Note: Descriptions are shown in the official language in which they were submitted.
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PEG-INTERFERON LAMBDA 1 CONJUGATES
Related Application:
[0001] The present application claims the benefit of Vietnamese Patent
application serial No.
VN1-2011-02222, filed August 25, 2011, which is incorporated herein by
reference.
Field of the Invention:
[0002] In one embodiment, the present application discloses pegylated
derivatives of recombinant
human interferon lambda 1 (PEG-interferon lambda 1 conjugates or PEG-IFNkl),
processes for
their preparation, pharmaceutical compositions containing these conjugates and
processes for
making the same.
Background of the Invention:
[0003] Hepatitis C virus (HCV) is a major health problem and the leading cause
of chronic liver
disease throughout the world. It is estimated that at least 180 million people
worldwide are
chronically infected with HCV. In Vietnam, the proportion of infected HCV
individuals in the
population is 4-9%. Approximately 55-85% of acutely infected HCV individuals
will convert to
chronic infection, 5-25% of these chronic carriers are at risk of developing
cirrhosis after 25-30
years and of those with cirrhosis, 30% are at risk of liver decompensation
over 10 years, and 1-3%
will develop liver cancer each year. According to epidemiological research,
HCV is the cause of
40% of individuals in final stage cirrhosis and 60% in hepatoma.
[0004] Currently, a-interferons (Al) are the therapies of choice for the
treatment of chronic HCV
infection. Al can give a persistent response to HCV in approximately 70% of
cases, however
these interferons cause many side-effects, even in the case of PEG-interferon
alpha. These side-
effects can sometimes limit treatment, leaving the treatment of patients
incomplete. Side-effects
include influenza-like symptoms and hematologic effects such as thalassemia
and anemia.
[0005] Interferons are currently used for the treatment of many viral diseases
such as hepatitis B,
hepatitis C, hepatitis D, condyloma acuminata, lepromatous leprosy, chronic
leukaemia and AIDS.
Al are also effective in reducing malignant tumors and treating Kaposi's
sarcoma, melanoma, and
renal cell carcinoma. Moreover, Al are applicable in the prevention and
treatment of diseases in
cattle and other livestock. For example, Al enhance the activity of vaccines
used in prophylaxis
and treatment of foot and mouth disease and porcine reproductive and
respiratory syndrome.
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[0006] Al have been produced from human cell lines incubated in tissue culture
media or
leukocytes derived from donors. However, these methods are time consuming,
labor intensive,
expensive, and not amenable to large scale manufacturing. Furthermore, there
is the risk of
septicaemia caused by infectious agents from the cell lines.
[0007] With the development of recombinant DNA technology, we can now
introduce Al genes
into microorganisms that enable production of large amounts of interferons.
However, these
methods also present certain advantages and difficulties, mainly in the steps
of expression and
large-scale protein production.
[0008] IL-29 is a member of the helical cytokine family and is a type III
interferon. It is also
known as interferon lambda 1 (IFNkl) and is highly similar in amino acid
sequence to IL-28, the
other type III interferon. IL-28 and IL-29 (IFNkl) were recently described as
members of a new
cytokine family that shares with type I interferon (IFN), the same Jak/Stat
signaling pathway
driving expression of a common set of genes. Accordingly, they have been named
IFNk. IFNsk
exhibit several common features with type I IFNs: antiviral activity,
antiproliferative activity and
in vivo antitumor activity. Importantly, however, IFNsk bind to a distinct
membrane receptor,
composed of IFNLR1 and IL1OR2.
[0009] The major disadvantage with the therapeutic use of most biologicals is
that they are
administered parenterally, e.g. intravenously (i.v.), subcutaneously (s.c.),
intramuscularly (i.m.)
etc. This means that delivery to the patient is associated with pain and
discomfort. Furthermore,
because of their usually very short half-lives, biologicals require frequent
administration to the
patient in order to maintain therapeutic blood serum or plasma levels of the
drug. Injections that
cannot be self-administered require frequent trips to the clinic and trained
medical personnel,
making such therapy inconvenient and expensive. Interferon alpha-2a (Roferon,
Roche) and
interferon alpha-2b (Intron A, Schering A G), the two recombinant forms of
human interferon
alpha used in the treatment of chronic hepatitis B and C, have a serum half-
life of less than 12h
(McHutchison, et al., Engl. J. Med. 1998, 339, 1485-1492; Glue, et al., Clin.
Pharmacol. Ther.
2000, 68, 556-567) and therefore require administration 3 times a week.
Repeated injections with
interferon beta-lb (Betaseron) are also required to treat the patients of
multiple sclerosis (MS).
[0010] One very successful and well accepted method of overcoming the above
requirement of
frequent high dose injections to maintain threshold levels of the drug in the
body is to increase the
in vivo half-life of the therapeutic protein by conjugating it with a polymer,
such as polyethylene
glycol (PEG or Peg). PEG molecules with their long chains not only create a
protective shield
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around the pegylated drug molecule in aqueous solution, thereby reducing the
immunogenicity of
protein drugs while also protecting them from the action of proteases, but
they further help
increase the circulation half-life of the drug by increasing its hydrodynamic
volume which reduces
its loss from the filtration mechanisms of the kidney glomeruli network. After
their separation
from the protein molecule, the PEG moieties are cleared without any structural
changes and their
clearance is proportional to their molecular weight.
[0011] Usually PEG moieties are attached to the protein by first activating
the PEG moiety and
then reacting the activated PEG agent with the side chains of an amino acid of
a protein, such as
the lysine residue and/or the N-terminal amino group on the protein. The most
frequently used
PEG is monofunctional PEG because this moiety resists cross-linking and
aggregation. One such
example has been disclosed by Davis et al. in U.S. Pat. No. 4,179,337.
Summary of the Invention:
[0012] PEG-interferon lambda 1 (PEG-IFNkl) is a pegylated derivative of human
recombinant
IFNkl (wherein polyethylene glycol is conjugated to IFNkl, also referred to as
the "conjugate")
that is useful in the treatment of chronic hepatitis C in adult patients. PEG-
IFNkl bypasses the
action of extracellular enzymes and resists filtration in the kidney after
injection into the patient's
body; therefore its half-life in circulation is extended. That is, the
conjugate has significantly
improved stability, better solubility, and enhanced circulating half-life and
plasma residence times
when compared to the corresponding non-PEG-conjugated IFNkl.
[0013] Interferon lambda 1 (IFN kl, Zcyto21 or IL-28A) is known in the art,
for example, from
U.S. Pat. Nos. 7,038,032, 6,927,040, 7,135,170, 7,157,559 and 7,351,689; and
PCT publication
Nos. WO 05/097165, WO 07/012,033, WO 07/013,944 and WO 07/041,713; all of
which are
herein incorporated by reference in their entirety.
[0014] In one embodiment, the present application discloses novel PEG-IFNkl
conjugates. In one
aspect, conjugates of the present application have a linear PEG chain
structure. As compared to
unmodified IFNkl (that is, the IFNkl that is not conjugated with PEG or mPEG),
these conjugates
have increased circulating half-life and persistence in plasma. Water soluble
PEGs include
polyethylene glycol (PEG), monomethoxy-PEG (mPEG), mono-C1_10 alkoxy-PEG and
mono-C1_3
alkoxy-PEG. These PEGs that may be employed may have a molecular weigth of
about 600 to
60,000 and include those, for example with about 10 kDa, 20 kDa, 30 kDa, 40
kDa, 50 kDa and 60
kDa. In one aspect, the PEG employed in the present conjugates are mPEG with a
molecular
weight of 40 kDa.
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[0015] In one embodiment, there is provided a physiologically active PEG-IFNkl
conjugate
comprising the formula I:
R0(CH2CH20)n L-X-INFIl
H I
m
wherein: R is H or Ci_3 alkyl; m is 1, 2, 3 or 4; n is a positive integer
selected in the range from
400 to 550; L is a C1_10 alkyl or heteroalkyl linker; X is ¨0-, -NH- or ¨S-;
and IFNkl is interferon
lambda 1; or a pharmaceutically acceptable salt thereof. In one aspect,
interferon lambda 1 is a
human recombinant interferon. In another aspect, the IFNkl may be a natural or
recombinant
protein. In another aspect, the IFNkl is a human protein derived from a source
such as tissues,
protein synthesis, or cell culture using natural cells or recombinant cells.
In a still another aspect,
IFNkl is a human recombinant protein. In another aspect, the conjugate
interferon lambda 1 of the
formula I is the SEQ ID 2. In one embodiment, the PEG chain is coupled to the
IFNkl via an
amide bond on a primary amino group of, for example, lysine, or the N-terminal
of IFNkl.
[0016] Heteroalkyl is defined as a C1_10 alkyl wherein at least one of the
carbon of the C1_10 alkyl is
replaced by an ¨0-, -C(=0)-, -NH- or combinations thereof. Such combinations
include, for
example, -0C(=0)-, -C(=0)0-, -NHC(=0)-, -C(=0)NH- and the like. In one aspect,
n is about
500 to 550. In another aspect, n is about 420, 520 or 455. In another aspect,
the molecular weight
of the Peg group is about 35 kDa to 45 kDa, or about 40 kDa. In another aspect
of the above, m is
1 or 2. A C1_10 alkyl or heteroalkyl may be a linear or a branched alkyl or
heteroalkyl group. In
one aspect, the C1_10 alkyl group is a ¨C(0)- group. In one embodiment, m is 1
and L-X- is
selected from the group consisting of the formulae:
o o s
H H
0
0 0 S
0
0 S
0
µ)HrS.ss . õ144)s
L A and \AsA
r ,
0
[0017] In one aspect of the above conjugate, m is 2 and L-X- is of the
formula:
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0
;s0iri-\IT
x)rt
0 HNIetet
0
[0018] In another aspect of the above conjugate, the two PEG groups, PEG,
alkyl-PEG or m-
PEGs, are attached to the two formamide groups (i.e. ¨C(0)NH-) of the above
formula. In another
aspect, X is -NH- or ¨0- and m is 2. In another aspect, the linker is attached
to two PEG groups.
In another aspect, X is ¨NH-, and the group attached to the linker is the
residue of a lysine on
IFNkl. In one aspect, the ¨NH- group (i.e., the amino group) attached to the
linker is the residue
of a histidine. In another aspect, X is ¨0-, and the group attached to the
linker may be derived
from the residue of a serine on IFNkl. In one variation of the above formula,
R is ¨CH3. In
another aspect, the linker is attached to the residue of a lysine, a serine, a
histidine or mixtures
thereof on the IFNkl. In another aspect, the linker is attached to a
positional isomer of the residue
of a lysine, a serine, a histidine or mixtures thereof on the IFNkl. In
another aspect of the
conjugate, R is H or ¨CH3, m is 1, L is ¨C(0)- and X is ¨NH-. In another
aspect, n is 500 to 550.
[0019] In another aspect, the conjugate (Nanogen PEG-IFNkl) comprises the
formula II:
0
II H
CH30(CH2CH20 C -N -INFX 1 II
wherein INFkl is interferon lambda 1; and n is 500 to 550. In one aspect, n is
a number of units of
ethylene glycol in the PEG structure and it is a positive integer selected
from any numbers such
that the molecular weight of PEG moiety is about 40 kDa, and INFkl is
interferon lambda 1. In
another embodiment of the above, the conjugate has a blood serum half-life and
persistence time
that are prolonged or extended when compared to IFNkl. In another aspect of
the conjugate, the
PEG is attached to a methionine at the N-terminal of the IFNkl. In yet another
aspect, the
conjugate is effective in the treatment of hepatitis B and hepatitis C.
[0020] In another embodiment, there is provided a process for the preparation
of a human
recombinant conjugate as disclosed above, the process comprises the step of
covalently binding
(ct-methoxy-co-(4-nitrophenoxy carbonyl)) polyoxyethylene (PEG-pNC) 40 kDa
with IFNkl
through a conjugation reaction as follows:
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0
ll
CH30(CH2CH20 ,).C-0 NO2 H2N¨Interferon Lambda 1
0
cH30(cH2cH20C¨N ¨Interferon Lambda 1 HO NO2
wherein, n is a positive integer selected such that the molecular weight of
PEG moiety is about 40
kDa; and isolating the conjugate. In one aspect, n is from about 500 to 550.
[0021] In another embodiment, there is provided a pharmaceutical composition
containing a
conjugate as disclosed above and pharmaceutically acceptable carriers and
excipients.
Conventional pharmaceutical formulations can be also prepared using the
compositions
comprising the conjugate of the present application. The formulations may
comprise a
therapeutically effective amount of the composition comprising the conjugate
together with
pharmaceutically acceptable carriers as known in the art. For example,
adjuvants, diluents,
preservatives and/or solubilizers, if needed, may be used. Pharmaceutical
compositions
comprising the conjugate may include diluents of various buffers (e.g., Tris-
HC1, acetate,
phosphate) having a range of pH and ionic strength, carriers (e.g., human
serum albumin),
solubilizers (e.g., polyoxyethylene sorbitan or TWEENC), polysorbate), and
preservatives (e.g.,
thimerosol, benzyl alcohol), as disclosed, for example, in U.S. Patent No.
4,496,537. In one
aspect, the pharmaceutical composition is formulated as a sterile lyophilized
powder for injection.
In another aspect, the composition comprises a combination of pharmaceutically
acceptable
vehicles, including saline, buffered saline and 5% dextrose in water. In
another aspect, the
pharmaceutical composition is formulated as a solution for injection in vials
or pre-filled syringes.
In one aspect of the above, the pharmaceutical composition is used in the
treatment of hepatitis B
and hepatitis C. Pharmaceutical formulations and methods for preparing such
formulations are
well known in the art and are disclosed, for example, in Remington, The
Science and Practice of
Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, Pa. 19th ed. 1995.
[0022] In another embodiment, there is provided a process for preparing a Peg-
IFNkl conjugate
comprising the formula I:
R0(CH2CH20)n L-X-INFIl
H I
m
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wherein: R is H or C1_3 alkyl; m is 1, 2, 3 or 4; n is a positive integer
selected in the range from
400 to 550; L is a C1_10 alkyl or heteroalkyl linker; X is ¨0-, -NH- or ¨S-;
and IFNkl is interferon
lambda 1; or a pharmaceutically acceptable salt thereof; the process
comprising: contacting the
IFNkl with a pre-activated Peg under conditions that are sufficient to
facilitate covalent
conjugation with an amino acid residue of the IFNkl. In one embodiment, there
is provided a
PEG-IFNkl conjugate prepared by the process as described herein. In one
aspect, there is
disclosed a method of preparing the above conjugate comprising contacting the
IFNkl with a
sufficient amount of an activated PEG or mPEG under conditions that are
sufficient to facilitate
covalent attachment of the PEG or mPEG on the IFNkl. In another aspect, the
activated mPEG is
mPEG-pNC. In another aspect, the attachment of the activated mPEG is on a
methionine at the N-
terminal of the IFNkl. In another aspect, the mPEG has a molecular weight of
about 40 kDa. In
another aspect, the activated oxycarbonyl agent is a mono- or di-activated
agent.
[0023] In another embodiment, there is provided a method for inhibiting the
proliferation of a
cancer cell in a patient comprising contacting the cancer cell with the
conjugate described above,
wherein the conjugate has a blood serum half-life and persistence time that
are prolonged or
extended when compared to IFNkl. In one aspect, the conjugate of the present
application has a
blood serum half-life that is extended by more than twice, three times, five
times, eight times or
more than 10 times the serum half life of the corresponding unconjugated
IFNkl.
[0024] In another embodiment, there is provided a method for treating a
proliferative disorder in a
mammal comprising administering to the mammal a therapeutically effective
amount of the above
conjugate. In one embodiment, the conjugate may be used for treating
interferon-susceptible
conditions or conditions which would respond positively or favorably to
interferon based therapy.
In one aspect, the treatment using the conjugate results in substantially
reduced or elimination of
side effects when compared to conventional treatment with interferons.
[0025] In one aspect, exemplary conditions which can be treated with the
conjugates of the present
application include, but are not limited to, cell proliferation disorders, in
particular cancer (e.g.,
hairy cell leukemia, Kaposi's sarcoma, chronic myelogenous leukemia, multiple
myeloma, basal
cell carcinoma and malignant melanoma, ovarian cancer and cutaneous T cell
lymphoma), and
viral infections. In another aspect, the conjugates may be used to treat
conditions which would
benefit from inhibiting the replication of interferon-sensitive viruses. Viral
infections which may
be treated with the conjugate of the present application include hepatitis A,
hepatitis B, hepatitis C,
other non-A/non-B hepatitis, herpes virus, Epstein-Barr virus (EBV),
cytomegalovirus (CMV),
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herpes simplex, human herpes virus type 6 (HHV-6)), papilloma, poxvirus,
picomavirus,
adenovirus, rhinovirus, human T lymphotropic virus-type 1 and 2 (HTLV-1/-2),
human rotavirus,
rabies, retroviruses including human immunodeficiency virus (HIV),
encephalitis and respiratory
viral infections.
[0026] In another embodiment, there is provided a method of treating a patient
infected or at risk
of infection with a viral infection, comprising administering to a patient in
need thereof, a
therapeutically effective amount of a conjugate of the formula I:
R0(CH2CH20)n L-X-INFX,1
H I
m
wherein: R is H or C1_3 alkyl; m is 1, 2, 3 or 4; n is a positive integer
selected in the range from
500 to 550; L is a C1_10 alkyl or heteroalkyl linker; X is ¨0-, -NH- or ¨S-;
and IFNkl is interferon
lambda 1; or a pharmaceutically acceptable salt thereof; or a pharmaceutical
formulation
comprising the conjugate of the formula I. In one aspect, the conjugate
interferon lambda 1 of the
formula I is the SEQ ID 2. In one aspect, the mammal is a human. In another
aspect of the
method, the viral infection is caused by a hepatitis C virus, or the viral
infection results in advance
liver cirrhosis. In a particular aspect, the patient is an HCV resistant or
refractory patient. In
another aspect of the above method, the PEG-IFNkl is administered in a dose of
about 0.5 ug/kg
to 10.0 ug/kg weekly. In one aspect of the method, the PEG-IFNkl is
administered in a dose of
about 2.5 ug/kg weekly. In another aspect, the PEG-IFNkl is administered for
about 8 weeks to
about 52 weeks. In another aspect, the PEG-IFNkl is administered for about 12
weeks, about 16
weeks, about 20 weeks or about 24 weeks. In another aspect, the PEG-IFNkl is
administered until
the patient is determined to be free of HCV RNA in blood serum. In another
aspect, the
administration of the conjugate provides significant improvement over the
standard PEG-INF-a
therapy because the method does not result in the significant reductions in
neutrophil counts,
platelet counts or hemoglobin levels. In yet another aspect of the above
method, the method
further comprises the administration of a nucleoside analogue selected from
ribavirin and
viramidine. In another aspect of the method, the ribavirin is administered
orally in a dose of 5
mg/kg to 25 mg/kg daily; or 15 mg/kg to 25 mg/kg daily. In one aspect, the
ribavirin is
administered in a dose of about 10 mg/kg to 30 mg/kg once or twice daily, or
about 15 mg/kg
daily once or twice daily. In one aspect of the above, the conjugate is
administered paRmccrally.
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[0027] In one embodiment, I-113V may be treated using a dose of about 200 ig
of the PEG-IFNkl
conjugate per week, combined with tenofovir (tenofovir disoproxil fumarate) at
about 300 mg per
day. Under this treatment, it is determined that most patients are clear of
HBV after about four
injections over about 30 days. Under these conditions, the HBV is found to be
suppressed, and the
HBsAg (virus surface antigen) is released, which triggers the immune system to
make the antibody
against this antigen, resulting in the optimal endpoint in the particular
treatment. The treatment
may be continued as disclosed herein, for about 12 weeks to 24 weeks,
depending on the patient's
initial viral load.
Definitions:
[00281 "Alkyl" means a straight or branched, saturated or unsaturated,
aliphatic radical having a
chain of carbon atoms, optionally substituted with oxygen (e.g., a C1 alkyl
may be -C(0)-),
nitrogen atoms (e.g., a C1 alkyl may be -C(NH)-) or sulfur atoms (e.g., a C2
alkyl may be
-CH2C(S)-),. A Cx alkyl and Cx_y alkyl, such as a C1_10 alkyl or a C1_6 alkyl,
are typically used
where X and Y indicate the number of carbon atoms in the chain. For example,
C1_6 alkyl includes
alkyls having between 1 and 6 carbons (e.g., methyl, ethyl, propyl, isopropyl,
butyl, isobutyl,
vinyl, isopropenyl, 1-butenyl, ethynyl, 1-propynyl and the like).
[0029] A "heteroalkyl" or "heteroalkylene" is an alkyl that may have an
oxygen, nitrogen or sulfur
between the carbon atoms. Examples of such heteroalkyl groups include ¨C(0)NH-
, -0C(0)-,
-CH2CH2C(0)-NH-, ¨CH2-0-CH2-CH2-, ¨CH2-NH-CH2-CH2-, ¨CH2-S-CH2-CH2- and ¨CH20-
CH2-CH3 and the like.
[0030] The term "PEG" as in "PEG-IFNkl" means polyethylene glycol as used in
the art, and
generally includes both alkyl-PEG such as mPEG (methoxy-polyethylene glycol)
and PEG, unless
specified otherwise.
[00311 A "therapeutically effective amount" is an amount of the PEG-IFNkl
conjugate that is
sufficient to produce a clinically significant change in the treated
condition, such as a clinically
significant change in the viral load or immune function, a significant
reduction in morbidity or a
significantly increased histological score, or combinations thereof.
[00321 As used herein, "treatment" or "treating" refers to a therapeutic
treatment and prophylactic
or preventive measures. Patients who are in need of treatment include patients
already infected
with hepatitis C virus as well as those in which the hepatitis C disease is to
be prevented.
[0033] In one aspect of the above method, the conjugate is administered by
injection or infusion.
In another aspect, the conjunte is administered intravenously,
intramuscularly, subcutaneously,
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intradermally or intraperitoneally. in another aspect, the conjugate is
administered to the patient in
a dose amount selected from less than 0.5 p.gfkg, 0.5 to 1,0 nYkg, 1.0 to 1.5
gglkg, 1.5 to 2.0
ng/kg, 2.0 to 2.5 gglkg, 2,5 to 3.0 rig/kg, 3.0 to 3,5 nYkg, 3.5 to 4.0 pg/kg,
4.0 to 4,5 pg/kg. 4.5 to
5.0 fkg, 5.0 to 5.5 pg/kg, 5.5 to 6.0 ngfkg, 6.0 to 6.5 ggfkg, 6.5 to 7.0
pg/kg, 7.0 to 7.5 j.tgikg,
7.5 to 8.0 pig/kg, 8.0 to 8.5 pg,fkg, 8.5 to 9.01.q?,/kg, 9.0 to 9.5 p.g/kg,
9.5 to 10.0 pykg, or greater
than 10.0 jig/kg. In another aspect, the conjugate is administered in a fixed
dose of about 60-80
us, 80-100 ps, 100-12011.g, 120.-140 p.g, 140-160 pg, 160-180 ng, 180.-
2001.113,,,, 200-220 t.g, 220-
240 pg, 240-260 p.g, 260-280 pg, or about 280-300 pg. In one embodiment, the
conjugate is
administered subcutaneously at 200 pg for 12 consecutive weeks.
[0034] In another embodiment, there is provided a pharmaceutical composition
containing the
above conjugate and pharmaceutically acceptable carriers and excipients. In
another aspect, the
pharmaceutical composition is used in treatment of hepatitis B and hepatitis
C. In another
embodiment, there is provided a process for the preparation of a
pharmaceutical composition
containing the above conjugate comprising mixing the conjugate with
pharmaceutically acceptable
carriers and excipients.
[0035] The conjugates of the application have similar effects or activities as
those of IFNkl. For
example, the conjugates may be used as anti-proliferative agents, antiviral
agents, or antitumor
agents. Specifically, the conjugates of the present application are effective
in treatment of
hepatitis B and hepatitis C, and they have a longer persistence time in blood
than IFNkl. In one
embodiment, pharmaceutical compositions containing the conjugates of the
present application are
prepared as sterile lyophilized powders for injection, or as solutions for
injection in vials or pre-
filled syringes. These pharmaceutical compositions may be formulated by mixing
the conjugates
with relevant pharmaceutically acceptable carriers and excipients.
[0036] In another embodiment, the present application provides processes for
the preparation of
human recombinant PEG-IFNkl conjugates. First, human recombinant IFNkl is
produced by
recombinant DNA technology in E.coli, then reacted with a pegylating agent
(such as ct-methoxy-
co-(4-nitrophenoxy carbonyl)) polyoxyethylene (PEG-pNC) to produce the PEG-
IFNkl. In one
aspect, the PEG-IFNkl is a linear chain PEG 40 kDa that is conjugated to
IFNkl. This product
bypasses the action of extracellular enzymes and kidney filtration when
injected into the patient's
body, therefore its blood serum half-life is extended.
[0037] The pegylation reaction between (ct-methoxy-co-(4-nitrophenoxy
carbonyl))
polyoxyethylene (PEG-pNC) 40 kDa and IFNkl to form the conjugate, is shown
below. In one
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aspect, the -NH2 group is a methionine residue at the N-terminal on a site of
the interferon lambda
1 molecule. In another aspect, the ¨NH2 group is the amine of a lysine residue
on a site of the
interferon lambda 1 molecule.
0
c1130(c112c020)jj¨C¨O NO2 + H2N-1NF21
0
H
CH30(CH2CH20C¨N HO = NO2
[0038] In one embodiment, the conjugates of the present application may be
prepared by
covalently binding Interferon lambda 1 with pre-activated PEG. In one
embodiment, PEG may be
activated by substituting the PEG hydroxyl group with a linking group to form
the coupling agent,
or an activated PEG agent that is (ct-methoxy-co-(4-nitrophenoxy carbonyl))
polyoxyethylene
(PEG-pNC). In one embodiment, the PEG-IFNkl conjugates may be prepared by the
preparation
of IFNkl and the pegylation of the IFNkl. Also disclosed are processes for
purifying and assaying
the conjugated products.
Brief description of the drawings:
[0039] Figure 1 exemplifies a nucleic acid sequence (SEQ ID 1) used to produce
human
recombinant IFNkl after the sequence was synthesized and introduced into the
expression vector
pNanogen 1-IL29.
[0040] Figure 2 is a representative amino acid sequence (SEQ ID 2) of human
recombinant IFNkl
produced by Nanogen Pharmaceutical Biotechnology Co., Ltd.
[0041] Figure 3 exemplifies a plasmid pNanogen 1-IL29 containing the gene
encoding human
IFN21 (interleukin-29).
[0042] Figure 4 depicts a result of analyzing plasmid pNanogen 1-IL29.
[0043] Figure 5 exemplifies a result of an electrophoresis process for
examining the ability of E.
coli containing pNanogen 1-IL29 used to produce IFNkl.
[0044] Figure 6 is a representative spectrum of the salt phase and SDS-PAGE
electrophoresis after
refolding protein. The spectrum of Figures 6, 7, 8 and 9 are all coomassi blue
stained.
[0045] Figure 7 exemplifies a spectrum and SDS-PAGE electrophoresis after
cation 1 phase.
[0046] Figure 8 exemplifies a spectrum and SDS-PAGE electrophoresis after
cation 2 phase.
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[0047] Figure 9 exemplifies a spectrum and SDS-PAGE electrophoresis after a
gel filtration
phase.
[0048] Figure 10 exemplifies a spectrum of the purification process and SDS-
PAGE
electrophoresis of PEG-interferon lambda 1.
[0049] Figure 11 exemplifies an identification results of IFNkl and PEG-IFNkl.
[0050] Figure 12 exemplifies a Maldi-Tof mass-spectrum of PEG-IFNkl produced
by Nanogen
Pharmaceutical Co., Ltd.
Detailed Description of the Invention
[0051] In one embodiment, the present application discloses processes for
preparing a
recombinant bacterial strain containing the gene encoding IFNkl, large scale
or industrial
manufacture of IFNkl, pegylation reaction of IFNkl, purification of the
produced the PEG-IFNkl,
and assays of PEG-IFNkl.
[0052] In one embodiment, the present application discloses an artificial
synthesis of the gene
encoding IFNkl based on the published sequence available from the National
Center for
Biotechnology Information (the encoding gene was modified to conform to the
industrial
production process on E.coli), creating of the gene transfer vectors,
introducing these vectors into
the bacteria, and selecting the bacterial strain that best produced IFNkl.
[0053] In one embodiment, the industrial manufacturing process for IFNkl
includes the steps of:
fermenting the initial material, collecting the solution of crude proteins and
purifying the IFNkl
protein. In a representative process, the fermentation process may be carried
out in a 10 liter
fermenting tank containing a nutrient medium and production of IFNkl was
induced by lactose.
The biomass obtained was separated and purified. IFNkl was collected and
refined through a
number of steps including: refolding the protein, separating the protein, for
example by ion
exchange chromatography (cation 1 and cation 2), and refining the protein on a
gel.
[0054] In one embodiment, the pegylation process comprises a reaction between
the linear chain
(ct-methoxy-co-(4-nitrophenoxy carbonyl)) polyoxyethylene (PEG-pNC- with a
molecular weight
of 40 kDa) and IFNkl. The resulting conjugate product may be purified by
chromatography, such
as using an HPLC system, and tested for quality and purity.
[0055] The present application will be more fully appreciated by reference to
the following
examples, which are to be considered merely illustrative and not limiting to
the scope of the
invention as claimed.
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Examples:
[0056] Example 1: Process for preparing E. coli strain containing the gene
encoding human
recombinant interferon lambda 1 (IFNkl)
[0057] The gene encoding IFNkl was artificially synthesized based on the
protein sequence data
available from NCBI or other databases. The novel method provided herein
reduces the time
required to isolate the gene but still provides a result as accurate as the
conventional method. The
nucleic acid sequence used to produce IFNkl in Nanogen Pharmaceutical
Biotechnology Co., Ltd.
is shown in Figure 1 and the amino acid sequence of this protein is shown in
Figure 2.
[0058] The expression vector pNanogen-1L29 (comprising the T7 transcription
promoter region,
the IFN2,1 transgene, the T7 reverse priming site, the T7 transcription
terminator, the fl origin, the
kanamycin resistance gene, and the pUC origin of replication) was specifically
designed to enable
high expression of the protein and facilitate fermentation for industrial
production of a large
quantity of IFNkl. Figures 3, 4 show the process for creation of vector
pNanogen 1-IL29.
[0059] Vector pNanogen 1-IL29 was then transferred into an E.coli strain
suitable for expression
of promoter T7. This strain has a genotype r ompT hsdSB (rgmli )gal dcm (DE3).
The strain
containing the IFNkl gene is termed E. Co/i-pNanogenl-IL29. It has the ability
to produce higher
than 100 mg of IFNkl per litre by fermentation (see Figure 5) and was
introduced into the original
strain bank.
[0060] Example 2: Process for fermentation of E. coli to produce human
recombinant IFNkl
[0061] The fermentation process was carried out in a 140 liter fermentation
tank with nutrient
medium at a temperature of 37 0.5 C, air pressure 0.5 m3/h, pH 7.0 0.2,
stirring rate of 300 rpm
and the pH was maintained at between 6.8-7.2 by adding H3PO4 or NH4OH. After 8
hours (when
E. coli grew in log phase is the time that cells develop most efficiently),
the temperature was
cooled to 30 0.5 C and the stirring rate was reduced to 200 rpm to start the
process for the
generation of IFNkl. The fermentation process was stopped after 4 hours and
the cold product
was centrifuged at 6000 rpm to obtain biomass.
[0062] The biomass was disrupted in a cell lysis solution (12 ml solution per
1 g wet biomass) by
homogenizing in a homogenizing device. The temperature was maintained at 4 C
for 1 hour, then
the cells were disrupted 2 times by an ultrasonic device. The resulting
suspension was centrifuged
at 6000 rpm for 30 minutes to give a pellet. The pellet was then washed with
an inclusion body
wash buffer (12 ml buffer per lg wet biomass), the resulting suspension was
kept at 4 C for 1
hour, then centrifuged twice at 13,000 rpm for 30 minutes to obtain a pellet.
The pellet was
13
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dissolved in 2M urea solution and incubated ice-cold for 1 hour, the
suspension was then
centrifuged at 13,000 rpm for 30 minutes to give the pellet. The pellet was
dissolved in a wash
solution and centrifuged at 13,000 rpm for 30 minutes to give a resulting
pellet. The pellet was
then dissolved in 6M guanidine solution, the suspension was kept ice-cold for
12-16 hours, and
centrifuged at 13,000 rpm for 30 minutes. The solution containing protein was
recovered and
purified in next step.
[0063] The components of culture medium and solutions used to separate IFNkl
are shown in
Table 1.
Table 1
Culture medium of E. coli - - 70 lag/m1 Kanamycin
pNanogen1 ¨ IL29 bacteria - 2 mM MgSO4
- 0.1% aspartate
25 mM Na2HPO4
25 mM KH2PO4
- 50 mM NH4C1
mM Na2SO4
- 0.5% Glycerol
- 0.05% Glucose
- 0.2% a-lactose
- 200 ug/nal each amino acid (18)
Cell lysis solution - NaC1 50 mM
- EDTA 1 mM
- Tris base 20 mM
Inclusion body wash solution - EDTA 1 mM
- Tris base, pH 8, 20 mM
- Triton X100 1%
Wash solution - EDTA 1 mM
- Tris base 20 mM
6M guanidine solution - EDTA 2 mM
- Tris base 50 mM
- Guanidine 6M
- Cysteine HC1 75 mM
[0064] Example 3: Process for purification of human recombinant IFNkl
[0065] IFNkl was refolded by dissolving the inclusion bodies in refolding
solution (25mM Tris
buffer, 1mM EDTA, 1.2M guanidine, pH 8.2) such that the final concentration of
the inclusion
bodies were 500 pg/ml. The mixture was then kept at 2-8 C for 16-24 hours.
The resulting
mixture was desalted before being subjected to a purification step on a
Sephadex G25 column.
The salt exchange buffer was a phosphate buffer (10mM, pH 8.0). In the step
"cation 1", the
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desalted mixture was loaded onto a Sephadex G25 column (this column was
prefilled with CM-
Sepharose FF gel and equilibrated in 10mM phosphate buffer pH 8.0), the
product was eluted
using 10mM sodium phosphate + 0.5M NaC1 pH 8Ø The resulting protein solution
was desalted
and chromatographed as above (step "cation 2"). The protein solution was then
filtered through a
gel column to give the product human recombinant IFNkl with purity greater
than 95% (see the
spectrum and electrophoresis results in Figures 5, 6, 7, 8 and 9).
[0066] Example 4: Pegylation Process for Preparing the Conjugate
[0067] The solution of 5 mg/ml human recombinant IFNkl (MW-20.1 kDa) in 50mM
sodium
borate-phosphate pH 8.0 was added (ct-methoxy-co-(4-nitrophenoxy carbonyl))
polyoxyethylene
(PEG-pNC) (MW-40 kDa) at a molar ratio of PEG-pNC:IFNkl about 3:1. The
reaction mixture
was kept at 2-4 C for 20 hours. The reaction was stopped by adjusting the pH
to 4.0 using
30%w/w acetic acid solution. The resulting mixture was then diluted 5-fold
with water.
[0068] In a general exemplary process, the reaction conditions for the
conjugation reaction of the
activated PEG or m-PEG reagent to the IFNkl further include conducting the
reaction using about
equi-molar to a relatively small molar excess of the activated PEG or m-PEG
with respect to
IFNkl. In one variation, the conjugation may be carried out with about 1-10
fold molar excess; or
about 1.5 to 7 fold molar excess; or about 1.75 to 5 fold molar excesses. In
one variation, the
conjugation reaction can be carried out at about room temperature, or about 20-
25 C. The
conjugation reaction may be allowed to proceed for about 1 to 10 hrs, 1 to 5
hrs, 1 to 3 hrs or about
1 to 2 hrs, before the reaction is terminated by quenching. In some cases, the
reaction conditions
provide a mixture of the PEG-IFNkl positional isomers. In one aspect, each
isomer contains a
single PEG-linker unit attached to the IFNkl via an amino acid residue as
disclosed herein. In
certain cases where more than one PEG-linker unit is attached to an IFNkl, if
desired, the resulting
composition containing these conjugates may be used or may be separated by
chromatography
using standard purification methods, including ultrafiltration, ion exchange
chromatography,
affinity chromatography and size exclusion chromatography. In one aspect, the
purification
method used for the separation and purification of the conjugates is cation
exchange
chromatography as described herein.
[0069] In certain conditions, the site of conjugation on the IFNkl may be
influenced by the pH of
the reaction medium. Modification of the particular pH of the conjugation
process will result in
certain preferred sites of conjugation. For example, under certain conditions,
the conjugation at
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basic pH values, such as pH of 7.5 or higher, 8.0 or higher, 8.5 or higher or
9.0 or higher, favors
the conjugation to a lysine group of the IFNkl.
[0070] In the above method, the pegylation reagent, suh as PEG-pNC, forms a
carbamate linker
between the PEG and IFNkl. Additional pegylation reagents that may be employed
in the above
process include oxycarbonyl-oxy-N-dicarboximide (such as succinimidyl
carbonate, succinimidyl
succinate), para-nitroaryl carbonates, para-nitrophenyl carbonates, carbonyl
di-imidazole,
benzotriazole carbonates, pyridyl carbonates, N-succinimide, N-phthalimide, N-
glutarimide, and
N-tetrahydrophthalimide as disclosed in U.S. Patent No. 5,122,614.
Representative activated PEG
or mPEG compounds that may be used to form the conjugate include PEG-2,4,6-
trichloro-S-
triazine, mPEG-2,4,6-trichloro-S-triazine, PEG-N-succinimidyl glutarate, mPEG-
N-succinimidyl
glutarate, PEG-N-succinimidyl succinate and mPEG-N-succinimidyl succinate.
[0071] Representative Compounds of the Examples:
[0072] The following Table provides a summary of selected compounds of the
Examples as
described herein:
R-0(CH2CH20)¨L -X ¨INFX 1 Ia
Variables
Conjugates R n -L- -X-
(kDa)
1 CH3- 900-945 -C(=0)- -NH-
(40)
2 CH3- 900-945 -C(=0)- -0-
(40)
3 CH3- 900-945 -C(=0)- -5-
(40)
4 CH3- 790-830 -C(=0)- -NH-
(35)
CH3- 790-830 -C(=0)- -0-
(35)
6 CH3- 790-830 -C(=0)- -5-
(35)
7 CH3- 1,010-1,060 -C(=0)- -NH-
(45)
8 CH3- 1,010-1,060 -C(=0)- -0-
(45)
9 CH3- 1,010-1,060 -C(=0)- -5-
(45)
CH3- 900-945 o N)a, -NH-
(40)
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Variables
Conjugates R n -L- -X-
(kDa)
11 CH3- 900-945
(40) \Ar?a,
12 CH3- 900-945
(40) \ArIA
13 CH3- 900-945-NH-
(40) \iyi,
14 CH3- 900-945-0-
(40) \j(?..4
15 CH3- 900-945-S-
(40) \ j(ii)i,
16 CH3- 900-945-NH-
(40) \joi,
17 CH3- 900-945-0-
(40) \joi,
18 CH3- 900-945-S-
(40) \i.);
19 CH3- 900-945-NH-
(40) \j(0),,
20 CH3- 900-945-0-
(40) \ joi,
21 CH3- 900-945 S-S-
(40)
22 CH3- 900-945 0 -NH-
(40) \)(sA
23 CH3- 900-945
(40)
24 CH3- 900-945
(40)
25 CH3- 900-945-NH-
(40) \isx
26 CH3- 900-945 \)s(sA -0-
(40)
27 CH3- 900-945-S-
(40) \is)i,
28 CH3- 900-945 0 -NH-
(40) vihrN)ss
0
29 CH3- 900-945 0
H
(40) vihr.N.ls
o
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Variables
Conjugates R n -L- -X-
(kDa)
30 CH3- 900-945 0 -S-
(40) 114)HrH
N ,,sos
0
31 CH3- 900-945 o -NH-
(40) 1,4,..Hroy
o
32 CH3- 900-945 0 -0-
(40) .5.4)Hroy
0
33 CH3- 900-945 o -S-
(40)
0
34 CH3- 900-945 0 -NH-
(40)
o
35 CH3- 900-945 o -0-
(40) ,1/4,1Hrsy
0
36 CH3- 900-945 0 -S-
(40) li..)Hro-sy
o
37 CH3- 900-945
(40) .14.õ; -NH-
xA
1
't
38 CH3- 900-945 o
-0-
(40) ,1YL'A A
x .
8
'.'
T
39 CH3- 900-945 o
-S-
(40) ,IY'lAx)C.
0
'T''''
40 H- 900-945 -C(=0)- -NH-
(40)
41 H- 900-945 -C(=0)- -0-
(40)
42 H- 900-945 -C(=0)-
(40)
43 H- 900-945 o
-NH-
(40) (1Y INI X A
8
't
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Variables
Conjugates R n -L- -X-
(kDa)
44 H- 900-945 0
-0-
(40) IYN
X
0 1-11,111,
45 H- 900-945 0
-S-
o
(40) fy }NI A
X
HI\ I
[0073] Processes for the purification of the conjugates of the formula Ia in
the above table are
performed using the methods described herein. The resulting PEG-IFNkl has a
purity that is
higher than about 95%. A spectrum and SDS-PAGE electrophoresis of the
conjugate after a gel
filtration phase is exemplified in Figure 9. A spectrum of the purification
process and SDS-PAGE
electrophoresis of the conjugate is exemplified in Figure 10. The conjugates
have antiviral EMC
activity on Hep-2C cell with ED50 in a range of about 10-50 ng/ml. Antiviral
activities of the
conjugates of the formula Ia in the above table at ED50 (ng/ml) are about
25.00 to 28.00; with a
Mean (ng/ml) of about 1.0 to about 30.0; SD of about 0.1 to about 1.0 and RSD
of about 3.0 to
7Ø
[0074] The conjugates of the formula Ia in the above table are administered to
patienst at 200 jug
(weekly subcutaneous injection) + ribavirin 15 mg/kg (daily). In the first 4
weeks, all patients are
determined to be free of HCV RNA (free virus in serum). The treatment
protoccols are continued
for 12 weeks. All patients achieve primary endpoint of total viral surpression
after 12 weeks
treatment and 12 weeks follow up.
[0075] Example 5: Purification of PEG-IFNkl
[0076] The solution containing PEG-IFNkl, quenched reagent and unmodified
IFNkl was
purified on a cation column (this column was prefilled with Sepharose CM gel
and equilibrated in
10mM sodium phosphate pH 6.0), eluted with a solution of 10mM sodium
phosphate, 0.5M NaC1
pH 6Ø The eluted fractions containing protein were transferred into
preservative buffer using a
solution of 10mM sodium phosphate pH 6Ø This product was then subjected to a
sterile filtration
process and stored at -20 C.
[0077] Figure 10 shows the spectrum of the purification process and SDS-PAGE
electrophoresis
of IFNkl. The resulting PEG-IFNkl had a purity that is higher than 95% and
antiviral EMC
activity on Hep-2C cell with ED50 about 10-50 ng/ml (see example 6).
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[0078] Example 6: Examination of antiviral activity of IFNkl and PEG-IFNkl
[0079] The examination was based on the antiviral activity in EMC viral model
and Hep-2C cell
according to the study of Ank et al. (J Virol. 2006 May; 80(9):4501-9). The
experiment was
carried out with 3 lots (IL290010111, 1290020311 and IL290030411). The results
indicated that
the antiviral activity of Nanogen's interferon lambda 1 (ED50 about 1-5 ng/ml)
is equivalent to the
study results of Sheppard et al. (Nat Immunol. 2003 Jan; 4(1):63-8.) (see
Table 2).
[0080] The antiviral activity of PEG-IFN21 was compared to IFNkl. The
experiments were
carried out with 5 lots (PIL290010111, PIL290020211, PIL290030311,
PIL290040411,
PIL290050511). Similar results were obtained in all lots, with an ED50 about
10-50 ng/ml (see
Table 3).
[0081] Patients' clinical information prior to treatment:
[0082] All patients in this treatment group were diagnosed with HCV chronic
have been
previously treated with Pegasys (PegInterferon alfa 2a) and PegIntron
(PegInterfeon alfa 2b)
combined with ribavirin (15 mg/kg) for over six months with no HCV RNA
reduction of more
than one log. HCV RNA >500,000 IU/ml serum; HCV Genotype 1-6; quantity 150;
Age 26-78
years old; median age 52 yr; some patient with high Ferritin, low Platelet
(<50,000/ml), low Hb.
Most patients have high level of fibrosis on the Fibro scale of F4 due to
being chronically infected
with HCV, where high AST/ALT ratio over 1 indication of liver fibrosis. Some
of the patients
were under insulin treatment for diabetes. All patients in the treatment group
that are over 50
years old have high blood pressure.
[0083] Treatment regimen:
[0084] Peglamda (PEG-IFNkl) 200 ug (weekly subcutaneous injection) + ribavirin
15 mg/kg
(daily). First 4 weeks, all patients were determined to be free of HCV RNA
(free virus in serum).
The treatment was continued for 12 weeks.
[0085] Results: All patients achieved primary endpoint of total viral
suppression after 12 weeks
treatment and 12 weeks follow up.
Table 2: Antiviral activity of Nanogen's interferon lambda 1 (PEG-IFNkl)
Lot 1L290010111 Lot 1L290020311 Lot 1L290030411
ED50 (ng/ml) 1.23 2.10 1.99
Mean (ng/ml) 1.97
SD 0.13
RSD 6.78
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Table 3: Antiviral activity of Nanogen's PEG-IFNal
Lot PIL Lot PIL Lot PIL Lot PIL Lot PIL
290010111 290020211 290030311 290040411 290050511
ED50(ng/m1) 26.94 28.70 26.27 26.00 27.31
Mean (ng/ml)
27.04
SD
1.06
RSD
3.92
[0086] Therapy for HCV Resistant Patients:
[0087] More than 50 patients who have been previously treated using standard
therapies, such as a
combination of PEGASYS (peginterferon alfa-2a) with ribavirin, were found to
be ineffective.
The non-responder patients, defined by the guidelines for HCV treatment from
AASLD as patients
who do not show a clearance of HCV RNA from serum from 24 weeks of therapy,
and the null-
responder patients, defined as those who show a failure to decrease HCV RNA at
the 12th week by
> 2 log, were enrolled in the treatment regiment using the PEG-IFNkl of the
present application.
[0088] After 4-12 weeks or 4-24 weeks of treatment with PEG-IFNkl using the
disclosed
treatment protocol, substantially all patients were tested and determined to
be HCV RNA negative;
or all patients had a sustained virological response (SVR), defined as having
no detectable virus 24
weeks after the final treatment dose.
[0089] In other studies using HCV resistant patients, the presently disclosed
treatment protocol
was found to be effective for greater than 80%, 85%, 90 % or greater than 95%
of the HCV
resistant patient population. Accordingly, the treatment methods using the PEG-
IFNkl
demonstrate efficacy in HCV including cases of resistance to current standard
therapy of
peginterferon alfa-2a with ribavirin. No significant side effects that are
typically associated with
the combination therapy of PEGASYS with ribavirin were observed.
[0090] Example 7: Identification of IFN2,1 and PEG-IFNal
[0091] Western blotting method was used to identify IFNkl and PEG-IFNkl using
anti-IFNkl
antibody. The protein solution after being analyzed on an SDS-PAGE gel was
transferred to a
nitrocellulose membrane and probed with the anti-IFNkl antibody. Antibody was
detected with
peroxidase coupled protein A and TMB substrate (see Figure 11).
[0092] Example 8: Molecular weight of PEG-IFNkl
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[0093] The MALDI-TOF assay was applied to determine the molecular weight of
PEG-IFNkl.
The result is provided in Figure 12. In the present example, the Nanogen's PEG-
IFNkl has a
molecular weight of approximately 62 kDa.
[0094] Example 9: Purity of PEG-IFNkl
[0095] Five lots (P1L290010111, P1L290020211, P1L290030311, P1L290040411,
PIL290050511)
were used to determine the purity of PEG-IFNkl by SDS-PAGE electrophoresis.
The
electrophoresis gel was stained with coomassie blue, destained and then
analyzed using Phoretix
software (TotalLab, England). All tested lots showed purity higher than 95%.
[0096] The following tests employ the PEG-IFNkl as prepared above:
[0097] Example 10: Toxicity of PEG-IFNal
[0098] Acute toxicity of PEG-IFNkl: The acute toxicity of PEG-IFNkl was
assessed in Swiss
mice and rats. Healthy ICR mice and Sprague-Dawley rats, at 5 week old, were
chosen for the
study. The animals were inspected for two weeks. PEG-IFNkl was administered at
three
different dosages (high dose 3 mg/kg, medium dose 0.3mg/kg, low dose 0.03
mg/kg and the
vehicle treatment (phosphate buffer saline, pH 7.2)) by subcutaneous or
intraperitoneal injection.
Animals were observed for clinical signs, body weight changes, and mortality
14 days after
treatment. At the end of the study, all animals were sacrificed, and their
tissues and organs were
examined for abnormalities. The results are summarized in table 4.
Table 4
Experiment Results
Number Administration Administration Dose Volume Body Clinical
Autopsy
Animal
of animals route period (mg/kg) (m1) weight signs
result
3 No
Swiss 5 males, Intraperitoneal
significant I
Once a week 0.3 0.5 None No
abs.
0.03
mice 5 females injection difference
(*)
3 No
males, Intraperitoneal significant
1
Rats Once a week 0.3 1.0 None No
abs.
5 females injection difference
0.03
(*)
3 No
Swiss 5 males, Subcutaneous
significant I
Once a week 0.3 0.5 None No
abs.
0.03
mice 5 females injection difference
(*)
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Experiment Results
A Number Administration Administration Dose Volume Body Clinical
Autopsy
nimal
of animals route period (mg/kg) (m1) weight signs
result
3 No
males, Subcutaneous significant
Rats Once a week 0.3 1.0 None No
abs.1
5 females injection difference
0.03
(*)
(*): ANOVA, single factor, compare to the vehicle treatment, (p>0.05)
1
means no abnormalities
[0099] All animals survived for the test period even at the highest dosage.
The body weight did
not significantly change in the treated animals compared to the control. There
were no clinical
signs or organ abnormalities observed in either group of the tested animals.
[00100] Based on these results, the lethal dose (LD50) of Nanogen's PEG-
IFNkl in mouse
and rat was greater than 3 mg/kg. Subacute toxicity of PEG-IFNkl: Animals (5
weeks old rats)
were administered PEG-IFNkl at three different dosages (high dose 3 mg/kg,
medium dose 0.3
mg/kg, low dose 0.03 mg/kg) by subcutaneous or intraperitoneal injection once
a day for 4 weeks.
[00101] The rats were examined throughout the study for any clinical and
behavioral
adverse effects caused by Nanogen's PEG-IFNkl administration. After the test
period, the
survived rats were sacrificed for autopsy and biochemical analyses. Blood
samples were also
collected from abdominal artery to conduct hematologic tests.
[00102] The test method and results are summarized in table 5.
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Table 5
Administration Dose Volume
Animals Administration route
period (mg/kg) (ml)
Rats 3
Once a day for 4
males, 5 Subcutaneous injection 0.3 1.0
weeks
females 0.03
Rats 3
Intraperitoneal Once a day for 4
5 males, 5 0.3 1.0
injection weeks
females 0.03
Examination/Analysis Male rats Female rats
Clinical signs none none
Body weight Normal Normal
Food consumption Normal Normal
Water consumption Normal Normal
Urinalysis Normal Normal
Hematology Normal Normal
Urinalysis Normal Normal
Hematology Normal Normal
Serum biochemistry Normal Normal
Absolute and relative organ weight Normal Normal
Autopsy result Normal Normal
Histopathological examination Normal Normal
[00103] There was no death in any groups during the entire study and no
clinical signs were
detected from the tested rats. The tested rats were normal in other
examination categories and
analyses even in the high dosage group. Therefore, the study shows that
Nanogen's PEG-IFNkl
does not have toxic effects in rats when it is administered repeatedly at the
dosage of 3 mg/kg.
[00104] Immunological toxicity of PEG-IFNkl: A study was carried out to
investigate
immunologic potential of Nanogen's PEG-IFNkl in guinea pigs. Healthy male
Hartley guinea
pigs with body weight of 300-500 gram were injected with PEG-IFNkl twice a
week for 3 weeks
either at a high dose (3 mg/kg) or low dose (0.03 mg/kg) and ovalbumin as
control. Fourteen days
after the final sensitization, the anaphylaxis test was performed by
intravenously injecting a high
dose of PEG-IFNkl. The study included PEG-IFNkl incorporated in Freund's
complete adjuvant
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PCT/US2012/027317
(FCA). The sensitized guinea pigs were observed for active systemic
anaphylaxis reactions after
injection of a high dose PEG-IFNkl. A list of indications was used as a sign
of anaphylactic
reaction and their occurrence was monitored in each tested animal.
1001051 Table 6 shows the study method and results.
Table 6
Administration Anaphylaxis No. of
Test groups Dose
route challenge animals
Negative control Intravenous injection
- Subcutaneous 5 males
(PBS) of PBS
Positive control Intravenous injection
2 mg/kg Subcutaneous 5 males
(ovalbumin) of ovalbumin
Intravenous injection
Low dose 0.03 mg/kg Subcutaneous of 3 mg/kg PEG- 5 males
IFNX,1
Intravenous injection
High dose 3 mg/kg Subcutaneous of 3 mg/kg PEG- 5 males
IFNX,1
Intravenous injection
3 mg/kg+
High dose + FCA Subcutaneous of 3 mg/kg PEG- 5 males
FCA
IFNX,1
Anaphylaxis Physical observation (1)
Animal
challenge
1 2 3 4 5 6 7 8
1 + -
2 - - + - - -
PBS 3 - - + - - -
4 - - - - - -
_ _ + _ _ _
1 - + + - + + + +
2 - + + - - - - -
OVALBUMIN
3 _ + + + + + + +
(2mg/kg)
4 - + + + + + + +
5 _ + + + _ _ _ _
CA 02846092 2014-02-20
WO 2013/028233 PCT/US2012/027317
1 - - - - - -
Peg-IFNkl 2 - - + - - -
(0.03mg/kg) 3 - - - - - -
4 - - + - - -
_ - + _ _ _
1 - - + - - -
Peg-IFNkl 2 - - + - - -
3 _ - + _ _ _
(3mg/kg)
4 - - - - - -
5 _ _ + _ _ _
1 - + + + - - - -
Peg-IFNkl 2 - +-- +-- - + - - -
3 _ + + + _ _ _
(3mg/kg) + FCA
4 - + + - + - - -
5 - + + _ _ _
(1)1. Licking nose, rubbing nose; 2. Ruffling fur;
3. Labored breathing; 4. Sneezing, coughing;
5. Evacuation of feces, micturition; 6. Convulsion; 7. Prostration;
8. Death. -, negative; +, positive
[00106] In the active systemic anaphylactic test, the guinea pigs slightly
sensitized with high
dose of PEG-IFNkl (3 mg/kg) incorporated in Freund's complete adjuvant (FCA)
showed some
indications of anaphylactic reaction. On the other hand, no guinea pigs
sensitized with low dose
and high dose of PEG-IFNkl (0.03 and 3 mg/kg) alone showed any anaphylactic
reaction. No
guinea pigs were dead after administration with Nanogen's PEG-IFNkl and
negative treatment
(PBS), but 3 pigs were dead after administration with ovalbumin (positive
treatment). Therefore,
it can be concluded that Nanogen's PEG-IFNkl does not induce systemic allergic
reaction when
administered alone in its clinical use.
[00107] It will be apparent to those skilled in the art that various
modifications and variations
can be made to the compounds, compositions, and methods of the present
invention without
departing from the spirit or scope of the invention. Thus, it is intended that
the present invention
cover the modifications and variations of this invention provided they come
within the scope of
the appended claims and their equivalents.
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