Note: Descriptions are shown in the official language in which they were submitted.
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GLYCEROL BRANCHED POLYETHYLENE GLYCOL HUMAN GROWTH HORMONE
CONJUGATES, PROCESS FOR THEIR PREPARATION, AND METHODS OF USE THEREOF
The present application claims priority under Title 35, United States Code,
119 to United States
Provisional application Serial No. 60/605,945, filed August 31, 2004, which is
incorporated by
reference in its entirety as if written herein.
FIELD OF THE INVENTION
[001] The present invention relates to PEGylation, of human Growth Hormone
(hGH) by which
the chemical and/or physiological properties of hGH can be changed. The
PEGylated hGH conjugate
may have an increased plasma residency duration, decreased clearance rate,
improved stability,
decreased antigenicity, decreased PEGylation heterogeneity or a combination
thereof. The present
invention also relates to processes for the modification of hGH. In addition,
the present invention
relates to pharmaceutical compositions comprising the modified hGH. A further
embodiment is the
use of the modified hGH for the treatment of growth and development disorders.
BACKGROUND OF THE INVENTION
[002] Native human growth hormone (hGH) is a protein comprising a single chain
of 191 amino
acids cross-linked by two disulphide bridges and the monomeric form has a
molecular weight of 22
kDa. Human GH is secreted by the pituitary gland and which also can be
produced by recombinant
genetic engineering. hGH will cause growth in all bodily tissues that are
capable of growth. hGH plays
an important role not only in promoting growth in the growing phase in human
beings but also in
maintaining normal body composition, anabolism, and lipid metabolism (K.
Barneis. And U. Keller,
Baillieres Clin. Endocrinlo. Metab. 10:337 (1996)).
[003] Recombinant hGH has been commercially available for several years. Two
types of
therapeutically useful recombinant hGH preparations are present on the market:
the authentic one,
e.g. GenotropinTM, or NutropinTM and an analogue with an additional methionine
residue at the N-
terminal end, e.g. SomatonormTM. hGH is used to stimulate linear growth in
patients with hypo
pituitary dwarfism also referred to as Growth Hormone Deficiency (GHD) or
Turner's syndrome but
other indications have also been suggested including long-term treatment of
growth failure in children
who were born short for gestational age (SGA), for treatment of patients with
Prader-Willi syndrome
(PWS), chronic renal insufficiency (CRI), AIDS wasting, and Aging. Adult GH
deficiency (aGHD)
patients have various problems, such as characteristic changes in body
composition including
increase in fat mass, decrease in lean body mass and extracellular fluid, and
reduction of bone
mineral density, metabolic abnormalities of lipids, and cardiovascular
dysfunction. Many of those
problems are improved by hGH replacement therapy (J. Verhelst J and R. Abs.
Drugs.;62:2399
(2002).
[004] A major biological effect of growth hormone (GH) is to promote growth in
young mammals
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and maintenance of tissues in older mammals. The organ systems affected
include the skeleton,
connective tissue, muscles, and viscera such as liver, intestine, and kidneys.
Growth hormones exert
their effect through interaction with specific receptors on the target cell's
membrane. hGH is a
member of a family of homologous hormones that include placental lactogens,
prolactins, and other
genetic and species variants or growth hormone (Nicoll, C. S., et al. (1986)
Endocrine Reviews 7:
169). hGH is unusual among these in that it exhibits broad species specificity
and binds to either the
cloned somatogenic (Leung, D. W., et al. [1987] Nature 330; 537) or prolactin
receptor (Boutin, J. M.,
et al. [1988] Cell; 53: 69). The cloned gene for hGH has been expressed in a
secreted form in
Escherichia coli (Chang, C. N., et al. [1987] Gene 55:189), and its DNA and
amino acid sequence has
been reported (Goeddel, et al. [1979) Nature 281: 544; Gray, et al. [1985]
Gene 39:247).
[005] Human growth hormone (hGH) participates in much of the regulation of
normal human
growth and development. This pituitary hormone exhibits a multitude of
biological effects including
linear growth (somatogenesis), lactation, activation of macrophages, insulin-
like and diabetogenic
effects among others (Chawla, R, K. (1983) Ann. Rev. Med. 34, 519; Edwards, C.
K. et al. (1988)
Science 239, 769; Thomer, M. 0., et al. (1988) J. Clin. Invest. 81:745).
Growth hormone deficiency in
children leads to dwarfism, which has been successfully treated for more than
a decade by
exogenous administration of hGH.
[006] In adults, as well as in children, hGH maintains a normal body
composition by increasing
nitrogen retention and stimulation of skeletal muscle growth, and by
mobilization of body fat. Visceral
adipose tissue is particularly responsive to hGH. In addition to enhanced
lipolysis, hGH decreases
the uptake of triglycerides into body fat stores. Serum concentrations of IGF-
I (insulin-like growth
factor-I), and IGFBP3 (insulin-like growth factor binding protein 3) are
increased by hGH.
[007] hGH is a potent anabolic agent, especially due to retention of nitrogen;
phosphorus,
potassium, and calcium. Treatment of hypophysectomized rats with GH can
restore at least a portion
of the growth rate of the rats. Moore et al., Endocrinology 122:2920-2926
(1988). Among its most
striking effects in hypo pituitary (GH-deficient) subjects is accelerated
linear growth of bone-growth-
plate-cartilage resulting in increased stature. Kaplan, Growth Disorders in
Children and Adolescents
(Springfield, IL: Charles C. Thomas, 1964).
[008] hGH causes a variety of physiological and metabolic effects in various
animal models
including linear bone growth, lactation, activation of macrophages, insulin-
like and diabetogenic
effects, and others (R. K. Chawla et al., Annu. Rev. Med. 34:519 (1983); 0. G.
P. Isaksson et al.,
Annu. Rev. Physiol. 47, 483 (1985); C. K. Edwards et al., Science 239, 769
(1988); M. 0. Thomer and
M. L. Vance, J. Clin. Invest. 82:745 (1988); J. P. Hughes and H. G. Friesen,
Ann. Rev. Physiol.
47:469 (1985)). It has been reported that, especially in women after
menopause, GH secretion
declines with age. Millard et al., Neurobiol. Aging, 11:229-235 (1990);
Takahashi et al.,
NeuroendocrinologyM, L6- 137-142 (1987). See also Rudman et al., J. Clin.
Invest., 67:1361-1369
(1981) and Blackman, Endocrinology and Aging, 16:981 (1987). Moreover, a
report exists that some
of the manifestations of aging, including decreased lean body mass, expansion
of adipose-tissue
mass, and the thinning of the skin, can be reduced by GH treatment three times
a week. See, e.g.,
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WO 2006/024953 PCT/IB2005/002939
Rudman et al., N. Eng. J. Med., 323:1-6 (1990) and the accompanying.article in
the same journal
issue by Dr. Vance (pp. 52-54). These biological effects derive from the
interaction between hGH and
specific cellular receptors. Two different human receptors have been cloned,
the hGH liver receptor
(D. W. Leung et al., Nature 330:537(1987)) and the human prolactin receptor
(J. M. Boutin et al., Mol.
Endocrinology. 3:1455 (1989)). However, there are likely to be others
including the human placental
lactogen receptor (M. Freemark, M. Comer, G. Komer, and S. Handwerger,
Endocrinol. 120:1865
(1987)). These homologous receptors contain a glycosylated extracellular
hormone binding domain, a
single transmembrane domain, and a cytoplasmic domain, which differs
considerably in sequence
and size. One or more receptors are assumed to play a determining role in the
physiological response
to hGH.
[009] It is generally observed that physiologically active proteins
administered into a body can
show their pharmacological activity only for a short period of time due to
their high clearance rate in
the body. Furthermore, the relative hydrophobicity of these proteins may limit
their stability and/or
solubility.
[0010] For the purpose of decreasing the clearance rate, improving stability
or abolishing
antigenicity of therapeutic proteins, some methods have been proposed wherein
the proteins are
chemically modified with water-soluble polymers. Chemical modification of this
type may block
effectively a proteolytic enzyme from physical contact with the protein
backbone itself, thus preventing
degradation. Chemical attachment of certain water-soluble polymers may
effectively reduce renal
clearance due to increased hydrodynamic volume of the molecule. Additional
advantages include,
under certain circumstances, increasing the stability and circulation time of
the therapeutic protein,
increasing solubility, and decreasing immunogenicity. Poly(alkylene oxide),
notably poly(ethylene
glycol) (PEG), is one such chemical moiety that has been used in the
preparation of therapeutic
protein products (the verb "pegylate" meaning to attach at least one PEG
molecule). The attachment
of poly(ethylene glycol) has been shown to protect against proteolysis, Sada,
et al., J. Fermentation
Bioengineering 71: 137-139 (1991), and methods for attachment of certain
poly(ethylene glycol)
moieties are available. See U.S. Pat. No. 4,179,337, Davis et al., Non-
Immunogenic Polypeptides,
issued Dec. 18, 1979; and U.S. Pat. No. 4,002,531, Royer, Modifying Enzymes
with Polyethylene
Glycol and Product Produced Thereby, issued Jan. 11, 1977. For a review, see
Abuchowski et aL, in
Enzymes as Drugs. (J. S. Holcerberg and J. Roberts, eds. pp. 367-383 (1981)).
[0011] Other water-soluble polymers have been used, such as copolymers of
ethylene
glycol/propylene glycol, carboxymethylcellulose, dextran, poly(vinyl alcohol),
poly(vinyl pyrrolidone),
poly(-1,3-dioxolane), poly(-1,3,6-trioxane), ethylene/maleic anhydride
copolymer, poly- amino acids
(either homopolymers or random copolymers).
[0012] For poly(ethylene glycol), a variety of means have been used to attach
the poly(ethylene
glycol) molecules to the protein. Generally, poly(ethylene glycol) molecules
are connected to the
protein via a reactive group found on the protein. Amino groups, such as those
on lysine residues or
at the N-terminus, are convenient for such attachment. For example, Royer
(U.S. Pat. No. 4,002,531,
above) states that reductive alkylation was used for attachment of
poly(ethylene glycol) molecules to
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an enzyme. Chamow et al., Bioconjugate Chem. 5: 133-140 (1994) report the
modification of CD4
immunoadhesin with monomethoxypoly(ethylene glycol) aldehyde via reductive
alkylation. U.S.
5,824,784 demonstrates PEGylating G-CSF including at the N-terminus under
reductive alkylation
conditions.
[0013] WO 93/00109 relates to a method for stimulating a mammal's or avian's
GH responsive
tissues comprising, maintaining a continuous, effective plasma GH
concentration for a period of 3 or
more days. One way of achieving such plasma concentration is stated to be by
use of GH coupled to
a macromolecular substance such as PEG (polyethylene glycol). The coupling to
a macromolecular
substance is stated to result in improved half-life. PEGylated human growth
hormone has been
reported in WO 93/00109 using mPEG aldehyde-5000 and mPEG N-hydroxysuccinmidyl
ester(mPEG-NHS-5000) to achieve a hydrodynamic volume greater than the 70K
molecular weight
cut-off of the kidney filtration as described (Knauf, M.J. et al, J. Biol.
Chem. 263:15064-15070,1988).
The use of mPEG-NHS resulted in heterogeneous mixtures of multiply PEGylated
forms of hGH. WO
93/00109 also discloses the use of mPEG-maleimide to PEGylate cysteine hGH
variants.
[0014] WO 99/03887 discloses a cysteine variant growth hormone that is
PEGylated. Designated
as BT-005, this conjugate is purported to be more effective at stimulating
weight gain in growth
hormone deficient rats and to have a longer half-life than hGH.
[0015] PEGylated human growth hormone has also been reported in Clark et al.
using
succinimidyl ester of carboxymethylated PEG (Journal of Biological
Chemistry.271:21969-21977,
1996). Clark et al. describes derivates of hGH of increasing size using mPEG-
NHS-5000, which
selectively conjugates to primary amines. Increasing levels of PEG
modification reduced the affinity
for its receptor and increased the EC50 in a cell-based assay up to 1500 fold.
Olson et al., Polymer
Preprints 38:568-569, 1997 discloses the use of N-hydroxysuccinimide (NHS)PEG
and succinimidyl
propionate (SPA)PEG to achieve multiply PEGylated hGH species.
[0016] WO 94/20069 prophetically discloses PEGylated hGH as part of a
formulation for
pulmonary delivery.
[0017] US 4,179,337 discloses methods of PEGylating enzymes and hormones to
obtain
physiologically active non-immunogenic, water-soluble polypeptide conjugates.
GH is mentioned as
one example of a hormone to be PEGylated.
[0018] EP 458064 A2 discloses PEGylation of introduced or naturally present
cysteine residues
in somatotropin. EP 458064 A2 further mentions the incorporation of two
cysteine residues in a loop
termed the omega loop stated to be located at residues 102-112 in wild type
bovine somatotropin,
more specifically EP 458064 A2 discloses the substitution of residues numbered
102 and 112 of
bovine somatotropin from Ser to Cys and Tyr to Cys, respectively.
[0019] WO 95/11987 suggests attachment of PEG to the thio group of a cysteine
residue being
either present in the parent molecule or introduced by site directed
mutagenesis. WO 95/11987
relates to PEGylation of protease nexin-1, however PEGylation in general of
hGH and other prbteins
is suggested as well.
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[0020] WO 99/03887 discloses, e.g., growth hormone modified by replacement
serine at position
25 with a cysteine residue and attachment of PEG to the introduced cysteine
residue.
[0021] WO 00/42175 relates to a method for making proteins containing free
cysteine residues
for attachment of PEG. WO 00/42175 discloses the following muteins of hGH:
T3C, S144C and
T148C and the cysteine PEGylation thereof.
[0022] WO 97/11178 (as well as US 5849535, US 6004931, and US 6022711) relates
to the use
of GH variants as agonists or antagonists of hGH. WO 97/11178 also discloses
PEGylation of hGH,
including lysine PEGylation and the introduction or replacement of lysine
(e.g. K168A and K172R).
WO 9711178 also discloses the substitution G120K.
[0023] WO 03/044056 discloses a variety of PEGylated hGH species including a
lysine branched
40K PEG aldehyde hGH conjugate.
[0024] US 2004/0127417 discloses lysine branched PEG butyraldehyde hGH
conjugates.
[0025] WO 04/46222, US 2005/0058620, JP 08-059818, JP 11-228685, and JP 2000-
001541
disclose polyalkylene glycol derivatives having a reactive group at the
primary carbon at the 1-position
of a giycerol skeleton and having polyalkylene glycol chains at the 2- and 3-
positions.
[0026] Currently administration of rhGH is daily for a long period of time,
and therefore a less
frequent administration would be highly desirable. An hGH molecule with a
longer circulation half-life
would decrease the number of necessary administrations and potentially provide
more optimal
therapeutic hGH levels with concomitant enhanced therapeutic effect.
[0027] Despite a number of attempts to develop a long lasting form of hGH,
including PEGylating
hGH, there is still an unmet need for a PEGylated hGH molecule with the
appropriate properties to be
a viable commercial product. The present invention provides PEG-hGH conjugates
having a single
PEG attached predominately at the N-terminal phenylalanine of hGH, which
provides advantages
over other PEG-hGH conjugates. The attachment of multiple low molecular weight
(5Kd) PEGs at a-
or E-amino sites (N-terminus and nine lysines in hGH) using mPEG aldehyde-5000
or mPEG N-
hydroxysuccinmidyl ester (mPEG-NHS-5000) has been described in WO 93/00109,
Clark et al.
(Journal of Biological Chemistry 271:21969-21977, 1996, and Olson et al.
(Polymer Preprints 38:568-
569, 1997). This results in a heterogeneous population. As an illustration hGH
with nine lysines may
have some molecules having ten PEGs attached, some with nine, some with eight,
some with seven,
some with six, some with five, some with four, some with three, some with two,
some with one and
some with zero. And, among the molecules with several, the PEG may not be
attached at the same
location on different molecules. This resulting heterogeneity is
disadvantageous when developing a
therapeutic product making conjugation, purification, and characterization
difficult, costly, and highly
irreproducible. Another approach (WO 00/42175) has been to use hGH variants
containing free
cysteine residues for attachment of PEG. However, this approach results in an
unnatural hGH variant
and can also lead to incorrectly folded protein having incorrectly paired
disulfide bonds resulting in a
heterogeneous PEGylated product that has the PEG attached at some or all of
the cysteines. Having
multiple PEGs attached to multiple sites may lead to molecules that have less
stable bonds between
the PEG and the various sites, which can become dissociated at different
rates. This makes it difficult
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to accurately predict the pharmacokinetics of the product resulting in
inaccurate dosing. A
heterogeneous product also poses unwanted problems in obtaining regulatory
approval for the
therapeutic product.
Therefore, it would be desirable to have a PEGylated hGH molecule that has a
single PEG attached
at a single site. The present invention addresses this need in a number of
ways.
SUMMARY OF THE INVENTION
[0028] The present invention relates to PEGylated hGH using a glycerol
branched poly(ethylene
glycol) moiety which may have at least one improved chemical or physiological
property selected from
but not limited to; decreased clearance rate, increased plasma residency
duration, increased stability,
improved solubility, and decreased antigenicity. Thus, as described below in
more detail, the present
invention has a number of aspects relating to chemically modifying hGH using a
glycerol branched
poly(ethylene glycol) moiety.
[0029] The present invention may also have one or more improved properties
compared to
lysine based branched PEG human growth hormone conjugates including but not
limited to: a)
increased stability of the glycerol skeleton, b) increased receptor binding,
c) decreased cost, d)
increased N-terminal selectivity of attachment, e) increased solubility, f)
decreased immunogenicity, g)
increased stability of the conjugate, h) increased manufacturability, and i)
decreased proteolysis.
[0030] The present invention also relates to methods of producing the
PEGylated hGH.
Particularly, the present invention relates to a method of producing a
PEGylated hGH using a glycerol
branched PEG.
[0031] The present invention also relates to compositions comprising the
PEGylated hGH alone
or in combination with another therapeutic agent. The present invention also
relates to the use of the
PEGylated hGH of the present invention, alone or in combination with another
therapeutic agent, in
the prevention and/or treatment of disorders and/or diseases in which GH
treatment is useful.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Figure 1 is a size exclusion HPLC tracing showing the elution profile
of the purified
monoPEGylated glycerol branched 43K PEG aldehyde hGH reaction product on a TSK
G4000PWXL
column.
[0033] Figure 2 is a HPLC tracing of tryptic map analysis of hGH and glycerol
branched 43K
PEG aldehyde hGH. The top panel is the tryptic map of hGH. The lower panel is
the tryptic map of
glycerol branched 43K PEG aldehyde hGH. T1 is the N-terminal tryptic fragment.
[0034] Figure 3 shows the amino acid sequence of human growth hormone (SEQ ID
NO:1).
[0035] Figure 4 shows the glycerol branched 43K PEG aldehyde hGH efficacy in
an eleven-day
Rat Weight Gain Assay. Hypophysectomized female Sprague-Dawley rats were
purchased at the
age of 4-5 weeks (85-110g) from Harlan Labs. Upon entering animal facilities,
the animals were
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maintained at a constant room temperature of 80 F. After 3 days' acclimation,
the rats were weighed
daily for 4-10 days in order to establish basal growth rates. Starting at day
0, rats (-100g) in control
groups then received one dailysubcutaneous injection of -0.3 mg/kg hGH (A), or
PBS vehicle (=) for
eleven consecutive days. The glycerol branched 43K P.EG aldehyde hGH test
group (.) received
single doses of 1.8 mg/kg of glycerol branched 43K PEG aldehyde hGH on days 0
and 6. There were
6 animals per group. Plotted values represent average weight gain SEM.
[0036] Figure 5 shows eleven-day tibia growth in response to glycerol branched
43K PEG
aidehyde hGH. Animals were those treated in Figure 4. Animals were sacrificed
after taking day 11
weights, the left tibias were X-rayed, and the bone length measured using a
caliper. Average length
+/- SEM is plotted. Asterisks denote significant differences from control
group (P<0.05). There were
6 animals per group.
[0037] Figure 6 shows eleven-day blood urea nitrogen levels in response to
glycerol branched
43K PEG aldehyde hGH. Blood samples were taken from animals treated in Figure
4. Serum was
prepared and urea nitrogen levels were measured. Average SEM is plotted (6
animals per group).
Asterisks denote significant differences from control group (P<0.05).
[0038] Figure 7 shows a six-day dose escalation efficacy study for glycerol
branched 43K PEG
aldehyde hGH. This growth study was performed in a similar manner to that
described in Figure 4
except that varied single doses of glycerol branched 43K PEG aldehyde hGH were
administered only
on day 0 and the study was run for 6 days. Control groups received once-daily
subcutaneous
injections of either 0.3 mg/kg hGH (*) or PBS vehicle (o) for six consecutive
days. The glycerol
branched 43K PEG aidehyde hGH test groups received a single dose of glycerol
branched 43K PEG
aldehyde hGH on day 0. The glycerol branched 43K PEG aldehyde hGH doses were
1.8 mg/kg (m),
0.6 mg/kg (X), 0.2 mg/kg, (+), 0.067 mg/kg (A). There were 6 animals per
group.
[0039] Figure 8 shows serum IGF-1 levels for six-day efficacy study. Animals
were treated as
described in Figure 7. Blood samples were taken at the various times plotted
and the serum IGF-1
levels determined by ELISA. Group (n=6) means were used to calculate the IGF-1
response using
one-way analysis of variance on the measured values and AUC dO-6 (ng/mL*24h)
values of 37,839,
28,1292, 22,958, and 20,040 were determined for the 1.8, 0.6, 0.2, and 0.067
mg/kg dosing cohorts,
respectively.
[0040] Figure 9 shows the PK/PD assessment following single dose
administration of glycerol
branched 43K PEG aldehyde hGH to hypophysectomized female rats. The effect of
single 1.8 mg/kg
SC dose administration of glycerol branched 43K PEG aldehyde hGH upon plasma
drug levels (a) or
plasma IGF-1 response (b).
DETAILED DESCRIPTION OF THE INVENTION
[0041] The present invention relates to glycerol branched polyethylene glycol-
human growth
hormone conjugates. In a specific embodiment the glycerol branched
polyethylene glycol derivative
has an aldehyde reactive group and optionally a linker between the
polyethylene glycol and the
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reactive functional group at the primary carbon at the 1-position of a
glycerol skeleton and having
polyalkylene glycol chains at the 2- and 3-positions as described in WO
04/46222 or US
2005/0058620 (incorporated by reference) to create hGH conjugates. The linker
is not particularly
limited as far as it is a covalent bond, but preferably includes an alkylene
group and an alkylene group
containing an ester bond, a urethane bond, an amide bond, an ether bond, a
carbonate bond, or a
secondary amino group. Preferable alkylene group includes a methylene group,
an ethylene group, a
trimethylene group, a propylene group, an isopropylene group, a tetramethylene
group, a butylene
group, an isobutylene group, a pentamethylene group, and a hexamethylene
group. ,
[0042] A specific embodiment of the present invention is a human growth
hormone-PEG
conjugate having the structure of the Formula:
H2C O(CH2CH2O)nCH2CH2CH2NHR
CH O(CH2CH2O)mCH3
H2C O(CH2CH2O)mCH3
wherein
n is an integer between 60 and 75;
m is an integer between 450 and 460; and
R is a human growth hormone polypeptide.
[0043] In a particular embodiment n is between about 64 and about 72.
[0044] In a particular embodiment the (CH2CH2O)õ moiety has an average
molecular weight
between about 2.6 and about 3.5Kd, and particularly the average molecular
weight is about 3Kd,
[0045] In a particular embodiment each (CH2CH2O)m moiety has an average
molecular weight
between about 17.6 and about 22Kd, and particularly the average molecular
weight is about 20Kd.
[0046] In a specific embodiment the (CH2CH2O)n moiety has an average molecular
weight of
about 3Kd and each (CH2CH2O)m moiety has an average molecular weight of about
20Kd.
[0047] The term "about" when used in connection with the molecular weight of a
PEG moiety
means that in preparations of polyethylene glycol, some molecules will weigh
more, some less, than
the stated molecular weight and the stated molecular weight refers to the
average molecular weight.
It is understood that there is some degree of polydispersity associated with
polymers such as
poly(ethylene glycol). It is preferable to use PEGs with low polydispersity.
In a specific embodiment
one of the terminal polymer hydroxyi end-groups is converted or capped with a
methyl group. As
used herein, the term "mPEG" refers to a PEG, which is capped at one end with
a methyi group. The
mPEG can be represented structurally as
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CH3O-(CH2CH2O)n--H
[0048] The term "human growth hormone polypeptide", "hGH polypeptide" or "hGH
protein",
when used herein, encompasses all hGH polypeptides, characterized by promoting
growth in the
growing phase and in maintaining normal body composition, anabolism, and lipid
metabolism.
Preferably, the term "hGH polypeptide" refers to the hGH polypeptide of SEQ ID
NO:1
[0049] The hGH polypeptides of the present invention can be prepared in any
suitable manner.
Such hGH polypeptides and fragments thereof may be purified from natural
sources, chemically
synthesized, produced by recombinant techniques including in vitro translation
techniques or
expression in a recombinant cell able to express hGH cDNA, or a combination of
these methods,
using techniques known to those skilled in the art (See, for example, "Methods
in Enzymology,
Academic Press, 1993" for a variety of methods for purifying proteins;
Creighton, (1983) Proteins:
Structures and Molecular Principles, W.H. Freeman & Co. 2nd Ed., T. E., New
York; and Hunkapiller
et al., (1984) Nature. 310(5973): 105-11 for chemical synthesis of proteins
and Davis et al. (1986)
Basic Methods in Molecular Biology, ed., Elsevier Press, NY for recombinant
techniques, which
disclosures are incorporated by reference in their entireties). The
polypeptides of the present
invention are preferably provided in an isolated form, and may be partially or
preferably substantially
purified.
[0050] A specific embodiment of the present invention is a human growth
hormone-PEG
conjugate wherein greater than 80%, more preferably 81%, more preferably 82%,
more preferably
83%, more preferably 84%, more preferably 85%, more preferably 86%, more
preferably 87%, more
preferably 88%, more preferably 89%, more preferably 90%, more preferably 91
%, more preferably
92%, more preferably 93%, more preferably 94%, more preferably 95%, more
preferably 96%, more
preferably 97, and more preferably 98% of the polyethylene glycol is
conjugated to the amino-terminal
phenylaianine of the human growth hormone of SEQ ID NO:1.
[0051] Another embodiment of the present invention is a substantially
homogenous preparation
of N-terminally PEGylated hGH optionally in a pharmaceutically acceptable
diluent, carrier or
adjuvant, said preparation being essentially free of hGH PEGylated at sites
other than the N-terminus.
The term "substantially homogenous preparation" means a preparation where
greater than 80%, more
preferably 81%, more preferably 82%, more preferably 83%, more preferably 84%,
more preferably
85%, more preferably 86%, more preferably 87%, more preferably 88%, more
preferably 89%, more
preferably 90%, more preferably 91 %, more preferably 92%, more preferably
93%, more preferably
94%, more preferably 95%, more preferably 96%, more preferably 97, and more
preferably 98% is
monoPEGylated.
[0052] In one embodiment of the invention secondary amine linkages are formed
between the N-
terminal primary a- amino group of a hGH polypeptide and a glycerol branched
chain PEG aldehyde
by reductive alkylation as described in Chamow et al., Bioconjugate Chem. 5:
133-140 (1994), US Pat
No. 4,002,531, WO 90/05534, and US Pat. No 5,824,784 with a suitable reducing
agent such as
9
CA 02577999 2007-02-23
WO 2006/024953 PCT/IB2005/002939
NaCNBH3, NaBH3, Pyridine Borane etc. The glycerol branched PEG aldehyde is
incubated with an
hGH polypeptide resulting in the addition of the PEG moiety to amino groups
via Schiff's base
formation. These linkages are converted to stable secondary amines by
reduction with a reducing
agent. The reductive alkylation process is depicted in the scheme below (from
Chamow et al.).
NH2 O
NH2 + C-CH2--OR
H
Amine- COOH
containing protein t I pH 6-9
NH2
6N CH-CH2-OR
COOH
i NaCNBH3
NH2
6 NH CHa-CH2-OR
COOH
[0053] Conjugation reactions, referred to as "PEGylation reactions", were
historically carried out
in solution with molar excess of polymer and without regard to where the
polymer will attach to the
protein. Such general techniques, however, have typically been proven
inadequate for conjugating
bioactive proteins to non-antigenic polymers while retaining sufficient
bioactivity. One way to maintain
the hGH bioactivity is to substantially avoid the conjugation of those hGH
reactive groups associated
with the receptor binding site(s) in the polymer coupling process. Another
aspect of the present
invention is to provide a process of conjugating poly(ethylene glycol) to hGH
maintaining high levels of
retained activity.
[0054] The chemical modification through a covalent bond may be performed
under any suitable
condition generally adopted in a reaction of a biologically active substance
with the activated
poly(ethylene glycol). The conjugation reaction is carried out under
relatively mild conditions to avoid
inactivating the hGH. Mild conditions include maintaining the pH of the
reaction solution in the range
of 3 to 10 and the reaction temperatures within the range of from about 0 -37
C. in the cases where
the reactive amino acid residues in hGH have free amino groups, the above
modification is preferably
carried out in a non-limiting list of suitable buffers (pH 4 to 10), including
phosphate, MES, citrate,
acetate, succinate or HEPES, for 1-48 hrs at 4 -37 C. In targeting N-terminal
amino groups with
CA 02577999 2007-02-23
WO 2006/024953 PCT/IB2005/002939
reagents such as PEG aldehydes pH 4-8 is preferably maintained. The activated
poly(ethylene
glycol) may be used in about 0.01-100 times, preferably about 0.01-2.5 times,
the molar amount of the
number of free amino groups of hGH.
[0055] Although the reaction conditions described herein can result in
significant amounts of
unmodified hGH, the unmodified hGH can be readily recycled into future batches
for additional
conjugation reactions. The processes of the present invention generate
surprisingly very little, i.e. less
than about 20% and more preferably, less than about 10%, of high molecular
weight species and
species containing more than one polymer strand per hGH. These reaction
conditions are to be
contrasted with those typically used for polymeric conjugation reactions
wherein the activated polymer
is present in several-fold molar excesses with respect to the target.
[0056] The conjugation reactions of the present invention initially provide a
reaction mixture or
pool containing mono- PEG-hGH conjugates, unreacted hGH, unreacted polymer,
and less than
about 20% high molecular weight species. The high molecular weight species
include conjugates
containing more than one polymer strand and/or polymerized PEG-hGH species.
After the unreacted
species and high molecular weight species have been removed, compositions
containing primarily
mono- PEGylated-hGH conjugates are recovered. Given the fact that the
conjugates for the most part
include a single polymer strand, the conjugates are substantially homogeneous.
These modified hGH
have at least about 0.1 % of the in vitro biological activity associated with
the native or unmodified
hGH as measured using standard FDC-P1 cell proliferation assays, (Clark et al.
Journal of Biological
Chemistry 271:21969-21977, 1996), receptor binding assay (US 5,057,417), or
hypophysectomized
rat growth (Clark et al. Journal of Biological Chemistry 271:21969-21977,
1996). In preferred aspects
of the invention, however, the modified hGH have about 25% of the in vitro
biological activity, more
preferably, the modified hGH have about 50% of the in vitro biological
activity, more preferably, the
modified hGH have about 75% of the in vitro biological activity, and most
preferably the modified hGH
have equivalent or improved in vitro biological activity.
[0057] The processes of the present invention preferably include rather
limited ratios of polymer
to hGH. Thus, the hGH conjugates have been found to be predominantly limited
to species containing
only one strand of polymer. Furthermore, the attachment of the polymer to the
hGH reactive groups is substantially less random than when higher molar
excesses of polymer linker are used. The
unmodified hGH present in the reaction pool, after the conjugation reaction
has been quenched, can
be recycled into future reactions using ion exchange or size exclusion
chromatography or similar
separation techniques.
[0058] A poly(ethylene glycol)-modified hGH may be purified from a reaction
mixture by
conventional methods which are used for purification of proteins, such as
dialysis, salting-out,
ultrafiltration, ion-exchange chromatography, hydrophobic interaction
chromatography (HIC), gel
chromatography and electrophoresis. Ion-exchange chromatography is
particularly effective in
removing unreacted poly(ethylene glycol) and hGH. In a further embodiment of
the invention, the
mono PEGylated-hGH species is isolated from the reaction mixture to remove
high.molecular weight
species, and unmodified hGH. Separation is effected by placing the mixed
species in a buffer solution
11
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WO 2006/024953 PCT/IB2005/002939
containing from about 0.5-10 mg/mL of the hGH-polymer conjugates. Suitable
solutions have a pH
from about 4 to about 10. The solutions preferably contain one or more buffer
salts selected from KCI,
NaCI, K2HPO4, KH2PO4, Na2HPO4, NaH2PO4, NaHCO3, NaBO4, CH3CO2H, and NaOH.
[0059] Depending upon the reaction buffer, the hGH polymer conjugate solution
may first have
to undergo buffer exchange/ultrafiltration to remove any unreacted polymer.
For example, the PEG-
hGH conjugate solution can be ultrafiltered across a low molecular weight cut-
off (10,000 to 30,000
Dalton) membrane to remove most unwanted materials such as unreacted polymer,
surfactants, if
present, or the like.
[0060] The fractionation of the conjugates into a pool containing the desired
species is preferably
carried out using an ion exchange chromatography medium. Such media are
capable of selectively
binding PEG-hGH conjugates via differences in charge, which vary in a somewhat
predictable
fashion. For example, the surface charge of hGH is determined by the number of
available charged
groups on the surface of the protein. These charged groups typically serve as
the point of potential
attachment of poly(alkylene oxide) polymers. Therefore, hGH conjugates will
have a different charge
from the other species to allow selective isolation.
[0061] Strongly polar anion or cation exchange resins such as quaternary amine
or sulfopropyl
resins, respectively, are used for the method of the present invention. Anion
exchange resins are
especially preferred. A non-limiting list of included commercially available
cation exchange resins
suitable for use with the present invention are SP-hitrap , SP Sepharose HP
and SP Sepharose
fast flow. Other suitable cation exchange resins e.g. S. and CM resins 'can
also be used. A non-
limiting list of anion exchange resins, including commercially available anion
exchange resins, suitable
for use with the present invention are Q-hitrap , Q Sepharose HP , and Q
sepharose fastfilow.
Other suitable anion exchange resins, e.g. DEAE resins, can also be used.
[0062] For example, the anion or cation exchange resin is preferably packed in
a column and
equilibrated by conventional means. A buffer having the same pH and osmolality
as the polymer
conjugated hGH solution is used. The elution buffer preferably contains one or
more salts selected
from KCI, NaCI, K2HPO4, KH2PO4, Na2HPO4, NaH2PO4, NaHCO3, NaBO4, and
(NH4)2CO3. The
conjugate-containing solution is then adsorbed onto the column with unreacted
polymer and some
high molecular weight species not being retained. At the completion of the
loading, a gradient flow of
an elution buffer with increasing salt concentrations is applied to the column
to elute the desired
fraction of polyalkylene oxide-conjugated hGH. The eluted pooled fractions are
preferably limited to
uniform polymer conjugates after the cation or anion exchange separation step.
Any unconjugated
hGH species can then be back washed from the column by conventional
techniques. If desired, mono
and multiply pegylated hGH species can be further separated from each other
via additional ion
exchange chromatography or size exclusion chromatography.
[0063] Techniques utilizing multiple isocratic steps of increasing
concentration of salt or pH can
also be used. Multiple isocratic elution steps of increasing concentration
will result in the sequential
elution of di- and then mono-hGH-polymer conjugates.
12
CA 02577999 2007-02-23
WO 2006/024953 PCT/IB2005/002939
[0064] The temperature range for elution is between about 4 C and about 25 C.
Preferably,
elution is carried out at a temperature of from about 4 C to about 22 C. For
example, the elution of
the PEG-hGH fraction is detected by UV absorbance at 280 nm. Fraction
collection may be achieved
through simple time elution profiles.
[0065] A surfactant can be used in the processes of conjugating the
poly(ethylene glycol)
polymer with the hGH moiety. Suitable surfactants include ionic-type agents
such as sodium dodecyl
sulfate (SDS). Other ionic surfactants such as lithium dodecyl sulfate,
quaternary ammonium
compounds, taurocholic acid, caprylic acid, decane sulfonic acid, etc. can
also be used. Non-ionic
surfactants can also be used. For example, materials such as poly(oxyethylene)
sorbitans (Tweens),
poly(oxyethylene) ethers (Tritons) can be used. See also Neugebauer, A Guide
to the Properties and
Uses of Detergents in Biology and Biochemistry (1992) Calbiochem Corp. The
only limitations on the
surfactants used in the processes of the invention are that they are used
under conditions and at
concentrations that do not cause substantial irreversible denaturation of the
hGH and do not
completely inhibit polymer conjugation. The surfactants are present in the
reaction mixtures in
amounts from about 0.01-0.5%; preferably from 0.05-0.5%; and most preferably
from about 0.075-
0.25%. Mixtures of the surfactants are also contemplated.
[0066] It is thought that the surfactants provide a temporary, reversible
protecting system during
the polymer conjugation process. Surfactants have been shown to be effective
in selectively
discouraging polymer conjugation while allowing lysine-based or amino terminal-
based conjugation to
proceed.
[0067] The present poly(ethylene glycol)-modified hGH has a more enduring
pharmacological
effect, which may be possibly attributed to its prolonged half-life in vivo.
[0068] Another embodiment of the invention relates to methods for the
prevention and/or
treatment of a disease or disorder in which use of GH, preferably hGH is
beneficial, comprising
administering to a patient in need thereof a therapeutically effective amount
of a poly(ethylene glycol)-
modified hGH of the invention or agonist variant thereof, alone or in
combination with another
therapeutic agent. The invention also relate to the use of a poly(ethylene
glycol)-modified hGH of the
invention or agonist variant thereof in the manufacture of a medicament for
the prevention and/or
treatment of a disease or disorder in which use of GH, preferably hGH is
beneficial. In addition, the
invention also relates to a pharmaceutical composition comprising a
poly(ethylene glycol)-modified
hGH of the invention or agonist variant thereof for the prevention and/or
treatment of a disease or
disorder in which use of GH, preferably hGH is beneficial.
[0069] Diseases or disorders in which the use of GH is beneficial include, but
are limited to,
growth hormone deficiency (GHD), adult growth hormone deficiency (aGHD),
Turner's syndrome,
growth failure in children who were born short for gestational age (SGA),
Prader-Willi syndrome
(PWS), chronic renal insufficiency (CRI), Aids wasting, Aging, end-stage Renal
Failure, Cystic
Fibrosis, Erectile dysfunction, HIV lipodystrophy, Fibromyalgia, Osteoporosis,
Memory disorders,
Depression, Crohn's disease, Skeletal dysplasias, Traumatic brain injury,
Subarachnoid
haemorrhage, Noonan's syndrome, Down's syndrome, Idiopathic short stature
(ISS), End stage renal
13
CA 02577999 2007-02-23
WO 2006/024953 PCT/IB2005/002939
disease (ESRD), Very low birth weight (VLBW), Bone marrow stem cell rescue,
Metabolic syndrome,
Glucocorticoid myopathy, Short stature due to glucocorticoid treatment in
children, and Failure of
growth catching for short premature children.
[0070] In a more specific embodiment of the invention, the poly(ethylene
glycol)-modified hGH of
the invention or agonist variants thereof are used in the prevention and/or
treatment of a disorders or
diseases selected from the group consisting of GHD, aGHD, SGA, PWS, Turner's
syndrome and CRI.
[0071] In another more specific embodiment of the invention, the poly(ethylene
glycol)-modified
hGH of the invention or agonist variants thereof are used in the prevention
and/or treatment of a
disorders or diseases selected from the group consisting of idiopathic short
stature, very low birth
weight, traumatic brain injury, metabolic syndrome, and Noonan's syndrome.
[0072] Another embodiment of the invention relate to pharmaceutical
compositions comprising a
poly(ethylene glycol)-modified hGH of the invention alone or in combination
with another therapeutic
agent, and at least one pharmaceutically acceptable excipient or carrier. The
present poly(ethylene
glycol)-modified hGH may then be formulated into pharmaceuticals containing
also a pharmaceutically
acceptable diluent, an agent for preparing an isotonic solution, a pH-
conditioner and the like in order
to administer them into a patient.
[0073] The above pharmaceuticals may be administered subcutaneously,
intramuscularly,
intravenously, pulmonary, intradermally, or orally, depending on a purpose of
treatment. A dose may
be also based on the kind and condition of the disorder of a patient to be
treated, being normally
between 0.1 mg and 5 mg by injection and between 0.1 mg and 50 mg in an
oral.administration for an
adult.
[0074] As used herein, the poly(ethylene glycol)-modified hGH or agonist
variants thereof of the
present invention may be used in combination with another therapeutic agent.
As used herein, the
terms "co-administration", "co-administered" and "in combination with",
referring to the compounds A
and one or more other therapeutic agents, is intended to mean, and does refer
to and include the
following :
o simultaneous administration of such combination of A and therapeutic
agent(s) to
a patient in need of treatment, when such components are formulated together
into a single dosage form which releases said components at substantially the
same time to said patient;
o substantially simultaneous administration of such combination of A and
therapeutic agent(s) to a patient in need of treatment, when such components
are
formulated apart from each other into separate dosage forms which are taken at
substantially the same time by said patient, whereupon said components are
released at substantially the same time to said patient;
o sequential administration of such combination of A and therapeutic agent(s)
to a
patient in need of treatment, when such components are formulated apart from
each other into separate dosage forms which are taken at consecutive times by
said patient with a significant time interval between each administration,
14
CA 02577999 2007-02-23
WO 2006/024953 PCT/IB2005/002939
whereupon said components are released at substantially different times to
said
patient; and
o sequential administration of such combination of A and therapeutic agent(s)
to a
patient in need of treatment, when such components are formulated together
into
a single dosage form which releases said components in a controlled manner
whereupon they are concurrently, consecutively, and/or overlappingly
administered at the same and/or different times by said patient.
[0075] Suitable examples of other therapeutic agents which may be used in
combination with A,
their pharmaceutically acceptable salts and/or their derived forms include,
but are by no mean limited
to: aromatase inhibitors such as exemestane, formestane, atamestane,
fadrozole, letrozole, vorozole
and anastrozole; free fatty acid regulators including fibric acid derivatives
(such as fenofibrate,
clofibrate, gemfibrozil, bezafibrate and ciprofibrate) and nicotinic acid
derivatives such as acipimox;
insulin sensitizing agents including but not limited to biguanides such as
metformin, PPAR gamma
insulin sensitizing agents and thiazolodeniones such as troglitazone and
rosiglitazone Troglitazone, 5-
[[4-[3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-]-benzopyran-2-yl)
methoxy]phenyl]methyl3 -2,4-
thiazolidinedione V411(DIABII, Glaucanin) Pioglitazone (ACTOS, AD 4833, U
72107, U 72107A, U
72107E, ZACTOS) Chemical Name: 2,4-Thiazolidinedione, 5-[[4-[2-(5-ethyl-2-
pyridinyl)
ethoxy]phenyl]methyl]-, monohydrochloride, (a/-); Rosiglitazone (Avandia, BRL
49653, BRL 49653C)
Chemical Name: 2,4 Thiazolidinedione, 5-[[4-[2- (methyl-2-
pyridinylarnino)ethoxy]phenyl]methyl]; 25
Bexarotene-oral (LGD 1069 oral, Targretin oral, Targretin, Targretyn oral
Targrexin oral) Chemical
Name: 4-[1-(3,5,5,8,8-Pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)
ethenyl]benzoic acid; ZD 2079, (ICI
D 2079) (Chemical Name: R)-N-[2-4- (Carboxymethyl) 30 phenoxy]ethyl)-N-(2-
hydroxy-2-phenethyl)
ammonium chloride: Netoglitazone, (Isaglitazone, MCC 555, RWJ 241947)
(Chemical Name: 5-[6(2-
Fluorobenzyloxy)naphthalen-2-ylmethyl]thiazolidine-2,4-dione) ; INS (D-chiro-
inositol) (Chemical
Name: D-1,2,3,4,5,6- Hexabydroxycyclohexane), ON 2344(DRF 2593); Dexlipotam,
Chemical Name:
5(R)-(1,2-Dithiolan-3-yI) pentaloic 35 acid; HQL 975, Chemical Name: 3-[4- [2-
(5-Methyl-2-
phenyloxazol-4-yl) ethoxy]phenyl]-2(S)-(propylamino) propionic acid; YM 268,
Chemical Name: 5,5'-
Methylene-bis(1,4-phenylene)bismethylenebis (thiazolidine-2,4-dione). I PPAR
agonists under
development include: Reglitazar (JTT 501, PNU 182716, PNU 716) (Chemical Name:
Isoxazolidien-3,
5-dione, i 4-[[4-(2-phenyi-5-methyl)-1,3-oxazolyl]ethoxyphenyl-4] methyl-,
(4RS)); I(RP 297, Chemical
Name: 10 5-(2,4-DioXothiazolidin-5-ylmethyl)-2-methoxy-N-[4-(trifluoromethyl)
benzylbenzamide; R
119702 (CI 1037, CS 011) ChemicalName: (/-)-5-[4-(5-Methoxy- 1H benzimidazol-2-
ylmethoxy)benzyl] thiazolin-2,4-dione; hydrochloride; 15 DRF 2189, Chemical
Name: 5-[[4-[2-(1-
Indolyl)ethoxy]phenyl]methyl] thiazolidine-2,4-dione; cortisol synthesis
inhibitors such as
Ketoconazole, econazole or miconazole; growth hormones such as somatropin or
somatonorm and
their derivatives such as human growth hormone fusion proteins such as
ALBUTROPIN; polyethylene
glycol growth hormones such as the cysteine-pegylated growth hormone, BT 005
(Bolder
BioTechnology Inc.); growth hormone secretagogues such as, for example, SM
130686 (Sumitomo)
capromorelin (Pfizer), mecasermin (Fujisawa), sermorelin {Salk Institute, Bio-
Technology General),
CA 02577999 2007-02-23
WO 2006/024953 PCT/IB2005/002939
somatrem, somatomedin (C Llorente; Pharmacia Corporation) examorelin,
tabimorelin; CP 464709
(Pfizer), LY 426410 and LY 444711 (Lilly); 8-(aminoalkoxyimino)-8H-
dibenzo[a,e]triazolo[4,5-
c]cycloheptenes as disclosed in W02002057241, 2-substituted dibenzo[a,e]1,2,3-
triazolo[4,5-
c][7]annulen-8-ones as described in W02002056873 growth hormone releasing
peptides GHRP-6
and GHRP-1 as described in U.S. Patent No. 4,411,890, and publications WO
89/07110, WO
89/07111, B-HT920, hexarelin and GHRP-2 as described in WO 93/04081 or growth
hormone
releasing hormone (GHRH, also designated GRF) and its analogs, somatomedins
including IGF-1
and IGF-2 and their derivatives such as SomatoKine - a recombinant fusion of
insulin-like growth
factor-1 and its binding protein, BP-3, alpha-2-adrenergic agonists such as
clonidine, xylazine,
detomidine and medetomidine or serotonin 5HTID agonists such as surnitriptan
or agents which
inhibit somatostatin or its release such as physostigmine and pyridostigmine,
ThGRF 1-44
(Theratechnologies); L 165166 (Merck & Company); dipeptide derivatives as
described in
W09858947, Inhibitors of dipeptidyl peptidase IV such as amino-acylpyrrolidine
nitrile as described in
US6521644, W095/15309 and W098/19998; Beta-amino heterocyclic dipeptidyl
peptidase inhibitors
such as those described in US20030100563 and W02003082817; growth hormone
releasing
compounds as described in US20030055261, US20030040483, EP 18 072, EP 83 864,
WO
89/07110, WO 89/01711, WO 89/10933, WO 88/9780, WO 83/02272, WO 91/18016, WO
92/01711,
WO 93/04081, WO 9514666, EP0923539, U.S. Patent Nos. 5,206,235, 5,283,241,
5,284,841,
5,310,737, 5,317,017, 5,374,721, 5,430,144, 5,434,261, 5,438,136, 5,494,919,
5,494,920, 5,492,916,
5,536,716 and 5,578,593, WO 94/13696, WO 94/19367, WO 95/03289, WO 95/03290,
WO 95/09633,
WO 95/11029, WO 95/12598, WO 95/13069, WO 95/14666, WO 95/16675, WO 95/16692,
WO
95/17422, WO 95/17423, WO 95/34311, and WO 96/02530, Piperidines, pyrrolidines
and hexahydro-
1 H-azepines as described in US5804578, US5783582, W02004007468, AMIDO
SPIROPIPERIDINES such as those described in W00104119, 2-amino-5-pyrimidine
acetic acid
compounds including 2-[(5,6-Dimetlhyl-2- benzoimidazolyl)amino]-4-hydroxy-6-
methyl-5-pyrimidine
acetic acid (2) and 2-[(5,6- Dim ethyl-2-benzoim idadazolyl)am ino]-4-hyd roxy-
6-m ethyl-5- pyrimidine
acetic acid, ethyl ester as described in US6329383, benzimidazoles as
described in EP1155014,
analogous peptidyl compounds related to GRF and the peptides of U.S. Patent
4,411,890,
antagonists of gonadotropin releasing hormone such as those described in
W00170228,
W00170227, W00170228, W00069433, W00004013, W0995156, W09951595, W09951231-4,
W09941251-2, W09921557, W09921553 and 6-AZAINDOLE COMPOUNDS as described in
W00053602, W00053185, W00053181, W00053180, W00053179, W00053178, US6288078;
IGF-
1 secretagogues; insulin-like growth factor-2 (IGF- 2 or somatomedin A) and
IGF-2 secretagogues;
myostatin antagonists and compounds which inhibit fibroblast growth factor
receptor-3,(FGFR-3)
tyrosine kinase.
[0076] The polymeric substances included are also preferably water-soluble at
room
temperature. A non-limiting list of such polymers include poly(alkylene oxide)
homopolymers such as
poly(ethylene glycol) or poly(propylene glycols), poly(oxyethylenated
polyols), copolymers thereof and
block copolymers thereof, provided that the water solubility of the block
copolymers is maintained.
16
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WO 2006/024953 PCT/IB2005/002939
[0077] As an alternative to PEG-based polymers, effectively non-antigenic
materials such as
dextran, poly(vinyl pyrrolidones), poly(acrylam ides), poly(vinyl alcohols),
carbohydrate-based
polymers, and the like can be used. Indeed, the activation of a- and E-
terminal groups of these
polymeric substances can be effected in fashions similar to that used to
convert poly(alkylene oxides)
and thus will be apparent to those of ordinary skill. Those of ordinary skill
in the art will realize that the
foregoing list is merely illustrative and that all polymer materials having
the qualities described herein
are contemplated. For purposes of the present invention, "effectively non-
antigenic" means all
materials understood in the art as being nontoxic and not eliciting an
appreciable immunogenic
response in mammals.
Definitions
[0078] The following is a list of abbreviations and the corresponding meanings
as used
interchangeably herein:
- g gram(s)
- mg milligram(s)
- ml or mL milliliter(s)
- RT room temperature
- PEG poly (ethylene glycol)
[0079] The complete content of all publications, patents, and patent
applications cited in this
disclosure are herein incorporated by reference as if each individual
publication, patent, or patent
application were specifically and individually indicated to be incorporated by
reference.
[0080] Although the foregoing invention has been described in some detail by
way of illustration
and example for the purposes of clarity of understanding, it will be readily
apparent to one skilled in
the art in light of the teachings of this invention that changes and
modifications can be made without
departing from the spirit and scope of the present invention. The following
examples are provided for
exemplification purposes only and are not intended to limit the scope of the
invention, which has been
described in broad terms above.
[0081] In the following examples, the hGH is that of SEQ ID NO:1. It is
understood that other
hGH polypeptides could also be PEGylated in a similar manner as exemplified in
the subsequent
examples.
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EXAMPLES
EXAMPLE 1
Glycerol branched 43K PEG aldehyde hGH
0
11
H2C O(CH2CH2O)nCH2CH2CH
CH O(CH2CH2O)111CH3
H2C O(CH2CH2O)mCH3
wherein
(CH2CH2O), has an average molecular weight of about 3Kd and each (CH2CH2O)m
has an average
molecular weight of about 20Kd
(GL3-400AL2)
[0082] This example demonstrates generation of N-terminally monoPEGylated hGH
by reductive
alkylation. The glycerol branched PEG aidehyde reagent of approximately 43,000
MW (GL3-400AL2
NOF corporation) was coupled via reductive alkylation to the N-terminus of hGH
by taking advantage
of the difference in the relative pKa value of the primary amine at the N-
terminus versus pKa values of
primary amines at the F,-amino position of lysine residues. hGH protein
dissolved at 4, 7, or 10 mg/mL
in 25 mM MES (Sigma Chemical, St. Louis, MO) pH 5.8 or 20mM HEPES pH 7.0 was
reacted with
glycerol branched 43K PEG aldehyde by addition of the reagent to yield a
relative PEG:hGH molar
ratio of 1.5:1, 2:1, 3.4:1, 4:1, or 5:1. Reactions were catalyzed by addition
of Pyridine Borane (Sigma
Chemical, St. Louis, MO), to a final concentration of 10 mM. Reactions were
carried out in the dark at
4 degrees C. for 16-87 hours. Reactions were stopped by dilution into
appropriate buffer for
purification.
[0083] Table 1 shows the percent of multi-PEGylated species, mono-PEGylated
conjugate, un-
reacted hGH, and final purification yield for glycerol branched 43K PEG
aldehyde hGH reacted for 63
hrs at a pH of 5.8 and a 1.5:1:1 molar ratio.
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Table 1
Glycerol branched 43K PEG aldehyde hGH conjugate Synthesis and Purification
Process
Synthesis and Purification yields for
glycerol branched 43K PEG aldehyde-hGH
Species in the reaction mix:
glycerol branched 43K PEG aldehyde-hGH
multi-PEG product 4%
mono-PEG product 68%
un-reacted hGH 28%
Purification yield 35%
EXAMPLE 2
Purification of PEGylated hGH
[0084] PEGylated hGH species were purified from the reaction mixture to >95%
(SEC analysis
Figure 1) using a single ion exchange chromatography step.
Anion exchange chromatography
[0085] The PEG hGH species were purified from the reaction mixture to >95%
(SEC analysis
Figure 1) using a single anion exchange chromatography step. Mono-PEGylated
hGH was purified
from unmodified hGH and multi-PEGylated hGH species using anion exchange
chromatography. A
typical glycerol branched 43K PEG aldehyde hGH reaction mixture (80 or 1500 mg
protein), as
described above, was purified on a Q-Sepharose Hitrap column (5 mL)(Amersham
Pharmacia
Biotech, Piscataway, NJ) or Q-Sepharose column (26/20, 70 mL bed
volume)(Amersham Pharmacia
Biotech, Piscataway, NJ) equilibrated in 25 mM HEPES, pH 7.3 (Buffer A). The
reaction mixture was
diluted 7X with buffer A and loaded onto the column at a flow rate of 2.5
mUmin. The column was
washed with 3-10 column volumes of buffer A. Subsequently, the various hGH
species were eluted
from the column in 20 column volumes of Buffer A and a linear NaCI gradient of
0-100 mM. The
eluant was monitored by absorbance at 280 nm (A280) and appropriate size
fractions were collected.
Fractions were pooled as to extent of PEGylation, e.g., mono, di, tri etc. (as
assessed in example 3).
The pool was then concentrated to 0.5-5 mg/mL in a Centriprep YM10
concentrator (Amicon,
Technology Corporation, Northborough, MA) or by diafiltration. Protein
concentration of pool was
determined by A280 using an extinction coefficient of 0.78.
EXAMPLE 3
Biochemical Characterization
[0086] The purified PEGylated hGH pools were characterized by non-reducing SDS-
PAGE, non-
denaturing Size Exclusion Chromatography, and peptide mapping.
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Size Exclusion High Performance Liquid Chromatography (SEC-HPLC)
[0087] The reaction mixture of glycerol branched 43K PEG aldehyde with hGH,
anion exchange
purification pools and final purified products were assessed using non-
denaturing SEC-HPLC.
Analytical non-denaturing SEC-HPLC was carried out using a column, TSK
G4000PWXL (Tosohaas)
or Shodex KW-804 (Waters Corp) in 20 mM Phosphate pH 7.2, 150 mM NaCI at a
flow rate of 0.5
mUminute (optionally Superdex 200 7.8 mm X 30 cm, Amersham Bioscience,
Piscataway, NJ).
PEGylation greatly increases the hydrodynamic volume of the protein resulting
in a shift to an earlier
retention time. New species were observed in the PEG aldehyde hGH reaction
mixtures along with
unmodified hGH. These PEGylated and non-PEGylated species were separated on Q-
Sepharose
chromatography, and the resultant purified mono PEG-Aldehyde hGH species were
subsequently
shown to elute as a single peak on non-denaturing SEC (> 95% purity, Figure
1). The Q-Sepharose
chromatography step effectively removed free PEG, hGH, and multi PEGylated hGH
species from the
mono-PEGylated hGH.
SDS-PAGE
[0088] SDS-PAGE was used to assess the reaction of glycerol branched 43K PEG
aldehyde
with hGH and the purified final products. SDS-PAGE was carried out on 1 mm
thick 10-NuPAGE gels
(Invitrogen, Carlsbad, CA) under reducing and non-reducing conditions and
stained using a Novex
Colloidal CoomassieTM G-250 staining kit (Invitrogen, Carlsbad, CA.
N-terminal Sequence
[0089] Automated Edman degradation chemistry is used to determine the NH2-
terminal protein
sequence. An Applied Biosystems Model 494 Procise sequencer (Perkin Elmer,
Wellesley, MA) is
employed for the degradation. The respective PTH-AA derivatives are identified
by RP-HPLC
analysis in an on-line fashion employing an Applied Biosystems Model 140C PTH
analyzer fitted with
a Perkin Elmer/Brownlee 2.1 mm i.d. PTH-C18 column.
Peptide Mapping
[0090] Tryptic digests were performed at a concentration of 1 mg/mL and
typically 25 ug of
material was used per digest. Trypsin was added such that the trypsin to PEG-
hGH ratio was 1:30
(w/w). Tris buffer was present at 30 mM, pH 7.5. ' Samples were incubated at
room temperature for
16 0.5 hours. Reactions were quenched by the addition of 50 pL of 1 N HCI
per mL of digestion
solution. Samples were diluted, prior to placing the samples in the
autosampler, to a final
concentration of 0.25 mg/mI in 6.25 % acetonitrile. Acetonitrile was added
first (to 19.8% acetonitrile),
mixed gently, and then water was added to final volume (four times the
starting volume). Extra
digestion solution may be removed and stored for up to 1 week at -20 C.
[0091] A Waters Alliance 2695 HPLC system was used for analysis, but other
systems should
produce similar results. An Astec C-4 polymeric 25 cm x 4.6 mm column with
5'Nm particles was
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used. Experiments were conducted at ambient temperature on a typical load of
50 pg of protein per
sample. Buffer A is 0.1% trifluoroacetic acid in water; buffer B was 0.085%
trifluoroacetic acid in
acetonitrile. Samples were eluted with a linear gradient of 0-45% B over 90
minutes
[0092] Peaks were detected using a Waters 996 PDA detector collecting data
between 210 and
300 nm. The extracted chromatogram at 214 nm was used for sample analysis.
[0093] Tryptic maps were performed for hGH, and glycerol branched 43K PEG
aldehyde reacted
at a molar ratio of 2:1 (PEG:hGH), (Figure 2). The N-terminal tryptic fragment
was referred to as T-1.
The percent of T-1 present compared to unPEGylated hGH suggests that greater
than 99% of the
PEG modification is at the N-terminus with remainder apparently linked to one
of several possible
lysine residues.
Table 2
T-1 present comparison
% T-1 present compared
to unPEGylated hGH
% T-1 pre
hGH 28.0% 100%
glycerol branched 43K
PEG aldehyde/hGH Less than 1% Less than 1%
Example 4
In vitro biology
The ability of the glycerol branched 43K PEG aldehyde hGH to recognize the
human receptor was
tested using a Biacore 3000 instrument in an assay configured to assess the
specific interactions
between the conjugate and the human growth hormone receptor (hGHR) (28kDa
extracellular
domain). Results from the surface plasmon resonance (SPR) experiments are
shown below in Table
3.
Table 3. Biacore assay results.
Avg. ka x 105 (M" Avg. kd KD relative Sample. 's"' stdev)a (s-' stdev)a
Kp=kd/ka, mM to hGH
hGH (n=9) 3.07 8.20 38.8 3.6 0.13 0.03 1.000
43K PEG-hGH 0.28 0.03 90.0 31.0 3.22 0.10 0.041
n=3
a ka (on-rate) and kd (off-rate) were determined at a flow rate of 50 pL/min
at 37 C in HEP-BES buffer
(0.01 M Hepes, pH 7.4, plus 0.15 M NaCI, 3 mM EDTA, and 0.005% Surfactant
P20), using human
growth hormone binding protein (28kDa, extracellular domain) labeled on a CM5
chip through amine
coupling chemistry at ARU=3000-5000. ka, expressed as per M per second, kd
expressed as per
second, both are an average value of at least 3 measurements on 1 chip
standard deviation. The
data assumes to measure hGH binding to the high affinity site 1 on GHBP at
1:.1 ratio.
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Example 5
Pharmacodynamic Studies
In vivo potency - Efficacy in 11-day Rat Assays (weight gain, tibia growth,
serum BUN depression)
Rat weight gain
[0094] Female Sprague Dawley rats, hypophysectomized at Harlan Labs, were
prescreened for
growth rate for a period of 4 to 10 days. Rats were divided into groups of
six. Starting, at day 0, control
rats received one daily subcutaneous injection of either 0.3 mg/kg hGH, or
vehicle, for eleven
consecutive days. The test group received single (once/weekly) doses of 1.8
mg/kg of glycerol
branched 43K PEG aldehyde hGH on days 0 and 6. The animals were weighed daily.
Figure 4 shows
the effects of hGH and glycerol branched 43K PEG aldehyde hGH on body weight
gain in a
representative study.
[0095] Combining the data represented by the study depicted in Figure 4 with
data from
historical 11-day growth studies for hGH (control) treated rats plus an
additional growth study using
the 43K glycerol branched PEG aldehyde hGH conjugate, the average incremental
weight gain for
animals treated once weekly with 1.8 mg/kg conjugate was 109% of that achieved
following daily hGH
administration (cumulative 3.3 mg/kg).
Rat tibia length
[0096] Animals in 11-Day weight gain studies at day 11 were sacrificed, left
tibias were removed
and X-rayed and bone lengths were measured using a caliper. Figure 5 shows
tibia length
measurements for Glycerol branched 43K PEG aidehyde hGH treated animals.
Rat BUN levels
[00971 As a biomarker for the metabolic effects following hGH-treatment, blood
urea nitrogen
levels were determined from day-11 blood samples. Figure 6 shows that both
daily hGH and
once/weekly glycerol branched 43K PEG aldehyde hGH treatment results in
significant reduction of
blood urea nitrogen.
Rat Weight Gain 6-Day Dose Escalation Study
[0098] Hypophysectomized rats were treated with various single doses of
glycerol branched 43K
PEG aldehyde hGH or else treated daily with hGH, and weight gain was monitored
for 6 days. Figure
7 shows the weight gain that was obtained for the various treatment groups.
Blood samples were
taken at the indicated times and the serum IGF-1 levels determined by ELISA.
Plotted are averages
+/- SEM. Group (n=6) means were used to calculate the IGF-1 response using one-
way analysis of
variance on the measured values and AUCdO-6 (ng-hr/mL) values of 20,040,
22,958, 28,129, and
37,839 were determined for the 0.067, 0.2, 0.6, and 1.8 mg/kg dosing cohorts,
respectively.
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Rat Weight Gain 11-Day Dose Escalation Study
[0099] In a second study, animals were treated with either 1.8 mg/kg or with a
higher dose, i.e.,
5.1 mg/kg of the 43K glycerol branched PEG aldehyde hGH conjugate, on day 0
and again on day 6.
Table 4 shows the dose-related weight gain at 6 days and 11 days compared to
that achieved
following the daily (QD 11) administration of either vehicle or daily hGH (at
0.3 mg/kg/d).
Table 4. Dose-related weight gain at day 6 and 11.
Day 0 BW Day 6 BW Day 11 BW
(g) (g) (g)
Vehicle (QD11) 106.2 2.3 106.2 2.8 106.7 2.8
(0.07 0.7 0.51 +0.6
hGH (0.3 mg/kg) (QD11) 109.2 1.3 123.1 1.4* 136.7 1.6
13.9 0.5) 27.5 0.7 *
Glycerol branched 43K PEG aldehyde hGH 1 120.6 1.1* 133.6 1.0
(1.8 m/k D0,6 06.8 1.3 (13.8 0.7) (26.7 0.8)*
Glycerol branched 43K PEG aldehyde hGH 1 127.1 1.3*t 142.5 1.4
(5.1 m/k D.0,6 06.1 1.4 21.0+1.0 36.4+1.2)*t
* p <0.05 vs. Vehicle, t p<0.05 vs. hGH; mean incremental weight gain (g) as
measured from day 0
BW, body weight; hGH, human growth hormone.
Values represent mean SEM (standard error of the mean).
Values in parenthesis represent change from Day 0 in mean SEM.
IGF-1 Studies
[00100] Animals from six-day weight gain studies were used. Blood samples were
taken at the
various times during the study and the serum IGF-1 levels determined by ELISA
as shown in Figure 8.
Rat IGF-1 levels were monitored by immunoassay kit (Diagnostic System
Laboratories).
Example 6
Pharmacokinetic Studies
[00101] Pharmacokinetic studies were conducted in normal, cannulated Sprague-
Dawley male
rats. Injections were made as a single intravenous dose of 1.0 mg/kg or a
single subcutaneous bolus
of 1.8 mg/kg hGH or glycerol branched 43K PEG aldehyde hGH using six rats per
group. Blood
samples are taken over one to five days as appropriate for assessment of
relevant PK parameters.
hGH and glycerol branched 43K PEG aldehyde hGH blood levels are monitored at
each sampling
using immuno-assay. Table 4 shows the rat PK parameters for glycerol branched
43K PEG aldehyde
hGH. The effect of PEGylation is evident in the observed half-life for
elimination as this parameter
exceeded 6 hours for the conjugate while data reported for hGH from similar
studies is reported as
1.35 0.2 (Clark ibid,) 0.77-1.7 (Jorgensen et.al., "Polyethylene glycol-
conjugated proteins", PSTT 1
(8), November 1998), or 1 hr (Genotropin (PNU-180307) Investigator Brochure).
hGH Immunoassay
[00102] hGH and glycerol branched 43K PEG aldehyde hGH protein concentration
levels in rat
plasma were determined using the hGH AutoDELFIA kit fluorescence immunoassay
(Perkin-Elmer).
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Table 5.
glycerol branched 43K PEG aldehyde
hGH
Route iv/sc
Dose mg/kg 1.0/1.8
T1/2 h 6.44 2.38
AUCO--, iv Ng=h/mL 489 16
AUCO--õ sc lag=h/mL
97.7 2.1
CLtotal mUhr/kg. 2.05 0.07
Vss m Ukg 27 2+2 8
Tmax h 12 0
Cmax p g/m l 1.96 0.05
F % 11.1 0.2
* AUCO--õ sc
normalize for 1.8
mpk
The relationship between plasma drug levels and the IGF-1 response was also
determined directly in
a comprehensive study design following the subcutaneous administration of a
single dose of the
glycerol branched 43K PEG aldehyde hGH (1.8 mg/kg) to hypophysectomized,
female rodents.
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