U.S. patent application number 11/607286 was filed with the patent office on 2007-08-02 for combination therapy for treating protein deficiency disorders.
Invention is credited to Jian-Qiang Fan.
Application Number | 20070178081 11/607286 |
Document ID | / |
Family ID | 32850826 |
Filed Date | 2007-08-02 |
United States Patent
Application |
20070178081 |
Kind Code |
A1 |
Fan; Jian-Qiang |
August 2, 2007 |
Combination therapy for treating protein deficiency disorders
Abstract
This application provides methods of improving protein
replacement therapy by combining protein replacement therapy with
active site-specific chaperones (ASSC) to increase the stability
and efficiency of the protein being administered. The application
further provides stable compositions comprising the purified
protein and an ASSC, and methods of treatment by administering the
compositions.
Inventors: |
Fan; Jian-Qiang; (Demarest,
NJ) |
Correspondence
Address: |
BAKER BOTTS L.L.P.
30 ROCKEFELLER PLAZA
44TH FLOOR
NEW YORK
NY
10112-4498
US
|
Family ID: |
32850826 |
Appl. No.: |
11/607286 |
Filed: |
December 1, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11317404 |
Dec 23, 2005 |
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11607286 |
Dec 1, 2006 |
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10771236 |
Feb 2, 2004 |
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11317404 |
Dec 23, 2005 |
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60444136 |
Jan 31, 2003 |
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Current U.S.
Class: |
424/94.61 ;
514/304; 514/327 |
Current CPC
Class: |
A61P 9/14 20180101; A61P
9/00 20180101; A61P 3/02 20180101; A61P 3/00 20180101; A61P 43/00
20180101; A61K 38/47 20130101; A61K 31/46 20130101; A61K 9/0019
20130101; A61K 31/445 20130101; A61P 3/08 20180101 |
Class at
Publication: |
424/094.61 ;
514/304; 514/327 |
International
Class: |
A61K 38/47 20060101
A61K038/47; A61K 31/445 20060101 A61K031/445; A61K 31/46 20060101
A61K031/46 |
Claims
1. A method of extending the half-life and prolonging the activity
in vivo of a purified protein in an individual who has been
administered the protein in a pharmaceutically acceptable carrier,
which method comprises contacting the protein with an effective
amount of an active site-specific chaperone in a pharmaceutically
acceptable carrier.
2. The method of claim 1, wherein the protein is co-administered
with the active site-specific chaperone.
3. The method of claim 1, wherein the protein is an enzyme and the
active site-specific chaperone is a reversible competitive
inhibitor of the enzyme.
4. The method of claim 3, wherein the enzyme is associated with a
lysosomal storage disorder.
5. The method of claim 4, wherein the enzyme is
.alpha.-galactosidase A.
6. The method of claim 4, wherein the enzyme is
.beta.-glucocerebrosidase.
7. The method of claim 5, wherein the reversible competitive
inhibitor is a compound of the following formula: ##STR1## wherein
R.sub.0 represents H or a C.sub.1-C.sub.12 alkyl chain; R.sub.1 and
R.sub.1' independently represent H, OH, a 1-4 carbon alkyl, alkoxy
or hydroxyalkyl group; R.sub.2 and R.sub.2' independently represent
H, OH or a C.sub.1-C.sub.12 alkyl group R.sub.4 and R.sub.4'
independently represent H or OH; and R.sub.7 represents H or
OH.
8. The method of claim 5, wherein the reversible competitive
inhibitor is a compound selected from the group consisting of
1-deoxygalactonojirimycin, .alpha.-allo-homonojirimycin,
.alpha.-galacto-homonojirimycin,
.alpha.-1-C-butyl-deoxynojirimycin, calystegine A.sub.3,
calystegine B.sub.2, N-methyl-calystegine A.sub.3, and
N-methyl-calystegine B.sub.2.
9. The method of claim 8, wherein the reversible competitive
inhibitor is 1-deoxygalactonojirimycin.
10. The method of claim 6 wherein the reversible competitive
inhibitor is a compound of the following formula: ##STR2## wherein
R.sub.0 represents H or a C.sub.1-C.sub.12 alkyl chain; R.sub.0'
represents H, a straight chain or branched saturated carbon chain
containing 1-12 carbon atoms, optionally substituted with a phenyl,
hydroxyl or cyclohexyl group; R.sub.1 and R.sub.1' independently
represent H, OH, a 1-4 carbon alkyl, alkoxy or hydroxyalkyl group;
and R.sub.2 and R.sub.2' independently represent H, OH or a
C.sub.1-C.sub.12 alkyl group.
11. The method of claim 6, wherein the reversible competitive
inhibitor is a compound selected from the group consisting of
isofagomine, N-dodecyl-isofagomine, N-nonyl-isofagomine,
N-dodecyl-deoxynojirimycin, calystegine A.sub.3, calystegine
B.sub.2, calystegine B.sub.3 and calystegine C.sub.1.
12. The method of claim 11, wherein the reversible competitive
inhibitor is isofagomine.
13. The method of claim 11, wherein the reversible competitive
inhibitor is N-dodecyl-isofagomine.
14. A method for improving treatment of an individual who has a
disorder characterized by a protein deficiency and who is being
administered a purified replacement protein, comprising
co-administering to the individual an effective amount of an active
site-specific chaperone for the replacement protein.
15. The method of claim 14, wherein the protein is an enzyme.
16. The method of claim 15, wherein the disorder is Fabry disease
and the replacement protein is .alpha.-galactosidase A, and the
active site-specific chaperone is selected from the group
consisting of 1-deoxygalactonojirimycin,
.alpha.-allo-homonojirimycin, .alpha.-galacto-homonojirimycin,
.alpha.-1-C-butyl-deoxynojirimycin, calystegine A.sub.3,
calystegine B.sub.2, N-methyl-calystegine A.sub.3, and
N-methyl-calystegine B.sub.2.
17. The method of claim 16 wherein the active site-specific
chaperone is 1-deoxygalactonojirimycin.
18. The method of claim 15, wherein the disorder is Gaucher disease
and the replacement protein is .beta.-glucocerebrosidase and the
active site-specific chaperone is selected from the group
consisting of isofagomine, N-dodecyl-isofagomine,
N-nonyl-isofagomine, N-dodecyl-deoxynojirimycin, calystegine
A.sub.3, calystegine B.sub.2, calystegine B.sub.3 and calystegine
C.sub.1.
19. The method of claim 10 wherein the active site-specific
chaperone is isofagomine.
20. The method of claim 15, wherein the disorder is Pompe disease
and the replacement protein is .alpha.-glucosidase, and the active
site-specific chaperone is 1-deoxynojirimycin.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 11/317,404, filed on Dec. 23, 2005 (pending), which is a
continuation of U.S. application Ser. No. 10/771,236, filed on Feb.
2, 2004 (abandoned), which claims priority benefit from U.S.
Provisional Application Ser. No. 60/444,136, filed Jan. 31, 2003
(abandoned), each of which is incorporated by reference herein in
its entirety.
FIELD OF THE INVENTION
[0002] This application provides methods of improving protein
replacement therapy by combining protein replacement therapy with
active site-specific chaperones (ASSC) to increase the stability
and efficiency of the protein being administered. The application
further provides compositions comprising the purified protein and
an ASSC.
BACKGROUND
Protein Deficiency
[0003] Proteins are synthesized intracellularly according to the
genomic nucleotide sequence of a particular gene through
transcription, translation, and other processes. Protein deficiency
can be caused by a mutation in the coding gene, which results in
(i) non-synthesis of the protein; (ii) synthesis of the protein
which lacks biological activity; or (iii) synthesis of the protein
containing normal or partial biological activity, but which cannot
be appropriately processed to reach the native compartment of the
protein. Protein deficiency disorders that result from genetic
mutations are also referred to as genetic disorders.
[0004] In addition to protein deficiencies resulting from genetic
mutations, some protein deficiencies arise due to a disease, or as
a side effect of a treatment for a disease (e.g., chemotherapy) or
as a result of nutritional insufficiency.
[0005] Current therapies. There are numerous disorders resulting
from protein deficiencies, some of which result from mutated,
misfolded proteins (conformational disorders-see infra). One
current therapy for treating protein deficiencies is protein
replacement therapy, which typically involves intravenous,
subcutaneous or intramuscular infusion of a purified form of the
corresponding wild-type protein, or implantation of the protein in
a bio-erodable solid form for extended-release. One of the main
complications with protein replacement therapy is attainment and
maintenance of therapeutically effective amounts of protein due to
rapid degradation of the infused protein. The current approach to
overcome this problem is to perform numerous costly high dose
infusions.
[0006] Protein replacement therapy has several additional caveats,
such as difficulties with large-scale generation, purification and
storage of properly folded protein, obtaining glycosylated native
protein, generation of an anti-protein immune response, and
inability of protein to cross the blood-brain barrier in diseases
having significant central nervous system involvement.
[0007] Gene therapy using recombinant vectors containing nucleic
acid sequences that encode a functional protein, or genetically
modified human cells that express a functional protein, is also
being used to treat protein deficiencies and other disorders that
benefit from protein replacement. Although promising, this approach
is also limited by technical difficulties such as the inability of
vectors to infect or transduce dividing cells, low expression of
the target gene, and regulation of expression once the gene is
delivered.
[0008] A third, relatively recent approach to treating protein
deficiencies involves the use of small molecule inhibitors to
reduce the natural substrate of deficient enzyme proteins, thereby
ameliorating the pathology. This "substrate deprivation" approach
has been specifically described for a class of about 40 related
enzyme disorders called lysosomal storage disorders or
glycosphingolipid storage disorders. These heritable disorders are
characterized by deficiencies in lysosomal enzymes that catalyze
the breakdown of glycolipids in cells, resulting in an abnormal
accumulation of lipids which disrupts cellular function. The small
molecule inhibitors proposed for use as therapy are specific for
inhibiting the enzymes involved in synthesis of glycolipids,
reducing the amount of cellular glycolipid that needs to be broken
down by the deficient enzyme. This approach is also limited in that
glycolipids are necessary for biological function, and excess
deprivation may cause adverse effects. Specifically, glycolipids
are used by the brain to send signals from the gangliosides of
neurons to another. If there are too few or too many glycolipids,
the ability of the neuron to send signals is impeded.
[0009] A fourth approach, discussed below as specific chaperone
strategy, rescues mutant proteins from degradation in the
endoplasmic reticulum.
Protein Processing in the Endoplasmic Reticulum
[0010] Proteins are synthesized in the cytoplasm, and the newly
synthesized proteins are secreted into the lumen of the endoplasmic
reticulum (ER) in a largely unfolded state. In general, protein
folding is governed by the principle of self assembly. Newly
synthesized polypeptides fold into their native conformation based
on their amino acid sequences (Anfinsen et al., Adv. Protein Chem.
1975; 29:205-300). In vivo, protein folding is complicated, because
the combination of ambient temperature and high protein
concentration stimulates the process of aggregation, in which amino
acids normally buried in the hydrophobic core interact with their
neighbors non-specifically. To avoid this problem, protein folding
is usually facilitated by a special group of proteins called
molecular chaperones which prevent nascent polypeptide chains from
aggregating, and bind to unfolded protein such that the protein
refolds in the native conformation (Hartl, Nature 1996;
381:571-580).
[0011] Molecular chaperones are present in virtually all types of
cells and in most cellular compartments. Some are involved in the
transport of proteins and permit cells to survive under stresses
such as heat shock and glucose starvation (Gething et al., Nature
1992; 355:33-45; Caplan, Trends Cell. Biol. 1999; 9:262-268; Lin et
al., Mol. Biol. Cell. 1993; 4:109-1119; Bergeron et al., Trends
Biochem. Sci. 1994; 19:124-128). Among the molecular chaperones,
Bip (immunoglobulin heavy-chain binding protein, Grp78) is the best
characterized chaperone of the ER (Haas, Curr. Top. Microbiol.
Immunol. 1991; 167:71-82). Like other molecular chaperones, Bip
interacts with many secretory and membrane proteins within the ER
throughout their maturation, although the interaction is normally
weak and short-lived when the folding proceeds smoothly. Once the
native protein conformation is achieved, the molecular chaperone no
longer interacts with the protein. Bip binding to a protein that
fails to fold, assemble or be properly glycosylated, becomes
stable, and leads to degradation of the protein through the
ER-associated degradation pathway. This process serves as a
"quality control" system in the ER, ensuring that only those
properly folded and assembled proteins are transported out of the
ER for further maturation, and improperly folded proteins are
retained for subsequent degradation (Hurtley et al., Annu. Rev.
Cell. Biol. 1989; 5:277-307).
[0012] Certain DNA mutations result in amino acid substitutions
that further impede, and in many cases preclude, proper folding of
the mutant proteins. To correct these misfoldings, investigators
have attempted to use various molecules. High concentrations of
glycerol, dimethylsulfoxide (DMSO), trimethylamine N-oxide (TMAO),
or deuterated water have been shown to suppress the degradation
pathway and increase the intracellular trafficking of mutant
protein in several diseases (Brown et al., Cell Stress Chaperones
1996; 1:117-125; Burrows et al., Proc. Natl. Acad. Sci. USA. 2000;
97:1796-801). These compounds are considered non-specific chemical
chaperones to improve the general protein folding, although the
mechanism of the function is still unknown. The high doses of this
class of compounds required for efficacy makes them difficult or
inappropriate to use clinically, although they are useful for the
biochemical examination of folding defect of a protein
intracellularly. These compounds also lack specificity.
Specific Chaperone Strategy
[0013] Previous patents and publications described a therapeutic
strategy for rescuing endogenous enzyme proteins, specifically
misfolded lysosomal enzymes, from degradation by the ER quality
control machinery. This strategy employs small molecule reversible
competitive inhibitors specific for a defective lysosomal enzyme
associated with a particular lysosomal disorder. The strategy is as
follows: since the mutant enzyme protein folds improperly in the ER
(Ishii et al., Biochem. Biophys. Res. Comm. 1996; 220: 812-815),
the enzyme protein is retarded in the normal transport pathway
(ER.fwdarw.Golgi apparatus.fwdarw.endosome.fwdarw.lysosome) and
rapidly degraded. Therefore, a functional compound which
facilitates the correct folding of a mutant protein will serve as a
site-specific chaperone for the mutant protein to promote the
smooth escape from the ER quality control system. Since some
inhibitors of an enzyme are known to occupy the catalytic center of
enzyme, resulting in stabilization of its conformation in vitro.
These specific chaperones may be designated active site-specific
chaperones (ASSC).
[0014] The strategy has been specifically demonstrated for enzymes
involved in the lysosomal storage disorders in U.S. Pat. Nos.
6,274,597, 6,583,158, 6,589,964, and 6,599,919, to Fan et al., and
in pending U.S. application Ser. No. 10/304,396 filed Nov. 26,
2002, which are hereby incorporated herein by reference in their
entirety. For example, a small molecule derivative of galactose,
1-deoxygalactonojirimycin (DGJ), a potent competitive inhibitor of
the mutant Fabry enzyme .alpha.-galactosidase A (.alpha.-Gal A),
effectively increased in vitro stability of a mutant .alpha.-Gal A
(R301Q) at neutral pH and enhanced the mutant enzyme activity in
lymphoblasts established from Fabry patients with R301Q or Q279E
mutations. Furthermore, oral administration of DGJ to transgenic
mice overexpressing a mutant (R301Q) .alpha.-Gal A substantially
elevated the enzyme activity in major organs (Fan et al., Nature
Med. 1999; 5: 112-115). Successful rescue of a misfolded protein
depends on achieving a concentration of the specific inhibitor in
vivo that is lower than necessary to completely inhibit the enzyme,
in contrast to the substrate deprivation approach in which enzyme
inhibitory concentrations are required.
[0015] In addition to the lysosomal storage disorders, a large and
diverse number of diseases are now recognized as conformational
diseases that are caused by adoption of non-native protein
conformations, which may lead to retardation of the protein in the
ER and ultimate degradation of the proteins (Kuznetsov et al., N.
Engl. J. Med. 1998; 339:1688-1695; Thomas et al., Trends Biochem.
Sci. 1995; 20:456-459; Bychkova et al., FEBS Lett. 1995; 359:6-8;
Brooks, FEBS Lett. 1997; 409:115-120). ASSC's have been shown to
rescue expression of mutant proteins other than enzymes. For
example; small synthetic compounds were found to stabilize the DNA
binding domain of mutant forms of the tumor suppressor protein p53,
thereby allowing the protein to maintain an active conformation
(Foster et al., Science 1999; 286:2507-10). Synthesis of receptors
has been shown to be rescued by small molecule receptor antagonists
and ligands (Morello et al., J. Clin. Invest. 2000; 105: 887-95;
Petaja-Repo et al., EMBO J. 2002; 21:1628-37.) Even pharmacological
rescue of membrane channel proteins and other plasma membrane
transporters has been demonstrated using channel-blocking drugs or
substrates (Rajamani et al., Circulation 2002; 105:2830-5; Zhou et
al., J. Biol. Chem. 1999; 274:31123-26; Loo et al., J. Biol. Chem
1997; 272: 709-12). All of the above references indicate that
ASSC's are capable of specific rescue of mutant proteins including,
but not limited to, enzymes, receptors, membrane channel proteins,
and DNA transcription factors.
[0016] In addition to mutant proteins, ASSC's have also been shown
to stabilize wild-type proteins, resulting in their enhanced
production and stability. As one example, it has been demonstrated
that a specific ASSC, DGJ, is able to increase the amount and
activity of wild-type .alpha.-Gal A in COS-7 cells transfected with
a vector coding the wild-type .alpha.-Gal A sequence. The ASSC
rescues the overexpressed wild-type enzyme, which is otherwise
retarded in the ER quality control system, because overexpression
and over production of the enzyme in the COS-7 cells exceeds the
capacity of the system and leads to aggregation and degradation
(see U.S. application Ser. No. 10/377,179, filed Feb. 28,
2003).
[0017] In summary, there is a need in the art for methods of
improving the biological and cost efficiency of protein replacement
therapy, such as for the treatment of protein deficiencies or other
disorders whereby replacement proteins are administered.
SUMMARY OF THE INVENTION
[0018] The present invention provides a method for enhancing the
stability of a purified protein, which method comprises contacting
the protein in a pharmaceutically acceptable carrier with an active
site-specific chaperone.
[0019] The purified protein can be a recombinant protein, and
either full-length or truncated while retaining activity.
[0020] The present invention also provides a method of increasing
in vitro the shelf-life of a protein by contacting the protein in a
pharmaceutically acceptable carrier with an active site-specific
chaperone.
[0021] The protein in the pharmaceutically acceptable carrier can
be lyophilized or an aqueous solution.
[0022] The present invention further provides a method of extending
the half-life and prolonging the activity in vivo of a purified
protein in an individual who has been administered the protein in a
pharmaceutically acceptable carrier, which method comprises
contacting the protein with an active site-specific chaperone in a
pharmaceutically acceptable carrier.
[0023] The present invention provides a method of treatment for an
individual having a disorder requiring protein replacement, (e.g.,
protein deficiency disorders) comprising administering to the
individual a purified replacement protein and an active
site-specific chaperone (ASSC) capable of stabilizing the
replacement protein.
[0024] In one embodiment, the replacement protein is a protein
associated with a conformational disorder.
[0025] In a preferred embodiment, the conformational disorder is a
lysosomal storage disorder.
[0026] In one embodiment, the lysosomal storage disorder is Fabry
disease.
[0027] In another embodiment, the lysosomal storage disorder is
Gaucher disease.
[0028] The invention also provides a method for enhancing the
stability of a mutant, endogenous protein that is deficient due to
defective folding or processing in the ER concurrently with protein
replacement therapy. Stability and, hence, activity of the
endogenous protein will be enhanced concurrently with the increased
stability of the administered replacement protein that corresponds
to the mutant protein.
[0029] The invention further provides a method for increasing the
production of recombinant protein by non-mammalian host cells by
contacting the host cell in a medium comprising an ASSC for the
protein.
[0030] The invention further provides a composition comprising a
purified protein and an ASSC for the purified protein in a
pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1 demonstrates improved stability of both wild type
.alpha.-Gal A purified from culture medium of Sf-9 cells infected
with recombinant baculovirus carrying human wild type .alpha.-Gal A
cDNA, and mutant .alpha.-Gal A collected as homogenates of hearts
of transgenic mice overexpressing human mutant (R301Q) .alpha.-Gal
A, respectively, using a site-specific chaperone
1-deoxygalactonojirimycin (DGJ, 1 .mu.M). The mice were treated
with 0.5 mM DGJ as drinking water for one week prior to the
experiment. The mutant (A) and wild type (B) enzymes were
pre-incubated with 0.1 M citrate-phosphate buffer (pH 7.0) at
37.degree. C. for the mutant enzyme and 42.degree. C. for the wild
type enzyme, respectively, in the presence of DGJ at a
concentration of 1 .mu.M (.largecircle.), 0.1 .mu.M
(.circle-solid.), 0.03 .mu.M (.diamond-solid.) or 0 .mu.M (no DGJ;
.diamond.). Enzyme activity is reported relative to the enzyme
without pre-incubation. DGJ can serve as a stabilizer to prevent
the denaturation/degradation of the mutant and wild type
enzymes.
DETAILED DESCRIPTION
[0032] The present invention advantageously improves the efficiency
of protein replacement therapy to treat diseases or disorders by
contacting the protein with an active site-specific chaperone
(ASSC). The advantages of the invention flow from (a) increased
efficiency of protein production from non-mammalian cells; (b)
increased stability of the therapeutic protein, manifested by
longer shelf life and better in vivo half life and activity; (c)
maintenance of protein active site structure during translocations
in vivo, including across cell membranes; and (d) rescue of
endogenous mutant protein that is misfolded during synthesis and
consequently cleared from the endoplasmic reticulum.
[0033] The present invention further provides formulations
comprising the protein and active site-specific chaperone (ASSC)
specific for the stabilization of the protein.
[0034] The invention is based on the discovery that ASSC's can be
used as a combination therapy with protein replacement therapy for
the treatment of genetic and other disorders. ASSC's can be
screened and identified using methods known in the art. Once a
specific ASSC useful for a particular disorder is identified, the
ASSC can be administered to a patient receiving protein replacement
therapy to enhance uptake of the replacement protein in the
appropriate cellular compartment, improve stability of the protein
in circulation and, if necessary, during transport into the cell.
The chaperone can stabilize the protein in its active form during
manufacture, storage and use in vivo.
DEFINITIONS
[0035] The terms used in this specification generally have their
ordinary meanings in the art, within the context of this invention
and in the specific context where each term is used. Certain terms
are discussed below, or elsewhere in the specification, to provide
additional guidance to the practitioner in describing the
compositions and methods of the invention and how to make and use
them.
[0036] Specific Definitions. The term "protein replacement" refers
to the introduction of a non-native, purified protein into an
individual having a deficiency in such protein. The administered
protein can be obtained from natural sources (such as human
gammaglobulin for treating RSV or mononucleosis) or by recombinant
expression (as described in greater detail below). The term also
refers to the introduction of a purified protein in an individual
otherwise requiring or benefiting from administration of a purified
protein, e.g., suffering from protein insufficiency. The introduced
protein may be a purified, recombinant protein produced in vitro,
or protein purified from isolated tissue or fluid, such as, e.g,
placenta or animal milk, or from plants.
[0037] The term "disorder characterized by a protein deficiency"
refers to any disorder that presents with a pathology caused by
absent or insufficient amounts of a protein. This term encompasses
protein folding disorders, i.e., conformational disorders, that
result in a biologically inactive protein product. Protein
insufficiency can be involved in infectious diseases,
immunosuppression, organ failure, glandular problems, radiation
illness, nutritional deficiency, poisoning, or other environmental
or external insults.
[0038] The term "stabilize a proper conformation" refers to the
ability of a compound or peptide or other molecule to associate
with a wild-type protein, or to a mutant protein that can perform
its wild-type function in vitro in, e.g., a formulation, and in
vivo, in such a way that the structure of the wild-type or mutant
protein can be maintained as its native or proper form. This effect
may manifest itself practically through one or more of (i)
increased shelf-life of the protein; (ii) higher activity per
unit/amount of protein; or (iii) greater in vivo efficacy. It may
be observed experimentally through increased yield from the ER
during expression; greater resistance to unfolding due to
temperature increases, or the present of chaotropic agents, and by
similar means.
[0039] As used herein, the term "conformational disorder" or
"conformational disease" refers to a disorder that is caused by
adoption of a protein conformation that is not normally formed by a
wild-type protein in a native condition with normal biological
activity, which may lead to retardation and destruction of a
protein in the ER. The decreased protein level results in a
physiological imbalance that manifests itself as a disease or
disorder. In a specific embodiment, the conformational disorder is
a lysosomal storage disorder.
[0040] As used herein, the term "active site" refers to the region
of a protein that has some specific biological activity. For
example, it can be a site that binds a substrate or other binding
partner and contributes the amino acid residues that directly
participate in the making and breaking of chemical bonds. Active
sites in this invention can encompass catalytic sites of enzymes,
antigen biding sites of antibodies, ligand binding domains of
receptors; binding domains of regulators, or receptor binding
domains of secreted proteins. The active sites can also encompass
transactivation, protein-protein interaction, or DNA binding
domains of transcription factors and regulators.
[0041] As used herein, the term "active site-specific chaperone"
refers to any molecule including a protein, peptide, nucleic acid,
carbohydrate, etc. that specifically interacts reversibly with an
active site of a protein and enhances formation of a stable
molecular conformation. As used herein, "active site-specific
chaperone" does not include endogenous general chaperones present
in the ER of cells such as Bip, calnexin or calreticulin, or
general, non-specific chemical chaperones such as deuterated water,
DMSO, or TMAO.
[0042] General Definitions. The term "purified" as used herein
refers to material that has been isolated under conditions that
reduce or eliminate the presence of unrelated materials i.e.,
contaminants, including native materials from which the material is
obtained. For example, a purified protein is preferably
substantially free of other proteins or nucleic acids with which it
is associated in a cell; a purified nucleic acid molecule is
preferably substantially free of proteins or other unrelated
nucleic acid molecules with which it can be found within a cell. As
used herein, the term "substantially free" is used operationally,
in the context of analytical testing of the material. Preferably,
purified material substantially free of contaminants is at least
95% pure; more preferably, at least 97% pure, and more preferably
still at least 99% pure. Purity can be evaluated by chromatography,
gel electrophoresis, immunoassay, composition analysis, biological
assay, and other methods known in the art. In a specific
embodiment, purified means that the level of contaminants is below
a level acceptable to regulatory authorities for administration to
a human or non-human animal.
[0043] In preferred embodiments, the terms "about" and
"approximately" shall generally mean an acceptable degree of error
for the quantity measured given the nature or precision of the
measurements. Typical, exemplary degrees of error are within 20
percent (%), preferably within 10%, and more preferably within 5%
of a given value or range of values. Alternatively, and
particularly in biological systems, the terms "about" and
"approximately" may mean values that are within an order of
magnitude, preferably within 10- or 5-fold, and more preferably
within 2-fold of a given value. Numerical quantities given herein
are approximate unless stated otherwise, meaning that the term
"about" or "approximately" can be inferred when not expressly
stated.
[0044] A "gene" is a sequence of nucleotides which code for a
functional "gene product". Generally, a gene product is a
functional protein. However, a gene product can also be another
type of molecule in a cell, such as an RNA (e.g., a tRNA or a
rRNA). For the purposes of the present invention, a gene product
also refers to an mRNA sequence which may be found in a cell.
[0045] The term "express" and "expression" means allowing or
causing the information in a gene or DNA sequence to become
manifest, for example producing RNA (such as rRNA or mRNA) or a
protein by activating the cellular functions involved in
transcription and translation of a corresponding gene or DNA
sequence. A DNA sequence is expressed by a cell to form an
"expression product" such as an RNA (e.g., a mRNA or a rRNA) or a
protein. The expression product itself, e.g., the resulting RNA or
protein, may also said to be "expressed" by the cell.
[0046] The term "transfection" means the introduction of a foreign
nucleic acid into a cell. The term "transformation" means the
introduction of a "foreign" (i.e., extrinsic or extracellular)
gene, DNA or RNA sequence into a host cell so that the host cell
will express the introduced gene or sequence to produce a desired
substance, in this invention typically an RNA coded by the
introduced gene or sequence, but also a protein or an enzyme coded
by the introduced gene or sequence. The introduced gene or sequence
may also be called a "cloned" or "foreign" gene or sequence, may
include regulatory or control sequences (e.g., start, stop,
promoter, signal, secretion or other sequences used by a cell's
genetic machinery). The gene or sequence may include nonfunctional
sequences or sequences with no known function. A host cell that
receives and expresses introduced DNA or RNA has been "transformed"
and is a "transformant" or a "clone". The DNA or RNA introduced to
a host cell can come from any source, including cells of the same
genus or species as the host cell or cells of a different genus or
species.
[0047] The terms "vector", "cloning vector" and "expression vector"
mean the vehicle by which a DNA or RNA sequence (e.g., a foreign
gene) can be introduced into a host cell so as to transform the
host and promote expression (e.g., transcription and translation)
of the introduced sequence.
[0048] The term "expression system" means a host cell and
compatible vector under suitable conditions, e.g. for the
expression of a protein coded for by foreign DNA carried by the
vector and introduced to the host cell. Common expression systems
include E. coli host cells and plasmid vectors, insect host cells
such as Sf9, Hi5 or S2 cells and Baculovirus vectors, and
expression systems, and mammalian host cells and vectors.
[0049] The terms "mutant" and "mutation" mean any detectable change
in genetic material, e.g., DNA, or any process, mechanism or result
of such a change. This includes gene mutations, in which the
structure (e.g., DNA sequence) of a gene is altered, any gene or
DNA. arising from any mutation process, and any expression product
(e.g., RNA, protein or enzyme) expressed by a modified gene or DNA
sequence.
[0050] As used herein the term "mutant protein" refers to proteins
translated from genes containing genetic mutations that result in
altered protein sequences. In a specific embodiment, such mutations
result in the inability of the protein to achieve its native
conformation under the conditions normally present in the ER. The
failure to achieve this conformation results in these proteins
being degraded, rather than being transported through their normal
pathway in the protein transport system to their proper location
within the cell. Other mutations can result in decreased. activity
or more rapid turnover.
[0051] A "wild-type gene" refers to a nucleic acid sequences which
encodes a protein capable of having normal biological functional
activity in vivo. The wild-type nucleic acid sequence may contain
nucleotide changes that differ from the known, published sequence,
as long as the changes result in amino acid substitutions having
little or no effect on the biological activity. The term wild-type
may also include nucleic acid sequences engineered to encode a
protein capable of increased or enhanced activity relative to the
endogenous or native protein.
[0052] A "wild-type protein" refers to any protein encoded by a
wild-type gene that is capable of having functional biological
activity when expressed or introduced in vivo. The term "normal
wild-type activity" refers to the normal physiological function of
a protein in a cell. Such functionality can be tested by any means
known to establish functionality of a protein.
[0053] The term "genetically modified" refers to cells that express
a particular gene product following introduction. of a nucleic acid
comprising a coding sequence which encodes the gene product, along
with regulatory elements that control expression of the coding
sequence. Introduction of the nucleic acid may be accomplished by
any method known in the art including gene targeting and homologous
recombination. As used herein, the term also includes cells that
have been engineered to express or overexpress an endogenous gene
or gene product not normally expressed by such cell, e.g., by gene
activation technology.
[0054] The phrase "pharmaceutically acceptable", whether used in
connection with the pharmaceutical compositions of the invention,
refers to molecular entities and compositions that are
physiologically tolerable and do not typically produce untoward
reactions when administered to a human. Preferably, as used herein,
the term "pharmaceutically acceptable" means approved by a
regulatory agency of the Federal or a state government or listed
in. the U.S. Pharmacopeia or other generally recognized
pharmacopeia for use in animals, and more particularly. in humans.
The term "carrier" refers to a diluent, adjuvant, excipient, or
vehicle with which the compound is administered. Such
pharmaceutical carriers can be sterile liquids, such as water and
oils. Water or aqueous solution saline solutions and aqueous
dextrose and glycerol solutions are preferably employed as
carriers, particularly for injectable solutions. Suitable
pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin, 18th Edition.
[0055] The terms "therapeutically effective dose" and "effective
amount" refer to the amount of the compound that is sufficient to
result in a therapeutic response. In embodiments where an ASSC and
protein are administered in a complex, the terms "therapeutically
effective dose" and "effective amount" may refer to the amount of
the complex that is sufficient to result in a therapeutic response.
A therapeutic response may be any response that a user (e.g., a
clinician) will recognize as an effective response to the therapy.
Thus, a therapeutic response will generally be an amelioration of
one or more symptoms of a disease or disorder.
[0056] It should be noted that a concentration of the ASSC that is
inhibitory during in vitro production, transportation, or storage
of the purified therapeutic protein may still constitute an
"effective amount" for purposes of this invention because of
dilution (and consequent shift in binding due to the change in
equilibrium), bioavailability and metabolism of the ASSC upon
administration in vivo.
Disorders Characterized by Protein Deficiencies
[0057] There currently are about 1100 known inherited disorders
characterized by protein deficiency or loss-of-function in specific
tissue. These disorders may be treatable by protein replacement
therapy in theory. The method of the present invention contemplates
co-therapy for proteins currently suited for use in protein
replacement therapy that is available now or will be in the future.
In such disorders, certain cells or all of the cells of an
individual lack a sufficient functional protein, contain an
inactive form of the protein or contain insufficient levels for
biological function.
[0058] Further, the list of diseases identified as being
conformational disorders, caused by mutations that alter protein
folding and retardation of the mutant protein in the ER, resulting
in protein deficiency, is increasing. These include cystic
fibrosis, .alpha.1-antitrypsin deficiency, familial
hypercholesterolemia, Fabry disease, Alzheimer's disease (Selkoe,
Annu. Rev. Neurosci. 1994; 17:489-517), osteogenesis imperfecta
(Chessler et al., J. Biol. Chem. 1993; 268:18226-18233),
carbohydrate-deficient glycoprotein syndrome (Marquardt et al.,
Eur. J. Cell. Biol. 1995; 66: 268-273), Maroteaux-Lamy syndrome
(Bradford et al., Biochem. J. 1999; 341:193-201), hereditary
blindness (Kaushal et al., Biochemistry 1994; 33:6121-8), Glanzmann
thrombasthenia (Kato et al., Blood 1992; 79:3212-8), hereditary
factor VII deficiency (Arbini et al., Blood 1996; 87:5085-94),
oculocutaneous albinism (Halaban et al., Proc. Natl. Acad. Sci.
USA. 2000; 97:5889-94) and protein C deficiency (Katsumi, et al.,
Blood 1996; 87:4164-75). Recently, one mutation in the X-linked
disease adrenoleukodystrophy (ALD), resulted in misfolding of the
defective peroxisome transporter which could be rescued by
low-temperature cultivation of affected cells (Walter et al., Am J
Hum Genet 2001; 69:35-48). It is generally accepted that mutations
take place evenly over the entire sequence of a gene. Therefore, it
is predictable that the phenotype resulting from misfolding of the
deficient protein exists in many other genetic disorders.
Lysosomal Storage Disorders
[0059] Many of the inherited protein deficient disorders are enzyme
deficiencies. As indicated above, a large class of inherited enzyme
disorders involves mutations in lysosomal enzymes and are referred
to as lysosomal storage disorders (LSDs). Lysosomal storage
disorders are a group of diseases caused by the accumulation of
glycosphingolipids, glycogen, mucopolysaccharides Examples of
lysosomal disorders include but are not limited to Gaucher disease
(Beutler et al., The Metabolic and Molecular Bases of Inherited
Disease, 8th ed. 2001 Scriver et al., ed. pp. 3635-3668,
McGraw-Hill, New York), GM1-gangliosidosis (id. at pp 3775-3810),
fucosidosis (The Metabolic and Molecular Bases of Inherited Disease
1995. Scriver, C. R., Beaudet, A. L., Sly, W. S. and Valle, D., ed
pp. 2529-2561, McGraw-Hill, New York), mucopolysaccharidoses (id.
at pp 3421-3452), Pompe disease (id. at pp. 3389-3420),
Hurler-Scheie disease (Weismann et al., Science 1970; 169, 72-74),
Niemann-Pick A and B diseases, (The Metabolic and Molecular Bases
of Inherited Disease 8th ed. 2001. Scriver et al. ed., pp
3589-3610, McGraw-Hill, New York), and Fabry disease (id. at pp.
3733-3774). A list of LSDs and their associated deficient enzymes
can be found in Table 1 infra. Two are discussed specifically
below.
Fabry Disease
[0060] Fabry disease is an X-linked inborn error of
glycosphingolipid metabolism caused by deficient lysosomal
.alpha.-galactosidase A (.alpha.-Gal A) activity (Desnick et al.,
The Metabolic and Molecular Bases of Inherited Disease, 8.sup.th
Edition Scriver et al. ed., pp. 3733-3774, McGraw-Hill, New York
2001; Brady et al., N. Engl. J. Med. 1967; 276, 1163-1167). This
enzymatic defect leads to the progressive deposition of neutral
glycosphingolipids with .alpha.-galactosyl residues, predominantly
globotriaosylceramide (GL-3), in body fluids and tissue lysosomes.
The frequency of the disease is estimated to be about 1:40,000 in
males, and is reported throughout the world within different ethnic
groups. In classically affected males, the clinical manifestations
include angiokeratoma, acroparesthesias, hypohidrosis, and
characteristic corneal and lenticular opacities (The Metabolic and
Molecular Bases of Inherited Disease, 8.sup.th Edition 2001,
Scriver et al., ed., pp. 3733-3774, McGraw-Hill, New York). The
affected male's life expectancy is reduced, and death usually
occurs in the fourth or fifth decade as a result of vascular
disease of the heart, brain, and/or kidneys. In contrast, patients
with the milder "cardiac variant" normally have 5-15% of normal
.alpha.-Gal A activity, and present with left ventricular
hypertrophy or a cardiomyopathy. These cardiac variant patients
remain essentially asymptomatic when their classically affected
counterparts are severely compromised. Recently, cardiac variants
were found in 11% of adult male patients with unexplained left
ventricular hypertrophic cardiomyopathy, suggesting that Fabry
disease may be more frequent than previously estimated (Nakao et
al., N. Engl. J. Med. 1995; 333: 288-293). The .alpha.-Gal A gene
has been mapped to Xq22, (Bishop et al., Am. J. Hum. Genet. 1985;
37: A144), and the full-length cDNA and entire 12-kb genomic
sequences encoding .alpha.-Gal A have been reported (Calhoun et
al., Proc. Natl. Acad. Sci. USA 1985; 82: 7364-7368; Bishop et al.,
Proc. Natl. Acad. Sci. USA 1986; 83: 4859-4863; Tsuji et al., Eur.
J. Biochem. 1987; 165: 275-280; and Kornreich et al., Nucleic Acids
Res. 1989; 17: 3301-3302). There is a marked genetic heterogeneity
of mutations that cause Fabry disease (The Metabolic and Molecular
Bases of Inherited Disease, 8.sup.th Edition 2001, Scriver et al.,
ed., pp. 3733-3774, McGraw-Hill, New York.; Eng et al., Am. J. Hum.
Genet. 1993; 53: 1186-1197; Eng et al., Mol. Med. 1997; 3: 174-182;
and Davies et al., Eur. J. Hum. Genet. 1996; 4: 219-224). To date,
a variety of missense, nonsense, and splicing mutations, in
addition to small deletions and insertions, and larger gene
rearrangements have been reported.
Gaucher Disease
[0061] Gaucher disease is a deficiency of the lysosomal enzyme
.beta.-glucocerebrosidase that breaks down fatty glucocerebrosides.
The fat then accumulates, mostly in the liver, spleen and bone
marrow. Gaucher disease can result in pain, fatigue, jaundice, bone
damage, anemia and even death. There are three clinical phenotypes
of Gaucher disease. Patients with, Type 1 manifest either early in
life or in young adulthood, bruise easily and experience fatigue
due to anemia, low blood platelets, enlargement of the liver and
spleen, weakening of the skeleton, and in some instances have lung
and kidney impairment. There are no signs of brain involvement. In
Type II, early-onset, liver and spleen enlargement occurs by 3
months of age and there is extensive brain involvement. There is a
high mortality rate by age 2. Type III is characterized by liver
and spleen enlargement and brain seizures. The
.beta.-glucocerebrosidase gene is located on the human 1q21
chromosome. Its protein precursor contains 536 amino acids and its
mature protein is 497 amino acids long.
[0062] Gaucher disease is considerably more common in the
descendants of Jewish people from Eastern Europe (Ashkenazi),
although individuals from any ethnic group may be affected. Among
the Ashkenazi Jewish population, Gaucher disease is the most common
genetic disorder, with an incidence of approximately 1 in 450
persons. In the general public, Gaucher disease affects
approximately 1 in 100,000 persons. According to the National
Gaucher Foundation, 2,500 Americans suffer from Gaucher
disease.
Other Enzyme Deficiency Disorders
[0063] Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the
most common X-linked human enzyme deficiency. The G6PD enzyme
catalyzes an oxidation/reduction reaction that is essential for the
production of ribose, which is an essential component of both DNA
and RNA. G6PD also involved in maintaining adequate levels of NADPH
inside the cell. NADPH is a required cofactor in many biosynthetic
reactions. Individuals with this deficiency have clinical symptoms
including neonatal jaundice, abdominal and/or back pain, dizziness,
headache, dyspnea (irregular breathing), and palpitations.
[0064] In addition to inherited disorders, other enzyme
deficiencies arise from damage to a tissue or organ resulting from
primary or secondary disorders. For example, damaged pancreatic
tissue, or pancreatitis, is caused by alcoholism results in a
deficiency in pancreatic enzymes necessary for digestion.
Pancreatitis is currently being treated using enzyme replacement
therapy. TABLE-US-00001 TABLE 1 Lysosomal Storage Disorders,
Associated Defective Enzymes and Small Molecule Active
Site-Specific Chaperones DISORDER DEFICIENT ENZYME REVERSIBLE
CHAPERONE Pompe disease .alpha.-Glucosidase 1-deoxynojirimycin
(DNJ) .alpha.-homonojirimycin castanospermine Gaucher disease Acid
.beta.-Glucosidase isofagomine (glucocerebrosidase) N-dodecyl-DNJ
calystegines A.sub.3, B.sub.1, B.sub.2 and C.sub.1 Fabry disease
.alpha.-Galactosidase A 1-deoxygalactonojirimycin (DGJ)
.alpha.-allo-homonojirimycin .alpha.-galacto-homonojirimycin
.beta.-1-C-butyl-deoxynojirimycin calystegines A.sub.2 and B.sub.2
N-methyl calystegines A.sub.2 and B.sub.2 G.sub.M1-gangliosidosis
Acid .beta.-Galactosidase 4-epi-isofagomine
1-deoxygalactonojirimycin Krabbe disease Galactocerebrosidase
4-epi-isofagomine 1-deoxygalactonojirimycin Morquio disease B Acid
.beta.-Galactosidase 4-epi-isofagomine 1-deoxygalactonojirimycin
.alpha.-Mannosidosis Acid .alpha.-Mannosidase 1-deoxymannojirimycin
Swainsonine Mannostatin A B-Mannosidosis Acid .beta.-Mannosidase
2-hydroxy-isofagomine Fucosidosis Acid .alpha.-L-fucosidase
1-deoxyfuconojirimycin .beta.-homofuconojirimycin
2,5-imino-1,2,5-trideoxy-L-glucitol 2,5-deoxy-2,5-imino-D-fucitol
2,5-imino-1,2,5-trideoxy-D-altritol Sanfilippo disease B
.alpha.-N-Acetylglucosaminidase 1,2-dideoxy-2-N-acetamido-
nojirimycin Schindler disease .alpha.-N-Acetylgalactosaminidase
1,2-dideoxy-2-N-acetamido- galactonojirimycin Tay-Sachs disease
.beta.-Hexosaminidase A 2-N-acetylamino-isofagomine
1,2-dideoxy-2-acetamido-nojirimycin nagstain Sandhoff disease
.beta.-Hexosaminidase B 2-N-acetamido-isofagomine
1,2-dideoxy-2-acetamido-nojirimycin nagstain Hurler-Scheie disease
.alpha.-L-Iduronidase 1-deoxyiduronojirimycin
2-carboxy-3,4,5-trideoxypiperidine Sly disease .beta.-Glucuronidase
6-carboxy-isofagomine 2-carboxy-3,4,5-trideoxypiperidine Sialidosis
Sialidase 2,6-dideoxy-2,6,imino-sialic acid Siastatin B Hunter
disease Iduronate sulfatase 2,5-anhydromannitol-6-sulphate
Niemann-Pick disease Acid sphingomyelinase desipramine,
phosphatidylinositol-4,5- diphosphate
Other Disorders Treated Using Protein Replacement
[0065] In addition to disorders characterized by protein
deficiencies, some disorders are treated by administration of
replacement proteins to enhance or stimulate biological processes.
For example, individuals with anemia are administered recombinant
erythropoietin (EPOGEN.RTM., PROCRIT.RTM., EPOIETIN.RTM.) to
stimulate red blood cell production and increase oxygen
transportation to tissues. In addition, recombinant interferons
such as interferon alpha 2b (INTRON A.RTM., PEG-INTRON.RTM., or
REBETOL.RTM.), and interferon beta 1a (AVONEX.RTM., BETASERON.RTM.)
are administered to treat hepatitis B and multiple sclerosis,
respectively. Still other proteins administered are recombinant
human deoxyribonuclease I (rhDNase-PULMOZYME.RTM.), an enzyme which
selectively cleaves DNA used to improve pulmonary function in
patients with cystic fibrosis; recombinant thyroid stimulating
hormone (THYROGEN.RTM.) developed for use in thyroid cancer
patients who have had near-total or total thyroidectomy, and who
must therefore take thyroid hormones; recombinant G-CSF
(NEUPOGEN.RTM.) for treating neutropenia from chemotherapy, and
digestive enzymes in individuals with pancreatitis. Another
significant area of protein therapy is in the treatment of
infectious diseases and cancer with antibodies, which have a highly
specific, well-defined active site. Antibody therapeutic products
include RESPIRGRAM.RTM. for respiratory syncitial virus,
HERCEPTIN.RTM., for breast cancer; REMICAID.RTM. and HUMIRA.RTM.,
for arthritis and inflammatory diseases, and others. ASSCs for
antibodies are well known, and either the target antigen or a
structurally related analog (e.g., a modified form of the active
target or a mimetic) can be employed. See Table 2 below for a list
of proteins currently on the market or being evaluated in clinical
trials for use as protein therapy. TABLE-US-00002 TABLE 2
Replacement Proteins Administered in Associated Disorders
Development Protein Trade name Therapeutic function phase
(rhuMAb-VEGF) Dynepo .TM. anemia associated with Phase III renal
disease .alpha.-L-iduronidase Aldurazyme .TM.
mucopolysaccharidosis-I Commercially available alronidase rDNA
insulin diabetes Phase III alteplase, Activase .RTM. acute
myocardial Commercially infarction; acute massive available
pulmonary embolism; ischemic stroke within 3 to 5 hours of symptom
onset darbepoetin alfa Aranesp .TM. anemia Commercially available
Deoxyribonuclease Pubnozyme cystic fibrosis Commercially I
available drotrecogin alfa Xigris .TM. severe sepsis Commercially
(activated protein available C) efalizumab Raptiva .RTM. moderate
to severe Commercially psoriasis available erythropoietin EPOGEN
.RTM. anemia Commercially available erythropoietin PROCRIT .RTM.
anemia Commercially available etanercept Enbrel .RTM. rheumatoid
Commercially arthritis; psoriatic arthritis available factor IX
BeneFIX .TM. hemophilia B Commercially available
follicle-stimulating Follistim .RTM. infertility Commercially
hormone available G-CSF Neupogen neutropenia resulted from
Commercially Chemotherapy available glucocerebrosidase Cerezyme
.TM. Gaucher's disease Commercially available GM-CSF KGF mucositis
Phase III completed (Repifermin) Growth hormone BioTropin .TM.
growth hormone deficiency Commercially in children available heat
shock protein Leukine .RTM. mucositis and melanoma Commercially
available Insulin Humalog .RTM. diabetes Commercially available
interferon Actimmune .RTM. idiopathic pulmonary Commercially
fibrosis available interferon alfa Enbrel .RTM. ankylosing
spondylitis, Commercially (enterecept) psoriasis available
interferon alfa-2a, Roferon .RTM.-A hairy cell Commercially
leukemia; Kaposi's available sarcoma; chronic recombinant
myelogenous leukemia; hepatitis C interferon alfa-n3 Actimmune
.RTM. systemic fungal infections Commercially available interferon
alfa-n3 Alferon N genital warts Commercially available interferon
beta-1a Avonex .RTM. relapsing multiple Commercially sclerosis
available interferon beta-1a Pegasys .RTM. chronic hepatitis C
Commercially available interferon beta-1b Betaseron .RTM.
relapsing, remitting Commercially multiple sclerosis available
interferon beta-1b Rebif .RTM. chronic hepatitis C Commercially
available interferon gamma Actimmune .RTM. chronic granulomatous
Commercially 1b disease; osteopetrosis available agalsidase beta
Fabrazyme .TM. Fabry disease Commercially available interleukin-2
Proleukin .RTM. renal cell carcinoma; Commercially metastatic
melanoma available keratinocyte Avastin .TM. colorectal cancer
Phase III completed growth factor lepirudin Refludan .TM.
heparin-induced Commercially (anticoagulant) thrombocytopenia type
II available omalizumab Xolair .RTM. allergy-related asthma
Commercially available rasburicase Elitek .RTM. hyperuricemia,
Commercially available reteplase (tissue Retavase .RTM. acute
myocardial infarction Commercially plasminogen available factor)
thyroid Thyrogen .RTM. thyroid cancer Commercially stimulating
available hormone TNF-alpha Oncophage .RTM. colorectal, renal cell
Phase III cancer, melanoma trastuzumab Herceptin .RTM. HER2
overexpressing Commercially metastatic breast cancer available
Treatment of Protein Deficiencies and Other Disorders
[0066] As mentioned briefly above, gene therapy, protein
replacement therapy, and small molecule inhibitor therapy have been
developed as therapeutic strategies for the treatment of genetic
disorders resulting from protein deficiencies and for disorders
that benefit from administration of replacement proteins.
[0067] Protein replacement therapy increases the amount of protein
by exogenously introducing wild-type or biologically functional
protein by way of infusion. This therapy has been developed for
many genetic disorders including Gaucher disease and Fabry disease,
as referenced above. The wild-type enzyme is purified from a
recombinant cellular expression system (e.g., mammalian cells or
insect cells-see U.S. Pat. No. 5,580,757 to Desnick et al.; U.S.
Pat. Nos. 6,395,884 and 6,458,574 to Selden et al.; U.S. Pat. No.
6,461,609 to Calhoun et al.; U.S. Pat. No. 6,210,666 to Miyamura et
al.; U.S. Pat. No. 6,083,725 to Selden et al.; U.S. Pat. No.
6,451,600 to Rasmussen et al.; U.S. Pat. No. 5,236,838 to Rasmussen
et al. and U.S. Pat. No. 5,879,680 to Ginns et al.), human
placenta, or animal milk (see U.S. Pat. No. 6,188,045 to Reuser et
al.). After the infusion, the exogenous enzyme is expected to be
taken up by tissues through non-specific or receptor-specific
mechanism. In general, the uptake efficiency is not high, and the
circulation time of the exogenous protein is short (Ioannu et al.,
Am. J. Hum. Genet. 2001; 68: 14-25). In addition, the-exogenous
protein is unstable and subject to rapid intracellular
degradation.
[0068] In addition to protein replacement and gene therapy, small
molecule therapy using enzyme inhibitors has been described for the
treatment of the LSD's, namely small molecule inhibitors useful for
substrate deprivation of the precursors of the deficient enzyme,
referenced above. Small molecule inhibitors have been described for
the treatment of LSD's including Fabry disease, Gaucher disease,
Pompe disease, Tay Sachs disease, Sandhoff disease, and G.sub.M2
gangliosidoses (see U.S. Pat. Nos. 5,472,969, 5,580,884, 5,798,366,
and 5,801,185 to Platt et al.).
Co-Therapy Using ASSC's and Protein Replacement
[0069] The present invention increases the effectiveness of protein
replacement therapy by increasing the stability of the purified
protein in vitro in a formulation or composition, and in vivo by
co-administration of an ASSC for the protein. Screening for an
appropriate ASSC for the target protein can be achieved using
ordinary methods in the art, for example, as described in U.S.
patent application Ser. No. 10/377,179, filed Feb. 28, 2003, which
is incorporated herein by reference.
Replacement Protein Production
[0070] Disorders that can be treated using the method of the
present invention include but are not limited to LSD's,
glucose-6-phosophate dehydrogenase deficiency, hereditary
emphysema, familial hypercholesterolemia, familial hypertrophic
cardiomyopathy, phenylketonuria, anemia, hepatitis B and multiple
sclerosis.
[0071] The replacement proteins useful for the methods of the
present invention can be isolated and purified using ordinary
molecular biology, microbiology, and recombinant DNA techniques
within the skill of the art. For example, nucleic acids encoding
the replacement protein can be isolated using recombinant DNA
expression as described in the literature. See, e.g., Sambrook,
Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual,
Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y.(herein "Sambrook et al., 1989"); DNA Cloning: A
Practical Approach, Volumes I and II (D. N. Glover ed. 1985);
Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid
Hybridization [B. D. Hames & S. J. EHiggins eds. (1985)];
Transcription And Translation [B. D. Hames & S. J. Higgins,
eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];
Immobilized Cells And Enzymes [IRL Press, (1986)]; B. EPerbal, A
Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al.
(eds.), Current Protocols in Molecular Biology, John Wiley &
Sons, Inc. (1994). The nucleic acid encoding the protein may be
full-length or truncated, as long as the gene encodes a
biologically active protein. For example, a biologically active,
truncated form of .alpha.-Gal A, the defective enzyme associated
with Fabry disease, has been described in U.S. Pat. No. 6,210,666
to Miyamura et al.
[0072] The identified and isolated gene encoding the target protein
can then be inserted into an appropriate cloning vector. A large
number of vector-host systems known in the art may be used.
Possible vectors include, but are not limited to, plasmids or
modified viruses, but the vector system must be compatible with the
host cell used. Examples of vectors include, but are not limited
to, E. coli, bacteriophages such as lambda derivatives, or plasmids
such as pBR322 derivatives or pUC plasmid derivatives, e.g., pGEX
vectors, pmal-c, pFLAG, etc. The insertion into a cloning vector
can, for example, be accomplished by ligating the DNA fragment into
a cloning vector which has complementary cohesive termini. However,
if the complementary restriction sites used to fragment the DNA are
not present in the cloning vector, the ends of the DNA molecules
may be enzymatically modified. Alternatively, any site desired may
be produced by ligating nucleotide sequences (linkers) onto the DNA
termini; these ligated linkers may comprise specific chemically
synthesized oligonucleotides encoding restriction endonuclease
recognition sequences. Production of the recombinant protein can be
maximized by genetic manipulations such as including a signal
peptide at the N terminus to facilitate secretion or a 3'
untranslated sequence containing a polyadenylation site.
[0073] In a preferred embodiment, the constructs used to transduce
host cells are viral-derived vectors, including but not limited to
adenoviruses, adeno-associated viruses, herpes virus, mumps virus,
poliovirus, retroviruses, Sindbis virus and vaccinia viruses.
[0074] Recombinant molecules can be introduced into host cells via
transformation, transfection, infection, electroporation, etc., so
that many copies of the gene sequence are generated. Preferably,
the cloned gene is contained on a shuttle vector plasmid, which
provides for expansion in a cloning cell, e.g., E. coli, and facile
purification for subsequent insertion into an appropriate
expression cell line, if such is desired.
[0075] Potential host-vector systems include but are not limited to
mammalian cell systems infected with virus (e.g., vaccinia virus,
adenovirus, etc.); insect cell systems infected with virus (e.g.,
baculovirus); microorganisms such as yeast containing yeast
vectors; or bacteria transformed with bacteriophage, DNA, plasmid
DNA, or cosmid DNA. The expression elements of vectors vary in
their strengths and specificities. Depending on the host-vector
system utilized, any one of a number of suitable transcription and
translation elements may be used. Different host cells have
characteristic and specific mechanisms for the translational and
post-translational processing and modification (e.g.,
glycosylation, cleavage [e.g., of signal sequence]) of proteins.
Appropriate cell lines or host systems can be chosen to ensure the
desired modification and processing of the foreign protein
expressed, such as glycosylation, sialyation and phosphorylation.
For example, expression in a bacterial system can be used to
produce an nonglycosylated core protein product. However, protein
expressed in bacteria may not be properly folded. Expression in
yeast can produce a glycosylated product. Expression in eukaryotic
cells can increase the likelihood of "native" glycosylation and
folding of a heterologous protein. Moreover, expression in
mammalian cells can provide a tool for reconstituting, or
constituting, protein. Furthermore, different vector/host
expression systems may affect processing reactions, such as
proteolytic cleavages, to a different extent. The expression
efficiency can be increased by use of a specific chaperone, as
described in U.S. Pat. No. 6,274,597, and related family members
disclosed above.
[0076] Purification of recombinantly expressed protein can be
achieved using methods known in the art such as by ammonium sulfate
precipitation, column chromatography containing hydrophobic
interaction resins, cation exchange resins, anion exchange resins,
and chromatofocusing resins. Alternatively, imunoaffinity
chromatography can be used to purify the recombinant protein using
an appropriate polyclonal or monoclonal antibody that binds
specifically to the protein, or to a tag that is fused to the
recombinant protein. In a preferred embodiment, the purity of the
recombinant protein used for the method of the present invention
with be at least 95%, preferably 97% and most preferably, greater
than 98%.
Replacement Protein Administration
[0077] Numerous methods can be employed to achieve uptake and
targeting of the replacement protein by the cells. Peptide
sequences have been identified that mediate membrane transport, and
accordingly provide for delivery of polypeptides to the cytoplasm.
For example, such peptides can be derived from the Antennapedia
homeodomain helix 3 to generate membrane transport vectors, such as
penetratin (PCT Publication WO 00/29427; see also Fischer et al.,
J. Pept. Res. 2000; 55:163-72; DeRossi et al., Trends in Cell Biol.
1998; 8:84-7; Brugidou et al., Biochem. Biophys. Res. Comm. 1995;
214:685-93), the VP22 protein from herpes simplex virus (Phelan et
al., Nat. Biotechnol. 1998; 16:440-3), and the HIV TAT
trascriptional activator. Protein transduction domains, including
the Antennapedia domain and the HIV TAT domain (see Vives et al.,
J. Biol. Chem. 1997; 272:16010-17), possess a characteristic
positive charge, which led to the development of cationic 12-mer
peptides that can be used to transfer therapeutic proteins and DNA
into cells (Mi et al., Mol. Therapy 2000; 2:339-47). The
above-mentioned protein transduction domains are covalently linked
to the target protein, either by chemical covalent cross-linking or
generation as a fusion protein. Further, a non-covalent, synthetic
protein transduction domain has been recently developed by Active
Motif Inc. (Carlsbad, Calif.). This domain associates with the
target protein through hydrophobic interactions, and advantageously
dissociates from the protein once inside the cell (Morris et al.,
Nat. Biotechnol. 2001; 19:1173-6). In addition, lipid carriers have
recently been shown to deliver proteins into cells in addition to
an established use for delivering naked DNA (Zelphati et al., J.
Biol. Chem. 2001; 276:35103-10). For an overview of protein
translocation techniques see Bonetta, The Scientist 2002;
16(7):38.
[0078] In specific embodiments, the replacement proteins used in
the method of the present invention are enzymes associated with
lysosomal storage disorders (see Table 1). Sequences of nucleic
acids encoding wild-type versions of such enzymes can be found in
the literature or in public databases such as GenBank, e.g., X14448
for .alpha.-Gal A (AGA), J03059 for human glucocerebrosidase (GCB),
M74715 for human .alpha.-L-iduronidase (IDUA), M34424 for human
acid .alpha.-glucosidase (GAA), AF011889 for human iduronate
2-sulfatase (IDS), and M59916 for human acid sphingomyelinase
(ASM).
[0079] Enzyme replacement in LSDs. Several replacement enzymes for
LSDs are currently available in Europe and the U.S. These include
Cerezyme.RTM., recombinant form of glucerebrosidase for the
treatment of Gaucher disease; Fabrazyme.RTM., recombinant form of
alpha galactosidase A; Aldurazyme.TM., a recombinant enzyme for the
treatment of MPS1, all from Genzyme Corp. and recombinant alpha
glucosidase for patients with Pompe disease (Van den Hout et al.,
Lancet 2000; 56:397-8).
Active Site-Specific Chaperones
[0080] ASSC's contemplated by the present invention include but are
not limited to small molecules (e.g., organic or inorganic
molecules which are less than about 2 kD in molecular weight, are
more preferably less than about 1 kD in molecular weight),
including substrate or binding partner mimetics; small
ligand-derived peptides or mimetics thereof; nucleic acids such as
DNA, RNA; antibodies, including Fv and single chain antibodies, and
Fab fragments; macromolecules (e.g., molecules greater than about 2
kD in molecular weight) and members of libraries derived by
combinatorial chemistry, such as molecular libraries of D- and/or
L-configuration amino acids; phosphopeptides, such as members of
random or partially degenerate, directed phosphopeptide libraries
(see, e.g., Songyang et al., Cell 1993; 72:767-778).
[0081] Synthetic libraries (Needels et al., Proc. Natl. Acad. Sci.
USA 1993; 90:10700-4; Ohlmeyer et al., Proc. Natl. Acad. Sci. USA
1993; 90:10922-10926; Lam et al., PCT Publication No. WO 92/00252;
Kocis et al., PCT Publication No. WO 94/28028) provide a source of
potential ASSC's according to the present invention. Synthetic
compound libraries are commercially available from Maybridge
Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.),
Brandon Associates (Merrimack, N.H.), and Microsource (New Milford,
Conn.). A rare chemical library is available from Aldrich
(Milwaukee, Wis.). Alternatively, libraries of natural compounds in
the form of bacterial, fungal, plant and animal extracts are
available from e.g. Pan Laboratories (Bothell, Wash.) or MycoSearch
(NC), or are readily producible. Additionally, natural and
synthetically produced libraries and compounds are readily modified
through Res. 1986; 155:119-29.
[0082] In a preferred embodiment, ASSC's useful for the present
invention are inhibitors of lysosomal enzymes and include glucose
and galactose imino-sugar derivatives as described in Asano et al.,
J. Med. Chem 1994; 37:3701-06; Dale et al., Biochemistry 1985;
24:3530-39; Goldman et al., J. Nat. Prod. 1996; 59:1137-42; Legler
et al, Carbohydrate Res. 1986; 155:119-29. Such derivatives include
but are not limited those compound listed in Table 1. Some of these
compounds can be purchased from commercial sources such as Toronto
Research Chemicals, Inc. (North York, On. Canada) and Sigma.
[0083] In a preferred embodiment, ASSC's useful for the present
invention are activators of cystic fibrosis transmembrane
conductance regulator (CFTR) which include benzo(c)quinolizinium
compounds as described in Dormer et al., J. Cell Sci. 2001; 114:
4073-81; and Ma et al., J. Biol. Chem. 2002; 277: 37235-41.
[0084] In another preferred embodiment, ASSC's useful for the
present invention are ligands of G protein-coupled receptors, such
as z,900 opioid receptor, V2 vasopressin receptor, and photopigment
rhodopsin, as described in Petaja-Repo et al., EMBO J 2002; 21:
1628-37; Morello et al., J. Clin. Invest. 2000; 105: 887-95; Saliba
et al., J. Cell Sci. 2002; 115: 2907-18.
[0085] In another preferred embodiment, ASSC's useful for the
present invention are compounds that stabilize the DNA binding
domain of p53, as described in Foster et al., Science 1999; 286:
2507-10; Friedler et al., PNAS 2002; 99: 937-42.
[0086] In yet another preferred embodiment, ASSC's useful for the
present invention are blockers of ion channel proteins, such as
HERG potassium channel in human Long QT syndrome, pancereatic
ATP-sensitive potassium (K.sub.ATP) channel in familial
hyperinsulinism, as described in Zhou et al., J Biol. Chem. 1999;
274: 31123-26; Taschenberger et al., J. Biol. Chem. 2002; 277:
17139-46.
FORMULATIONS
[0087] In one embodiment, the ASSC and replacement protein are
formulated in a single composition. Such a composition enhances
stability of the protein during storage and in vivo administration,
thereby increasing therapeutic efficacy. The formulation is
preferably suitable for parenteral administration, including
intravenous subcutaneous, and intraperitoneal, however,
formulations suitable for other routes of administration such as
oral, intranasal, or transdermal are also contemplated.
[0088] In another embodiment, the replacement protein and the
ASSC's are formulated in separate compositions. In this embodiment,
the chaperone and the replacement protein may be administered
according to the same route, e.g., intravenous infusion, or
different routes, e.g., intravenous infusion for the replacement
protein, and oral administration for the ASSC. The pharmaceutical
formulations suitable for injectable use include sterile aqueous
solutions (where water soluble) or dispersions and sterile powders
for the extemporaneous preparation of sterile injectable solutions
or dispersion. In all cases, the form must be sterile and must be
fluid to the extent that easy syringability exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and polyethylene glycol, and
the like), suitable mixtures thereof, and vegetable oils. The
proper fluidity can be maintained, for example, by the use of a
coating such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of
surfactants. The preventions of the action of microorganisms can be
brought about by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, benzyl alchohol, sorbic
acid, and the like. In many cases, it will be preferable to include
isotonic agents, for example, sugars or sodium chloride. Prolonged
absorption of the injectable compositions can be brought about by
the use in the compositions of agents delaying absorption, for
example, aluminum monosterate and gelatin.
[0089] Sterile injectable solutions are prepared by incorporating
the purified protein and ASSC in the required amount in the
appropriate solvent with various of the other ingredients
enumerated above, as required, followed by filter or terminal
sterilization. Generally, dispersions are prepared by incorporating
the various sterilized active ingredients into a sterile vehicle
which contains the basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and the
freeze-drying technique which yield a powder of the active
ingredient plus any additional desired ingredient from previously
sterile-filtered solution thereof.
[0090] Preferably the formulation contains an excipient.
Pharmaceutically acceptable excipients which may be included in the
formulation are buffers such as citrate buffer, phosphate buffer,
acetate buffer, and bicarbonate buffer, amino acids, urea,
alcohols, ascorbic acid, phospholipids; proteins, such as serum
albumin, collagen, and gelatin; salts such as EDTA or EGTA, and
sodium chloride; liposomes; polyvinylpyrollidone; sugars, such as
dextran, mannitol, sorbitol, and glycerol; propylene glycol and
polyethylene glycol (e.g., PEG-4000, PEG-6000); glycerol; glycine
or other amino acids; and lipids. Buffer systems for use with the
formulations include citrate; acetate; bicarbonate; and phosphate
buffers. Phosphate buffer is a preferred embodiment.
[0091] The formulation also preferably contains a non-ionic
detergent. Preferred non-ionic detergents include Polysorbate 20,
Polysorbate 80, Triton X-100, Triton X-114, Nonidet P-40, Octyl
.alpha.-glucoside, Octyl .beta.-glucoside, Brij 35, Pluronic, and
Tween 20.
[0092] For lyophilization of protein and chaperone preparations,
the protein concentration can be 0.1-10 mg/mL. Bulking agents, such
as glycine, mannitol, albumin, and dextran, can be added to the
lyophilization mixture. In addition, possible cryoprotectants, such
as disaccharides, amino acids, and PEG, can be added to the
lyophilization mixture. Any of the buffers, excipients, and
detergents listed above, can also be added.
[0093] Formulations for inhalation administration may contain
lactose or other excipients, or may be aqueous solutions which may
contain polyoxyethylene-9-lauryl ether, glycocholate or
deoxycocholate. A preferred inhalation aerosol is characterized by
having particles of small mass density and large size. Particles
with mass densities less than 0.4 gram per cubic centimeter and
mean diameters exceeding 5 .mu.m efficiently deliver inhaled
therapeutics into the systemic circulation. Such particles are
inspired deep into the lungs and escape the lungs' natural
clearance mechanisms until the inhaled particles deliver their
therapeutic payload. (Edwards et al., Science 1997; 276:
1868-1872). Replacement protein preparations of the present
invention can be administered in aerosolized form, for example by
using methods of preparation and formulations as described in, U.S.
Pat. Nos. 5,654,007, 5,780,014, and 5,814,607, each incorporated
herein by reference. Formulation for intranasal administration may
include oily solutions for administration in the form of nasal
drops, or as a gel to be applied intranasally.
[0094] Formulations for topical administration to the skin surface
may be prepared by dispersing the composition with a dermatological
acceptable carrier such as a lotion, cream, ointment, or soap.
Particularly useful are carriers capable of forming a film or layer
over the skin to localize application and inhibit removal. For
topical administration to internal tissue surfaces, the composition
may be dispersed in a liquid tissue adhesive or other substance
known to enhance adsorption to a tissue surface. Alternatively,
tissue-coating solutions, such as pectin-containing formulations
may be used.
[0095] In preferred embodiments, the formulations of the invention
are supplied in either liquid or powdered formulations in devices
which conveniently administer a predetermined dose of the
preparation; examples of such devices include a needle-less
injector for either subcutaneous or intramuscular injection, and a
metered aerosol delivery device. In other instances, the
preparation may be supplied in a form suitable for sustained
release, such as in a patch or dressing to be applied to the skin
for transdermal administration, or via erodable devices for
transmucosal administration. In instances where the formulation,
e.g., the ASSC is orally administered in tablet or capsule form,
the preparation might be supplied in a bottle with a removable
cover or as blister patches.
[0096] In vitro stability. Ensuring the stability of a
pharmaceutical formulation during its shelf life is a major
challenge. Prior to development of a protein pharmaceutical,
inherent or latent instabilities within the active ingredients must
be explored and addressed. Instability of protein and peptide
therapeutics is classified as chemical instability or physical
instability. Examples of chemical instability are hydrolysis,
oxidation and deamidation. Examples of physical instability are
aggregation, precipitation and adsorption to surfaces. In addition,
a protein may be subjected to stresses such as pH, temperature,
shear stress, freeze/thaw stress and combinations of these
stresses.
[0097] One of the most prevalent formulation problems is product
aggregation, resulting in a loss in bioactivity. The addition of
excipients may slow the process but may not completely prevent it.
Activity losses may or may not be detected by physical assays and
are only evident in bioassays or potency assays with large
(sometimes 15-20%) coefficients of variation, making it difficult
to determine actual losses.
[0098] ASSC have been shown to enhance enzyme activity by
preventing degradation of enzymes and aggregation of enzyme
proteins (Fan et al., Nat. Med. 1999; 5: 112-5; FIG. 1). In the
embodiment where the ASSC and the replacement protein are in the
same composition, the formulated compositions of the invention may
be provided in containers suitable for maintaining sterility, and
importantly, protecting the activity of the replacement protein
during proper distribution and storage. In addition to stabilizing
the administered protein in vivo, the ASSC reversibly binds to and
stabilizes the conformation of the replacement protein in vitro,
thereby preventing aggregation and degradation, and extending the
shelf-life of the formulation. Analysis of the ASSC/replacement
protein interaction may be evaluated using techniques well-known in
the art, such as, for example, differential scanning calorimetry,
or circular dichroism.
[0099] For example, where an aqueous injectable formulation of the
composition is supplied in a stoppered vial suitable for withdrawal
of the contents using a needle and syringe, the presence of an ASSC
inhibits aggregation of the replacement protein. The vial could be
for either single use or multiple uses. The formulation can also be
supplied as a prefilled syringe. In another embodiment, the
formulation is in a dry or lyophilized state, which would require
reconstitution with a standard or a supplied, physiological diluent
to a liquid state. In this instance, the presence of an ASSC would
stabilize the replacement protein during and post-reconstitution to
prevent aggregation. In the embodiment where the formulation is a
liquid for intravenous administration, such as in a sterile bag for
connection to an intravenous administration line or catheter, the
presence of an ASSC would confer the same benefit.
[0100] In addition to stabilizing the replacement protein to be
administered, the presence of an ASSC may enable the pharmaceutical
formulation to be stored at a neutral pH of about 7.0-7.5. This
will confer a benefit to proteins that normally must be stored at a
lower pH to preserve stability. For example, lysosomal enzymes,
such as those listed in Table 1, retain a stable conformation at a
low pH (e.g., 5.0 or lower). However, extended storage of the
replacement enzyme at a low pH may expedite degradation of the
enzyme and/or formulation.
[0101] Separate formulations. Where the replacement enzyme and ASSC
are in separate formulations, the ASSC can be in a form suitable
for any route of administration, including all of the forms
described above, e.g., as sterile aqueous solution or in a dry
lyophilized powder to be added to the formulation of the
replacement protein during or immediately after reconstitution to
prevent aggregation in vitro prior to administration.
Alternatively, the ASSC can be formulated for oral administration
in the form of tablets or capsules prepared by conventional means
with pharmaceutically acceptable excipients such as binding agents
(e.g., pregelatinized maize starch, polyvinylpyrrolidone or
hydroxypropyl methylcellulose); fillers (e.g., lactose,
microcrystalline cellulose or calcium hydrogen phosphate);
lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well known in the art. Liquid preparations for
oral administration may take the form of, for example, solutions,
syrups or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate. Preparations for oral administration may be
suitably formulated to give controlled release of the active
compound.
ADMINISTRATION
[0102] The route of administration may be oral or parenteral,
including intravenous, subcutaneous, intra-arterial,
intraperitoneal, ophthalmic, intramuscular, buccal, rectal,
vaginal, intraorbital, intracerebral, intradermal, intracranial,
intraspinal, intraventricular, intrathecal, intracisternal,
intracapsular, intrapulmo nary, intranasal, transmucosal,
transdermal, or via inhalation.
[0103] Administration of the above-described parenteral
formulations may be by periodic injections of a bolus of the
preparation, or may be administered by intravenous or
intraperitoneal administration from a reservoir which is external
(e.g., an i.v. bag) or internal (e.g., a bioerodable implant, a
bioartificial organ, or a population of implanted cells that
produce the replacement protein). See, e.g., U.S. Pat. Nos.
4,407,957 and 5,798,113, each incorporated herein by reference.
Intrapulmonary delivery methods and apparatus are described, for
example, in U.S. Pat. Nos. 5,654,007, 5,780,014, and 5,814,607,
each incorporated herein by reference. Other useful parenteral
delivery systems include ethylene-vinyl acetate copolymer
particles, osmotic pumps, implantable infusion systems, pump
delivery, encapsulated cell delivery, liposomal delivery,
needle-delivered injection, needle-less injection, nebulizer,
aeorosolizer, electroporation, and transdermal patch. Needle-less
injector devices are described in U.S. Pat. Nos. 5,879,327;
5,520,639; 5,846,233 and 5,704,911, the specifications of which are
herein incorporated by reference. Any of the formulations described
above can administered in these methods.
[0104] Subcutaneous injections the replacement protein and/or ASSC
have the advantages allowing self-administration, while also
resulting in a prolonged plasma half-life as compared to
intravenous administration. Furthermore, a variety of devices
designed for patient convenience, such as refillable injection pens
and needle-less injection devices, may be used with the
formulations of the present invention as discussed herein.
[0105] Timing. When the replacement protein and ASSC are in
separate formulations, administration may be simultaneous, or the
ASSC may be administered prior to, or after the replacement
protein. For example, where the replacement protein is administered
intravenously, the ASSC may be administered during a period from 0
h to 6 h later. Alternatively, the chaperone may be administered
from 0 to 6 h prior to the protein.
[0106] In a preferred embodiment, where the ASSC and replacement
protein are administered separately, and where the ASSC has a short
circulating half-life (e.g., small molecule), the ASSC may be
orally administered continuously, such as daily, in order to
maintain a constant level in the circulation. Such constant level
will be one that has been determined to be non-toxic to the
patient, and optimal regarding interaction with a target
replacement protein during the time of administration to confer a
non-inhibitory, therapeutic effect.
[0107] In another embodiment, the ASSC is administered during the
time period required for turnover of the replacement protein (which
will be extended by administration of the ASSC).
[0108] Regardless of the timing, the administration must be such
that the concentrations of the protein and ASSC must be such that
the chaperone stabilizes, but does not prevent or inhibit the
protein's activity in vivo. This also applies where the replacement
protein and ASSC are administered in the same formulation.
[0109] In vivo stability. As described above for the in vitro
formulations, the presence of an ASSC for the replacement protein
will have the benefit of prolonging in plasma the half-life,
thereby maintaining effective replacement protein levels over
longer time periods, resulting in increased exposure of clinically
affected tissues to the replacement protein and, thus, increased
uptake of protein into the tissues. This confers such beneficial
effects to the patient as enhanced relief, reduction in the
frequency, and/or reduction in the amount administered. This will
also reduce the cost of treatment.
[0110] In addition to stabilizing wild-type replacement proteins,
the ASSC will also stabilize and enhance expression of endogenous
mutant proteins that are deficient as a result of mutations that
prevent proper folding and processing in the ER, as in
conformational disorders such as the LSDs.
Dosages
[0111] The amount of ASSC effective to stabilize the administered
protein and endogenous mutant protein can be determined on a
case-by-case basis, depending on the protein and corresponding
ASSC, by those skilled in the art. Pharmacokinetics and
pharmacodynamics such as half-life (t.sub.1/2), peak plasma
concentration (c.sub.max), time to peak plasma concentration
(t.sub.max), exposure as measured by area under the curve (AUC),
and tissue distribution for both the replacement protein and the
ASSC, as well as data for ASSC-replacement protein binding
(affinity constants, association and dissociation constants, and
valency), can be obtained using ordinary methods known in the art
to determine compatible amounts required to stabilize the
replacement protein, without inhibiting its activity, and thus
confer a therapeutic effect.
[0112] Data obtained from cell culture assay or animal studies may
be used to formulate a therapeutic dosage range for use in humans
and non-human animals. The dosage of compounds used in therapeutic
methods of the present invention preferably lie within a range of
circulating concentrations that includes the ED50 concentration
(effective for 50% of the tested population) but with little or no
toxicity. The particular dosage used in any treatment may vary
within this range, depending upon factors such as the particular
dosage form employed, the route of administration utilized, the
conditions of the individual (e.g., patient), and so forth.
[0113] A therapeutically effective dose may be initially estimated
from cell culture assays and formulated in animal models to achieve
a circulating concentration range that includes the IC.sub.50,
[0114] The IC.sub.50 concentration of a compound is the
concentration that achieves a half-maximal inhibition of symptoms
(e.g., as determined from the cell culture assays). Appropriate
dosages for use in a particular individual, for example in human
patients, may then be more accurately determined using such
information.
[0115] Measures of compounds in plasma may be routinely measured in
an individual such as a patient by techniques such as high
performance liquid chromatography (HPLC) or gas chromatography.
[0116] Toxicity and therapeutic efficacy of the composition can be
determined by standard pharmaceutical procedures, for example in
cell culture assays or using experimental animals to determine the
LD.sub.50 and the ED.sub.50. The parameters LD.sub.50 and ED.sub.50
are well known in the art, and refer to the doses of a compound
that is lethal to 50% of a population and therapeutically effective
in 50% of a population, respectively. The dose ratio between toxic
and therapeutic effects is referred to as the therapeutic index and
may be expressed as the ratio: LD.sub.50/ED.sub.50. ASSCs that
exhibit large therapeutic indices are preferred.
[0117] According to current methods, the concentration of
replacement protein is between 0.05-5.0 mg/kg of body weight,
typically administered weekly or biweekly. The protein can be
administered at a dosage ranging from 0.1 .mu.g/kg to about 10
mg/kg, preferably from about 0.1 mg/kg to about 2 mg/kg. For
example, for the treatment of Fabry disease, the dose of
recombinant .alpha.-Gal A administrated is typically between
0.1-0.3 mg/kg and is administered weekly or biweekly. Regularly
repeated doses of the protein are necessary over the life of the
patient. Subcutaneous injections maintain longer term systemic
exposure to the drug. The subcutaneous dosage is preferably 0.1-5.0
mg of the .alpha.-Gal A per kg body weight biweekly or weekly. The
.alpha.-Gal A is also administered intravenously, e.g., in an
intravenous bolus injection, in a slow push intravenous injection,
or by continuous intravenous injection. Continuous IV infusion
(e.g., over 2-6 hours) allows the maintenance of specific levels in
the blood.
[0118] The optimal concentrations of the ASSC will be determined
according to the amount required to stabilize the recombinant
protein in vivo, in tissue or circulation, without preventing its
activity, bioavailability of the ASSC in tissue or in circulation,
and metabolism of the ASSC in tissue or in circulation. For
example, where the ASSC is an enzyme inhibitor, the concentration
of the inhibitor can be determined by calculating the IC.sub.50
value of the specific chaperone for the enzyme. Taking into
consideration bioavailability and metabolism of the compound,
concentrations around the IC.sub.50 value or slightly over the
IC.sub.50 value can then be evaluated based on effects on enzyme
activity, e.g., the amount of inhibitor needed to increase the
amount of enzyme activity or prolong enzyme activity of the
administered enzyme. As an example, the IC.sub.50 value of the
compound deoxygalactonojiromycin (DGJ) for the .alpha.-Gal A enzyme
is 0.04 /.mu.M, indicating that DGJ is a potent inhibitor.
Accordingly, it is expected that the intracellular concentration of
.alpha.-Gal A would be much lower than that of the .alpha.-Gal A
administered. See Examples below.
EXAMPLES
[0119] The present invention is further described by means of the
examples, presented below. The use of such examples is illustrative
only and in no way limits the scope and meaning of the invention or
of any exemplified term. Likewise, the invention is not limited to
any particular preferred embodiments described herein. Indeed, many
modifications and variations of the invention will be apparent to
those skilled in the art upon reading this specification and can be
made without departing from its spirit and scope. The invention is
therefore to be limited only by the terms of the appended claims
along with the full scope of equivalents to which the claims are
entitled.
Example 1
In Vitro Stabilization of (.alpha.-Gal A With ASSCs
[0120] Methods. The wild type .alpha.-Gal A was purified from
culture medium of Sf-9 cells infected with recombinant baculovirus
carrying human wild type .alpha.-Gal A cDNA and the mutant
.alpha.-Gal A was collected as homogenates of hearts of transgenic
mice overexpressing human mutant (R301Q) .alpha.-Gal A. The mice
were treated with 0.5 mM DGJ as drinking water for one week prior
to the experiment. The mutant and wild type enzymes were
pre-incubated with 0.1 M citrate-phosphate buffer (pH 7.0) at
37.degree. C. for the mutant enzyme and 42.degree. C. for the wild
type enzyme, respectively, in the presence of DGJ at a
concentration of 1 .mu.M, 0.1 .mu.M, 0.03 .mu.M or no DGJ. The wild
type and mutant (R301Q) .alpha.-Gal A were incubated for a period
of time in the absence or presence of DGJ (various concentrations),
and the remaining enzyme activity was determined with
4-MU-.alpha.-Gal A as a substrate, after diluting the mixture with
5-volume of 0.1 M citrate buffer (pH 4.5). Enzyme activity is
reported relative to the enzyme without pre-incubation.
[0121] Results. As shown in FIG. 1, the mutant enzyme was not
stable at neutral pH after incubation at 37.degree. C. for 20 min
without incubation with DGJ (FIG. 1A). The wild type enzyme also
lost significant enzyme activity at neutral pH at 42.degree. C.
without incubation with DGJ (FIG. 1B). The stability of both
enzymes can be improved by inclusion of DGJ at 1 .mu.M
concentration, i.e., more than 80% of enzyme activity was remained
in the reaction mixture for 60 min. This indicates that the ASSC
(DGJ) can serve as a stabilizer to prevent the
denaturation/degradation of the mutant and wild type enzymes.
Example 2
Intracellular Enhancement of Wild-Type .alpha.-Gal A With ASSCs
[0122] Methods. Human wild type .alpha.-Gal A purified from insect
cells transfected with recombinant baculovirus or from recombinant
CHO cells can be conjugated to .alpha.-2-macroglobulin
(.alpha.-2-M), according to the previous reference (Osada et al.,
Biochem Biophys Res Commun. 1993; 142: 100-6). Since the conjugate
of .alpha.-Gal A from coffee beans and .alpha.-2-M can be
internalized by cultured fibroblasts derived from Fabry
hemizygotes, the conjugate of .alpha.-Gal A and .alpha.-2-M is
expected to be internalized by the cells as well. Alternatively,
the wild type .alpha.-Gal A can be added into the culture medium of
skin fibroblasts derived from Fabry patient with no residual enzyme
activity as described in Blom et al., Am J Hum Gen. 2003; 72:
23-31.
[0123] Results. The half-life of the coffee bean .alpha.-Gal A is
about 2 hr as described previously (Osada et al., Biochem Biophys
Res Commun. 1987;143: 954-8). It is expected that the half-life of
the .alpha.-Gal A/.alpha.-2-M conjugate or .alpha.-Gal A added into
the culture medium can be extended by inclusion of DGJ into the
culture medium, since the DGJ has been shown to be effective in
stabilize the enzyme in vitro (FIG. 1). This will indicate that the
DGJ can prolong the exogenous .alpha.-Gal A taken up by the cells
intracellularly.
Example 3
Co-Administration of DGJ to Fabry Mice Treated by Infusion of
Replacement Enzyme
[0124] Enzyme replacement therapy for Fabry disease has been
developed by Genzyme Corporation as described above. It is expected
that co-administration of DGJ to Fabry knock-out (KO) mice treated
by infusion of the replacement enzyme increases the stability,
e.g., half-life of the replacement enzyme in vivo, because the ASSC
DGJ stabilizes the enzyme and prevents degradation. DGJ is orally
administered to the KO mice after infusion of the wild type
.alpha.-Gal A according to the protocol described previously
(Ioannu et al., Am J Hum Genet. 2001; 68:14-25). The .alpha.-Gal A
activity in various tissues including heart, kidney, spleen, liver,
and lung as well as serum is determined over a period of time, and
compared with those from the control mice that do not receive DGJ,
and mice that receive only DGJ but no enzyme. The extended time
will indicate that co-administration of ASSC can improve the
efficiency of enzyme replacement therapy.
[0125] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
[0126] Patents, patent applications, publications, procedures, and
the like are cited throughout this application, the disclosures of
which are incorporated herein by reference in their entireties.
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