U.S. patent application number 17/549816 was filed with the patent office on 2022-04-07 for method of treating glycogen storage disease.
This patent application is currently assigned to Duke University. The applicant listed for this patent is Duke University. Invention is credited to Yuan-Tsong Chen, Priya KISHNANI, Baodong SUN.
Application Number | 20220106579 17/549816 |
Document ID | / |
Family ID | |
Filed Date | 2022-04-07 |
United States Patent
Application |
20220106579 |
Kind Code |
A1 |
Chen; Yuan-Tsong ; et
al. |
April 7, 2022 |
Method of Treating Glycogen Storage Disease
Abstract
The disclosure relates, in general, to Glycogen Storage Disease
and, in particular, to a method of treating Glycogen Storage
Disease and to compounds and compositions suitable for use in such
a method.
Inventors: |
Chen; Yuan-Tsong; (Durham,
NC) ; KISHNANI; Priya; (Durham, NC) ; SUN;
Baodong; (Durham, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Duke University |
Durham |
NC |
US |
|
|
Assignee: |
Duke University
Durham
NC
|
Appl. No.: |
17/549816 |
Filed: |
December 13, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16843436 |
Apr 8, 2020 |
11214782 |
|
|
17549816 |
|
|
|
|
15659549 |
Jul 25, 2017 |
10647969 |
|
|
16843436 |
|
|
|
|
14701470 |
Apr 30, 2015 |
9850474 |
|
|
15659549 |
|
|
|
|
12737394 |
Jan 7, 2011 |
9050333 |
|
|
PCT/US2009/003993 |
Jul 8, 2009 |
|
|
|
14701470 |
|
|
|
|
61129612 |
Jul 8, 2008 |
|
|
|
International
Class: |
C12N 9/26 20060101
C12N009/26; A61K 38/47 20060101 A61K038/47 |
Claims
1-18. (canceled)
19. A method of treating glycogen storage disease type XI or
cardiac glycogenosis, the method comprising: administering to a
human in need thereof a composition comprising acid
.alpha.-glucosidase.
20. The method of claim 19, wherein the amount of the acid
.alpha.-glucosidase administered is from about 1 mg to about 40 mg
of acid .alpha.-glucosidase per kilogram of body weight.
21. The method of claim 19, wherein the acid .alpha.-glucosidase is
a recombinant acid .alpha.-glucosidase, a precursor of recombinant
acid .alpha.-glucosidase, or a combination thereof.
22. The method of claim 19, wherein the acid .alpha.-glucosidase is
administered intravenously.
23. The method of claim 19, wherein the acid .alpha.-glucosidase is
administered intrathecally.
24. The method of claim 19, further comprising administering an
immunosuppressant, an immunotherapeutic agent, or a combination
thereof, concurrently, or sequentially.
25. The method of claim 19, wherein the acid .alpha.-glucosidase is
administered daily.
26. The method of claim 19, wherein the acid .alpha.-glucosidase is
administered weekly.
27. The method of claim 19, wherein the acid .alpha.-glucosidase is
administered twice weekly.
28. The method of claim 19, wherein the acid .alpha.-glucosidase is
administered monthly.
29. The method of claim 19, wherein the acid .alpha.-glucosidase is
administered bi-monthly.
30. The method of claim 19, wherein the acid .alpha.-glucosidase is
administered orally, intramuscularly, intraventricularly, or a
combination thereof.
31. The method of claim 21, wherein the recombinant acid
.alpha.-glucosidase or the precursor of recombinant acid
.alpha.-glucosidase is produced in Chinese hamster ovary cells.
32. The method of claim 19, wherein the acid .alpha.-glucosidase is
human.
33. A method of treating an individual who has been diagnosed as
having glycogen storage disease type XI or cardiac glycogenosis,
the method comprising: administering by injection to an individual
following diagnosis of glycogen storage disease type XI or cardiac
glycogenosis of human acid .alpha.-glucosidase at a regular
interval sufficient to effect the treatment.
34. The method of claim 33, wherein the regular interval is
daily.
35. The method of claim 33, wherein the regular interval is
weekly.
36. The method of claim 33, wherein the regular interval is twice
weekly.
37. The method of claim 33, wherein the regular interval is
monthly.
38. The method of claim 33, wherein the regular interval is
bi-monthly.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 16/843,436, filed on Apr. 8, 2020, which is a divisional of
U.S. application Ser. No. 15/659,549, filed on Jul. 25, 2017 (now
U.S. Pat. No. 10,647,969), which is a divisional of U.S.
application Ser. No. 14/701,470, filed on Apr. 30, 2015 (now U.S.
Pat. No. 9,850,474), which is a divisional of U.S. application Ser.
No. 12/737,394, filed on Jan. 7, 2011 (now U.S. Pat. No.
9,050,333), which is the U.S. national phase of International
Application No. PCT/US2009/003993, filed on Jul. 8, 2009, which
designated the U.S. and claims priority from U.S. Provisional
Application No. 61/129,612, filed on Jul. 8, 2008, the entire
contents of each application of which are incorporated herein by
reference.
REFERENCE TO THE SEQUENCE LISTING
[0002] The Sequence Listing submitted 13 Dec. 2021 as a .txt file
named "15_667_DIV_CON_SEQUENCE_LISTING", created on 13 Dec. 2021
and having a size of 9 kilobytes, is hereby incorporated by
reference pursuant to 37 C.F.R. .sctn. 1.52(e)(5).
TECHNICAL FIELD
[0003] The present invention relates, in general, to Glycogen
Storage Disease and, in particular, to a method of treating
Glycogen Storage Disease-type-III and to compounds and compositions
suitable for use in such a method.
BACKGROUND
[0004] Glycogen debranching enzyme (GDE) is a multifunctional
enzyme acting as 1,4-.alpha.-D-glucan: 1,4-.alpha.-D-glucan
4-.alpha.-D-glycosyltransferase (E.C 2.4.1.25) and
amylo-1,6-glucosidase (E.C 3.2.1.33) in glycogen degradation. The
two activities of the debranching enzyme are believed to reside at
separate sites on a single polypeptide chain with a molecular mass
of 174 kDa. The structure-function domain has not been studied in
detail (Chen and Burchell, Glycogen storage disease. In: The
Metabolic and Molecular Bases of Inherited Disease, C. R. Scriver
et al, eds., 7.sup.th Edition McGraw-Hill/New York, pp. 935-965
(1995); Bates et al, FEBS Lett. 58:181-185 (1975); Gillard et al,
Biochemistry 16:3978-3987 (1977); Chapter 71 Kishnani P S; Koeberl
D, Chen Y T. Glycogen Storage Diseases, in The Online Metabolic
& Molecular Bases of Inherited Disease, Valle D, Beaudet A L,
Vogelstein B, Kinzler K W, Antonarakis S E, Ballabio A, Scriver C
R, Sly W S, Childs B, Editors (2008)). The predominant form of cDNA
that encodes human debrancher has a 4596 bp coding region and a
2371 bp 3'nontranslated region (Yang et al, J. Biol. Chem.
267:9294-9299 (1992)). Tissue specific debrancher mRNAs exist.
These isoforms differ at the 5' nontranslated region and are
believed to be generated by differential RNA transcription and
splicing from a single debrancher gene (Bao et al, Gene 197:389-398
(1997)). The human gene is localized to chromosome 1p21 (Yang-Feng
et al, Genomics 13:931-934 (1992)). The genomic structure of the
human GDE gene has been determined and consists of 35 exons
spanning .about.85 kb of DNA.
[0005] Debranching enzyme, together with phosphorylase, is
responsible for complete degradation of glycogen. Liver and muscle
are the two major organs most active in glycogen metabolism. The
primary function of glycogen in these organs is different. In
muscle, glycogen provides a local fuel store for short-term energy
consumption. In liver, it maintains glucose homeostasis.
[0006] Genetic deficiency of glycogen debranching enzyme (Glycogen
Storage Disease-type III, GSD-III) causes an incomplete
glycogenolysis resulting in accumulation of glycogen with
abnormally short outer chain in various organs. The commonly
affected organs in GSD-III are liver, skeletal muscle and heart.
The disease is characterized by hepatomegaly, hypoglycemia, short
stature, variable myopathy and cardiomyopathy. Patients with this
disease vary remarkably, both clinically and enzymatically
(Markowitz et al, Gastroenterology 105:1882-1885 (1993); Shen et
al, J. Clin. Invest. 98:352-357 (1996); Telente et al, Annals.
Intern. Med. 120:218-226 (1994)). Most patients have disease
involving both liver and muscle (type IIIa), some patients (-15% of
all GSD-III patients) have only liver involvement (type IIIb), and,
in rare cases, there is a selective loss of only one of the two GDE
activities (glucosidase, (type IIIc) or transferase (type IIId)).
Liver symptoms in GSD-III can improve with age and may disappear
after puberty. Overt liver cirrhosis has been seen in some
patients, some have developed hepatocellular carcinoma. Muscle
weakness, though minimal during childhood, may become predominant
in adults with onset in the third or fourth decade. These patients
have slowly progressive proximal weakness and distal muscle wasting
and some patients become wheelchair bound. Even within the subgroup
of patients who develop myopathy/cardiomyopathy there is clinical
variability. Some patients have asymptomatic cardiomyopathy, some
have early symptomatic cardiomyopathy leading to death, and some
have only muscle and no apparent heart involvement. An abnormal
electrocardiogram (ECG) with ventricular hypertrophy is a frequent
finding and does not correlate with clinical severity. Normal serum
creatine kinase levels do not rule out muscle enzyme deficiency.
The biochemical subtypes do not predict clinical severity.
[0007] There appears to be no correlation between the amount of
debrancher protein and clinical severity (Yang et al, Am. J. Hum.
Genet. 41:A28 (1992)). To predict accurately at initial diagnosis
whether myopathy or cardiomyopathy may occur, one must determine
whether debranching enzyme activity is deficient in muscle (Chen
and Burchell, Glycogen storage disease. In: The Metabolic and
Molecular Bases of Inherited Disease, C. R. Scriver et al, eds.,
7.sup.th Edition McGraw-Hill/New York, pp. 935-965 (1995)). It
appears that muscle disease will not develop in patients with GDE
activity retained in muscle (Coleman et al, Annals of Internal
Medicine 116:896-900 (1992)).
[0008] The variable phenotype is, in part, explained by differences
in tissue-specific expression of the defective enzyme. As pointed
out above, in type IIIa, enzyme is deficient in both liver and
muscle, in Mb there is enzyme deficiency only in liver. Unlike
phosphorylase, which has tissue-specific isoenzymes encoded by
different genes, at the protein level and at the molecular level it
appears that there are no tissue-specific GDE isoenzymes in
different tissues. Until now, it has not been understood in GSD-III
how a single GDE gene, normally expressed in all tissues, can
change expression in different tissues (Chen and Burchell, Glycogen
storage disease. In: The Metabolic and Molecular Bases of Inherited
Disease, C. R. Scriver et al, eds., 7.sup.th Edition
McGraw-Hill/New York, pp. 935-965 (1995)). Two mutations (17delAG
and G6X), both located in exon 3 at amino acid codon 6, are
exclusively found in the GSD-IIIb (Shen et al, J. Clin. Invest.
98:352-357 (1996)) suggesting that exon 3 is important in
controlling tissue-specific expression of the GDE gene. Histology
of the liver in these patients is characterized by a universal
distension of hepatocytes by glycogen and the presence of fibrous
septa. Electron microscopy studies on muscle specimens have shown
presence of accumulated glycogen beneath the sarcolemma and between
myofibrils; the excess glycogen not only disperses in the
cytoplasm, but is also seen in the lysosomes (Cornelio et al, Arch.
Neurol. 41:1027-1032 (1984), Miranda et al, Ann. Neurol. 9:283-288
(1981)).
[0009] The detailed structural biology of GDE is not known,
although several functional domains of glycogen debranching enzyme
have been proposed from enzymological studies and sequence
comparison to other enzymes with similar catalytic function (Yang
et al, J. Biol. Chem. 267:929409299 (1992), Liu et al, Archives of
Biochemistry and Biophysics 306:232-239 (1993), Liu et al,
Biochemistry 34:7056-7061 (1995), Jespersen et al, Journal of
Protein Chemistry 12(6):791-805 (1993)). A region at the
COOH-terminal of the debranching enzyme could be a candidate for
glycogen binding site (Yang et al, J. Biol. Chem. 267:9294-9299
(1992)), and 4 regions at N-terminal half of the enzyme bear
sequence homology to the catalytic sites identified or proposed in
other amylolytic enzymes (Liu et al, Archives of Biochemistry and
Biophysics 306:232-239 (1993), Jespersen et al, Journal of Protein
Chemistry 12(6):791-805 (1993)). Aspartate at position 549 has been
identified as the catalytic nucleophile in the transferase site of
rabbit muscle glycogen debranching enzyme (Braun et al,
Biochemistry 35:5458-5463 (1996)).
[0010] Currently there is no effective treatment for the disease.
Hypoglycemia can be controlled by frequent meals high in
carbohydrates with cornstarch supplements or nocturnal gastric drip
feedings. Patients with myopathy have been given diets high in
protein during the daytime plus overnight enteral infusion. In some
patients, transient improvement in symptoms has been documented but
there are no long-term data demonstrating that the high protein
diet prevents or treats the progressive myopathy (Chen and
Burchell, Glycogen storage disease. In: The Metabolic and Molecular
Bases of Inherited Disease, C. R. Scriver et al, eds., 7.sup.th
Edition McGraw-Hill/New York, pp. 935-965 (1995)). The progressive
myopathy and/or cardiomyopathy is a major cause of morbidity in
adults and patients with progressive liver cirrhosis and hepatic
carcinoma have been reported. While gene therapy delivery of a
normal, functional gene into the diseased organ could ultimately
cure the disease, an ideal gene delivery vehicle that is reliable
is currently not available. There is no living animal model for
this disease. Dogs affected with GSD-III have been reported
(Gregory et al, J. Vet. Intern. Med. 21(1):40-46 (2007)), and a
breeding colony is currently being established.
[0011] Enzyme replacement therapy has been effective in diseases in
which the responsible enzymes/proteins exert their functions in
extracellular fluids, such as adenosine deaminase deficiency,
hemophilia, and al-antitrypsin deficiency, or in a lysosomal
location such as a lysosomal storage disease. Enzyme replacement
has not been explored in diseases in which the defective enzyme is
present in cytosol (such as the debranching enzyme in GSD-III),
presumably due to the lack of an efficient and specific cellular
uptake mechanism that delivers exogenous enzyme across the plasma
membrane into the cytoplasm. Liposomes can fuse with plasma
membrane and deliver their content, however, the use of liposomes
is compromised by lack of organ-specific tropism and clearance by
the reticulo-endothelial system (Mumtaz et al, Glycobiology
1(5):505-510 (1991)). For an effective treatment for GSD-III, the
enzyme should be able to target muscle and heart as well as
liver.
[0012] Cytoplasmic glycogen is normally digested by phosphorylase
and debranching enzyme; excess glycogen taken up by lysosomes
through autophagy can be digested by lysosomal acid
.alpha.-glucosidase (GAA). Deficiency of debranching enzyme
activity results in massive accumulation of glycogen having
abnormally short outer branches. The excess glycogen in GSD-III
resides not only in the cytoplasm but also in the lysosomes
(cytoplasm >lysosome) (Cornelio et al, Arch. Neurol.
41:1027-1032 (1984), Miranda et al, Ann. Neurol. 9:283-288 (1981)).
This suggests that the "normal" GAA activity in GSD-III may not be
sufficient to digest all the excess glycogen. GAA is a lysosomal
exo 1,4-.alpha.-D-glucosidase that hydrolyzes both a .alpha.-1,4
and .alpha.-1,6 linkages of glycogen and can completely digest
glycogen with and without abnormally short outer branches (Onodera
et al, J. Biochem. 116:7-11 (1994)). GAA thus acts on glycogen with
abnormally short outer branches such as accumulates in GSD-III. As
this glycogen in GSD-III accumulates both in cytoplasm and
lysosomes, providing more GAA may help to digest lysosomal glycogen
and hence also cytoplasmic glycogen. It is postulated that, as the
lysosomes are cleared of glycogen, glycogen from the cytoplasm
shuffles into them thus decreasing the total amount of accumulated
glycogen in GSD-III patients.
[0013] Deficiency of GAA causes Pompe disease (type II glycogen
storage disease), a fatal metabolic myopathy with accumulation of
glycogen in lysosome and cytoplasm (lysosome >cytoplasm)
(Hirschhorn, Glycogen Storage Disease Type II: Acid
.alpha.-glucosidase (Acid Maltase) Deficiency. In: The Metabolic
and Molecular Bases of Inherited Disease, C. R. Scriver et al,
eds., 7.sup.th Edition McGraw-Hill/New York, pp. 2443-2464 (1995)).
Enzyme replacement therapy with mannose-6-Phosphate (man-6-P)-rich
precursor recombinant human GAA results in efficient man-6-P
receptor mediated endocytosis of the enzyme followed by reduction
of both lysosomal and cytoplasmic glycogen in fibroblasts (Van Hove
et al, Proc. Natl. Acad. Sci. 93:65-70 (1996)). In vivo, this
enzyme targets heart and muscle as well as liver and spleen
following intravenous injection in animals and in human Pompe
patients. The rapid clearance of glycogen in Pompe fibroblasts when
cultured in glucose-free medium suggests a ready mobilization of
glycogen from both lysosomal and cytoplasmic compartments (Van Hove
et al, Proc. Natl. Acad. Sci. 93:65-70 (1996), DiMauro et al,
Pedatr. Res. 7:739-744 (1973)). It is contemplated that cytoplasmic
glycogen continuously shuffles through lysosomes by autophagy for
degradation.
[0014] The present invention results, at least in part, from the
realization that administered GAA can reduce lysosomal glycogen in
GSD-III patients and ultimately also reduce cytoplasmic glycogen.
Some of the administered GAA may also go directly into the cytosol
and reduce the glycogen. The invention provides a method of
treating GSD-III (as well as GSD-IV, --VI, --IX, XI and cardiac
glycogenosis due to AMP-activated protein kinase gamma subunit 2
deficiency) based on the use of GAA.
SUMMARY OF THE INVENTION
[0015] The present invention relates generally to GSD. More
specifically, the invention relates to methods of treating GSD-III
and to compounds and compositions suitable for use in such
methods.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1. The light microscopic appearance of the skeletal
muscle biopsy from a GSD-IIIa patient. The glycogen is present as
cytoplasmic lakes within myocytes. The glycogen is not membrane
bound within lysosomes but is free flowing within the
cytoplasm.
[0017] FIG. 2. Cytoplasmic glycogen forming lakes within myocyte
under EM in a patient with GSD-III.
[0018] FIG. 3. Lysosomal glycogen was seen within myocyte of a
patient with GSD-III.
[0019] FIGS. 4A and 4B. Pattern of glycogen accumulation in muscle
cells derived from a normal subject and 3 GSD-III patients.
Duplicate cultures were harvested at times indicated. Cellular GAA
activity (FIG. 4A) and glycogen content (FIG. 4B) were determined
in duplicates for each culture (mean+SD).
[0020] FIGS. 5A and 5B. Glycogen depletion in untreated muscle
cells by glucose starvation. GAA activity (FIG. 5A) and glycogen
content (FIG. 5B) were analyzed in GSD-III muscle cells after
48-hour culturing (from Day 13 to Day 15) in differentiation medium
with (w/) glucose or without (w/o) glucose. (*, p value <0.01;
**, p<0.001).
[0021] FIGS. 6A and 6B. rhGAA uptake and glycogen reduction in
GSD-III muscle cells after 48-hour treatment (from Day 13 to Day
15) with 100 .mu.g of rhGAA. Muscle cells from normal subject were
treated from Day 7 to Day 10. (*, p value <0.05; **, p<0.005;
***, p<0.001).
DETAILED DESCRIPTION OF THE INVENTION
[0022] The present invention relates to a method of treating GSD,
particularly GSD-III, by administering GAA to an individual
suffering from the disease. The invention also relates to the use
of the enzyme, GAA, in the manufacture of a medicament for the
treatment of GSD (e.g., GSD-III). As described herein, patients
suffering from, for example, GSD-III can be treated by
administering GAA on, for example, a regular basis. Patients thus
treated can be expected to demonstrate improvement of hypoglycemia,
hepatomegaly, hepatic function, cardiac status, and/Or muscular
strength, as well as a reduction of tissue glycogen levels.
[0023] The invention makes possible the treatment of GSD-III,
including GSD-type IIIa, type IIIb, type Inc or type IIId. The
invention also makes possible the treatment of other forms of GSD,
including, but not limited to, GSD-IV, --VI, --IX, XI and cardiac
glycogenosis due to AMP-activated protein kinase gamma subunit 2
deficiency.
[0024] The terms, "treat" and "treatment," as used herein, refer to
amelioration of one or more symptoms associated with the disease,
prevention or delay of the onset of one or more symptoms of the
disease, and/or lessening of the severity or frequency of one or
more symptoms of the disease. For example, treatment can refer to
improvement of hypoglycemia, growth retardation, hepatomegaly, and
hepatic function (e.g., reduction of SGOT, SGPT); cardiac status
(e.g., reduction, amelioration or prevention of the progressive
cardiomyopathy, arrhythmia and other cardiac manifestations that
can be found, for example, in GSD-III), myopathy (e.g., exercise
tolerance), reduction of glycogen levels in tissue (e.g., liver and
muscle) of the individual affected by the disease, or any
combination of these effects. Further, the treatment may prevent
long term complications such as liver cirrhosis, hepatocellular
carcinoma due to clearance of glycogen with an abnormal structure.
In one preferred embodiment, treatment includes improvement of
liver symptoms, particularly, in reduction or prevention of GSD
(e.g., GSD-III)-associated hypoglycemia, hepatomegaly and abnormal
liver function. The terms, "improve," "prevent" or "reduce," as
used herein, indicate values that are relative to a baseline
measurement, such as a measurement in the same individual prior to
initiation of the treatment described herein, or a measurement in a
control individual (or multiple control individuals) in the absence
of the treatment described herein. A control individual is an
individual afflicted with the same form of the disease (e.g.,
GSD-III) as the individual being treated, who is about the same age
as the individual being treated (to ensure that the stages of the
disease in the treated individual and the control individual(s) are
comparable).
[0025] The individual being treated can be an individual (infant,
child, adolescent, or adult human) having GSD-III. The individual
can have residual GDE activity, or no measurable activity. In
another preferred embodiment, the individual is an individual who
has been recently diagnosed with the disease. Early treatment
(treatment commencing as soon as possible after diagnosis) is
important to minimize the effects of the disease and to maximize
the benefits of treatment.
[0026] While the invention is described in detail with reference to
GSD-III, the methods described herein can also be used to treat
individuals suffering from other GSDs, including, but not limited
to, GSD-IV, -VI, -IX and -XI. The methods described herein can also
be used in the treatment of individuals suffering from cardiac
glycogenosis due to AMP-activated protein kinase gamma subunit 2
deficiency.
[0027] In the methods of the invention, GAA (preferably, human GAA)
is administered to the individual. The GAA is in a form that, when
administered, targets tissues such as the tissues affected by the
disease (e.g., liver, heart or muscle). In one preferred
embodiment, human GAA is administered in its precursor form, as the
precursor contains motifs that allow efficient receptor-mediated
uptake of GAA. Alternatively, a mature form of human GAA that has
been modified to contain motifs to allow efficient uptake of GAA
into cells, can be administered. In a particularly preferred
embodiment, the GAA is the precursor form of recombinant human
GAA.
[0028] GAA is obtainable from a variety of sources. In a
particularly preferred embodiment, recombinant human acid
.alpha.-glucosidase (rhGAA) produced in Chinese hamster ovary (CHO)
cell cultures is used (see, e.g., Fuller, M. et al., Eur. J.
Biochem. 234:903 909 (1995); Van Hove, J. L. K. et al., Proc. Natl.
Acad. Sci. USA 93:65 70 (1996) and U.S. Pat. No. 7,056,712).
Production of GAA in CHO cells yields a product having
glycosylation that allows significant and efficient uptake of GAA
in tissues such as heart and muscle. MYOZYME (alglucosidase alpha)
Genzyme Corp.), or other recombinant human GAA, can be used in
accordance with the invention.
[0029] The GAA has a specific enzyme activity in the range of about
1.0-8.0 .mu.mol/min/mg protein, preferably in the range of about
4.0-8.0 .mu.mol/min/mg protein. In one preferred embodiment, the
GAA has a specific enzyme activity of at least about 1.0
.mu.mol/min/mg protein; more preferably, a specific enzyme activity
of at least about 4.0 .mu.mol/min/mg protein; even more preferably,
a specific enzyme activity of at least about 6.0 .mu.mol/min/mg
protein; and still more preferably, a specific enzyme activity of
at least about 8.0 .mu.mol/min/mg protein.
[0030] GAA can be administered alone, or in compositions or
medicaments comprising the GAA, as described herein. The
compositions can be formulated with a physiologically acceptable
carrier or excipient to prepare a pharmaceutical composition. The
carrier and composition can be sterile. The formulation should suit
the mode of administration.
[0031] Suitable pharmaceutically acceptable carriers include but
are not limited to water, salt solutions (e.g., NaCl), saline,
buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable
oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates
such as lactose, amylose or starch, sugars such as mannitol,
sucrose, or others, dextrose, magnesium stearate, talc, silicic
acid, viscous paraffin, perfume oil, fatty acid esters,
hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as
combinations thereof. The pharmaceutical preparations can, if
desired, be mixed with auxiliary agents, e.g., lubricants,
preservatives, stabilizers, wetting agents, emulsifiers, salts for
influencing osmotic pressure, buffers, coloring, flavoring and/or
aromatic substances and the like which do not deleteriously react
with the active compounds. In a preferred embodiment, a
water-soluble carrier suitable for intravenous administration is
used.
[0032] The composition or medicament, if desired, can also contain
minor amounts of wetting or emulsifying agents, or pH buffering
agents. The composition can be a liquid solution, suspension,
emulsion, tablet, pill, capsule, sustained release formulation, or
powder. The composition can also be formulated as a suppository,
with traditional binders and carriers such as triglycerides. Oral
formulation can include standard carriers such as pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, polyvinyl
pyrollidone, sodium saccharine, cellulose, magnesium carbonate,
etc.
[0033] The composition or medicament can be formulated in
accordance with the routine procedures as a pharmaceutical
composition adapted for administration to human beings. For
example, in a preferred embodiment, a composition for intravenous
administration typically is a solution in sterile isotonic aqueous
buffer. Where necessary, the composition can also include a
solubilizing agent and a local anesthetic to ease pain at the site
of the injection. Generally, the ingredients are supplied either
separately or mixed together in unit dosage form, for example, as a
dry lyophilized powder or water free concentrate in a hermetically
sealed container such as an ampule or sachette indicating the
quantity of active agent. Where the composition is to be
administered by infusion, it can be dispensed with an infusion
bottle containing sterile pharmaceutical grade water, saline or
dextrose/water. Where the composition is administered by injection,
an ampule of sterile water for injection or saline can be provided
so that the ingredients may be mixed prior to administration.
[0034] The GAA can be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include those formed with free
amino groups such as those derived from hydrochloric, phosphoric,
acetic, oxalic, tartaric acids, etc., and those formed with free
carboxyl groups such as those derived from sodium, potassium,
ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0035] GAA (or composition or medicament containing GAA) is
administered by an appropriate route. In one embodiment, the GAA is
administered intravenously. In other embodiments, GAA is
administered by direct administration to a target tissue, such as
heart or muscle (e.g., intramuscular). In yet another embodiment,
GAA is administered orally. More than one route can be used
concurrently, if desired.
[0036] GAA (or composition or medicament containing GAA) can be
administered alone, or in conjunction with other agents, such as
antihistamines (e.g., diphenhydramine) or immunosuppressants or
other immunotherapeutic agents which counteract anti-GAA
antibodies. Possible immunomodulation strategies include preventive
tolerance induction either with initiation of therapy, or tolerance
modulation after the development of inhibitory antibodies. The
term, "in conjunction with," indicates that the agent is
administered at about the same time as the GAA (or composition
containing GAA). For example, the agent can be mixed into a
composition containing GAA, and thereby administered
contemporaneously with the GAA; alternatively, the agent can be
administered contemporaneously, without mixing (e.g., by
"piggybacking" delivery of the agent on the intravenous line by
which the GAA is also administered, or vice versa). In another
example, the agent can be administered separately (e.g., not
admixed) but within a short time frame (e.g., within 24 hours) of
administration of the GAA. In one embodiment, GAA (or composition
containing GAA) is administered in conjunction with an
immunosuppressive or immunotherapeutic regimen designed to reduce
amounts of, or prevent production of, anti-GAA antibodies. For
example, a protocol similar to those used in hemophilia patients
(Nilsson, I. M. et al., N. Engl. J. Med. 318:947 50 (1988)) can be
used to reduce anti-GAA antibodies. In a particularly preferred
embodiment, the immunosuppressive or immunotherapeutic regimen is
begun prior to the first administration of GAA, in order to
minimize the possibility of production of anti-GAA antibodies. As
an example, use of Rituximab, which eliminates mature B cells
expressing CD20, methotrexate, which acts on both B and T cells, or
different combinations of such agents is possible (Mendehlson et
al, N. Engl. J. Med. 360(2):194-195 (2009)).
[0037] GAA (or composition or medicament containing GAA) is
administered in a therapeutically effective amount (i.e., a dosage
amount that, when administered, for example, at regular intervals,
is sufficient to treat the disease, such as by ameliorating
symptoms associated with the disease, preventing or delaying the
onset of the disease, and/or also lessening the severity or
frequency of symptoms of the disease, as described above). The
amount that will be therapeutically effective in the treatment the
disease will depend on the nature and extent of the disease's
effects, and can be determined by standard clinical techniques. In
addition, in vitro or in vivo assays can optionally be employed to
help identify optimal dosage ranges. The precise dose to be
employed can also depend on the route of administration, and the
seriousness of the disease, and should be decided according to the
judgment of a practitioner and each patient's circumstances.
Effective doses can be extrapolated from dose-response curves
derived from in vitro or animal model test systems. In a preferred
embodiment, the therapeutically effective amount is less than about
40 mg enzyme/kg body weight of the individual, preferably in the
range of about 1-40 mg enzyme/kg body weight, and even more
preferably about 20 mg enzyme/kg body weight or about 10 mg
enzyme/kg body weight. The effective dose for a particular
individual can be varied (e.g., increased or decreased) over time,
depending on the needs of the individual. For example, in times of
physical illness or stress, or if anti-GAA antibodies become
present or increase, or if disease symptoms worsen, the amount can
be increased.
[0038] The therapeutically effective amount of GAA (or composition
or medicament containing GAA) can be administered at regular
intervals, depending on the nature and extent of the disease's
effects, and on an ongoing basis. Administration at a "regular
interval," as used herein, indicates that the therapeutically
effective amount is administered periodically (as distinguished
from a one-time dose). The interval can be determined by standard
clinical techniques. In preferred embodiments, GAA is administered
monthly, bimonthly, weekly, twice weekly, or daily. The
administration interval for a single individual need not be a fixed
interval but can be varied over time, depending on the needs of the
individual. For example, in times of physical illness or stress, if
anti-GAA antibodies become present or increase, or if disease
symptoms worsen, the interval between doses can be decreased.
[0039] In one preferred embodiment, a therapeutically effective
amount of 20 mg enzyme/kg body weight is administered bi-monthly.
In another preferred embodiment, a therapeutically effective amount
of 10 mg enzyme/kg body weight is administered weekly or 5 mg
enzyme/kg body weight is administered twice weekly.
[0040] The invention additionally pertains to a pharmaceutical
composition comprising human GAA, as described herein, in a
container (e.g., a vial, bottle, bag for intravenous
administration, syringe, etc.) with a label containing instructions
for administration of the composition for treatment of GSD-III,
such as by the methods described herein.
[0041] Certain aspects of the invention are described in greater
detail in the non-limiting Examples that follow. (See also U.S.
Pat. No. 7,056,712.)
Example 1
[0042] GAA is a lysosomal exo 1,4-.alpha.-D-glucosidase that
hydrolyzes both .alpha.-1,4 and, .alpha.-1,6 linkage of glycogen. A
highly efficient system has been developed for producing human GAA
which targets heart and muscle and corrects glycogen accumulation
in patients with Pompe disease. The excess glycogen in GSD-III
resides not only in the cytoplasm but also in the lysosome
(Cornelio et al, Arch. Neurol. 41:1027-1032 (1984), Miranda et al,
Ann. Neurol. 9:283-288 (1981)). This suggests that excess
cytoplasmic glycogen is shuffled more effectively into the
lysosomes than can be cleared by the "normal" GAA activity in
GSD-III cells. It is hypothesized that cytoplasmic glycogen
continuously shuffles through lysosomes by autophagy for
degradation, and that the administered GAA can reduce lysosomal
glycogen in GSD-III and ultimately also reduce cytoplasmic
glycogen.
[0043] Source of the Enzyme
[0044] High GAA producing CHO cells can be grown in expanded
culture and recombinant enzyme purified from the medium. Milligram
quantities of purified GAA is available weekly as part of an
ongoing project on the development of enzyme replacement therapy
for Pompe disease. MYOZYME (Genzyme Corp.), or other rhGAA
(preferably CHO-produced), is suitable for use in the instant
invention.
[0045] Tissue Source
[0046] Muscle samples can be obtained from patients with GSD-IIIa
whose disease has been previously diagnosed by demonstrating
debrancher activity deficiency in both liver and muscle. Needle
muscle biopsy from patients with the diagnosis can be performed. It
is expected that at least 3 patients with a confirmed diagnosis of
GSD-IIIa will be studied. The needle muscle biopsy can obtain 50-70
mg of tissues sufficient for analysis. The procedure is less
invasive than an open biopsy. Currently, skin fibroblasts from 9
GSD-IIIa patients are available. An IRB approved protocol is
available that makes it possible to obtain muscle and skin tissue
from patients with GSD III.
[0047] Cell Culture
[0048] Cultured skin fibroblasts and muscle cells can be used to
test the feasibility of using GAA to treat GSD-III. The focus will
be particularly on Ina patients who have, in addition to
hepatomegaly, progressive myopathy (increasing muscle weakness by
muscle strength testing in 6 months to 1 year follow-up and/or
clinical complaints of a decrease in muscle strength) and
cardiomyopathy (increase in left ventricular mass in a 6-12 month
period on follow-up), and also patients with progressive liver
disease. Deficiency of debranching enzyme and accumulation of
glycogen are present in skin fibroblasts and muscle cells grown in
culture from GSD-III patients (Miranda et al, Ann. Neurol.
9:283-288 (1981), DiMauro et al, Pedatr. Res. 7:739-744 (1973),
Yang et al, Am. J. Hum. Genet. 47:735-739 (1990), Brown, Diagnosis
of glycogen storage disease, In: Wapnir P A, (ed), Congenital
Metabolic Disease Diagnosis and Treatment, Dekker, New York, pp.
227-250 (1985)). Cultured muscle cells also reflect muscle biopsy
findings in that both cytoplasmic and lysosomal glycogen are
observed (Cornelio et al, Arch. Neurol. 41:1027-1032 (1984),
Miranda et al, Ann. Neurol. 9:283-288 (1981)). The skin fibroblasts
and muscle cultures can, therefore, be used to improve
understanding of the pathogenesis of the disease and to evaluate
approaches to therapy. Muscle cultures can be established from
muscle biopsy samples using a protocol similar to the isolation of
quail myoblasts (Konigsberg, Methods in Enzymology 45:511-527
(1979)). Replating of the primary cultures can be performed to
select against fibroblasts. If the biopsy sample is too small, a
more efficient myoblasts isolation method using the
fluorescence-activated cell sorter can be performed (Webster,
Experimental Cell Research 174:252-265 (1988)). Myoblasts can be
allowed to differentiate to myotubes by changing the medium to 2%
horse serum. Muscle cells in the appropriate stage will be used for
conduct of the experiments.
[0049] Effect on Glycogen with GAA Treatment
[0050] Glycogen concentrations in skin fibroblasts vary with cell
confluence and passage number (DiMauro et al, Pedatr. Res.
7:739-744 (1973)). Experiments can be performed at near confluence,
and each patient can serve as his/her own control by concurrent
duplicate testing with and without GAA treatment. Dose response
(from 500 to 5000 nmol/hr/ml of GAA) and time course (1 to 10 days)
can be tested for the effect of glycogen reduction. Glycogen
content can be measured in total cell homogenates and also
individually in cytosol and crude lysosomal fractions (van der
Ploeg et al, J. Neurol. Sci. 79:327-336 (1987)). Electromicroscopic
examination can be performed for evidence of clearance in both
cytoplasm and lysosomes.
[0051] To avoid obscuring of the results by the continuous glycogen
synthesis that occurs in cells cultured in the presence of glucose
in the medium, cells can be shifted to glucose-free medium 24 hours
after GAA treatment. Deprivation of glucose resulted in greater
than 80% drop of glycogen in the normal cells, but only 30%
reduction in GSD-III cells (Yang et al, Am. J. Hum. Genet.
47:735-739 (1990), Brown, Diagnosis of glycogen storage disease,
In: Wapnir P A, (ed), Congenital Metabolic Disease Diagnosis and
Treatment, Dekker, New York, pp. 227-250 (1985)). Thus sufficiently
high levels of glycogen persist which allow accurate evaluation of
GAA treatment. The persistent glycogen in GSD-III cells have short
outer brancher which can be assessed using glucose-1-phosphate
formed from endogenous polysaccharide by phosphorylase (Yang et al,
Am. J. Hum. Genet. 47:735-739 (1990)).
[0052] Example 2 below includes a description of studies that have
been undertaken.
Example 2
[0053] Primary human skeletal muscle cultures from GSD-IIIa
patients have been established as an in vitro model to evaluate
efficacy of rhGAA for treatment of GSD-III. Myoblasts were isolated
from three GSD-IIIa patients and one healthy volunteer.
Histopathology of these muscle biopsies was examined by light and
electronic microscopy (EM) to confirm abnormal glycogen
accumulation in these GSD-IIIa patients. The light microscopic
appearance of the skeletal muscle biopsies from GSD-IIIa patients
showed abundant glycogen accumulation in cytoplasmic pools (FIG.
1). Under EM, the vast majority of the glycogen was found free in
the cytoplasm along with small amount of membrane-bound glycogen
(FIGS. 2 and 3).
[0054] Differentiation of myoblasts into mature myotubes
(myogenesis) was induced by incubation of the muscle cells in
low-serum differentiation medium (low-glucose DMEM (GIBCO)
containing 2% Hyclone H1 horse serum (Sigma), 0.5 mg/ml Fetuin, 0.5
mg/ml BSA, 0.025 mg/ml gentamycin and 0.125 .mu.g/ml Amphotericin B
(Clonetics). Glycogen was stored in fully differentiated GSD-III
muscle cells when sufficient glucose was supplied in the medium
(FIG. 4). Incomplete glycogenolysis was seen in GSD-III cells, but
not in normal control cells after 48-hour glucose starvation (FIG.
5), indicating lack of debranching enzyme activity in the GSD-III
patients. Fully differentiated GSD-III myotubes were treated for 48
hours by adding 100 .mu.g of rhGAA (i.e., MYOZYME (Genzyme)) into
the culture medium. GAA enzyme activity and glycogen content were
analyzed biochemically and histologically in these cells. Treatment
with rhGAA significantly reduced glycogen level by 48%, 35% and
17%, respectively, in the three GSD-IIIa patient muscle cells (FIG.
6). These data suggest the role of GAA in glycogen clearance in
conditions where the primary defect results in cytoplasmic glycogen
accumulation.
Sequence CWU 1
1
11952PRThomo sapiens 1Met Gly Val Arg His Pro Pro Cys Ser His Arg
Leu Leu Ala Val Cys1 5 10 15Ala Leu Val Ser Leu Ala Thr Ala Ala Leu
Leu Gly His Ile Leu Leu 20 25 30His Asp Phe Leu Leu Val Pro Arg Glu
Leu Ser Gly Ser Ser Pro Val 35 40 45Leu Glu Glu Thr His Pro Ala His
Gln Gln Gly Ala Ser Arg Pro Gly 50 55 60Pro Arg Asp Ala Gln Ala His
Pro Gly Arg Pro Arg Ala Val Pro Thr65 70 75 80Gln Cys Asp Val Pro
Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys 85 90 95Ala Ile Thr Gln
Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro 100 105 110Ala Lys
Gln Gly Leu Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe 115 120
125Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser
130 135 140Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr
Phe Phe145 150 155 160Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp Val
Met Met Glu Thr Glu 165 170 175Asn Arg Leu His Phe Thr Ile Lys Asp
Pro Ala Asn Arg Arg Tyr Glu 180 185 190Val Pro Leu Glu Thr Pro Arg
Val His Ser Arg Ala Pro Ser Pro Leu 195 200 205Tyr Ser Val Glu Phe
Ser Glu Glu Pro Phe Gly Val Ile Val His Arg 210 215 220Gln Leu Asp
Gly Arg Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe225 230 235
240Phe Ala Asp Gln Phe Leu Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr
245 250 255Ile Thr Gly Leu Ala Glu His Leu Ser Pro Leu Met Leu Ser
Thr Ser 260 265 270Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu Ala
Pro Thr Pro Gly 275 280 285Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr
Leu Ala Leu Glu Asp Gly 290 295 300Gly Ser Ala His Gly Val Phe Leu
Leu Asn Ser Asn Ala Met Asp Val305 310 315 320Val Leu Gln Pro Ser
Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile 325 330 335Leu Asp Val
Tyr Ile Phe Leu Gly Pro Glu Pro Lys Ser Val Val Gln 340 345 350Gln
Tyr Leu Asp Val Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly 355 360
365Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr
370 375 380Arg Gln Val Val Glu Asn Met Thr Arg Ala His Phe Pro Leu
Asp Val385 390 395 400Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg
Arg Asp Phe Thr Phe 405 410 415Asn Lys Asp Gly Phe Arg Asp Phe Pro
Ala Met Val Gln Glu Leu His 420 425 430Gln Gly Gly Arg Arg Tyr Met
Met Ile Val Asp Pro Ala Ile Ser Ser 435 440 445Ser Gly Pro Ala Gly
Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg 450 455 460Gly Val Phe
Ile Thr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val465 470 475
480Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu
485 490 495Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp Gln Val
Pro Phe 500 505 510Asp Gly Met Trp Ile Asp Met Asn Glu Pro Ser Asn
Phe Ile Arg Gly 515 520 525Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu
Glu Asn Pro Pro Tyr Val 530 535 540Pro Gly Val Val Gly Gly Thr Leu
Gln Ala Ala Thr Ile Cys Ala Ser545 550 555 560Ser His Gln Phe Leu
Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly 565 570 575Leu Thr Glu
Ala Ile Ala Ser His Arg Ala Leu Val Lys Ala Arg Gly 580 585 590Thr
Arg Pro Phe Val Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg 595 600
605Tyr Ala Gly His Trp Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu
610 615 620Ala Ser Ser Val Pro Glu Ile Leu Gln Phe Asn Leu Leu Gly
Val Pro625 630 635 640Leu Val Gly Ala Asp Val Cys Gly Phe Leu Gly
Asn Thr Ser Glu Glu 645 650 655Leu Cys Val Arg Trp Thr Gln Leu Gly
Ala Phe Tyr Pro Phe Met Arg 660 665 670Asn His Asn Ser Leu Leu Ser
Leu Pro Gln Glu Pro Tyr Ser Phe Ser 675 680 685Glu Pro Ala Gln Gln
Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala 690 695 700Leu Leu Pro
His Leu Tyr Thr Leu Phe His Gln Ala His Val Ala Gly705 710 715
720Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser
725 730 735Thr Trp Thr Val Asp His Gln Leu Leu Trp Gly Glu Ala Leu
Leu Ile 740 745 750Thr Pro Val Leu Gln Ala Gly Lys Ala Glu Val Thr
Gly Tyr Phe Pro 755 760 765Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val
Pro Ile Glu Ala Leu Gly 770 775 780Ser Leu Pro Pro Pro Pro Ala Ala
Pro Arg Glu Pro Ala Ile His Ser785 790 795 800Glu Gly Gln Trp Val
Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val 805 810 815His Leu Arg
Ala Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr 820 825 830Thr
Thr Glu Ser Arg Gln Gln Pro Met Ala Leu Ala Val Ala Leu Thr 835 840
845Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser
850 855 860Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln Val Ile Phe
Leu Ala865 870 875 880Arg Asn Asn Thr Ile Val Asn Glu Leu Val Arg
Val Thr Ser Glu Gly 885 890 895Ala Gly Leu Gln Leu Gln Lys Val Thr
Val Leu Gly Val Ala Thr Ala 900 905 910Pro Gln Gln Val Leu Ser Asn
Gly Val Pro Val Ser Asn Phe Thr Tyr 915 920 925Ser Pro Asp Thr Lys
Val Leu Asp Ile Cys Val Ser Leu Leu Met Gly 930 935 940Glu Gln Phe
Leu Val Ser Trp Cys945 950
* * * * *