Note: Descriptions are shown in the official language in which they were submitted.
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
CHEMICALLY-MODIFIED HUMAN GROWTH HORMONE
CONJUGATES
The present application claims priority under Title
35, United States Code, X119 to United States
Provisional application Serial No. 60/331,907, filed
November 20, 2001, which is incorporated by reference
in their entirety as if written herein.
FIELD OF THE INVENTION
The present invention relates to a chemical
modification of human Growth Hormone (hGH) and agonist
variants thereof by which the chemical and/or
physiological properties of hGH can be changed. The
PEGylated hGH may have an increased plasma residency
duration, decreased clearance rate, improved
stability, decreased antigenicity, 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
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
1
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
growth. 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.
Genotropin'~'t, or NutropinTN' 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.
A major biological effect of growth hormone (GH)
is to promote growth in young mammals 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
2
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
(Goeddel, et al. [1979) Nature 281: 544; Gray, et al.
[1985] Gene 39:247).
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.
Human growth hormone (hGH) is a single-chain
polypeptide consisting of 191 amino acids (molecular
weight 21,500). Disulfide bonds link positions 53 and
165 and positions 182 and 189. Niall, Nature, New
Biology, 230:90 (1971). 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).
hGH causes a variety of physiological and
metabolic effects in various animal models including
linear bone growth, lactation, activation of
3
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
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., Neuroendocrinology M, 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., 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.
4
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
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.
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 polyethylene 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 polyethylene glycol)
has been shown to protect against proteolysis, Sada,
et al., J. Fermentation Bioengineering 71: 137-139
(1991), and methods for attachment of certain
polyethylene 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
5
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
as Drugs. (J. S. Holcerberg and J. Roberts, eds. pp.
367-383 (1981)).
Other water-soluble polymers have been used, such
as copolymers of ethylene glycol/propylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol),
polyvinyl pyrrolidone), poly(-1,3-dioxolane), poly(-
1,3,6-trioxane), ethylene/maleic anhydride copolymer,
poly- amino acids (either homopolymers or random
copolymers).
A number of examples of pegylated therapeutic
proteins have been described. ADAGEN~, a pegylated
formulation of adenosine deaminase, is approved for
treating severe combined immunodeficiency disease.
ONCASPARC?, a pegylated L-asparaginase has been
approved for treating hypersensitive ALL patients.
Pegylated superoxide dismutase has been in clinical
trials for treating head injury. Pegylated Cc-
interferon (U. S. 5,738,846, 5,382,657) has been
approved for treating hepatitis; pegylated
glucocerebrosidase and pegylated hemoglobin are
reported to have been in preclinical testing. Another
example is pegylated IL-6, EF 0 442 724, entitled,
"Modified hIL-6," which discloses polyethylene
glycol) molecules added to IL-6.
Another specific therapeutic protein, which has
been chemically modified, is granulocyte colony
stimulating factor, (G-CSF). G-CSF induces the rapid
proliferation and release of neutrophilic granulocytes
to the blood stream, and thereby provides therapeutic
effect in fighting infection. European patent
publication EP 0 401 384, published Dec. 12, 1990,
entitled, "Chemically Modified Granulocyte Colony
Stimulating Factor," describes materials and methods
for preparing G-CSF to which polyethylene glycol)
6
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
molecules are attached. Modified G-CSF and analogs
thereof are also reported in EP 0 473 268, published
Mar. 4, 1992, entitled "Continuous Release
Pharmaceutical Compositions Comprising a Polypeptide
Covalently Conjugated To A Water Soluble Polymer,"
stating the use of various G-CSF and derivatives
covalently conjugated to a water soluble particle
polymer, such as polyethylene glycol). A modified
polypeptide having human granulocyte colony
stimulating factor activity is reported in EP 0 335
423 published Oct. 4, 1989. Provided in U.S. 5,824,784
are methods for N-terminally modifying proteins or
analogs thereof, and resultant compositions, including
novel N-terminally chemically modified G-CSF
compositions. U.S. 5,824,778 discloses chemically
modified G-CSF.
For polyethylene glycol), a variety of means
have been used to attach the polyethylene glycol)
molecules to the protein. Generally, polyethylene
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 eacample, Royer (U. S. Pat. No. 4,002,531, above)
states that reductive alkylation was used for
attachment of polyethylene glycol) molecules to an
enzyme. EP 0 539 167, published Apr. 28, 1993, Wright,
"Peg Imidates and Protein Derivatives Thereof" states
that peptides and organic compounds with free amino
groups) are modified with an imidate derivative of
PEG or related water-soluble organic polymers. US
5,298,643 and US 5,637,749 disclose PEG aryl imidates
Chamow et al., Bioconjugate Chem. 5: 133-140
(1994) report the modification of CD4 immunoadhesin
with monomethoxypoly(ethylene glycol) aldehyde via
7
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
reductive alkylation. The authors report that 500 of
the CD4-Ig was MePEG-modified under conditions
allowing control over the extent of pegylation. Id. at
page 137. The authors also report that the in vitro
binding capability of the modified CD4-Ig (to the
protein gp 120) decreased at a rate correlated to the
extent of MePEGylation Ibid. U.S. Pat. No. 4,904,584,
Shaw, issued Feb. 27, 1990, relates to the
modification of the number of lysine residues in
proteins for the attachment of polyethylene glycol)
molecules via reactive amine groups.
Many methods of attaching a polymer to a protein
involve using a moiety to act as a linking group. Such
moieties may, however, be antigenic. A tresyl
chloride method involving no linking group is
available, but this method may be difficult to use to
produce therapeutic products as the use of tresyl
chloride may produce toxic by-products. See Francis et
al., In: Stability of protein pharmaceuticals: in vivo
pathways of degradation and strategies for protein
stabilization (Eds. Ahern, T. and Manning, M. C.)
Plenum, New York, 1991) Also, Delgado et al.,
"Coupling of PEG to Protein By Activation With Tresyl
Chloride, Applications In Immunoaffinity Cell
Preparation", in Separations Using Aqueous Phase
Systems, Applications In Cell Biology and
Biotechnology, Fisher et al., eds. Plenum Press, New
York, N.Y., 1989 pp. 211-213.
See also, Rose et al., Bioconjugate Chemistry 2:
154-159 (1991) which reports the selective attachment
of the linker group carbohydrazide to the C-terminal
carboxyl group of a protein substrate (insulin).
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
8
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
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).
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.
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.
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 ECSO 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.
WO 94/20069 prophetically discloses PEGylated hGH
as part of a formulation for pulmonary delivery.
US 4,179,337 discloses methods of PEGylating
9
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
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.
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.
WO 95/11987 suggests attachment of PEG to the
thiol 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 proteins is suggested as well.
WO 99/03887 discloses, e.g., growth hormone
modified by insertion of additional cysteine for
serine residues and attachment of PEG to the
introduced cysteine residues.
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.
WO 9711178 (as well as US 5849535, US 6004931,
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
and US 6022711) relates to the use of GH variants as
agonists or antagonists of hGH. WO 9711178 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.
The previous reports of PEGylated hGH require the
attachment of multiple PEGS, which results in
undesirable product heterogeneity, 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).
A GH 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.
The present invention provides chemically
modified hGH conjugates having decreased
heterogeneity, decreased clearance rate, increased
plasma residency duration, improved solubility,
increased stability, decreased antigenicity, or
combinations thereof.
SUt~IARY OF THE INVENTION
The present invention relates to chemically
modified hGH and agonist variants thereof, which have
at least one improved chemical or physiological
property selected from but not limited to decreased
clearance rate, increased plasma residency duration,
11
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
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 and
agonist variants thereof as well as specific
modifications using a variety of polyethylene glycol)
moieties.
The present invention also relates to methods of
producing the chemically modified hGH and agonist
variants thereof.
The present invention also relates to
compositions comprising the chemically modified hGH
and agonist variants thereof.
The modified hGH and agonist variants thereof of
the present invention may be useful in the treatment
of, but not limited to, dwarfism (GHD), Adult GHD,
Turner's syndrome, 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, Aging, End-stage
Renal Failure, and Cystic Fibrosis.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a reproduction of a reducing and non-
reducing SDS-PAGE analysis of the products of the
reaction of hGH and 20K PEG-ALD and the anion exchange
purified 20K PEG-ALD hGH. Lane 1. MW Protein
standards; Lane 2. reduced hGH-10 ug; Lane 3. reduced
20 K linear PEG-ALD hGH reaction mix-10 ug; Lane 4.
reduced anion exchange purified 20 K linear PEG-ALD
hGH-10 ug Lane 5. Blank; Lane 6. non-reduced hGH-10
ug; Lane 7. non-reduced 20 K linear PEG-ALD hGH
reaction mix-10 ug; Lane 8. non-reduced anion
12
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
exchange purified 20 K linear PEG-ALD hGH-10 ug; Lane
9. Blank; Lane 10. MW Protein standards.
Figure 2 is a reproduction of a non-reducing SDS-PAGE
analysis of various anion exchange purified pegylated
hGH molecules. Lane 1. MV~T Protein standards; Lane 2.
hGH-10 ug; Lane 2. 4-6 x 5K PEG-SPA hGH-10 ug; Lane 3.
20 K linear PEG-ALD hGH-10 ug; Lane 4. 20 K branched
PEG-ALD hGH-10 ug Lane 5. 40 K branched PEG hGH-10
ug .
Figure 3 shows reproductions of RP-HPLC elution
profiles for trypsin digests of hGH, 40K Br PEG-ALD
hGH and 40K Br PEG-NHS hGH. PEG coupled primarily to
the N-terminus of hGH (as shown in the 40K Br ALD hGH)
results in a reduction in the N-terminal (T1) fragment
peak with generation of a new PEGylated T1 peak.
Figure 4 compares the in vivo bioactivity of
unPEGylated hGH dosed daily (0.3 mg/Kg/day) to mono-
PEGylated hGH dosed subcutaneously(SC) once every six
days(1.8 mg/Kg) by illustrating the weight gain in
hypophysectomized rats during a period of 11 days.
Figure 5 compares the in vivo bioactivity of
unPEGylated hGH dosed SC daily (0.3 mg/Kg/day) to 4-6
x 5K PEG-SPA-hGH, mono-PEGylated 20K branched PEG-ALD
hGH, and mono-PEGylated 40K branched PEG-ALD hGH each
dosed SC once every six days (1.8 mg/Kg) by
illustrating the weight gain in hypophysectomized rats
during a period of 11 days.
Figure 6 compares the in vivo bioactivity of
unPEGylated hGH dosed SC daily (0.3 mg/Kg/day) to 4-6
x 5K PEG-CMHBA-hGH, mono-PEGylated 20K linear ALD,
13
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
mono-PEGylated 30K linear ALD, mono-PEGylated 20K
branched PEG-ALD hGH, and mono-PEGylated 40K branched
PEG-ALD hGH each dosed SC once every six days (1.8
mg/Kg) by illustrating the increase in tibial bone
growth in hypophysectomized rats during a period of 11
days.
Figure 7 compares the in vivo bioactivity of a single
1.8 mg/Kg SC dose of unPEGylated hGH, mono-PEGylated
5K linear PEG-ALD hGH, mono-PEGylated 20K linear PEG-
ALD hGH, mono-PEGylated 20K branched PEG-ALD hGH,
mono-PEGylated 20K linear PEG-Hydrazide hGH, mono-
PEGylated 30K linear PEG-ALD hGH, mono-PEGylated 40K
branched PEG-ALD hGH, 4-6 x 5K PEG SPA hGH, 4-6 x 5K
PEG-CMHBA hGH by illustrating the increase in plasma
IGF-1 levels in hypophysectomized rats during a period
of 9 days .
DETAILED DESCRIPTION
hGH and agonist variants thereof are members of a
family of recombinant proteins, described in US
4,658,021 and US 5,633,352. Their recombinant
production and methods of use are detailed in US
4,342,832, 4,601,980; US 4,898,830; US 5,424,199; and
US 5,795,745.
Any purified and isolated hGH or agonist variant
thereof, which is produced by host cells such as E.
coli and animal cells transformed or transfected by
using recombinant genetic techniques, may be used in
the present invention. Additional hGH variants are
described in U.S. Ser. No. 07/715,300 filed Jun. l4,
1991 and Ser. No. 07/743,614 filed Aug. 9, 1991, and
WO 92/09690 published Jun. 11, 1992. Among them, hGH
or agonist variant thereof, which is produced by the
14
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
transformed E. coli, is particularly preferable. Such
hGH or agonist variant thereof may be obtained in
large quantities with high purity and homogeneity. For
example, the above hGH or agonist variant thereof may
be prepared according to a method disclosed in US
4,342,832, 4,601,980; US 4,898,830; US 5,424,199; and
US 5,795,745. The term "substantially has the
following amino acid sequence" means that the above
amino acid sequence may include one or more amino-acid
changes (deletion, addition, insertion or replacement)
as long as such changes will not cause any
disadvantageous non-similarity in function to hGH or
agonist variant thereof. It is more preferable to use
the hGH or agonist variant thereof substantially
having an amino acid sequence, in which at least one
lysine, aspartic acid, glutamic acid, unpaired
cysteine residue, a free N-terminal a-amino group or a
free C-terminal carboxyl group, is included.
According to the present invention, polyethylene
glycol) is covalently bound through amino acid
residues of hGH or agonist variant thereof. A variety
of activated polyethylene glycol)s having a number of
different functional groups, linkers, configurations,
and molecular weights are known to one skilled in the
art, which may be used to create PEG-hGH conjugates or
PEG-hGH agonist variant conjugates (for reviews see
Roberts M.J. et al., Adv. Drug Del. .Rev. 54:459-476,
2002), Harris J.M, et al., Drug Delivery Sytems
40:538-551, 2001) The amino acid residue may be any
reactive ones) having, for example, free amino,
carboxyl, sulfhydryl (thiol), hydroxyl, guanidinyl, or
imidizoyl groups, to which a terminal reactive group
of an activated polyethylene glycol) may be bound.
The amino acid residues having the free amino groups
may include lysine residues and/or N-terminal amino
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
acid residue, those having a free carboxyl group may
include aspartic acid, glutamic acid and/or C-terminal
amino acid residues, those having a free sulfhydryl
(thiol) such as cysteine, those having a free hydroxyl
such as serine or tyrosine, those having a free
guanidinyl such as arginine, and those having a free
imidizoyl such as histidine.
In another embodiment, oxime chemistries (Lemieux
& Bertozzi Tib Tech 16:506-513, 1998) are used to
target N-terminal serine residues.
The polyethylene glycol) used in the present
invention is not restricted to any particular form or
molecular weight range. The polyethylene glycol)
molecular weight may between 500 and 100,000.
Normally, a molecular weight of 500-60,000 is used and
preferably of from 1,000-40,000. More preferable, the
molecular weight is greater than 5,000 to about
40,000.
In another embodiment the polyethylene glycol)
is a branched PEG having more than one PEG moiety
attached. Preferred examples of branched PEGs are
described in U.S. 5,932,462; U.S. 5,342,940; U.S.
5,643,575; U.S. 5,919,455; U.S. 6,113,906; U.S.
5,183,660; WO 02/09766; Kodera Y., Bioconjugate
Chemistry 5:283-288 (1994); and Yamasaki et al.,
Agric. Biol. Chem., 52:2125-2127, 1998. In a preferred
embodiment the molecular weight of each polyethylene
glycol) of the branched PEG is 5,000-20,000.
Poly(alkylene oxides, notably polyethylene
glycol)s, are bound to hGH or agonist variant thereof
via a terminal reactive group, which may or may not
leave a linking moiety (spacer) between the PEG and
the protein. In order to form the hGH conjugates or
agonist variant thereof of the present invention,
polymers such as poly(alkylene oxide) are converted
16
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
into activated forms, as such term is known to those
of ordinary skill in the art. The reactive group, for
example, is a terminal reactive group, which mediates
a bond between chemical moieties on the protein, such
as amino, carboxyl or thiol groups, and polyethylene
glycol). Typically, one or both of the terminal
polymer hydroxyl end-groups, (i.e. the alpha and omega
terminal hydroxyl groups) are converted into reactive
functional groups, which allows covalent conjugation.
This process is frequently referred to as "activation"
and the polyethylene glycol) product having the
reactive group is hereinafter referred to as "an
activated polyethylene glycol)". Polymers containing
both a and ~ linking groups are referred to as "bis-
activated poly(alkylene oxides)'° and are referred to
as "bifunctional". Polymers containing the same
reactive group on a and ~ terminal hydroxyls are
sometimes referred to as "homobifunctional" or
"homobis-activated°'. Polymers containing different
reactive groups on a and ~ terminal hydroxyls are
sometimes referred to as "heterobifunctional" (see for
example WO 01/26692) or "heterobis-activated".
Polymers containing a single reactive group are
referred to as "mono-activated°' polyalkylene oxides or
"mono-functional". Other substantially non-antigenic
polymers are similarly "activated" or
"functionalized".
The activated polymers are thus suitable fo.r
mediating a bond between chemical moieties on the
protein, such as a- or ~-amino, carboxyl or thiol
groups, and polyethylene glycol). Bis-activated
polymers can react in this manner with two protein
molecules or one protein molecule and a reactive small
molecule in another embodiment to effectively form
17
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
protein polymers or protein-small molecule conjugates
through cross linkages.
Functional groups capable of reacting with either
the amino terminal a-amino group or E-amino groups of
lysines found on the hGH or agonist variant thereof
include: N-hydroxysuccinimidyl esters, carbonates such
as the p-nitrophenyl, or succinimidyl (US 5,808,096,
5,612,460, US 5,324,844, US 5,5,122,614); carbonyl
imidazole; azlactones (US 5,321,095, US 5,567,422);
cyclic imide thiones (US 5,405,877, 5,349,001);
isocyanates or isothiocyanates (Greenwald R.B., J.
0rg. Chem., 60:331-336, 1995); tresyl chloride (EP 714
402, EP 439 508); halogen formiates (WO 96/40792), and
aldehydes.
Functional groups capable of reacting with
carboxylic acid groups, reactive carbonyl groups and
oxidized carbohydrate moieties on hGH or agonist
variant thereof include; primary amines; and hydrazine
and hydrazide functional groups such as the acyl
hydrazides, carbazates, semicarbamates,
thiocarbazates, etc (WO 01/70685).
Mercapto groups, if available on the hGH or
agonist variant thereof, can also be used as
attachment sites for suitably activated polymers with
reactive groups such as thiols; maleimides, sulfones,
and phenyl glyoxals; see, for example, U.S. Pat. No.
5,093,531, the disclosure of which is hereby
incorporated by reference. Other nucleophiles capable
of reacting with an electrophilic center include, but
are not limited to, for example, hydroxyl, amino,
carboxyl, thiol, active methylene and the like.
Also included are polymers including lipophilic
and hydrophilic moieties disclosed in US 5,359,030 and
US 5,681,811; US 5,438,040; and US 5,359,030.
18
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
As well halogenated PEGS are disclosed on WO
98/32466 that can react with amino, thiol groups, and
aromatic hydroxy groups, which directly covalently
attach the PEG to the protein.
In one preferred embodiment of the invention secondary
amine or amide linkages are formed using the N-
terminal a-amino group or ~-amino groups of lysine of
hGH or agonist variant thereof and the activated PEG.
In another preferred aspect of the invention, a
secondary amine linkage is formed between the N-
terminal primary a- or g-amino group of hGH or agonist
variant thereof and single or branched chain PEG
aldehyde by reduction with a suitable reducing agent
such as NaCNBH3, NaBH3, Pyridine Borane etc. as
described in Chamow et al., Bioconjugate Chem. 5: 133-
140 (1994) and US Pat. No 5,824,784.
In a preferred embodiment at least 70%,
preferably at least 80%, preferably at least 81%,
preferably at least 820, preferably at least 83~,
preferably at least 840, preferably at least 850,
preferably at least 860, preferably at least 87o,
preferably at least 880, preferably at least 890,
preferably at least 900, preferably at least 91~,
preferably at least 920, preferably at least 930,
preferably at least 940, preferably at least 95%,
preferably at least 960, preferably at least 970, and
most preferably at least 980 of the polyethylene
glycol) is on the amino terminal a-amino group.
In another preferred embodiment of the invention,
polymers activated with amide-forming linkers such as
succinimidyl esters, cyclic imide thiones, or the like
are used to effect the linkage between the hGH or
agonist variant thereof and polymer, see for example,
U.S. Pat. No. 5,349,001; U.S. Pat. No. 5,405,877; and
Greenwald, et al., Crit. Rev. Ther. Drug Carrier Syst.
19
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
17:101-161, 2000, which are incorporated herein by
reference. One preferred activated polyethylene
glycol), which may be bound to the free amino groups
of hGH or agonist variant thereof includes single or
branched chain N-hydroxysuccinylimide polyethylene
glycol) may be prepared by activating succinic acid
esters of polyethylene glycol) with N-
hydroxysuccinylimide.
Other preferred embodiments of the invention
l0 include using other activated polymers to form
covalent linkages of the polymer with the hGH or
agonist variant thereof via ~-amino or other groups.
For example, isocyanate or isothiocyanate forms of
terminally activated polymers can be used to form urea
or thiourea-based linkages with the lysine amino
groups (Greenwald R.B., J. Org. Chem., 60:331-336,
1995) .
In another preferred aspect of the invention,
carbamate (urethane) linkages are formed with protein
amino groups as described in U.S. Pat. Nos. 5,122,614,
5,324,844, and 5,612,640, which are hereby
incorporated by reference. Examples include N-
succinimidyl carbonate, para-nitrophenyl carbonate,
and carbonyl imidazole activated polymers. In another
preferred embodiment of this invention, a
benzotriazole carbonate derivative of PEG is linked to
amino groups on hGH or agonist variant thereof.
Another aspect of the invention represents a
prodrug or sustained release form of hGH or agonist
variant thereof, comprised of a water soluble polymer,
such as polyethylene glycol), attached to an hGH or
agonist variant thereof molecule by a functional
linker that can predictably break down by enzymatic or
pH directed hydrolysis to release free hGH or agonist
variant thereof or other hGH or agonist variant
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
thereof derivative. The prodrug can also be a "double
prodrug" (Bundgaard in Advanced Drug Delivery Reviews
3:39-65, 1989) involving the use of a cascade
latentiation. In such systems, the hydrolytic reaction
involves an initial rate-limiting (slow) enzymatic or
pH directed step and a second step involving a rapid
non-enzymatic hydrolysis that occurs only after the
first has taken place. Such a releasable polymer
provides protein conjugates, which are impermanent and
could act as a reservoir, that continually discharge
hGH or agonist variant thereof. Such functional
linkers are described in US 5,614,549; US 5,840,900;
US 5,880,131; US 5,965,119; us 5,965,565; US
6,011,042; US 6,153,655; US 6,180,095 B1; US
6,413,507; Greenwald R.B. et al., J. Med. Chem.
42;3657-3667, 1999; Lee, S. et al., Bioconjugate Chem
12:163-169, 2001; Garman A.J. et al., FEBS Lett.
223:361-365, 1987; Woghiren C. et al., Bioconjucate
Chem. 4:314-318, 1993; Roberts M.J. et al., J. Pharm.
Sci. 87;1440-1445, 1998; Zhao X., in Ninth Int. Symp.
Recent Adv. Drug Delivery Syst. 199; Greenwald R.B. et
al., J. Med. Chem. 43:475-487, 2000; and Greenwald
R.B. Crit. Rev. Ther. Drug Carrier Syst. 17:101-161,
2000. Zalipsky et al., 28th Int. Symp. On controlled
Release of Bioactive Materials 1; 73-74,2001
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 or agonist
21
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
variant thereof bioactivity is to substantially avoid
the conjugation of those hGH or agonist variant
thereof reactive groups associated with the receptor
binding sites) in the polymer coupling process.
Another aspect of the present invention is to provide
a process of conjugating polyethylene glycol) to hGH
or agonist variant thereof maintaining high levels of
retained activity.
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 polyethylene
glycol). The conjugation reaction is carried out under
relatively mild conditions to avoid inactivating the
hGH or agonist variant thereof. 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 or
agonist variant thereof have free amino groups, the
above modification is preferably carried out in a non-
limiting list of suitable buffers (pH 3 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 reagents such as PEG
aldehydes pH 4-8 is preferably maintained. The
activated polyethylene glycol) may be used in about
0.05-100 times, preferably about 0.01 -2.5 times, the
molar amount of the number of free amino groups of hGH
or agonist variant thereof. On the other hand, where
reactive amino acid residues in hGH or agonist variant
thereof have the free carboxyl groups, the above
modification is preferably carried out in pH from
about 3.5 to about 5.5, for example, the modification
with poly(oxyethylenediamine) is carried out in the
22
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
presence of carbodiimide (pH 3.5-5) for 1-24 hrs at 4°
-37°C. The activated polyethylene glycol) may be used
in 0.05-300 times the molar amount of the number of
free carboxyl groups of hGH or agonist variant
thereof.
In separate embodiments, the upper limit for the
amount of polymer included in the conjugation
reactions exceeds about 1:1 to the extent that it is
possible to react the activated polymer and hGH or
agonist variant thereof without forming a substantial
amount of high molecular weight species, i.e. more
than about 200 of the conjugates containing more than
about one strand of polymer per molecule of hGH or
agonist variant thereof. For example, it is
contemplated in this aspect of the invention that
ratios of up to about 6:1 can be employed to form
significant amounts of the desired conjugates which
can thereafter be isolated from any high molecular
weight species.
In another aspect of this invention,
bifunctionally activated PEG derivatives may be used
to generate polymeric hGH or agonist variant thereof-
PEG molecules in which multiple hGH or agonist variant
thereof molecules are crosslinked via PEG. Although
the reaction conditions described herein can result in
significant amounts of unmodified. hGH or agonist
variant thereof, the unmodified hGH or agonist variant
thereof 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 30o and more preferably,
less than about 100, of high molecular weight species
and species containing more than one polymer strand
per hGH or agonist variant thereof. These reaction
conditions are to be contrasted with those typically
23
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
used for polymeric conjugation reactions wherein the
activated polymer is present in several-fold molar
excesses with respect to the target. In other aspects
of the invention, the polymer is present in amounts of
from about 0.1/amino group to about 50 equivalents per
equivalent of hGH or agonist variant thereof. In other
aspects of the invention, the polymer is present in
amounts of from about 1 to about 10 equivalents per
equivalent of hGH or agonist variant thereof.
The conjugation reactions of the present
invention initially provide a reaction mixture or pool
containing mono- and di-PEG-hGH conjugates, unreacted
hGH, unreacted polymer, and usually less than about
20o high molecular weight species. The high molecular
weight species include conjugates containing more than
one polymer strand and/or polymerized PEG-hGH or
agonist variant thereof species. After the unreacted
species and high molecular weight species have been
removed, compositions containing primarily mono- and
di-polymer-hGH or agonist variant thereof 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 or agonist variant thereof have at least
about 0.10 of the in vitro biological activity
associated with the native or unmodified hGH or
agonist variant thereof 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
pref erred aspects of the invention, however, the
modified hGH or agonist variant thereof have about 250
of the in vitro biological activity, more preferably,
24
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
the modified hGH or agonist variant thereof have about
50% of the in vitro biological activity, more
preferably, the modified hGH or agonist variant
thereof have about 750 of the in vitro biological
activity, and most preferably the modified hGH or
agonist variant thereof have equivalent or improved in
vitro biological activity.
The processes of the present invention preferably
include rather limited ratios of polymer to hGH or
agonist variant thereof. Thus, the hGH or agonist
variant thereof 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 or agonist variant thereof reactive
groups is substantially less random than when higher
molar excesses of polymer linker are used. The
unmodified hGH or agonist variant thereof 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.
A polyethylene glycol)-modified hGH or agonist
variant thereof, namely chemically modified protein
according to the present invention, 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 polyethylene glycol) and hGH or
agonist variant thereof. In a further embodiment of
the invention, the mono- and di-polymer-hGH or agonist
variant thereof species are isolated from the reaction
mixture to remove high molecular weight species, and
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
unmodified hGH or agonist variant thereof. Separation
is effected by placing the mixed species in. a buffer
solution containing from about 0.5-10 mg/mL of the hGH
or agonist variant thereof-polymer conjugates.
Suitable solutions have a pH from about 4 to about 8.
The solutions preferably contain one or more buffer
salts selected from KCl, NaCl, KzHP04, KHZP04, Na~HP04,
NaH2P04, NaHCO3, NaB04, CH3CO~H, and NaOH.
Depending upon the reaction buffer, the hGH or
agonist variant thereof polymer conjugate solution may
first have to undergo buffer exchange/ultrafiltration
to remove any unreacted polymer. For example, the PEG-
hGH or agonist variant thereof 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.
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 or
agonist variant thereof conjugates via differences in
charge, which vary in a somewhat predictable fashion.
For example, the surface charge of hGH or agonist
variant thereof 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 or agonist variant thereof
conjugates will have a different charge from the other
species to allow selective isolation.
Strongly polar anion or ration exchange resins
such as quaternary amine or sulfopropyl resins,
respectively, are used for the method of the present
invention. Ion exchange resins are especially
26
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
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~ fast flow. Other suitable anion exchange
resins, e.g. DEAE resins, can also be used.
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 or agonist
variant thereof solution is used. The elution buffer
preferably contains one or more salts selected from
KC1, NaCl, K2HP04, KH~P04, Na~HP04, NaHZP04, NaHC03,
NaB04, and (NH4)2C03. 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 or agonist variant thereof. The eluted pooled
fractions are preferably limited to uniform polymer
conjugates after the cation or anion exchange
separation step. Any unconjugated hGH or agonist
variant thereof species can then be back washed from
the column by conventional techniques. If desired,
mono and multiply pegylated hGH or agonist variant
thereof species can be further separated from each
27
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
other via additional ion exchange chromatography or
size exclusion chromatography.
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 or agonist variant thereof-
polymer conjugates.
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 or
agonist variant thereof fraction is detected by UV
absorbance at 280 nm. Fraction collection may be
achieved through simple time elution profiles.
A surfactant can be used in the processes of
conjugating the polyethylene glycol) polymer with the
hGH or agonist variant thereof 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 or agonist
variant thereof and do not completely inhibit polymer
conjugation. The surfactants are present in the
28
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
reaction mixtures in amounts from about 0.01-0.5%;
preferably from 0.05-0.50; and most preferably from
about 0.075-0.25%. Mixtures of the surfactants are
also contemplated.
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 aggregates while allowing lysine-based or
amino terminal-based conjugation to proceed.
The present polyethylene glycol)-modified hGH or
agonist variant thereof has a more enduring
pharmacological effect, which may be possibly
attributed to its prolonged half-life in vivo.
Furthermore,'the present polyethylene glycol)-
modified hGH or agonist variant thereof may be useful
for the treatment of hypo pituitary dwarfism (GHD),
Adult Growth Hormone Deficiency, 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, and
Aging.
The present polyethylene glycol)-modified hGH or
agonist variant thereof may 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.
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
29
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
injection and between 0.1 mg and 50 mg in an oral
administration for an adult
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 polyethylene glycol) or
polypropylene glycols), poly(oxyethylenated polyols),
copolymers thereof and block copolymers thereof,
provided that the water solubility of the block
copolymers is maintained.
As an alternative to PEG-based polymers,
effectively non-antigenic materials such as dextran,
polyvinyl pyrrolidones), poly(acrylamides),
polyvinyl alcohols), carbohydrate-based polymers, and
the like can be used. Indeed, the activation of a- and
C~-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
The following is a list of abbreviations and the
corresponding meanings as used interchangeably herein:
g grams)
mg milligrams)
ml or mL milliliter(s)
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
RT room temperature
PEG poly (ethylene glycol)
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.
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.
In the following examples, the hGH is that of SEQ
ID N0:1. It is understood that other members of the
hGH or agonist variant thereof family of polypeptides
could also be pegylated in a similar manner as
exemplified in the subsequent examples.
All references, patents or applications cited
herein are incorporated by reference in their entirety
as if written herein.
The present invention will be further illustrated
by referring to the following examples, which however,
are not to be construed as limiting the scope of the
present invention.
31
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
EXAMPLE 1
Straight Chain 20,000 MW PEG-ALD hGH
O
mPEG O-CH2CH2 CH
This example demonstrates a method for generation of
substantially homogeneous preparations of N-terminally
monopegylated hGH by reductive alkylation. Methoxy-
linear PEG-propionaldehyde reagent of approximately
20,000 MW (Shearwater Corp.) was selectively coupled
via reductive amination 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 ~-amino position
of lysine residues. hGH protein dissolved at 10 mg/mL
in 25 mM MES (Sigma Chemical, St. Louis, MO) pH 6.0,
mM Hepes (Sigma Chemical, St. Louis, MO) pH 7.0, or'
in 10 mM Sodium Acetate (Sigma Chemical, St. Louis,
20 MO) pH 4.5, was reacted with Methoxy-PEG-
propionaldehyde, M-PEG-ALD, (Shearwater Corp.,
Huntsville, AL) by addition of M-PEG-ALD to yield a
relative PEG:hGH molar ratio of 0.1:0.7 per amine
(optionally 8o acetonitrile may also be added).
25 Reactions were catalyzed by addition of stock 1M
NaCNBH4 (Sigma Chemical, St. Louis, MO), dissolved in
H20, to a final concentration of 10-50 mM. Reactions
were carried out in the dark at 4°C to RT for 18-24
hours. Reactions were stopped by addition of 1 M Tris
(Sigma Chemical, St. Louis, MO) ~pH 7.6 to a final
Tris concentration of 50 mM or diluted into
appropriate buffer for immediate purification.
EXAMPLE 2
32
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
Straight Chain 30,000 MW PEG-ALD hGH
Methoxy-linear 30,000 MW PEG-propionaldehyde reagent
(Shearwater Corp.) was coupled to the N-terminus of
hGH using the procedure described for Example 2.
EXAMPLE 3
Straight chain 5, 000 MV~T PEG-ALD hGH
Methoxy-linear 5,000 MW PEG-propionaldehyde reagent
(Fluka) was coupled to the N-terminus of hGH using the
procedure described for Example 1.
EXAMPLE 4
Branched chain 40,000 MW PEG-ALD hGH
Methoxy-branched 40,000 MW PEG-aldehyde (PEG2-ALD)
reagent (Shearwater Corp.) was coupled to the N-
terminus of hGH using the procedure described for
Example 1.
EXAMPLE 5
Branched chain 20,000 MW PEG-ALD hGH
33
O
II
mPEG-O-'C-Ii H
i Hz
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
Methoxy-branched 20,000 MW PEG-aldehyde (PEG2-ALD)
reagent (Shearwater Corp.) was coupled to the N-
terminus of hGH using the procedure described for
Example 1 using PEG to hGH molar ratios of 0.1-0.5 per
amine.
EXAMPLE 6
Straight chain 30,000 MW SPA-PEG hGH
O
This example demonstrates a method for generation of
substantially homogeneous preparations of mono-
pegylated hGH using N-hydroxysuccinimidyl (NHS) active
esters. hGH protein stock solution was dissolved at 10
mg/mL in 0.25 M HEPES buffer, pH 7.2 (optionally 80
acetonitrile may also be added). The solution was
then reacted with Methoxy-PEG-succinimidyl propionate
(SPA-PEG) by addition of SPA-PEG to yield a relative
PEG:hGH molar ratio of 0.1-0.65 per amine. Reactions
were carried out at 4°C to RT for from 5 minutes to 1
hour. Reactions were stopped by lowering the pH to 4.0
with 0.1 N acetic acid or by adding a 5X molar excess
of Tris HC1.
EXAMPLE 7
Straight chain 20,000 MW SPA-PEG hGH
34
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
Straight chain 20,000 MW SPA-PEG reagent (Shearwater
Corp.) was coupled to the N-terminus of hGH using the
procedure described for Example 6
EXAMPLE 8
Straight chain 3,400 MW Biotin-SPA-PEG-hGH
O
O O
H2CH2CH2CH2~CONH-PEG-CO
to
3,400 MW Biotin-PEG-C02-NHS reagent (Shearwater Corp.)
is coupled to hGH using the procedure described for
Example 6.
EXAMPLE 9
Branched 10,000 MW NHS-PEG-hGH
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
O
mPE~
O
10,000 MW branched PEG2-NHS (Shearwater Corp.) is
coupled to hGH using the procedure described for
Example 6.
EXAMPLE 10
Branched 20,000 MW NHS-PEG-hGH
20,000 MW branched PEG2-NHS (Shearwater Corp.) is
coupled to hGH using the procedure described for
Example 6.
EXAMPLE 111
Branched 40,000 MW NHS-PEG-hGH
40,000 MW branched PEG2-NHS (Shearwater Corp.) was
coupled to hGH using the procedure described for
Example 6.
EXAMPLE 12
Straight chain 20,000 MW PEG-BTC-hGH
36
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
O
PEG-O-C-O N~ / N
N
20,000 MW PEG-BTC (Shearwater Corp.} is coupled to hGH
using the procedure described for Example 6. This
example demonstrates a method for generation of
substantially homogeneous preparations of pegylated
hGH using benzotriazole carbonate derivatives of PEG.
EXAMPLE 13
Straight chain 5,000 MW PEG-SS-hGH
O
O O
PEG-OCCH2CH2C0 N
O/
5,000 MW succinimidyl succinate-PEG (SS-
PEG)(Shearwater Corp.) is coupled to hGH using the
procedure described for Example 6. This example
demonstrates a method for generation of substantially
homogeneous preparations of pegylated hGH using a
hydrolyzable linkage.
EXAMPLE 14
Straight chain 20,000 MW PEG-CM-HBA-hGH
37
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
o
O O
II
PEG-OCH2C0 ~ HCH2C0 N
CH3
O~
20,000 MW carboxymethyl hydroxybutyric acid-PEG (CM-
HBA-PEG)(Shearwater Corp.) was coupled to hGH using
the procedure described for Example 6. This example
demonstrates a method for generation of substantially
homogeneous preparations of pegylated hGH using a
hydrolyzable linkage.
EXAMPLE 15
Straight chain 2-4x 5,000 MW PEG-CM-HBA-hGH
5,000 MW PEG-CM-HBA (Shearwater Corp.) was coupled to
hGH using the procedure described for Example 13.
EXAMPLE 16
Straight chain 20,000 MW HZ-PEG hGH
PEG-OCH2CONHNH2
This example demonstrates a method for generation of
substantially homogeneous preparations of pegylated
hGH using 20,000 MW methoxy-PEG-hydrazide, HZ-PEG
(Shearwater Corp.). hGH protein stock solution was
dissolved at 10 mg/mL in 10 mM MES, pH 4Ø The
solution was then reacted with HZ-PEG by addition of
solid to yield a relative PEG:hGH molar ratio of 0.1-
5.0 per carboxyl group. Reactions were catalyzed with
carbodiimide (EDC, EOAC, EDEC) at a final
concentration of 2 mM to 4 mM. Reactions were carried
38
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
out at 4°C for 2 hours to overnight or room
temperature from 10 minutes to overnight. Reactions
were stopped by Removing the unconjugated PEG and the
carbodiimide by purification on can on exchange.
EXAMPLES 17
Multi-pegylated species
Modified hGHs having two or more PEGS (multi-
pegylated) attached were also obtained from Examples 1
and 4 and were separated from the mono-pegylated
species using anion exchange chromatography. Modified
hGHs having two or more PEGS (multi-pegylated)
attached are also separated from mono-PEGylated
species using cation exchange chromatography.
Modified hGHs having two or more PEGS (multi
pegylated) attached are also obtained in examples
2,3,5-13 and are purified in similar fashion to
examples 1 and 4.
EXAMPLE 18
Purification of Pegylated hGH
Pegylated hGH species were purified from the reaction
mixture to >950 (SEC analysis) using a single ion
exchange chromatography step
Anion exchange chromatography
The PEG hGH species were purified from the reaction
mixture to >950 (SEC analysis) 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
39
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
typical 20K aldehyde hGH reaction mixture (5-100 mg
protein), as described above, was purified on a Q-
Sepharose Hitrap column (1 or 5 mL)(Amersham Pharmacies
Biotech, Piscataway, NJ) or Q-Sepharose fast flow
column (26120, 70 mL bed volume)(Amersham Pharmacies
Biotech, Piscataway, NJ) equilibrated in 25 mM HEPES,
pH 7.3 (Buffer A). The reaction mixture was diluted
5-10X with buffer A and loaded onto the column at a
flow rate of 2.5 mL/min. The column was washed with 8
column volumes of buffer A. Subsequently, the various
hGH species were eluted from the column in 80-100
column volumes of Buffer A and a linear NaCl gradient
of 0-100 mM. The eluant was monitored by absorbance
at 280 nm (Azso) and 5 mL fractions were collected.
Fractions were pooled as to extent of pegylation,
e.g., mono, di, tri etc. (as assessed in example 15).
The pool was then concentrated to 0.5-5 mg/mL in a
Centriprep YM10 concentrator (Amicon, Technology
Corporation, Northborough, MA). Protein concentration
of pool was determined by Ago using an extinction
coefficient of 0.78. Total yield of purified mono 20
K PEG-aldehyde hGH from this process was 25-30 0.
Cation Exchange Chromatography
Cation exchange chromatography is carried out on an SP
Sepharose high performance column (Pharmacies XK 26/20,
70 ml bed volume) equilibrated in 10 mM sodium acetate
pH 4.0 (Buffer B). The reaction mixture is diluted
10X with buffer B and loaded onto the column at a flow
rate of 5 mL/min. Next the column is washed with 5
column volumes of buffer B, followed by 5 column
volumes of 12o buffer C (10 mM acetate pH 4.5, 1 M
NaCl). Subsequently, the PEG-hGH species is eluted
from the column with a linear gradient of 12 to 270
buffer C in 20 column volumes. The eluant is
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
monitored at 280 nm and 10 mL fractions are collected.
Fractions are pooled according to extent of pegylation
(mono, di, tri etc.), exchanged into 10 mM acetate pH
4.5 buffer and concentrated to 1-5 mg/mL in a stirred
cell fitted with an Amicon YM10 membrane. Protein
concentration of pool is determined by A280 nm using
an extinction coefficient of 0.78. Total yield of
monopegylated hGH from this process is 10 to 500.
EXAMPLE 19
Biochemical Characterization
The purified pegylated hGH pools were characterized by
reducing and non-reducing SDS-PAGE, non-denaturing and
denaturing Size Exclusion Chromatography, analytical
Anion Exchange Chromatography, N-terminal Sequencing,
Hydrophobic Interaction Chromatography, and Reversed
Phase HPLC.
Size Exclusion High Performance Liquid Chromatography
(SEC-HPLC)
Non-denaturing SEC-HPLC
The reaction of Methoxy-PEG of various attachment
chemistries, sizes, linkers, and geometries 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 Tosohaas G4000PWXL column, 7.8 mm X 30 cm,
(Tosohaas Amersham Bioscience, Piscataway, NJ) or
Superdex 200 (Amersham Bioscience, Piscataway, NJ) in
20 mM Phosphate pH 7.2, 150 mM NaCl at a flow rate of
0.5 mL/minute. PEGylation greatly increases the
hydrodynamic volume of the protein resulting in a
41
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
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).
The Q-Sepharose chromatography step effectively
removed free PEG, hGH, and multi PEGylated hGH species
from the mono-Pegylated hGH. Non-denaturing SEC-HPLC
demonstrated that the effective size of the various
PEGylated-hGH was much greater than their respective
theoretical molecular weights (Table 1)
Tab1 a 1
Size Exclusion Chromatography (SEC)
MW Size (SEC)
(Theoretical)
hGH 22,000 21,000
4-6x5K PEG-SPA GH 47,000 128,000
2-4x5K PEG-CMHBA (NHS) 37,000 71,000
GH
20K PEG-ALD GH 42,000 120,000
20K Branched PEG-ALD GH 4f,000 114,000
20K PEG-CMHBA (NHS) GH 42,000 115,000
20k PEG-Hydrazide GH 42,000 125,000
2x20K PEG-ALD GH 62,000 250,000
30K PEG-ALD GH 52,000 231,000
30K PEG-SPA GH 52,000 183,000
2x30K PEG-SPA GH 82,000 569,000
40K Branched PEG-ALD GH 62,000 330,000
40K Branched PEG-NHS GH 62,000 253,000
42
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
Denaturing SEC-HPLC
The reaction of the various Methoxy-PEGS with hGH,
anion exchange purification, and final purified
products were assessed using denaturing SEC-HPLC.
Analytical denaturing SEC-HPLC was carried out using a
Tosohaas 3000SV~1XL column 7.8 mm X 30cm (Tosohaas
Pharmacia Biotech, Piscataway, NJ) in 100 mM Phosphate
pH 6.8, 0.1o SDS at a flow rate of 0.8 mL/minute.
PEGylation greatly increases the hydrodynamic volume
of the protein resulting in a shift to an earlier
retention time. New species were observed in the 20K
PEG aldehyde hGH reaction mixture along with
unmodified hGH. These PEGylated and non-PEGylated
species were separated on Q-Sepharose chromatography,
and the resultant purified mono 20K PEG-Aldehyde hGH
was subsequently shown to elute as a single peak on
denaturing SEC(> 95o purity). The Q-Sepharose
chromatography step effectively removed free PEG, hGH,
and multi PEGylated hGH species from the mono-
Pegylated hGH.
SDS PAGE/PVDF transfer
SDS-PAGE was used to assess the reaction of the
various PEG reagents with hGH and the purified final
products. Examples of this technique are shown with
mono 20K linear and branched 20K and 40K PEG aldehyde,
and 4X6 5K SPA PEG. (Figures 1 & 2). SDS-PAGE was
carried out on 1 mm thick 10-20o Tris tricine gels
(Invitrogen, Carlsbad, CA) under reducing and non-
reducing conditions and stained using a Novex
Colloidal CoomassieTM G-250 staining kit (Invitrogen,
Carlsbad, CA). Purified mono PEG-aldehyde hGH species
migrate as one major band on SDS-PAGE. Bands were
blotted onto PVDF membrane for subsequent N-terminal
43
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
sequence identification.
Analytical anion exchange HPLC
Analytical anion exchange HPLC was used to assess the
reaction of various mPEGs with hGH, anion exchange
purification fractions and final purified products.
Analytical anion exchange HPLC was carried out using a
Tosohaas ~5PW or DEAE-PW anion exchange column, 7.5 mm
x 75 mm (Tosohaas Pharmacia Biotech, Piscataway, NJ)
in 50 mM Tris ph 8.6 at a flow rate of 1 mL/min.
Samples were eluted with a linear gradient of 5-200 mM
NaCl.
Reversed phase HPLC (RP-HPLC)
PEG-GH reaction mixtures and purified PEGylated
products were analyzed by RP HPLC to elucidate hGH
P species, mono and multiply PEGylated hGH species, and,
to monitor oxidized hGH forms, as well as, PEG hGH
isoforms having a single PEG linked at different sites
(e. g. N-terminus vs Lysine ~-amino groups). RP-HPLC
was carried out utilizing a Zorbax SB-CN 150 or 250 mm
x 4.6 mm (3.5 mm or 5 mm) reversed phase HPLC column.
Experiments were conducted at ambient temperature on a
typical load of 10 mg of protein per sample. Buffer A
is 0.1o triflouroacetic acid in water; Buffer B is
0.1o trifluoroacetic acid in acetonitrile. The
gradient, which results in a 1/ZO increase in B per
minute, is as follows:
44
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
Step Time Flow %A oB Step
0 0 1 60 40 0
1 3 1 60 40 0
2 20 1 50 50 1
3 2 1 60 40 1
4 6 1 60 40 0
N-terminal Sequence and Peptide Mapping
Automated Edman degradation chemistry was used to
determine the NH2-terminal protein sequence. An
Applied Biosystems Model 494 Procise sequencer (Perkin
Elmer, Wellesley, MA) was employed for the
degradation. The respective PTH-AA derivatives were
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. 20K linear and 20 and 40K
branched PEG-ALD hGH Protein bands transferred to PVDF
membranes or solutions of purified 20K linear and
branched 20 and 40K PEG-ALD hGH were sequenced.
Purified 20K linear PEG-hGH yielded a major signal
(approximately 88o yield) was observed that had the
expected sequence for hGH except for the absence of
the N-terminal amino acid. This result is as expected
for a protein N-terminally PEGylated via the aldehyde
chemistry. The residue of the first cycle is
unrecoverable due to the attached PEG moiety. A minor
signal (approximately 12o yield) had the correct N-
terminal amino acid sequence. Considering that the
peak collected off the RP-HPLC is 1000 PEGylated,
these data suggest that approximately 880 of the PEG
modification is at the N-terminus with remainder
apparently linked to one of several possible lysine
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
residues.
Tryptic digests were performed at a concentration
of 1 mg/mL and, typically, 50 ug of material is 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 ~,L of 1N HCl per mL of
digestion solution. Samples were diluted, prior to
placing the samples in the autosampler, to a final
concentration of 0.25 mg/ml in 6.25 o acetonitrile.
Acetonitrile is added first (to 19.80 acetonitrile),
mixed gently, and then water is 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.
A Waters HPLC system was used
Alliance for
2695
analysis , but other systemsshould produce similar
results.The column used s an Astec C-4 polymeric
wa
cm x 4.6 mm column with ~.m particles.
5
Experime nts were conducted t ambient temperature
a on a
typical load of 50 ~,g of tein per sample. Buffer
pro A
is 0.10 trifluoroacetic in water; buffer B is
acid
25 0.0850 rifluoroacetic acidin acetonitrile. The
t
gradient is as follows:
Time A% Bo Co Do Flow Curve
0.00 0.0 0.0 100.0 0.0 1.000 1
90.00 0.0 0.0 55.0 45.0 1.000 6
90.10 0.0 0.0 0.0 100.0 1.000 6
91.00 0.0 0.0 0.0 100.0 1.000 6
91.10 0.0 0.0 100.0 0.0 1.000 6
95.00 0.0 0.0 100.0 0.0 1.000 6
46
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
The column is heated to 40~C using a heat jacket.
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 to
determine the extent of n-terminal Pegylation (loss of
T-1 fragment) as shown in Table 2.
Table 2
oT-1 Present
o T-1 o T-1 compared to
Sample present Lost control
Aldehyde
5K ALD 2.0 98.0 7.4
20K 0.0 100.0 0.0
2x20K 0.0 100.0 0.0
30K 1.3 98.7 4.5
40K
Branched 1.9 98.1 6.8
Nf3S
4-6x5 SPA 1.3 98.7 4.7
2-4X5 CM 0.0 100.0 0.0
20K CM 23.1 76.9 84.1
30K 18.2 81.8 63.9
2x30K 5.7 94.3 19.9
40K
branched 20.9 79.1 73.5
Example 20
Pharmacodynamic Studies
Efficacy studies in Hypophysectomized rats
Female Sprague Dawley rats, hypophysectomized at
Harlan Labs, were prescreened for growth rate for a
period of 7 to 10 days. Subsequently, growth studies
were carried out for 11 days. Rats were divided into
groups of six to eight. Group 1 consisted of rats
given either daily or day 0 and day 6 subcutaneous
doses) of vehicle. Group 2 were given daily
47
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
subcutaneous doses of GH (0.3 mg/kg/dose). Group 3
were given subcutaneous doses of GH on day 0 and day
6(1.8 mg/kg/dose). Group 4 were given subcutaneous
doses of PEG-GHs on day 0,6 (1.8 mg/kg/dose).
Hypophysectomized rats were monitored for weight gain
by weighing at least every other day during the study.
Weight gains (average +/- SEM) for 20K PEG-ALD hGH,
20K and 40K branched PEG-ALD hGH, and 4-6a~ SPEG-SPA
hGH dosed once a week were similar to those for daily
dosing of hGH (Figures 3& 4) Table 3 summarizes total
weight gain (average +/- SEM) at day 11 for once per
week dosing of various Pegylated hGH molecules
relative to daily dosing of hGH.
Table 3
Murine weight gain
Compound Single Daily weighto weight
Weekly gain in gain
Dose grams/day relative
(mpk) (d0-d11) to
(Avg. + SEM)daily hGH
gain (Avg.)
hGH (un-pegylated) 1.8 0.97 + 0.12 39~
5K Linear PEG-ALD 1.8 0.96 + 0.27 360
GH
20K Linear PEG-ALD 1.8 1.99 + 0.13,730
GH 1.43 + 0.08,
1.7 + 0.10
20K Linear CM-HBA 1.8' 2.36 + 0.11 990
PEG GH
20K Linear PEG-HYD 1.8 2.62 + 0.22 990
GH
20K Branched PEG-ALD1.8 2.24 + 0.07 87a
GH
30K Linear PEG-ALD 1.8 2.11+ 0.06; 940
GH 1.85+ 0.14
30K Linear PEG-SPA 1.8 2.6 +/- 0.1 1170
GH
40K Branched PEG-ALD1.8 2.57 + 0.08 1000
GH
40K Branched PEG-NHS1.8 2.53 + 0.09 1210
GH
2x 20K PEG-ALD GH 1.8 2.66 + 0.10 1280
4-6x5K SPA-PEG GH 1.8 3.18 + 0.10 124%
2-4x5K CM-HBA-PEG 1.8 3.5 1340
GH 4 + 0.15
2x 30K Linear PEG-SPA1.8 ~ _ 134
GH 3.1 + 0.1
Upon completion of each growth study, animals were
sacrificed and bone (tibia) lengths were analyzed.
Figure 6 shows the change in tibial bone length
(average +/- SEM) at day 11 in response to various
48
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
PEG-GH conjugates dosed on day 0 and day 6 or hGH
dosed daily.
IGF-.Z Levels in hypophysectomized rats
Experiments were carried out as above for the weight
gain studies, however blood samples were taken at day
0,1,2,3,4,5 and upon sacrifice of the animals at day
9. IGF-1 levels were determined by ELISA. Figure 7
compares increases in serum IGF-1 levels (average +/-
SEM) in hypophysectomized rats following either daily
dosing of hGH or single dose of hGH or day 0, day 6
dosing of Pegylated hGH.
Pharmacokinetic Studies
Pharmacokinetic studies were conducted in normal,
Sprague-Dawley male rats, mice, and cynomolgus
monkeys. Injections were made, either as a single
subcutaneous bolus of 1.8 mg/kg or as a single iv dose
at 1.0 mg/kg GH or PEG-GH in rats and mice, using six
rats and up to 60 mice per group. In cynomolgus
monkeys a 0.18 mg/kg GH or PEG-GH dose was used for
both single subcutaneous bolus and iv, using 2-4
monkeys per group. Blood samples were taken over one
to five days as appropriate for assessment of relevant
PK parameters (Table 4). (t1~2) - Terminal half life,
(C1) - clearance, (Tmax) - time to maximum
concentration, ZTss = volume distribution (apparent) at
steady state, and (Cmax) - maximum concentration GH
and PEG-GH blood levels were monitored at each
sampling using immuno-assay.
hGH Immunoassay
49
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
hGH and pegylated hGH protein concentration
levels in rat, mouse, and cynomolgus monkey plasma
were determined using the hGH AutoDELFIA kit
fluorescence immunoassay (PerkinElmer Life Sciences),
using the appropriate PEG hGH to generate standard
curve.
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
Table 4
40K 40K 30K 20K 4-6 x
Br Br
S ParametersALD NHS ALD ALD hGH 5K SPA
ecies hGH hGH hGH
dose iv iv 1.0 iv 1.0 iv 1.0 iv 1.0
1.0
mouse(mglkg) sc sc 1.8 sc 1.8 sc 1.0 sc 1.8
1.8
CL
(ml/hr/kg)2.29 2.12 4.43 7.89 4.53
Vss
(ml/kg) 18 16 24 17 51
Tvz, iv
(hr) 4.3 3.8 2.8 1.8 11
T1/z, S
(hr) 4 6.2 3.7 2.5 9
Tmax, sc
(hr) 11 9 6 3 12
SC AUC
( ug/ml*hr)682 577 160 31 668
SC
Bioavailability
87 67 39 24 167
dose iv Iv 1.0 iv 1.0 iv 1.8 iv 1.0
1.0
rat (mglkg) sc sc 1.8 sc 1.8 sc 1.8 sc 1.8
1.8
CL
(ml/hr/kg)1.36 1.75 5.75 9.9 2.9
Vss
(ml/kg) 19 25 44 33 36
Ti/z, iv
(hr) 5.4 5.8 3.6 2.2 24
Ti/z, S
(hr) 5.8 7.1 6.7 2.9 29
Tmax, sc
(hr) 24 22 12 9 20
SC AUC
(ug/ml*hr)398 344 97 70 249
SC
Bioavailability
30 33 31 39 40
dose iv iv 0.18Iv 0.18iv 0.18 iv 0.18
0.18
cyno (mg/kg) sc sc 0.18sc 0.18sc 0.18 sc 0.18
0.18
CL
(mUhr/kg) 1.83 0.78 1.94 2.19 0.49
Vss
(ml/kg) 57 20 29 44 25
Ti/z, iv
(hr) 21 13.6 14.9 7.3 38
Ti/z, s
(hr) 19 21 12 8.3 35
Tmax, sc
(hr) 22 22 10 8 32
SC AUC
( ug/ml*hr)100 483 125 38 242
SC
Bioavailability
64 77 97 44 66
51
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
03582 1 PCT.ST25.txt
SEQUENCE LISTING
<110> Pharmacia Corporation
Finn, Rory
Liao, Wei
Siegel, Ned
<120> CHEMICALLY-MODIFIED HUMAN GROWTH HORMONE CONJUGATES
<130> 03582/1/PCT
<150> US 60/331907
<151> 2001-11-20
<160> 1
<170> PatentIn version 3.1
<210> 1
<211> 191
<212> PRT
<213> homo Sapiens
<400> 1
Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asp Ala Met Leu Arg
1 5 10 15
Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu
20 25 30
Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asp Pro
35 40 45
Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asp Arg
50 55 60
Page 1
CA 02467731 2004-05-17
WO 03/044056 PCT/US02/37270
03582 1 PCT.ST25.txt
Glu Glu Thr Gln Gln Lys Ser Asp Leu Glu Leu Leu Arg Ile Ser Leu
65 70 75 80
Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Ser Leu Arg Ser Val
85 90 95
Phe Ala Asp Ser Leu Val Tyr Gly Ala Ser Asp Ser Asp Val Tyr Asp
100 105 110
Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu
115 120 125
Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser
130 135 140
Lys Phe Asp Thr Asp Ser His Asp Asp Asp Ala Leu Leu Lys Asp Tyr
145 150 155 160
Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe
165 170 175
Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe
180 185 190
Page 2
