Title: Method for treating
disorders and diseases treatable with human fibroblast interferon
United States Patent: 7,357,925
Issued: April 15, 2008
Inventors: El-Tayar; Nabil
(Milton, MA), Roberts; Michael J. (Madison, AL), Harris; Milton (Hunstville,
AL), Sawlivich; Wayne (Wilmington, MA)
Seronosa (Coinsins, Vaud, CH)
Filed: August 28, 2003
PEG-IFN-.beta. conjugates, where a PEG
moiety is covalently bound to Cys.sup.17 of human IFN-.beta., are produced
by a process of site specific PEGylation with a thiol reactive PEGylating
agent. A pharmaceutical composition and a method for treating infections,
tumors and autoimmune and inflammatory diseases are also provided. The
invention further relates to a method for the stepwise attachment of PEG
moieties in series to a polypeptide, and more particularly to IFN-.beta..
Description of the
FIELD OF THE INVENTION
The invention relates to polyol-IFN-.beta. conjugates wherein a polyol
unit is covalently bound to Cys.sup.17. Further objects of the present
invention are the process for their site-specific production as well as
their use in the therapy, prognosis or diagnosis of bacterial infections,
viral infections, autoimmune diseases and inflammatory diseases. The
present invention further relates to a method for the stepwise attachment
of two or more PEG moieties to a polypeptide.
BACKGROUND OF THE INVENTION
Human fibroblast interferon (IFN-.beta.) has antiviral activity and can
also stimulate natural killer cells against neoplastic cells. It is a
polypeptide of about 20,000 Da induced by viruses and double-stranded RNAs.
From the nucleotide sequence of the gene for fibroblast interferon, cloned
by recombinant DNA technology, Derynk et al. (Nature, 285:542-547, 1980)
deduced the complete amino acid sequence of the protein. It is 166 amino
Shepard et al. (Nature, 294:563-565, 1981) described a mutation at base
842 (Cys-Tyr at position 141) that abolished its anti-viral activity, and
a variant clone with a deletion of nucleotides 1119-1121.
Mark et al. (Proc. Natl. Acad. Sci. U.S.A., 81(18):5662-5666, 1984)
inserted an artificial mutation by replacing base 469 (T) with (A) causing
an amino acid switch from Cys--Ser at position 17. The resulting IFN-.beta.
was reported to be as active as the `native` IFN-.beta. and stable during
long-term storage (-70.degree. C.).
Covalent attachment of the hydrophilic polymer polyethylene glycol, (PEG),
also known as polyethylene oxide, (PEO), to molecules has important
applications in biotechnology and medicine. In its most common form, PEG
is a linear polymer having hydroxyl groups at each terminus:
This formula can be represented in brief as HO--PEG--OH, where it is meant
that --PEG-- represents the polymer backbone without the terminal groups:
PEG is commonly used as methoxy-PEG--OH, (m-PEG), in which one terminus is
the relatively inert methoxy group, while the other terminus is a hydroxyl
group that is subject to chemical modification.
Branched PEGs are also in common use. The branched PEGs can be represented
as R(--PEG--OH).sub.m in which R represents a central core moiety such as
pentaerythritol or glycerol, and m represents the number of branching
arms. The number of branching arms (m) can range from three to a hundred
or more. The hydroxyl groups are subject to chemical modification.
Another branched form, such as that described in PCT patent application WO
96/21469, has a single terminus that is subject to chemical modification.
This type of PEG can be represented as (CH.sub.3O--PEG--).sub.pR--X,
whereby p equals 2 or 3, R represents a central core such as lysine or
glycerol, and X represents a functional group such as carboxyl that is
subject to chemical activation. Yet another branched form, the "pendant
PEG", has reactive groups, such as carboxyl, along the PEG backbone rather
than at the end of PEG chains.
In addition to these forms of PEG, the polymer can also be prepared with
weak or degradable linkages in the backbone. For example, Harris has shown
in U.S. patent application Ser. No. 06/026,716 that PEG can be prepared
with ester linkages in the polymer backbone that are subject to
hydrolysis. This hydrolysis results in cleavage of the polymer into
fragments of lower molecular weight, according to the reaction scheme:
According to the present invention, the term polyethylene glycol or PEG is
meant to comprise all the above described derivatives.
The copolymers of ethylene oxide and propylene oxide are closely related
to PEG in their chemistry, and they can be used instead of PEG in many of
its applications. They have the following general formula:
HO--CH.sub.2CHRO(CH.sub.2CHRO).sub.nCH.sub.2CHR--OH wherein R is H or
PEG is a useful polymer having the property of high water solubility as
well as high solubility in many organic solvents. PEG is also non-toxic
and non-immunogenic. When PEG is chemically attached (PEGylation) to a
water insoluble compound, the resulting conjugate generally is water
soluble, as well as soluble in many organic solvents.
PEG-protein conjugates are currently being used in protein replacement
therapies and for other therapeutic uses. For example, PEGylated adenosine
deaminase (ADAGEN.RTM.) is being used to treat severe combined
immunodeficiency disease (SCIDS), PEGylated L-asparaginase (ONCAPSPAR.RTM.)
is being used to treat acute lymphoblastic leukemia (ALL), and PEGylated
interferon-.alpha. (INTRON(R) A) is in Phase III trials for treating
For a general review of PEG-protein conjugates with clinical efficacy see
N. L. Burnham, Am. J. Hosp. Pharm., 15:210-218, 1994.
A variety of methods have been developed to PEGylate proteins. Attaching
PEG to reactive groups found on the protein is typically done utilizing
electrophilically activated PEG derivatives. Attaching PEG to the .alpha.-
and .epsilon.-amino groups found on lysine residues and the N-terminus
results in a conjugate consisting of a mixture of products.
Generally, such conjugates consist of a population of the several PEG
molecules attached per protein molecule ("PEGmers") ranging from zero to
the number of amino groups in the protein. For a protein molecule that has
been singly modified, the PEG unit may be attached at a number of
different amine sites.
This type of non-specific PEGylation has resulted in a number of
conjugates that become almost inactive. Reduction of activity is typically
caused by shielding the protein's active binding domain as is the case
with many cytokines and antibodies. For example, Katre et al. in U.S. Pat.
No. 4,766,106 and U.S. Pat. No. 4,917,888 describe the PEGylation of IFN-.beta.
and IL-2 with a large excess of methoxy-polyethylene glycolyl N-succinimidyl
glutarate and methoxy-polyethylene glycolyl N-succinimidyl succinate. Both
proteins were produced in microbial host cells, which allowed the
site-specific mutation of the free cysteine to a serine. The mutation was
necessary in microbial expression of IFN-.beta. to facilitate protein
folding. In particular, the IFN-.beta. used in these experiments is the
commercial product Betaseron.RTM., in which Cys.sup.17 residue is replaced
with a serine. Additionally, the absence of glycosylation reduced its
solubility in aqueous solution. Non-specific PEGylation resulted in
increased solubility, but a major problem was the reduced level of
activity and yield.
European Patent Application EP 593 868, entitled PEG-Interferon
Conjugates, describes the preparation of PEG-IFN-.alpha. conjugates.
However, the PEGylation reaction is not site-specific, and therefore a
mixture of positional isomers of PEG-IFN-.alpha. conjugates are obtained
(see also Monkarsh et al., ACS Symp. Ser., 680:207-216, 1997).
Kinstler et al. in European Patent Application EP 675 201 demonstrated the
selective modification of the N-terminal residue of megakaryocyte growth
and development factor (MGDF) with mPEG-propionaldehyde. This allowed for
reproducible PEGylation and pharmacokinetics from lot to lot. Gilbert et
al. in U.S. Pat. No. 5,711,944 demonstrated that PEGylation of IFN-.alpha.
with an optimal level of activity could be produced. In this instance a
laborious purification step was needed to obtain the optimal conjugate.
The majority of cytokines, as well as other proteins, do not possess a
specific PEG attachment site and, apart from the examples mentioned above,
it is very likely that some of the isomers produced through the PEGylation
reaction be partially or totally inactive, thus causing a loss of activity
of the final mixture.
Site-specific mono-PEGylation is thus a desirable goal in the preparation
of such protein conjugates.
Woghiren et al. in Bioconjugate Chem., 4(5):314-318, 1993, synthesized a
thiol-selective PEG derivative for such a site-specific PEGylation. A
stable. thiol-protected PEG derivative in the form of an parapyridyl
disulfide reactive group was shown to specifically conjugate to the free
cysteine in the protein, papain. The newly formed disulfide bond between
papain and PEG could be cleaved under mild reducing conditions to
regenerate the native protein.
Citation of any document herein is not intended as an admission that such
document is pertinent prior art, or considered material to the
patentability of any claim of the present application. Any statement as to
content or a date of any document is based on the information available to
applicants at the time of filing and does not constitute an admission as
to the correctness of such a statement.
SUMMARY OF THE INVENTION
In the present invention, polyol-IFN-.beta. conjugates, and particularly
PEG-IFN-.beta. conjugates, are provided wherein a polyol unit is
covalently bound to Cys.sup.17. The specific conjugation is obtained by
allowing a thiol-reactive polyol agent to react with the Cys.sup.17
residue in IFN-.beta.. Such conjugates are expected to show increased
effectiveness in vivo. The aim is to obtain increased solubility at
neutral pH, increased stability (decreased aggregation), decreased
immunogenicity, and no loss of activity with respect to `native` IFN-.beta..
The results of such conjugation would decrease the number of doses for an
intended effect, simplify and stabilize the formulation of a
pharmaceutical composition, and possibly increase the long-term efficacy.
The present invention further provides a method for the stepwise
attachment of PEG moieties in series to a polypeptide.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based on the discovery that the attachment of a
polyol moiety, more specifically a PEG moiety, to the Cys.sup.17 residue
of human IFN-.beta. unexpectedly increased (or at least retained and did
not result in a decrease) the IFN-.beta. biological activity from that of
native human interferon-.beta.. Thus, not only does IFN-.beta. with a
polyol moiety attached to the Cys.sup.17 residue exhibit the same or
increased IFN-.beta. biological activity but this polyol-IFN-.beta.
conjugate also provides the desirable properties conferred by the polyol
moiety, such as increased solubility.
"IFN-.beta.", as used herein, means human fibroblast interferon, as
obtained by isolation from biological fluids or as obtained by DNA
recombinant techniques from prokaryotic or eukaryotic host cells as well
as its salts, functional derivatives, precursors and active fractions,
provided that they contain the cysteine residue appearing at position 17
in the naturally occurring form.
The polyol moiety in the polyol-IFN-.beta. conjugate according to the
present invention can be any water-soluble mono- or bifunctional
poly(alkylene oxide) having a linear or branched chain. Typically, the
polyol is a poly(alkylene glycol) such as poly(ethylene glycol) (PEG).
However, those of skill in the art will recognize that other polyols, such
as, for example poly (propylene glycol) and copolymers of polyethylene
glycol and polypropylene glycol, can be suitably used.
As used herein, the term "PEG moiety" is intended to include, but is not
limited to, linear and branched PEG, methoxy PEG, hydrolytically or
enzymatically degradable PEG, pendant PEG, dendrimer PEG, copolymers of
PEG and one or more polyols, and copolymers of PEG and PLGA (poly(lactic/glycolic
The definition "salts" as used herein refers both to salts of the
carboxyl-groups and to the salts of the amino functions of the compound
obtainable through known methods. The salts of the carboxyl-groups include
inorganic salts as, for example, sodium, potassium, calcium salts and
salts with organic bases as those formed with an amine as triethanolamine,
arginine or lysine. The salts of the amino groups included for example,
salts with inorganic acids as hydrochloric acid and with organic acids as
The definition "functional derivatives" as herein used refers to
derivatives which can be prepared from the functional groups present on
the lateral chains of the amino acid moieties or on the terminal N- or
C-groups according to known methods and are included in the present
invention when they are pharmaceutically acceptable, i.e., when they do
not destroy the protein activity or do not impart toxicity to the
pharmaceutical compositions containing them. Such derivatives include for
example esters or aliphatic amides of the carboxyl-groups and N-acyl
derivatives of free amino groups or O-acyl derivatives of free
hydroxyl-groups and are formed with acyl-groups as for example alcanoyl-
The "precursors" are compounds which are converted into IFN-.beta. in the
human or animal body.
As "active fractions" of the protein, the present invention refers to any
fragment or precursor of the polypeptidic chain of the compound itself,
alone or in combination with related molecules or residues bound to it,
for example, residues of sugars or phosphates, or aggregates of the
polypeptide molecule when such fragments or precursors show the same
activity of IFN-.beta. as medicament.
The conjugates of the present invention can be prepared by any of the
methods known in the art. According to an embodiment of the invention, IFN-.beta.
is reacted with the PEGylating agent in a suitable solvent and the desired
conjugate is isolated and purified, for example, by applying one or more
"Chromatographic method" means any technique that is used to separate the
components of a mixture by their application on a support (stationary
phase) through which a solvent (mobile phase) flows. The separation
principles of the chromatography are based on the different physical
nature of stationary and mobile phase.
Some particular types of chromatographic methods, which are well-known in
the literature, include: liquid, high pressure liquid, ion exchange,
absorption, affinity, partition, hydrophobic, reversed phase, gel
filtration, ultrafiltration or thin-layer chromatography.
The "thiol-reactive PEGylating agent", as used in the present application,
means any PEG derivative which is capable of reacting with the thiol group
of the cysteine residue. It can be, for example, PEG containing a
functional group such as orthopyridyl disulfide, vinylsulfone, maleimide,
iodoacetimide, and others. According to a preferred embodiment of the
present invention, the thiol-reactive PEGylating agent is the orthopyridyl
disulfide (OPSS) derivative of PEG.
The PEGylating agent is used in its mono-methoxylated form where only one
terminus is available for conjugation, or in a bifunctional form where
both termini are available for conjugation, such as for example in forming
a conjugate with two IFN-.beta. covalently attached to a single PEG
moiety. It has preferably a molecular weight between 500 and 100,000.
A typical reaction scheme for the preparation of the conjugates of the
invention is presented below -- see Original Patent.
The second line of the above scheme reports a method for cleaving the
PEG-protein linkage. The mPEG-OPSS derivative is highly selective for free
sulphydryl groups and reacts rapidly under acidic pH conditions where the
IFN-.beta. is stable. The high selectivity can be demonstrated from the
reduction of the conjugate to the native form of IFN-.beta. and PEG.
The disulfide bond that is produced between the protein and PEG moieties
has been shown to be stable in the circulation, but it can be reduced upon
entering the cell environment. Therefore it is expected that this
conjugate, which does not enter the cell, will be stable in the
circulation until it is cleared.
It should be noted that the above reaction is site-specific because the
other two Cys residues appearing at positions 31 and 141 in the naturally
occurring form of human IFN-.beta. do not react with the thiol-reactive
PEGylating agent since they form a disulfide bridge.
The present invention is also directed to a method for the stepwise
attachment of two or more PEG moieties to a polypeptide. This method is
based upon the recognition that a low molecular weight activated PEG
reacts more completely with a sterically hindered reaction site on a
protein than does a high molecular weight activated PEG. PEG-modification
of expensive therapeutic proteins must be cost effective in order for the
production of the PEG conjugate to be practical. In addition, in order to
reduce glomerular filtration and optimize the pharmacological properties
of the PEG-protein conjugate, the conjugate should have an effective size
equivalent to that of a protein with a molecular weight of 70 kDa. This
means that for a site specific modification where one PEG will be
attached, a PEG derivative having a molecular weight of greater than 20
kDa is preferably attached. If the site of modification is sterically
crowded, the reactive group on the large PEG moiety may have difficulty
reaching the modification site and thus will lead to low yields. A
preferred method of PEGylating a polypeptide according to the present
invention increases the yield of site-specific PEGylation by first
attaching a small hetero or homobifunctional PEG moiety that, due to its
relatively smaller size, can react with sterically crowded sites.
Subsequent attachment of a large molecular weight PEG derivative to the
small PEG results in high yield of the desired PEGylated protein.
The method for stepwise attachment of two or more PEG moieties in series
to a polypeptide according to the present invention includes attaching a
low molecular weight heterbifunctional or homobifunctional PEG moiety
first to the polypeptide and then attaching a monofunctional or
bifunctional PEG moiety to the free terminus of the low molecular weight
PEG moiety that is attached to the polypeptide. Following the stepwise
attachment of two or more PEG moieties in series to a polypeptide, which
polypeptide is preferably IFN-.beta. and where Cys.sup.17, located in a
sterically crowded site, is the preferred site of PEG attachment, the
PEG-polypeptide conjugate can be purified using one or more of the
purification techniques such as ion exchange chromatography, size
exclusion chromatography, hydrophobic interaction chromatography, affinity
chromatography, and reverse phase chromatography.
The low molecular weight PEG moiety has the formula:
W--CH.sub.2CH.sub.2O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2--X, where W
and X are groups that independently react with an amine, sulfhydryl,
carboxyl or hydroxyl functional group to attach the low molecular weight
PEG moiety to the polypeptide. W and X are preferably independently
selected from orthopyridyl disulfide, maleimides, vinyl sulfones,
iodoacetamides, amines, thiols, carboxyls, active esters, benzotriazole
carbonates, p-nitrophenol carbonates, isocyanates, and biotin. The low
molecular weight PEG moiety preferably has a molecular weight in the range
of about 100 to 5,000 daltons.
The monofunctional or bifunctional PEG moiety for attachment to the free
terminus of a low molecular weight PEG that is attached to the polypeptide
has preferably a molecular weight in the range of about 100 daltons to 200
kDa and is preferably a methoxy PEG, branched PEG, hydrolytically or
enzymatically degradable PEG, pendant PEG, or dendrimer PEG. The
monofunctional or bifunctional PEG furthermore has the formula:
Y--CH.sub.2CH.sub.2O(CH.sub.2CH.sub.2O).sub.MCH.sub.2CH.sub.2--Z where Y
is reactive to a terminal group on the free terminus of the low molecular
weight PEG moiety that is attached to the polypeptide and Z is --OCH.sub.3
or a group reactive with to form a bifunctional conjugate.
The PEG-polypeptide conjugate produced by the above method for stepwise
attachment of two or more PEG moieties can be used to produce a medicament
or pharmaceutical composition for treating diseases or disorders for which
the polypeptides is effective as an active ingredient.
Another object of the present invention is to provide the conjugates in
substantially purified form in order for them to be suitable for use in
pharmaceutical compositions, as active ingredient for the treatment,
diagnosis or prognosis of bacterial and viral infections as well as
autoimmune, inflammatory diseases and tumors. Such pharmaceutical
compositions represent a further object of the present invention.
Non-limiting examples of the above-mentioned diseases include: septic
shock, AIDS, rheumatoid arthritis, lupus erythematosus and multiple
Further embodiments and advantages of the invention will be evident in the
An embodiment of the invention is the administration of a
pharmacologically active amount of the conjugates of the invention to
subjects at risk of developing one of the diseases reported above or to
subjects already showing such pathologies.
Any route of administration compatible with the active. principle can be
used. Parenteral administration, such as subcutaneous, intramuscular or
intravenous injection is preferred. The dose of the active ingredient to
be administered depends on the basis of the medical prescriptions
according to age, weight and the individual response of the patient.
The dosage can be between 10 .mu.g and 1 mg daily for an average body
weight of 75 kg, and the preferable daily dose is between 20 .mu.g and 200
The pharmaceutical composition for parenteral administration can be
prepared in an injectable form comprising the active principle and a
suitable vehicle. Vehicles for the parenteral administration are well
known in the art and include, for example, water, saline solution, Ringer
solution and/or dextrose. The vehicle can contain small amounts of
excipients in order to maintain the stability and isotonicity of the
pharmaceutical preparation. The preparation of the solutions can be
carried out according to the ordinary modalities.
Claim 1 of 19 Claims
1. A method for treating viral
infections, lupus erythematosus, or rheumatoid arthritis or for
stimulating natural killer cells against neoplastic cells, comprising
administering an effective amount of a polyol-human fibroblast interferon
(interferon-.beta.) conjugate having a polyol moiety covalently bound to
Cys.sup.17 of human fibroblast interferon to a subject in need thereof.
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