII.
[0035] FIG. 3. MALDI-TOF analysis of monosubstituted
pentasaccharide-insulin conjugate 6.
[0036] FIG. 4. HP-SEC analysis of monosubstituted
pentasaccharide-insulin conjugate 6 on Superdex 30.
[0037] FIG. 5. hATIII binding of reference pentasaccharide-spacer
residues 7, 8 and 9 (BIA study).
[0038] FIG. 6. hATIII binding of insulin-pentasaccharide conjugates
24, 28 and 29 (BIA study).
[0039] FIG. 7. Mass spectrometric analysis (ESI-QTOF) of
insulin-pentasaccharide conjugate 24
[0040] FIG. 7A. Comparison of an experimentally determined and
calculated typical isotope distribution of an
insulin-pentasaccharide conjugate (ESI-QTOF, M.sup.5+, compound
24)
[0041] FIG. 8. hATIII binding of compounds 31, 32, 36, 37 (Biacore
study).
[0042] FIG. 9. hATIII binding of compound 39 (as determined by
BIA).
[0043] FIG. 10. hATIII binding profile of compound 41 (as
determined by BIA).
[0044] FIG. 11. hATIII binding profile of compound 44 (as
determined by BIA).
[0045] FIG. 12. SDS-PAGE and Western blot analyses of compound
47
[0046] FIG. 13. Mean plasma levels (mean.+-.s.e.m.) determined by
measurement of the insulin concentration after i.v. administration
of 3.5 nmol/kg recH insulin (open circles) or
pentasaccharide-insulin conjugate 6 (triangles).
[0047] FIG. 14. Mean plasma levels (mean.+-.s.e.m.) expressed as %
of the concentration measured at T=1 minute after i.v.
administration of .sup.125I-labeled conjugate 29 (open squares), 24
(open circles) and 28 (open triangles) and of recH insulin itself
(closed bullets).
[0048] FIG. 15. Mean plasma levels (mean.+-.s.e.m.) expressed as %
of the concentration measured at T=1 minute after i.v.
administration of .sup.125I labeled conjugate 31 (open squares) and
32 (closed triangles). (Data not corrected for dehalogenation).
[0049] FIG. 16. Mean plasma levels (mean.+-.s.e.m.) expressed as %
of the concentration measured at T=1 minute after i.v.
administration of .sup.125I labeled conjugate 39 (open squares) and
of .sup.125I labeled ADM(27-52) (closed triangles). (data not
corrected for dehalogenation).
[0050] FIG. 17. Mean plasma levels (mean.+-.s.e.m.) expressed as %
of the concentration measured at T=1 minute after i.v.
administration of .sup.125I labeled conjugate 41 (closed triangles)
and of .sup.125I labeled GLP-1 (open squares).
[0051] FIG. 18. Detection of the pentasaccharide-insulin conjugate
6-ATIII complex with anti-human and anti-rabbit ATIII
antibodies.
[0052] FIG. 19. Mean glucose levels (mean.+-.s.e.m.) after i.v.
administration of 7 nmol/kg pentasaccharide-insulin conjugate 6
(open triangles) or 3.5 nmol/kg recH-insulin (open circles).
[0053] FIG. 20. Mean glucose levels (mean.+-.s.e.m.) after i.v.
administration of 12 nmol/kg pentasaccharide-insulin conjugate 26
(closed triangles) or 24 (closed circles) compared to the glucose
levels after treatment with 9 nmol/kg recH-insulin (open
circles).
[0054] FIG. 21. Mean glucose levels (mean.+-.s.e.m.) after i.v.
administration of 24 nmol/kg pentasaccharide-insulin conjugate 27
(closed squares) or 25 (closed diamonds) compared to the glucose
levels after treatment with 9 nmol/kg recH-insulin (open circles)
or 48 nmol/kg of Insulin Detemir (open triangles).
[0055] FIG. 22. Mean glucose levels (mean.+-.s.e.m.) after i.v.
administration of 24 nmol/kg pentasaccharide-insulin conjugate 24
(closed triangles), 28 (closed circles) or 29 (closed squares)
compared to the glucose levels after treatment with 9 nmol/kg
recH-insulin (open circles) or 24 nmol/kg of Insulin Detemir (open
diamonds).
EXAMPLES
[0056] Abbreviations used: ACN acetonitrile AcOH acetic acid ADM
adrenomedullin (h)ATIII (human) anti-thrombin III AUC area under
the curve BIA biomolecular interaction analysis Boc.sub.2O
di-tert-butyl dicarbonate Cl clearance DCCI
N,N'-dicyclohexylcarbodiimide DIPEA diisopropylethylamine DMF
N,N'-dimethylformamide DMSO dimethyl sulfoxide EDTA ethylenediamine
tetra-acetate ELISA enzyme-linked immunosorbent assay Equiv.
equivalents ESI electron spray ionization GLP-1 glucagon-like
peptide I GMB gamma-maleimidobutyryl GMBS gamma-maleimidobutyric
acid N-hydroxy succinimic ester HBS-EP hepes buffered saline
containing EDTA and polyethylene glycol HPLC high performance
liquid chromatography HP-SEC high performance size exclusion
chromatography HRP horse-radish peroxidase i.v. intravenous IL-2
interleukin-2 MALDI-TOF matrix assisted laser disorption ionisation
time of flight MoAb monoclonal antibody MRT mean residence time MS
mass spectrometry NMM N-methyl morpholin NMR nuclear magnetic
resonance PAGE polyacrylamide gel electrophoresis PBS
phosphate-buffered saline PS pentasaccharide Q-TOF quadropole time
of flight recH recombinant human RT room temperature Rt retention
time SDS sodium docecyl sulfate TBTU
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate
TCA trichloroacetic acid TEA triethylamine TFA trifluoroacetic acid
THF tetrahydrofuran Vss volume of distribution at steady state
Materials and Methods
[0057] .sup.1H-NMR spectra were recorded at 400 MHz on a Bruker
DRX-400 (ultra shield). Chemical shifts in organic solvents are
reported in ppm (.delta.) relative to tetramethylsilane.
[0058] DMF, 1,4-dioxane, NMM, ammonium acetate, Boc.sub.2O, TFA,
DMSO, DCCI (Acros), hydroxylamine (50 wt. % in H.sub.2O),
iodoacetic anhydride, GMBS (Sigma Aldrich), recH insulin
(Diosynth), TEA, 6-aminocaproic acid, N-hydroxysuccinimide
(Janssen), THF (Biosolve), 2-mercapto-[S-acetyl]acetic acid
N-hydroxysuccinimide ester (13), TBTU (Fluka), ACN (Merck), AcOH
and Na.sub.2HPO.sub.4 (J. T. Baker) were used as received from the
commercial suppliers mentioned.
Column chromatography was performed on MP Biomedicals Germany GmbH
kieselgel 60 (MP silica 32-63, 60 .ANG.) and on Merck LiChroprep
RP-18 (40-63 .mu.m). TLC analysis was performed on Merck TLC plates
kieselgel 60 F.sub.254. Compounds were visualized by UV absorption
(254 nm) and/or charring with USUI reagent (phosphor molybdenic
acid/AcOH/H.sub.2SO.sub.4 in EtOH). MALDI spectra were obtained
with a Voyager DE PRO (Applied Biosystems, Framingham, Mass., USA)
in linear, delayed extraction mode in positive and negative ion
mode. Re-crystallized alpha-cyano hydroxy cinnamic acid (CHCA, 3
g/L in 500 mL/L ACN/1 mL/L TFA) was used as matrix. Molecular
weights were measured using a two point calibration (e.g. by
assigning recH insulin and one of its fragment chains, or myoglobin
at m/z 16953 and m/z 8477). Q-TOF spectra were obtained with a PE
Sciex API Q-star Pulsar in positive ion mode with an ESI-source.
Samples were dissolved in H.sub.2O and desalted by use of reversed
phase Zip Tip.RTM.. Default analytical HPLC was conducted on a
Gilson 234 autosampler with three Gilson pumps (305) gradient
system using a Luna.TM. C18(2) column (reversed phase,
150.times.4.6 mm, 5 .mu.m). A Gilson UV detector (118) was used for
detection at 210 nm. Gradient elution was performed at a column
temperature of 40.degree. C. and a flow rate of 1 mL/min by
starting with 95% of eluent A (0.1% TFA in H.sub.2O/ACN, 9:1) and
5% eluent B (0.1% TFA in ACN) for 5 min., then applying a linear
gradient from 15 to 50% ACN in 25 min. Analytical (HPLC) analysis
of compound 4 was conducted on a Shimadzu SCL-10A vp (system
controller) two way pump gradient system using a Luna.TM. C18(2)
column (reversed phase, 150.times.2.0 mm, 5 .mu.m). A Shimadzu
diode array UV detector (SPD-M10A vp) was used for detection at 214
nm. Gradient elution was performed at a column temperature of
40.degree. C. and a flow rate of 0.4 mL/min. Preparative HPLC was
conducted on a Waters 2769 sample manager with a single pump
(Waters 600) gradient system using a Luna.TM. C18(2) column
(reversed phase, 250.times.50 mm, 10 .mu.m). A Gilson UV detector
(2996, photodiode array) was used for detection at 210 nm. Gradient
elution was performed at a flow rate of 50 mL/min by starting with
90% eluent A (0.1% TFA in H.sub.2O/ACN, 9:1) and 10% eluent B (0.1%
TFA in ACN) for 10 min., then applying a linear gradient from 15 to
50% eluent B in 50 min. Analytical anion exchange chromatography
was conducted on a Pharmacia Akta Explorer with a Pharmacia pump
(P-900) gradient system using a Pharmacia Biotech MonoQ HR 5/5
column. A Pharmacia UV detector (UV 900) was used for detection at
210 nm in combination with a Pharmacia pH and conductivity detector
(pH/C 900). An AD detector (AD900) was used in combination with a
Chiralyser (IBZ). Furthermore a Pharmacia fraction collector (Frac
950) and a Pharmacia autosampler (A 900) were used. Default elution
was performed with a flow rate of 1 mL/min starting with 74% eluent
A (ACN/H.sub.2O 2:8) and 26% eluent B (ACN/2M NaCl 2:8) for 5 min.
and then applying a gradient to 20% eluent A and 80% eluent B in 15
min.
[0059] Preparative anion exchange chromatography was conducted on a
Pharmacia system with a Pharmacia pump (P-50) and a Pharmacia
gradient mixer (LKB GP-10) gradient system using a Pharmacia
Biotech XK16 Q-sepharose Fast-Flow column. A Pharmacia UV detector
(LKB-UV-MII) was used for detection at 214 nm in combination with a
Biotechnics conductivity detector. Default gradient elution was
performed at a flow rate of 4.6 mL/min by starting with 80% eluent
A (H.sub.2O) and 20% eluent B (2M NaCl in H.sub.2O) for 20 min.,
then applying a gradient to 20% eluent A and 80% eluent B in 210
min..
Preparative gel filtration on Sephadex G25 (desalting) was
conducted on a Pharmacia system with a Watson-Marlow pump (101V)
using a Pharmacia Biotech XK26 Sephadex-G25 fine column. A
Pharmacia UV detector (LKB-UV-MII) was used for detection at 214 nm
in combination with a Biotechnics conductivity detector. Isocratic
elution was performed with H.sub.2O at a flow rate of 1 mL/min
H.sub.2O for 10 h. Analytical HP-SEC with compound 6 was carried
out on a Pharmacia Superdex.TM. 30 HR 10/30 column mounted in a HP
1100 chromatography system. Elution was performed with 0.2 mol/L
sodium phosphate buffer pH 7.0 at a flow rate of 0.4 mL/L.
Analytical HP-SEC with the other conjugates was conducted on the
Akta Explorer system as described for the analytical Q-sepharose
chromatography, with a Pharmacia Superdex.TM. 75 HR 10/30 column.
An isocratic elution was performed with 50 mM ammonium acetate at a
flow rate of 1 mL/min. Preparative HPSEC was conducted on the same
Akta Explorer system as described for the analytical Q-sepharose
chromatography, with a Pharmacia Superdex.TM. 75 XK26 Hiload 26/60
prep-grade column. An isocratic elution was performed with 50 mM
ammonium acetate at a flow rate of 1.32 mL/min. Binding studies of
pentasaccharide conjugates to ATIII, anti-insulin antibody and
insulin receptor were performed using BIAtechnology. The
sensorgrams and report points were analysed with blanc flow cell
subtraction using BIAevaluation 3.2. IC.sub.50 values were
calculated using graphpad Prism 3.0. A.sub.280 measurements were
performed on a Nano Drop.RTM. ND-1000 UV-VIS spectrophotometer.
##STR00010##
Example 1
6-(2-Iodo-acetylamino)-hexanoic acid (2)
[0060] To a suspension of 6-amino-hexanoic acid (1) (0.37 g, 2.8
mmol) in 1,4-dioxane (60 mL) was added iodoacetic anhydride (0.50
g, 1.4 mmol). The reaction mixture was stirred at 50.degree. C. for
3 h and for 16 h at ambient temperature after which TLC analysis
(CH.sub.2Cl.sub.2/MeOH/AcOH, 98/10/1, v/v/v) revealed complete
conversion of compound 1 into a less lypophilic product. EtOAc (100
mL) was added and the reaction mixture was washed with 0.10 M
aqueous HCl solution (50 mL). The organic layer was then washed
twice with brine (50 mL) and the combined water layers were
extracted twice with EtOAc (75 mL). The combined organic layers
were dried (MgSO.sub.4) and concentrated in vacuo. The residue was
chromatographed on silica gel (CH.sub.2Cl.sub.2/MeOH/AcOH, 98/10/1,
v/v/v) to give 6-(2-iodo-acetylamino)-hexanoic acid (2) (0.45
g,>100%). .sup.1H NMR (MeOD): .delta. 3.67 (s, 2H), 3.17 (t,
2H), 2.29 (t, 2H), 1.66-1.33 (m, 6H).
Example 2
6-(2-Iodo-acetylamino)-hexanoic acid 2,5-dioxo-pyrrolidin-1-yl
ester (3)
[0061] To a solution of 6-(2-iodo-acetylamino)-hexanoic acid (2)
(0.20 g, 0.67 mmol) in THF (10 mL) was added N-hydroxysuccinimide
(85 mg, 0.74 mmol) and N,N'-dicyclohexylcarbodiimide (0.21 g, 1.0
mmol). The reaction mixture was stirred in the dark for 16 h. When
TLC analysis (EtOAc/Hep/AcOH, 80/20/1, v/v/v) revealed complete
conversion into the activated ester 3, seven drops of acetic acid
were added. The mixture was then stored in the freezer overnight
(-20.degree. C.). The crude mixture was filtered and the filtrate
was concentrated in vacuo. The crude product was purified by column
chromatography (EtOAc/Hep/AcOH, 40/60/5.fwdarw.20/80/5, v/v/v) and
concentration of the appropriate fractions gave
N-hydroxysuccinimide ester derivative 3 (0.18 g, 67%). .sup.1H NMR
(MeOD): .delta. 3.67 (s, 2H), 3.18 (t, 2H), 2.82 (s, 4H), 2.63 (t,
2H), 2.0-1.0 (m, 6H).
Example 3
Compound 4
[0062] To a suspension of recH insulin (50 mg, 8.6 .mu.mol) in DMF
(15 mL) was added H.sub.2O (9.0 mL) until the solution became
clear. The solution was stirred for 15 min. to adjust to room
temperature. The pH of the solution was adjusted to 10 by adding
dropwise 0.1 M NaOH in H.sub.2O. After this, the reaction flask was
wrapped in tin foil. A solution of 3 (5.0 mg, 8.6 .mu.mol) in DMF
(1.0 mL) was added dropwise to the reaction mixture in 1 min. The
reaction mixture was stirred by using a magnetic stirring bar and
the pH was kept at 10. After 30 min an excess of 0.1% TFA in
H.sub.2O (5.0 mL) was added to quench the reaction. H.sub.2O (200
mL) was added and the reaction mixture was lyophilised to give 4
(60 mg, >100%, max. 8.6 .mu.mol). HPLC (Shimadzu, reversed
phase) analysis by starting with 80% eluent A (0.1% TFA in
H.sub.2O) and 20% eluent B (ACN) for 5 min., then applying a
gradient to 20% eluent A and 80% eluent B in 30 min. revealed the
presence of 45% of the monosubstituted product (recH insulin Rt:
12.84 min; compound 4 Rt: 13.54 min; B29/A1 disubstituted product:
Rt 14.16 min). The crude product was used without purification in
the next reaction.
Example 4
Compound 6
[0063] Crude compound 4 (60 mg) was dissolved in a degassed (by
passing through N.sub.2) 0.05 M solution of NH.sub.2OH in 0.1 M
Na.sub.2HPO.sub.4 buffer (25 mL, pH 7.0). The reaction mixture was
stirred by using a magnetic stirring bar and degassed for 30 min
(by passing through N.sub.2). Then the pentasaccharide-spacer
compound 5 (95 mg, 43 .mu.mol), which was prepared as described in
Angew. Chem. Intl. Ed. (1996), 35, 331-333, was added as a solid
and the reaction mixture was stirred under a nitrogen atmosphere
for 16 h.
##STR00011## ##STR00012## ##STR00013##
Purification of the Pentasaccharide-Insulin Conjugate 6.
[0064] From the reaction mixture of the previous paragraph,
monosubstituted pentasaccharide-insulin conjugate 6 was purified to
near homogeneity by anion exchange chromatography (capture step)
and size exclusion chromatography (polishing step).
[0065] The conjugate-containing solution was applied on a
Q-Sepharose FF column, equilibrated in 20 mmol/L sodium phosphate
buffer PH 8.0. After the unretained protein fraction had passed the
column, an extensive wash with equilibration buffer was carried out
until the A.sub.280 had returned to baseline level. Bound,
unreacted insulin was eluted at around 0.4 mol/L NaCl and the
monosubstituted pentasaccharide-insulin conjugate at 0.7 mol/L NaCl
as determined by analytical HP-SEC on Superdex 30 and MALDI-TOF-MS.
The conjugate fraction was concentrated either by ultrafiltration
or anion exchange chromatography using a 2 mol/L NaCl bump as
elution step and was applied on a preparative Superdex 30 column
equilibrated in phosphate-buffered saline. Fractions containing
pure monoconjugate as determined by HP-SEC and MALDI-MS were
pooled. The final product was stored at -70.degree. C. after
snapfreezing in an ethanol/dry ice mixture. The conjugate
concentration was estimated by A.sub.280 measurement using an
absorbance coefficient of 0.8 for 1 mg/mL.
Characterization of Compound 6
[0066] The identity of the purified pentasaccharide-insulin
conjugate 6 was determined by an ELISA for insulin and by
Biomolecular Interaction Analysis (BIA) using the human insulin
receptor and human ATIII as analytes. Purity and monomericity were
assessed by HP-SEC on Superdex 30 and MALDI-MS.
As is shown in FIG. 1, two batches of pentasaccharide-insulin
conjugate 6 are recognized by the insulin-specific ELISA, which
demonstrates the presence of an immunoreactive insulin moiety. From
BIA experiments in the Biacore it can be concluded that
pentasaccharide-conjugated insulin is still able to bind to the
human insulin receptor (FIG. 2A). In these experiments a MoAb to
human insulin (clone M3222213, 10-130, batch 223, Fitzgerald
Industries International, designated MoAb 13) was immobilized on a
CM5 sensor chip using standard amino coupling. HBS-EP (Biacore,
cat. No. 22-0512-44) was used as running buffer at a flow rate of 5
.mu.L/min. Injection of the insulin conjugate resulted in binding
of the conjugate to the immobilised antibody. The immunobound
insulin conjugate was able to react with the human insulin receptor
as well as with ATIII, the latter being indicative for a covalently
attached pentasaccharide (FIG. 2B).
[0067] MALDI-TOF spectra of the pentasaccharide-insulin conjugate 6
were obtained as described under Materials and Methods. Prior to
analysis, the samples were desalted and concentrated on
.mu.C18-ZipTips (Millipore Corporation, Billerica Mass., USA).
Elution was directly onto a stainless steel MALDI target in 1 .mu.L
of a solution containing 10 g/L alpha-cyano in 500 mL/L ACN/1 mL/L
TFA. FIG. 3 represents a typical MALDI-TOF MS profile of a
monosubstituted pentasaccharide-insulin conjugate with peaks around
m/z 6700 and 7400. The apparent heterogeneity is caused by
laser-induced desulfatation of the pentasaccharide moiety resulting
in a serial loss of 80 Da. No disubstituted pentasaccharide-insulin
conjugate was found in the MALDI-TOF MS analysis since no peaks in
the range of m/z 9400 characteristic of disubstituted insulin were
found. It should be noticed that also peaks for unreacted insulin
are absent (m/z 5808).
[0068] HP-SEC analysis (FIG. 4) shows a major peak with a retention
time of 22 min. The purity was estimated at 97%. (Unreacted insulin
and disubstituted conjugate appear to be absent).
Example 5
Compound 11
[0069] Pentasaccharide 7 (46 mg) [which may be obtained by coupling
of the derivatised monosaccharide 5, described in WO 2001/42262,
with the tetrasaccharide that was obtained by conducting the
synthetic route towards tetrasaccharide 30 described in Bioorg.
Med. Chem. (1994), 2, 1267-1280 in which the reducing end
monosaccharide building block 12 was replaced with methyl
2,3-di-O-benzyl-6-O-methyl-.alpha.-D-glucose, using methods similar
to those described in these publications, including deprotection
and sulfation] and glycol derivative 10 (18 mg, 1.6 equiv.) were
dissolved in DMF (5 mL) under a nitrogen atmosphere. NMM (61 .mu.L,
5 equiv.) was added and the reaction mixture was stirred overnight
at ambient temperature. The solvent was evaporated in vacuo and the
remaining residue was purified by preparative anion exchange
chromatography. The appropriate fractions were combined and
desalted on a preparative G25-column. The combined fractions were
lyophilized to give 11 (29 mg, 57%) as a white powder. Purity
>98% (analytical anion exchange, UV.sub.210 nm). .sup.1H-NMR
(D.sub.2O, 400 MHz, HH-COSY): .delta. 5.31 (d, 1H), 5.23 (m, 1H),
4.91 (m, 1H), 4.45-4.35 (m, 1H), 4.48-3.93 (m, 11H), 3.87-3.62 (m,
9H), 3.60-3.45 (m, 39H), 3.41-3.34 (m, 15H), 3.33-3.23 (m, 7H),
3.18-3.08 (m, 2H), 2.97 (t, 2H), 2.23 (s, 3H).
Example 6
Compound 12
[0070] Pentasaccharide 8 (0.2 g), which was prepared as described
in WO 2001/42262, and glycol derivative 10 (53 mg, 1.3 equiv.),
which was prepared as described in Angew. Chem. Intl. Ed. (1996),
35, 331-333, were dissolved in DMF (5.0 mL). NMM (61 .mu.L, 5
equiv.) was added and the reaction mixture was stirred overnight at
ambient temperature. The solvent was evaporated in vacua and the
residue was purified by preparative anion exchange chromatography.
The appropriate fractions were combined and desalted on a
preparative G25-column. The combined fractions were lyophilized to
give 12 (0.13 g, 55%) as a white powder. Purity >95% (analytical
anion exchange, UV.sub.210 nm). .sup.1H-NMR (D.sub.2O, 400 MHz,
HH-COSY): .delta. 5.12 (d, 1H), 5.03 (d, 1H), 4.70 (d, 1H),
4.34-4.18 (m, 2H), 4.09-4.03 (m, 1H), 3.98-3.90 (m, 5H), 3.85-3.74
(m, 6H), 3.66-3.48 (m, 7H), 3.43-3.24 (m, 41H), 3.23-3.15 (m, 13H),
3.12 (m, 2H), 3.10-3.01 (m, 7H), 2.99-2.88 (m, 3H), 2.78 (t, 2H),
2.04 (s, 3H).
Example 7
Compound 14
[0071] Pentasaccharide 9 (100 mg) [which may be obtained by
coupling of the derivatised monosaccharide 5 described in WO
01/42262 with the tetrasaccharide 48 described in US 2004/0024197
using methods similar to those described in these patent
applications, including deprotection and sulfation] and compound 13
(18 mg, 1.5 equiv.) were dissolved in DMF. NMM (15 .mu.L, 2.5
equiv.) was added and the reaction mixture was stirred overnight at
ambient temperature. The solvent was evaporated in vacua and the
remaining residue was purified on a preparative G25-column. The
appropriate fractions were combined and lyophilized to give 14 (84
mg, 79%) as a white powder. Purity: >95% (analytical anion
exchange, UV.sub.210 nm). .sup.1H-NMR (D.sub.2O, 400 MHz, HH-COSY):
.delta. 5.12 (d, 1H), 5.09 (d, 1H), 4.82 (d, 1H), 4.40-4.26 (m,
1H), 4.10-3.87 (m, 8H), 3.82-3.73 (m, 4H), 3.67-3.43 (m, 11H),
3.41-3.35 (m, 14H), 3.31-3.26 (m, 13H), 3.24-3.15 (m, 8H),
3.14-3.02 (m, 5H), 3.01-2.87 (m, 3H), 2.08 (s, 3H).
##STR00014## ##STR00015##
Example 8
Compound 15
[0072] RecH insulin (779 mg) was dissolved in anhydrous DMSO (25
mL) and AcOH (465 .mu.L). Boc.sub.2O (73 mg, 2.5 equiv) was added
to the solution and the resulting mixture was stirred for 5 h at
ambient temperature. The reaction was quenched by the addition of
0.1% TFA in H.sub.2O/ACN (9/1, v/v, 150 mL) and the solution was
lyophilized four times. The residue was dissolved in 0.1% TFA in
H.sub.2O/ACN (9/1)/ACN (3:1) and the main product was isolated by
preparative HPLC. The appropriate fractions were combined and
lyophilized to give Al,BI-diBoc insulin 15 (200 mg, 26%) as a white
powder. Purity: 98% (analytical HPLC). MS calcd. for
C.sub.267H.sub.399N.sub.65O.sub.81S.sub.6=6008, found on MALDI-TOF
6008 (using recH insulin as internal reference standard).
Example 9
Compound 16
[0073] RecH insulin (752 mg) was dissolved in anhydrous DMSO (20
mL) and TEA (0.75 mL). Boc.sub.2O (66 mg, 2.5 equiv.) in DMSO (5
mL) was added to the solution, and the reaction was stirred at
ambient temperature for 1.5 h. The reaction was quenched by
addition of 0.1% TFA in H.sub.2O/ACN (9/1, v/v, 150 mL) and the
mixture was lyophilized three times. The resulting residue was
dissolved in 0.1% TFA in H.sub.2O/ACN (9/1) and was subjected to
preparative HPLC. The appropriate fractions were combined and
lyophilized to give Al,B29-diBoc insulin 16 as a white powder (332
mg, 43%). Purity: >98% (analytical HPLC). MS calcd. for
C.sub.267H.sub.399N.sub.65 O.sub.81S.sub.6=6008, found on MALDI-TOF
6008 (using recH insulin as internal reference standard).
Example 10
Compound 17
[0074] A1,B1-diBoc insulin 15 (200 mg) was dissolved in anhydrous
dimethyl sulfoxide (5 mL) and triethylamine (145 .mu.L). GMBS (45
mg, 5 equiv.) was added and the reaction mixture was stirred for 30
min at ambient temperature. The reaction mixture was quenched by
the addition of 0.1% TFA in H.sub.2O/ACN (9/1, v/v, 150 mL) and the
resulting mixture was lyophilized to give A1,B1-diBoc-B29-GMB
insulin 17 (0.5 g, crude), which was used in the next step without
further purification.
Example 11
Compound 18
[0075] A1,B29-diBoc insulin 16 (330 mg) was dissolved in anhydrous
DMSO (5 mL) and TEA (332 .mu.L). GMBS (230 mg, 15 equiv.) was added
and the reaction mixture was stirred for 30 min at ambient
temperature. The reaction mixture was quenched by the addition of
0.1% TFA in H.sub.2O/ACN (9/1, v/v, 150 mL) and the resulting
solution was lyophilized to give A1,B29-diBoc-B1-GMB insulin 18
(0.6 g, crude), which was used in the next step without further
purification.
Example 12
Compound 19
[0076] A1,B1-diBoc-B29-GMB insulin 17 (0.5 g, crude) was dissolved
in TFA (5 mL) and stirred for 10 min at ambient temperature. The
TFA was removed under reduced pressure, the residue was dissolved
in 0.1% TFA in H.sub.2O/ACN (2/1, v/v) and the solution immediately
subjected to preparative HPLC. The appropriate fractions were
combined and lyophilized to give B29-GMB insulin 19 as a white
powder (93 mg, 47%). Purity >99% (analytical HPLC).
Example 13
Compound 20
[0077] A1,B29-diBoc-B1-GMB insulin 18 (0.6 g, crude) was dissolved
in TFA (5 mL) and the mixture was stirred for 10 min at ambient
temperature. The TFA was evaporated in vacuo, the crude product was
dissolved in 0.1% TFA in H.sub.2O/ACN (9/2, v/v) and the resulting
solution was immediately subjected to preparative HPLC. The
appropriate fractions were combined and lyophilized to give B1-GMB
insulin 20 as a white powder (127 mg, 40%). Purity >99%
analytical HPLC). MS calcd. for
C.sub.267H.sub.399N.sub.65O.sub.81S.sub.6=5973, found on MALDI-TOF
5973 (using recH insulin as internal reference standard).
Examples 14-19
[0078] General Procedure for the Conjugation of GMB-Insulin with
Pentasaccharide GMB-insulin 19 or 20 (25 mg) was dissolved in a 0.1
M Na.sub.2HPO.sub.4 buffer (12 mL, pH 7.0, degassed by passing
N.sub.2 through the solution). The solution was stirred by using a
magnetic stirring bar and was degassed for another 30 min.. Then
pentasaccharide 5, 11, 12 or 14 (23 mg, 2.5 equiv.) was added as a
solid, followed by the addition of NH.sub.2OH (50 .mu.L, 0.05M).
The reaction mixture was stirred under a nitrogen atmosphere at
ambient temperature. After 16 hours the reaction mixture was
subjected to preparative HPSEC (S75). The appropriate fractions
were combined and lyophilized to give insulin-penta conjugates 24,
25, 26, 27, 28, 29 as a white powder in a typical yield of 30%-50%.
Yields were determined by A.sub.280 measurements using the same
molar extinction coefficient as for recH insulin.
Example 14
Compound 24
[0079] B29-GMB insulin 19 (25 mg) was conjugated to pentasaccharide
5 (23 mg) according to the general procedure to give
B29-pentasaccharide insulin derivative 24 (15 mg, 45%). ESI-MS
calcd. for C.sub.320H.sub.487N.sub.67O.sub.137S.sub.14=7913, found
on Q-TOF 2638.7 M.sup.3+; 1979.2 M.sup.4+; 1583.6 M.sup.5+, 1319.8
M.sup.6+. Purity:>98% (analytical HPSEC, analytical anion
exchange).
Example 15
Compound 25
[0080] B1-GMB insulin 20 (25 mg) was conjugated to pentasaccharide
5 (23 mg) according to the general procedure to give
B1-pentasaccharide insulin derivative 25 (16 mg, 47%). ESI-MS
calcd. for C.sub.320H.sub.487N.sub.67O.sub.137S.sub.14=7913, found
on Q-TOF 2637 M.sup.3+; 1978 M.sup.4+; 1583 M.sup.5+. Purity:
>95% (analytical HPSEC), >98% (analytical anion
exchange).
Example 16
Compound 26
[0081] B29-GMB insulin 19 (13 mg) was conjugated to pentasaccharide
14 (12 mg) according to the general procedure to give
B29-pentasaccharide insulin derivative 26 (5 mg, 31%). ESI-MS
calcd. for C.sub.312H.sub.471N.sub.67O.sub.133S.sub.14=7737, found
on Q-TOF 2578 M.sup.3+; 1934 M.sup.4+. Purity: >98% (analytical
HPSEC).
Example 17
Compound 27
[0082] B1-GMB insulin 20 (15 mg) was conjugated to pentasaccharide
14 (13 mg) according to the general procedure to give
B1-pentasaccharide insulin derivative 27 (8 mg, 40%). ESI-MS calcd.
for C.sub.312H.sub.471N.sub.67O.sub.133S.sub.14=7737, found on
Q-TOF 2578 M.sup.3+; 1934 M.sup.4+. Purity: >98% (analytical
HPSEC).
Example 18
Compound 28
[0083] B29-GMB insulin 19 (15 mg) was conjugated to pentasaccharide
12 (13 mg) according to the general procedure to give
B29-pentasaccharide insulin derivative 28 (6 mg, 30%). ESI-MS
calcd. for C.sub.321H.sub.489N.sub.67O.sub.134S.sub.13=7847, found
on Q-TOF 1962 M.sup.4+; 1570 M.sup.5+; 1308 M.sup.6+. HPSEC purity:
>98%.
Example 19
Compound 29
[0084] B29-GMB insulin 19 (15 mg) was conjugated to pentasaccharide
11 (13 mg) according to the general procedure to give
B29-pentasaccharide insulin derivative 29 (7 mg, 33%). ESI-MS
calcd. for C.sub.322H.sub.491N.sub.67O.sub.131S.sub.12=7782, found
on Q-TOF 2593 M.sup.3+; 1945 M.sup.4+; 1556 M.sup.5+. Purity:
>98% (analytical HPSEC).
##STR00016## ##STR00017## ##STR00018##
Characterisation
Analytical Size Exclusion Chromatography
[0085] Compounds 24-29 were subjected to analytical HP-SEC analysis
on a Superdex 75 26/10 column. Elution was performed with 50 mM
ammonium acetate at a flow rate of 1.0 mL/min.
TABLE-US-00001 TABLE 1 HPSEC analyses of insulin conjugates 24-29
HPSEC (Superdex 75) Compound Rt Purity 24 12.7 min >98% 25 12.5
min >95% 26 12.7 min >98% 27 12.5 min >98% 28 12.7 min
>98% 29 12.8 min >98% recH insulin 15.0 min >98%
[0086] All conjugates were observed as single peaks (at least
>95% purity) illustrating the absence of aggregated forms of
insulin-pentasaccharide.
[0087] N-Terminal Sequence Analysis
[0088] The insulin-pentasaccharide conjugates 24-29, as well as the
corresponding precursors 15, 16, 19 and 20 were subjected to
N-terminal sequence analysis (Edman degradation). In each of the
cycles carried out, the B29 substituted insulin derivatives 15, 19,
24, 26, 28 and 29 yielded equimolar amounts of both A- and B-chain
amino acids to a level comparable with that of the initial amounts
of the conjugates. This indicates full accessibility of both
N-termini and thus absence of conjugate moieties which are
therefore confined to the B29 position. In contrast, only A chain
amino acids were found during N-terminal sequencing of the
B1-substituted insulin derivatives 16, 20, 25 and 27, demonstrating
conjugation at the B1 position with as a consequnce inhibition of
Edman degradation at the N-terminus of the B-chain.
Competitive hATIII Binding assay Using Biomolecular Interaction
Analysis
[0089] Principle and aim of the test: Biomolecular Interaction
Analysis (BIA) studies the interaction between (bio)molecules by
covalent immobilisation of one of the interactants to a sensor chip
surface, and injection of the other interactant in the continuous
buffer flow over this surface. Binding is registered as a change in
refractive index on this surface and is proportional to the
molecular weigth (Mw) of the interactants.
To study the interaction between hATIII and pentasaccharide
conjugates, compound 9 is covalently coupled to the sensor chip
surface. Binding of hATIII to the pentasaccharide generates a
strong signal as a resultant of the difference in Mw between the
bound (small) pentasaccharide ligand and the (large) hATIII
analyte. Preincubated samples containing a constant concentration
of hATIII and variable concentrations of free pentasaccharide or
conjugate were injected over the surface. Binding of
pentasaccharide or conjugate to ATIII during preincubation will
result in a reduction of ATIII binding to the immobilised
pentasacchariede. This competitive binding assay allows
determination of IC.sub.50 values for each pentasaccharide
conjugate. Experimental procedure: Compound 9 is covalently coupled
to a CM5 sensor chip by amine coupling at pH 8.5. The sensor chip
is activated by EDC/NHS for 15 min. and subsequently compound 9 is
injected at a concentration of 100 .mu.g/mL. The unreacted
hydroxysuccinimide groups are reacted with ethanolamine for 7 min..
The surface is regenerated by three short injections of 5 .mu.L 5
mol/L NaCl, at a flow rate of 25 .mu.AL/min. Immobilisation of the
pentasaccharide can not be detected, however, binding of hATIII to
a surface treated as described was found specific, demonstrating
the presence of pentasaccharide on the surface. A concentration
series of hATIII was tested to estimate a sensitive concentration
for inhibition (at 80% of maximum binding). At a flow rate of 20
.mu.L/min three minute injections were carried out at both blanc
and immobilised surface in series at 25.degree. C. A report point
is generated at 170 s. The surface is regenerated by a 12s
injection of 5 mol/L NaCl. The samples were tested against a
constant concentration of hATIII (i.e. 15 nmol/L) and
concentrations of pentasaccharide conjugate ranging between
0.78-100 nM. The hATIII injection or report point without
pentasaccharide conjugate is set at 100% binding. The relative %
binding of the pentasaccharides and the conjugates compared to
maximum binding was used to generate a sigmoidal curve (with
variable slope) by plotting Log [concentration] vs % binding from
which the IC.sub.50 values were derived.
TABLE-US-00002 TABLE 2 IC.sub.50 values expressing ATIII binding
potential in a competitive binding assay (BIA study) BIA
(competitive ATIII binding) Compound IC.sub.50 95% confidence
interval 7 (reference) 96 nM 53.1-173 nM 8 (reference) 58 nM
33.7-99.1 nM 9 (reference) 5.5 nM 5.1-5.8 nM 24 8.5 nM 7.9-9.3 nM
25 9.1 nM 8.2-10.0 nM 26 4.5 nM 4.2-4.7 nM 27 15 nM 13.3-17.0 nM 28
96 nM 50.6-183 nM 29 68 nM 33.9-137 nM
CONCLUSION
[0090] The difference in IC.sub.50's between the reference carrier
pentasaccharides 7-9 (FIG. 5, Table 2) confirms that their
competitive binding potential to ATIII, a measure for the binding
affinity for ATIII, can be tuned by changing the number of sulfate
groups contained in these molecules. The (competitive) binding
potential (IC.sub.50) of all corresponding insulin-pentasaccharide
conjugates (24-29) to hATIII fall in the same range when compared
to the parent reference pentasaccharides 7-9 (FIG. 6, Table 2).
These data indicate that the pharmacokinetic properties of the
conjugates may be tuned by using alternative carrier
pentasaccharides with different binding affinity for ATIII.
Mass Spectrometry
[0091] A typical mass spectrometric analysis of a pentasaccharide
conjugate is depicted in FIG. 7. For instance, compound 24 with the
bruto formula C.sub.320H.sub.487N.sub.67O.sub.137S.sub.14 and a
calculated mono isotopic mass of 7913, has been analysed with an
ESI-QTOF system. In the ESI-MS spectrum multiple charged ions at
m/z ratios 1319.8 (6+), 1583.6 (5+), 1979.2 (4+), 2638.7 (3+) in
line with the charge distribution of recti insulin have been
encountered. In addition, the isotope distribution of a randomly
selected multiple charged peak (e.g. 5+) for
C.sub.320H.sub.487N.sub.67O.sub.137S.sub.14 (compound 24) is in
agreement with the theoretically calculated isotope distribution
with the programme Isopro (see dotted lines in FIG. 7A).
General Procedure for the Conjugation of GnRH Antagonistic
Decapeptides with Pentasaccharide (Examples 20, 21, 24, 25)
[0092] Ganirelix-dervative 30 or 35 (70 mg) was dissolved in DMF
(20 mL) under a nitrogen atmosphere. TBTU (14 mg, 1.05 equiv.) and
NMM (25 .mu.L, 5 equiv.) were added and the mixture was stirred for
1 hour at ambient temperature. Pentasacchardie 8 or 9 (88 mg, 1.1
equiv.) was dissolved in DMF (10 mL) to give a suspension. This
suspension was added to the reaction mixture and the remaining
mixture was stirred for 16 hours at ambient temperature. The
reaction mixture was diluted with water (200 mL) and was
lyophilized. The resulting residue was purified on reversed phase
silica (Cl 8) with 0.01 M ammonium acetate (pH 7) and a gradient of
10 to 50% ACN in block-elutions. The appropriate fractions were
combined and lyophilized to give the ganirelix pentasaccharide
conjuagte 31, 32, 36 or 37 as a white powder. Analytical HPLC was
performed with a gradient elution by starting with 90% eluent A
(0.01 M ammonium acetate) and 10% eluent B (ACN) for 5 min., then
applying a gradient to 100% eluent B in 30 min..
Example 20
Compound 31
[0093] Ganirelix-derivative 30 (70 mg), which was prepared by solid
phase peptide synthesis as described in J. Med. Chem. 1992, 35,
3942-3948, was conjugated to pentasaccharide 8 (88 mg) according to
the general procedure to give 31 (16 mg, 40%). Purity 96%
(analytical HPLC), 97% (analytical anion exchange). ESI-MS calcd.
for C.sub.126H.sub.191ClN.sub.18O.sub.62S.sub.6=3175.0359, found
792.7390 [M-4H].sup.4-, 1057.3258 [M-3H].sup.3-, 1062.9883
[M+NH.sub.3-3H].sup.3-. .sup.1H-NMR (D.sub.2O, 400 MHz, HH-COSY):
.delta. 8.56 (m, 1H), 8.53 (m, 1H), 8.10 (m, 1H), 7.76 (m, 2H),
7.68 (m, 2H), 7.47 (m, 1H), 7.39 (m, 2H), 7.16 (m, 3H), 7.04 (m,
2H), 6.95 (m, 2H), 6.63 (m, 2H), 5.35-5.25 (m, 2H), 4.95 (m, 1H),
4.68-4.61 (m, 1H), 4.61-4.51 (m, 2H), 4.50-4.38 (m, 2H), 4.38-4.25
(m, 3H), 4.24-3.96 (m, 15H), 3.90-3.84 (m, 1H), 3.84-3.67 (m, 8H),
3.67-3.20 (m, 53H), 3.19-3.09 (m, 4H), 3.09-2.98 (m, 12H),
2.98-2.84 (m, 8H), 2.77 (m, 2H), 2.13 (m, 1H), 1.96-1.72 (m, 5H),
1.68-1.42 (m, 5H), 1.42-1.20 (m, 7H), 1.18-0.89 (m, 13H), 0.85-0.71
(m, 5H).
Example 21
Compound 32
[0094] Ganirelix-derivative 30 (70 mg) was conjugated to
pentasaccharide 9 (88 mg) according to the general procedure to
give 32 (52 mg, 35%). Mass calcd. for
C.sub.125H.sub.189ClN.sub.18O.sub.65S.sub.7=3240.9770; found on
ESI-QTOF 809.2219 [M-4H].sup.4-, 1079.2994 [M-3H].sup.3-, 1084.9739
[M+NH.sub.3].sup.3-, 1090.6340, [M+2NH.sub.3-3H].sup.3-.
.sup.1H-NMR (D.sub.2O, 400 MHz, HH-COSY): .delta. 8.45 (m, 1H),
8.38 (m, 1H), 7.90 (m, 1H), 7.78 (d, 1H), 7.73 (m, 2H), 7.59 (m,
1H), 7.50 (m, 1H), 7.43 (m, 2H), 7.20 (m, 3H), 7.05 (d, 2H), 6.97
(m, 2H), 6.66 (m, 2H), 5.40-5.30 (m, 2H), 5.09 (d, 1H), 4.67-4.48
(m, 2H), 4.40-3.92 (m. 18H), 3.92-3.75 (m, 7H), 3.77-3.17 (m, 56H),
3.12-3.02 (m, 11H), 3.02-2.98 (m, 8H), 2.97-2.83 (m, 4H), 2.81-2.75
(m, 2H), 2.14 (m, 1H), 1.99-1.86 (m, 5H), 1.82-1.23 (m, 12H),
1.22-0.90 (m, 13H), 0.87-0.72 (m, 5H). Purity: 95% (analytical
HPLC), 97% (analytical anion exchange purity).
Example 22
Compound 34
[0095] Compound 30 (100 mg) was dissolved in DMF, TBTU (36 mg, 2
equiv.) and NMM (60 .mu.L, 10 equiv.) were added and the reaction
mixture was stirred for 1 hour at ambient temperature. Then
compound 33 (34 mg, 2 equiv.), which was prepared as described in
WO 2005090382, was added and the reaction mixture was stirred for
16 hours at ambient temperature. The solvent was removed in vacuo
and the remaining residue was dissolved in water/CAN, the resulting
solution which was subjected to preparative HPLC (gradient: 80%
eluent A (0.1% TFA in H.sub.2O) and 20% eluent B (ACN) to 20%
eluent A and 80% eluent B in 45 min.). The appropriate fractions
were combined to give, after lyophilization, compound 34 (60 mg,
60%) as a white powder. Analytical HPLC was performed by applying a
gradient starting with 75% eluent A (0.1% TFA in H.sub.2O) and 25%
eluent B (CH.sub.3CN) to 20% eluent A and 80% eluent B in 15 min..
Mass calcd. for C.sub.94H.sub.139ClN.sub.18O.sub.19=1859, found on
MALDI-TOF 1860 [M+H].sup.+ and 1882 [M+Na].sup.+. Purity: >90%
(analytical HPLC).
Example 23
Compound 35
[0096] Compound 34 (60 mg) was dissolved in H.sub.2O/TFA/ACN (7 mL,
5:1:1) and was stirred for 2 hours at ambient temperature. An extra
amount of TFA (2.5 mL) was added and the reaction mixture was
stirred for another 22 hours. The TFA was evaporated in vacuo and
the remaining solution was lyophilized to give 35 (45 mg, 77%) as a
white powder. Mass calcd. for
C.sub.90H.sub.131ClN.sub.18O.sub.19=1803, found on MALDI-TOF 1804
[M+H].sup.-and 1826 [M+Na].sup.+. Purity: >95% (analytical
HPLC).
Example 24
Compound 36
[0097] Ganirelix-derivative 35 (17 mg) was conjugated to
pentasaccharide 8 (19 mg) according to the general procedure.
Additional purification was performed by preparative HPLC
(gradient: 90% eluent A (0.01 M ammonium acetate) and 10% eluent B
(CH.sub.3CN) for 5 min., then to 100% B in 50 min.). The
appropriate fractions were combined and lyophilized to give 36 (2
mg, 6%). Purity: 94% (analytical HPLC), >98% (analytical anion
exchange).
Example 25
Compound 37
[0098] Ganirelix-derivative 35 (17 mg) was conjugated to
pentasaccharide 9 (19 mg) according to the general procedure.
Additional purification was performed by preparative HPLC as
described for compound 36. The appropriate fractions were combined
and lyophilized to give 37 (1.16 mg, 3%). Purity: 88% (analytical
HPLC). Analytical anion exchange purity: >95%.
##STR00019## ##STR00020## ##STR00021##
CONCLUSION
[0099] The differences in competitive binding potential to ATIII
between on the one hand conjugates 31 and 32, and on the other hand
conjugates 36 and 37 (as depicted in FIG. 8) reveal that,
irrespective of the length of the linking residue, the affinity for
ATIII can be tuned by changing the number of sulfate groups
contained in these molecules. These data further indicate that the
in-vivo pharmacokinetic properties of these conjugates may be
tunable by the choice of the pentasaccharide (see pharmacokinetic
study below). Furthermore, the binding of the conjugates to ATIII
is specific, since the non-conjugated parent peptide ganirelix
shows no competitve binding to ATIII.
Example 24
Compound 39
[0100] Compound 38 (26.5 mg, 8.4 .mu.mol), supplied by NeoMPS
(Strasbourg, France), was dissolved in degassed 0.1 M
Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 buffer (16 mL, pH 7.0).
Pentasaccharide 5 (45.5 mg, 21 .mu.mol, 2.5 equiv.) was added under
a nitrogen atmosphere and the resulting mixture was stirred for
approximately 10 min.. Then an aqueous solution of NH.sub.2OH (50
wt %, 69 .mu.L) was added and the reaction mixture was stirred for
16 h. The product was purified on a Q-Sepharose column (2M
NaCl.sub.(aq)/H.sub.2O/ACN, 10/40/1.fwdarw.40/10/1, v/v/v).
Desalting of the appropriate fractions was carried out using G25
sephadex chromatography as described above to yield compound 39
(15.7 mg, 35%). The yield was determined by A.sub.280 measurement
using a theoretical absorbance of 0.48 for a 1 mg/mL solution.
Calcd. mass for C.sub.196H.sub.309N.sub.41O.sub.101S.sub.8=5108.8;
found on ESI-Q-TOF=1740.6[M+5Na].sup.3+, 1311.2 [M-1H+6Na].sup.4+,
1053.5 [M-2H+7Na].sup.5+
##STR00022##
CONCLUSION
[0101] As depicted in FIG. 9 the competitive binding potential to
ATIII of conjugate 39 is conserved when compared to the parent
pentasaccharide-spacer residue (see FIG. 5, compound 9). These data
suggest that a significant extension of the in-vivo half-life of
the peptide can be achieved by conjugation to an ATIII binding
carrier pentasaccharide (see pharmacokinetic study below).
Furthermore, the binding of the conjugate to ATIII is specific,
since the non-conjugated parent peptide ADM(27-52) shows no
significant competitive binding to ATIII.
Example 25
Compound 41
[0102] Compound 40 (15 mg, 4.2 .mu.mol), prepared by NeoMPS
(Strasbourg, France), was dissolved in degassed 0.1 M
Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 buffer (8 mL, pH 7.0).
Pentasaccharide 5 (22.7 mg, 10.4 .mu.mol, 2.5 equiv.) was added
under a nitrogen atmosphere and the mixture was stirred for
approximately 10 min. Then an aqueous solution of NH.sub.2OH (50 wt
%, 0.14 mL) was added and the reaction mixture was stirred for 16
h. The product was purified as described for compound 39 to yield
compound 41 (1.38 mg, 6%). The yield was determined by A.sub.280
measurement using a theoretical absorbance of 1.22 for a 1 mg/mL
solution. Mass calcd. for
C.sub.218H.sub.342N.sub.44O.sub.106S.sub.8=5528; found on ESI
Q-TOF=1843.7 [M+3H].sup.3+, 1383.0 [[M+4H].sup.4+, 1107
M+5H].sup.5+.
CONCLUSION
[0103] The competitive binding potential to ATIII of conjugate 41
is conserved when compared to the parent pentasaccharide-spacer
residue compound 9 (see FIG. 10). These data suggest that a
significant extension of in-vivo half-life of the peptide may be
attained by conjugation to an ATIII binding carrier pentasaccharide
(see pharmacokinetic study below).
##STR00023##
Example 26
Compound 44
[0104] Octreotide (compound 42, 50 mg, 0.04 mmol), which can be by
obtained at commercial suppliers such as Bachem (Weil am Rhein,
Germany), was dissolved in DMSO (5 mL). AcOH (7 .mu.L) was added to
generate a slightly acidic ssolution. Subsequently GMBS (11.2 mg,
0.04 mmol, 1.0 equiv.) was added and the resulting solution was
stirred under nitrogen for 1 h. LC-MS analysis showed a near
complete conversion to GMB octreotide 43 in which the GMB moiety is
introduced to the N-terminal Phe residue in a highly regioselective
manner. The reaction was cooled to .about.5.degree. C. after which
a solution of NaH.sub.2PO.sub.4/Na.sub.2HPO.sub.4 (20 mL, pH 7.0)
was added. After 10 min. the mixture was allowed to reach to RT and
N.sub.2 was lead through the solution for 10 min.. Then
pentasaccharide 5 (21.8 mg, 0.01 mmol, 0.25 equiv.) was added as a
solid under a nitrogen atmosphere, followed by an aqueous solution
of NH.sub.2OH (0.11 mL, 50 wt %) and the reation mixture was
stirred for 16 h. The product was purified by ion exchange
chromatography as described above, to yield compound 44 (6.3 mg,
19%). Mass calcd. for
C.sub.112H.sub.170N.sub.12O.sub.70S.sub.10=3122.7; found on
ESI-Q-TOF=1562.3 [M+2H].sup.2+, 1041.9 [M+3H].sup.3+. .sup.1H-NMR
(D.sub.2O, 400 MHz, HH-COSY): .delta. 7.52-7.29 (m, 10H), 7.26-7.14
(m, 4H), 7.03 (s, 1H), 5.44 (m, 1H), 5.37 (m, 1H), 5.14 (m, 1H),
5.12-4.89 (m, 2H), 4.77-4.60 (m, 3H), 4.45-3.97 (m, 18H), 3.95-3.76
(m, 13H) 3.75-3.35 (m, 57H), 3.33-2.59 (m, 18H), 2.22-1.16 (m, 2H),
1.85-1.76 (m, 3H), 1.71-1.38 (m, 3H), 1.32-1.15 (m, 7H), 0.81-0.61
(M, 2H).
##STR00024## ##STR00025##
CONCLUSION
[0105] The competitive binding potential to ATIII of conjugate 44
is conserved when compared to the parent pentasaccharide-spacer
residue compound 9 (see FIG. 11). These data are indicative for a
significant extension of in-vivo half-life of the peptide by
conjugation to an ATIII binding carrier pentasaccharide.
Example 27
Compound 46
[0106] Pentasaccharide 9 (100 mg) and GMBS (22 mg, 1.5 equiv.) were
dissolved in DMF (10 mL). DiPEA (18 .mu.L, 2 equiv.) was added and
the reaction mixture was stirred overnight at ambient temperature.
The solvent was evaporated in vacuo and the residue was purified on
a preparative G25-column. The combined fractions were lyophilized
to give 46 (50 mg, 46%) as a white powder. ESI-MS calcd. for
C.sub.53H.sub.77N.sub.2O.sub.55S.sub.7Na.sub.9=2052; found on
ESI-Q-TOF=1027 M.sup.2+.
Example 28
Compound 47
[0107] Pentasaccharide maleimide derivative 46 was conjugated to
the free Cys.sup.125 of native recH-IL2 (R&D systems,
202-IL/CF). Prior to conjugation recH-IL-2 was analysed by HPSEC,
SDS-PAGE and MALDI-TOF MS indicating a predominant monomeric
composition. RecH-IL2 (1 mg, 800 .mu.L, 1.26 mg/mL in PBS
containing 0.5% SDS) was treated overnight at RT on a roller bank
with an excess of compound 46 (59 .mu.L of a 10 mg/mL aq. solution,
5 equiv.) to achieve complete conversion of starting material.
Next, unreacted maleimide 46 was blocked with a 5 times molar
excess of cysteamine (16 .mu.L, 10 mg/mL) for 3 h. The reaction
mixture was dialysed (cut off 6-8 kDa) against 0.5% SDS in PBS to
remove excess cysteamine and 46 and the final amount of product as
determined by A.sub.280 was 0.69 mg.
The final conjugate 47 was characterized by SDS-PAGE (4-12%) and
Western blot (see FIG. 12). From lanes 3 and 4 it is concluded that
compound 47 has a higher Mw relative to recH-IL2 (corresponding to
the presence of a pentasaccharide moiety). Western blot analysis
with subsequent incubations of a) 10 .mu.g/mL hATIII (HAT
950A2L-Kordia); b) a-hATIII (MoAb HATIII 200--Kordia); c) GAM-HRP
(W402B 20373201--Promega) and final detection with DAB reagent
clearly revealed specific ATIII binding to
pentasaccharide-containing IL2 (lane 7 vs 8).
##STR00026##
Pharmacology
Determination of Pharmacokinetics of Conjugated (Poly)Peptides
[0108] The pharmacokinetic properties of representative examples of
compounds of the invention were determined as described in the
following paragraphs.
Human Insulin ELISA for Determination of Insulin in rat Plasma
[0109] The human Enzyme-Linked Immuno-Sorbent Assay (ELISA) has
been developed to measure human insulin in human and rat plasma or
buffer system. In this way the ELISA could be used to determine the
insulin concentration in plasma samples derived from
pharmacokinetic experiments.
The assay is based on the immunochemical "sandwich" principle using
two monoclonal antibodies, i.e. a solid phase bound, capture
antibody and a detection antibody which is labeled with horseradish
peroxidase (HRP). For the determination of the pharmacokinetic
properties of pentasaccharide-insulin conjugate 6 and recombinant
human insulin (recH insulin, batch SIHR017, from Diosynth, The
Netherlands), rat plasma samples were incubated for 1 h at room
temperature, while shaking at 10 Hz. During this incubation,
pentasaccharide-insulin conjugate 6 or recH insulin binds to the
immobilized anti-insulin antibody. After a second washing
procedure, detection antibody anti-insulin conjugated with HRP was
added to bind to the immobilized insulin-complex. The plate was
washed to remove unbound enzyme-labeled antibody and subsequently
the 3,3',5,5'-tetramethylbenzidine/H.sub.2O.sub.2 substrate
solution was added. The reaction was stopped with 0.5 M sulphuric
acid and the microtiter plate was read spectrophotometrically at
450 nm. The intensity of colour is directly proportional to the
concentration of insulin. Then for each plasma sample the mean
concentration (mol/L) of insulin was determined.
Determination of Pharmacokinetic Properties of Compound 6
[0110] The pharmacokinetic behavior of insulin conjugate 6 and recH
insulin were studied in male Wistar rats of 300-400 gr. The rats
were anaesthetized by inhalation of a mixture of
O.sub.2/N.sub.2O/isoflurane, after which the right jugular vein was
cannulated. The next day rats were treated i.v. with doses of 3.5
nmol/kg of compound 6 or recH insulin, after which blood was
sampled at several time intervals. Blood was centrifuged after
which the plasma was siphoned off and stored at -20.degree. C.
until use. The concentration of the tested compounds in the plasma
samples were determined using the human ELISA against a calibration
curve which was made of the stock solution of the tested compound
itself. The concentration in the samples was expressed in nmol/L
and the kinetic parameters were calculated with the noncompartment
model of WinNonlin.
CONCLUSION
[0111] As can be seen in FIG. 13 and Table 3 the pharmacokinetic
properties of pentasaccharide-insulin conjugate 6 were strongly
improved compared to those of the parent recH insulin.
TABLE-US-00003 TABLE 3 Pharmacokinetic parameters after i.v.
administration of compound 6 or recH insulin (3.5 nmol/kg) in rat.
Experiment performed in n = 3/treatment. Pentasaccharide- insulin
conjugate 6 recH insulin Mean .+-. s.e.m. Mean .+-. s.e.m. T1/2 eli
(h) 2.8 .+-. 0.1 0.033 .+-. 0.001 AUCinf (h nmol/L) 72.5 .+-. 6.4
4.6 .+-. 0.8 Vss (L/kg) 0.11 .+-. 0.01 0.012 .+-. 0.002 Cl (L/h/kg)
0.049 .+-. 0.004 0.80 .+-. 0.12 MRT (h) 2.1 .+-. 0.1 0.014 .+-.
0.1
Determination of Pharmacokinetics after Labeling with .sup.125I
[0112] Pentasaccharide conjugates 24, 28, 29, 31, 32, 39 and 41
were labeled with .sup.125I using the lactoperoxidase method,
according to Machalonis et al. (Biochem J. 1969, 113: 299-305).
After labeling, the conjugate was purified by gel filtration on
Sephadex G25 and anion exchanger HiTrap Q10. Kinetic experiments
were repeated as described for compound 6 but using
.sup.125I-labeled conjugates instead. In order to prevent
accumulation of .sup.125I.sup.- in the thyroid, rats were orally
treated with 10 mg/kg potassium iodide prior to administration of
compound.
The correct determination of the fate of .sup.125I-labeled
conjugates might be affected aversely by metabolic intracellular
endogenous dehalogenation to give free .sup.125I.sup.- in
circulation. Since the free .sup.125I.sup.- itself showed an
elimination half-life of 3.2 h in rats (control, data not shown),
the measured overall half-life of .sup.125I labeled compounds with
a relatively short half-life might be prolonged and the
experimentally determined half-life of compounds with a relatively
long half life may be shorter. Indeed, the observed elimination
half-lives of conjugates 31, 32 and 39 demonstrate in a qualitative
manner that conjugation of the peptides to a carrier
pentasaccharide leads to prolonged residence times. Adaptation of
the above described method by measuring the radioactivity in pellet
(0.1 mL) after precipitation with a 40 times higher volume of TCA
(10% final concentration) yielded pharmacokinetic parameters of
compounds 24, 28, 29 and 41 which were corrected for competing
endogenous .sup.125I-dehalogenation. Pharmacokinetics of Insulin
Pentasaccharide Conjugates 24, 28 and 29 Compared to recH
Insulin
TABLE-US-00004 TABLE 4 Pharmacokinetic parameters after i.v.
administration of insulin conjugates 24, 28 and 29. Experiment
performed in n = 3/treatment. For comparison recH insulin was
tested in n = 1 (doses expressed in cpm were normalized). Compound
24 Compound 28 Compound 29 recH insulin Mean .+-. s.e.m. Mean .+-.
s.e.m. Mean .+-. s.e.m. value (n = 1) Correlation -1.00 -1.00 -1.00
-1.00 T1/2 eli 3.0 .+-. 0.2 3.9 .+-. 0.1 5.5 .+-. 0.3 0.74 AUCinf
30447 .+-. 4034 13756 .+-. 590 14397 .+-. 517 481 (h cpm/0.1 mL)
Vss (L/kg) 0.192 .+-. 0.022 0.428 .+-. 0.028 0.570 .+-. 0.018 2.225
Cl (L/h/kg) 0.068 .+-. 0.009 0.146 .+-. 0.007 0.139 .+-. 0.05 4.16
MRT (h) 2.8 .+-. 0.1 2.9 .+-. 0.1 4.1 .+-. 0.2 0.5
CONCLUSION
[0113] The pharmacokinetic properties of insulin-pentasaccharide
conjugate 24 as determined by the .sup.125I-labeling method (FIG.
14, Table 4) were in agreement with those obtained with a similar
insulin-pentasaccharide conjugate (compound 6) determined by the
ELISA method (FIG. 13, Table 3). The differences in kinetic
parameters for recH insulin as determined by two analytical methods
can be explained by differences in curve extrapolation (to
calculate Vss and Cl) due to the fast disappearance of label from
the circulation during the first 15 min. Furthermore, the observed
differences in AUC, Cl and Vss of the insulin pentasaccharide
conjugates 24, 28, 29 (FIG. 14, Table 4) clearly demonstrate that
the pharmacokinetic properties of the conjugates can be tuned by
using alternative carrier pentasaccharides with different binding
affinity for ATIII (which is in agreement with the findings of the
BIA study, see FIG. 6 and Table 2).
Pharmacokinetics of Pentasaccharide Conjugates 31 and 32
TABLE-US-00005 [0114] TABLE 5 Pharmacokinetic parameters after i.v.
administration of the two conjugates 31 and 32. Experiment
performed in n = 3/treatment. (Data not corrected for
dehalogenation). Compound 31 Compound 32 Mean .+-. s.e.m. Mean .+-.
s.e.m. Correlation -1.00 -1.00 T1/2 eli (h) 7.1 .+-. 0.3 9.4 .+-.
0.6 Vss (ml/rat) 66.7 .+-. 2.3 52.5 .+-. 2.6 Cl (ml/h/rat) 7.9 .+-.
0.1 4.2 .+-. 0.3 MRT (h) 8.4 .+-. 0.2 12.6 .+-. 0.7
CONCLUSION
[0115] The half-life of free ganirelix (T1/21.4 h in rat, i.v.,
Chan et al. Drug. Metab. Dispos 1991, 19, 858) is significantly
extended when conjugated to a carrier pentasaccharide (FIG. 15,
Table 5). Comparison of the pharmacokinetics of compound 31 to that
of 32 shows that an improvement in Vss, Cl and T1/2 elimination is
obtained by using a pentasaccharide with a higher affinity for
ATIII. These data in combination with the findings of the BIA study
(see FIG. 8) indicate that the pharmacokinetic properties of the
conjugates can be tuned by using alternative carrier
pentasaccharides with different binding affinity for ATIII.
Pharmacokinetics of Pentasaccharide Conjugate 39 and ADM(27-52)
TABLE-US-00006 [0116] TABLE 6 Pharmacokinetic parameters after i.v.
administration of ADM(27-52) and compound 39. Experiment performed
in n = 3/treatment. (data not corrected for dehalogenation).
ADM(27-52) Compound 39 Mean .+-. s.e.m. Mean .+-. s.e.m.
Correlation -1.00 -1.00 T1/2 eli (h) 4.9 .+-. 0.1 11.1 .+-. 0.2 Vss
(ml/rat) 556 .+-. 48 80 .+-. 3.3 Cl (ml/h/) 112 .+-. 9 5.0 .+-. 0.1
MRT (h) 5.0 .+-. 0.1 14.5 .+-. 0.3
CONCLUSION
[0117] The pharmacokinetic properties of ADM(27-52) were improved
by conjugation to an ATIII binding carrier pentasaccharide
(compound 39, FIG. 16, Table 6). The T1/2 of ADM(27-52) per se may
have been overestimated since data have not been corrected for
dehalogenation. Moreover, the half-life of free adrenomedullin is
only 22 min in human (Meeran et al. J. Clin. Endocrin. Met. 1997,
82, 95-100). These and earlier observations in the BIA study (FIG.
9) support the conclusion that an improvement of pharmacokinetic
properties of a (poly)peptide can be achieved by conversion into a
(carrier) conjugate with specific binding affinity to circulating
ATIII.
Pharmacokinetics of Pentasaccharide Conjugate 41 Compared with
[D-Ala.sup.8]-GLP-1(7-36)
TABLE-US-00007 TABLE 7 Pharmacokinetic parameters after i.v.
administration of GLP-1 and compound 41 Experiment performed in n =
3/treatment (data expressed in cpm were normalized)
[D-Ala.sup.8]-GLP-1(7-36) Compound 41 Mean .+-. s.e.m. Mean .+-.
s.e.m. Correlation -0.98 -0.99 T1/2 eli (h) 2.0 .+-. 0.4 9.8 .+-.
0.4 AUCinf (h cpm/0.1 mL 1154 .+-. 59 108658 .+-. 4858 Vss (ml/kg
rat) 2696 .+-. 456 205 .+-. 13 Cl (ml/h/kg) 1732 .+-. 86 18 .+-.
0.8 MRT (h) 1.6 .+-. 0.1 11.2 .+-. 0.3
CONCLUSION
[0118] The pharmacokinetic properties of [D-Ala.sup.8]-GLP1(7-36)
were improved by conjugation to an ATIII binding carrier
pentasaccharide (compound 41, FIG. 17, Table 7). The Cl of compound
41 was decreased.about.100 fold and Vss 13 fold, resulting in
a.about.100 fold increase in AUC (exposure) compared to the
non-conjugated peptide. Combined with the BIA data (FIG. 10) these
observations support the conclusion that an improvement of
pharmacokinetic properties of a (poly)peptide can be achieved by
conversion into an ATIII binding conjugate.
Determination of (Poly)Peptide-Pentasaccharide-ATIII Complex in rat
Plasma
[0119] To ensure that (poly)peptide pentasaccharide conjugates bind
to Antithrombin III in vivo, a sandwich-type ELISA employing an
anti-insulin Mab as capture and a HRP-conjugated anti-ATIII
antibodies as detector was carried out on plasma samples from the
pharmacokinetic experiment of compound 6. Obviously, only intact
pentasaccharide-insulin-ATIII complex can be detected in this type
of assay in which recH insulin was used as a negative control).
From the plasma sample obtained 1 min after i.v. administration of
3.5 nmol/kg compound 6 or recH insulin in rat, the binding of
pentasaccharide-insulin conjugate 6 and recH insulin to rat ATIII
was determined. The results are shown in FIG. 18.
CONCLUSION
[0120] Pentasaccharide-insulin conjugate 6 is bound to ATIII, in
contrast with recH-insulin which cannot form a complex with ATIII.
Although the anti-rabbit ATIII antibody was less sensitive, both
ATIII antibodies were able to detect the pentasaccharide-insulin
conjugate 6-ATIII complex. These results demonstrate that compound
6 is bound to ATIII in circulation and that the prolonged half-life
of ATIII-binding pentasaccharides is the result of this
complexation Therefore, it can be concluded that the improvement of
the pharmacokinetic properties of (poly)peptide-pentasaccharide
conjugates (such as compound 6) compared to those of the original
non-conjugated (poly)peptide can be ascribed to specific binding of
the conjugate to ATIII.
Glucose Suppression Test in vivo in rats
[0121] The biological activities of insulin and the insulin
conjugates were tested in a rat model by measuring the glucose
depression levels. The animals were fasted overnight (16 hours)
prior to the experiment. In the morning blood was sampled from all
the rats by cutting a little piece of the tail, after which the
blood was dropped on a test strip and the glucose levels were
measured with an ACCU-Check Sensor blood glucose monitor (Roche
Diagnostics). The pentasaccharide-insulin conjugate 6 and insulin
were i.v. administered in the tail vein after pre-heating of the
rats in a heating box at 39.degree. C. during 10 min.. The applied
doses were 7 nmol/kg for pentasaccharide-insulin conjugate 6 and
3.5 nmol/kg for recH-insulin. At various time intervals blood
samples were taken by removing the crusted blood, after which the
glucose content was determined immediately as described.
Pharmacodynamics of Pentasaccharide Insulin Conjugate 6
[0122] The improved pharmacokinetic properties of
pentasaccharide-insulin conjugate 6 compared to those of
recH-insulin are confirmed by the prolonged glucose suppression
levels after i.v. administration (see FIG. 19).
In the experiments performed with compounds 24, 25, 26, 27 and
insulin detemir (control), the rats were starved just prior to
administration of compound (to warrant consistant glucose levels in
control group).
[0123] Comparison of the B29-insulin conjugate 24 with B29-insulin
conjugate 26 (FIG. 20), and B1-insulin conjugate 25 with B1-insulin
conjugate 27 (FIG. 21), reveals that similar prolonged glucose
suppression activities are obtained irrespective of spacer
length.
[0124] Surprisingly, it was found that the onset of action of all
insulin-conjugates tested was slower than that of recH insulin or
insulin detemir (i.v. administration) and that the exposure was
enhanced by the longer duration of action.
Direct comparison of B29-insulin conjugates 24, 28 and 29 within
one experiment at the dose of 24 nmol/kg substantiates the
difference in duration of action of their blood glucose lowering
activities (see FIG. 22). Thus, suppression of glucose levels with
compound 24 lasted beyond 7 h, while conjugates 28 and 29 were no
longer active, than 5.5 h after i.v. administration. The
pharmacodynamic differences correspond with the earlier mentioned
pharmacokinetic differences in distribution volume and clearance of
compound 24 compared to compound 28 and 29, respectively. Finally,
insulin detemir was tested as a comparative example showing less
pronounced and less prolonged activity at doses of 24 and 48
nmol/kg (FIGS. 21, 22).
* * * * *