U.S. patent application number 11/889457 was filed with the patent office on 2008-07-24 for treatment of glycogen storage disease type ii.
This patent application is currently assigned to DUKE UNIVERSITY. Invention is credited to Yuan-Tsong Chen.
Application Number | 20080175833 11/889457 |
Document ID | / |
Family ID | 22818451 |
Filed Date | 2008-07-24 |
United States Patent
Application |
20080175833 |
Kind Code |
A1 |
Chen; Yuan-Tsong |
July 24, 2008 |
Treatment of glycogen storage disease type II
Abstract
Methods of treating glycogen storage disease type II, by
administering acid .alpha.-glucosidase, are described, as are
compositions for use in treatment of glycogen storage disease type
II.
Inventors: |
Chen; Yuan-Tsong; (Chapel
Hill, NC) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Assignee: |
DUKE UNIVERSITY
Durham
NC
|
Family ID: |
22818451 |
Appl. No.: |
11/889457 |
Filed: |
August 13, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11039281 |
Jan 20, 2005 |
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11889457 |
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09902461 |
Jul 10, 2001 |
7056712 |
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11039281 |
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60219237 |
Jul 18, 2000 |
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Current U.S.
Class: |
424/94.61 |
Current CPC
Class: |
A61K 38/00 20130101;
C12Y 302/0102 20130101; A61P 3/10 20180101; A61P 37/06 20180101;
A61P 3/00 20180101; A61P 9/00 20180101; A61P 43/00 20180101; A61P
9/04 20180101; A61P 21/00 20180101; A61P 9/02 20180101; A61K 45/06
20130101; A61K 38/47 20130101; A61K 38/47 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/94.61 |
International
Class: |
A61K 38/47 20060101
A61K038/47; A61P 3/10 20060101 A61P003/10 |
Claims
1. A method of treating glycogen storage disease type II in a human
individual having glycogen storage disease type II, comprising
administering to the individual human acid .alpha.-glucosidase
produced in chinese hamster ovary cell cultures, in a
therapeutically effective amount that is sufficient to increase
end-diastolic and/or end-systolic ventricular volume over baseline
measurement of said volume in the individual.
2. The method of claim 1, wherein the end-diastolic and/or
end-systolic ventricular volume is increased at least about 2 to 3
fold over baseline measurement of said volume in the
individual.
3. The method of claim 2, wherein the ventricular volume is
measured by 2-D echocardiography.
4. The method of claim 2, wherein the ventricular volume is
measured by M-mode echocardiography.
5. The method of claim 2, wherein the ventricular volume is
measured by Doppler echocardiography.
6. A method of treating glycogen storage disease type II in a human
individual having glycogen storage disease type II, comprising
administering to the individual human acid .alpha.-glucosidase
produced in chinese hamster ovary cell cultures, in a
therapeutically effective amount that is sufficient to decrease
left ventricular mass from baseline measurement of said mass in the
individual.
7. The method of claim 6, wherein the left ventricular mass is
decreased to about 60-70% of baseline measurement of said mass in
the individual.
8. The method of claim 7, wherein the ventricular mass is measured
by 2-D echocardiography.
9. The method of claim 7, wherein the ventricular mass is measured
by M-mode echocardiography.
10. The method of claim 7, wherein the ventricular mass is measured
by Doppler echocardiography.
11. A method of treating glycogen storage disease type II in a
human individual having glycogen storage disease type II,
comprising administering to the individual human acid
.alpha.-glucosidase produced in chinese hamster ovary cell
cultures, in a therapeutically effective amount that is sufficient
to increase pulmonary function over baseline capacity in the
individual.
12. The method of claim 11, wherein the increase in pulmonary
function is indicated by an increase in crying vital capacity over
baseline measurement of crying vital capacity in the
individual.
13. The method of claim 12, wherein the crying vital capacity is
increased at least about 28% over baseline measurement in the
individual.
14. The method of claim 11, wherein the increase in pulmonary
function is indicated by normalization of oxygen saturation during
crying over baseline measurement of oxygen saturation during crying
in the individual.
15. The method of claim 11, wherein the increase in pulmonary
function is indicated by increased respiratory muscle strength over
baseline measurement of respiratory muscle strength in the
individual.
16. A method of treating glycogen storage disease type II in a
human individual having glycogen storage disease type II,
comprising administering to the individual human acid
.alpha.-glucosidase produced in chinese hamster ovary cell
cultures, in a therapeutically effective amount sufficient to
improve motor development over baseline measurement of motor
development in the individual.
17. The method of claim 16, wherein the improvement in motor
development is. indicated by an increase over baseline measurement
in Alberta Infant Motor Scale (AIMS) score in the individual.
18. A method of treating glycogen storage disease type II in a
human individual having glycogen storage disease type II,
comprising administering to the individual human acid
.alpha.-glucosidase produced in chinese hamster ovary cell
cultures, in a therapeutically effective amount sufficient to
improve neurodevelopment over baseline measurement of
neurodevelopment in the individual.
19. The method of claim 18, wherein improvement in neurodevelopment
is indicated by an increase over baseline measurement in
personal-social, language, or fine motor developmental domains as
measured by Denver Developmental evaluation in the individual.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 09/902,461, filed Jul. 10, 2001, which claims the benefit of
U.S. Provisional Application No. 60/219,237, filed Jul. 18, 2000.
The entire teachings of the above applications are incorporated
herein by reference.
BACKGROUND OF THE INVENTION
[0002] Glycogen storage disease type II (GSD-II) (also known as
Pompe disease or acid maltase deficiency) is a fatal genetic muscle
disorder caused by a deficiency of acid .alpha.-glucosidase (GAA),
a glycogen degrading lysosomal enzyme (Hirschhorn, R., "Glycogen
storage disease type II: acid .alpha.-glucosidase (acid maltase)
deficiency", in Scriver, C. R. et al., (eds) The Metabolic and
Molecular Basis of Inherited disease, 7.sup.th Ed., McGraw-Hill,
New York, 1995, pp. 2443-2464). The deficiency results in lysosomal
glycogen accumulation in almost all tissues of the body, with
cardiac and skeletal muscle being the most seriously affected. The
combined incidence of all forms of GSD-II is estimated to be
1:40,000, and the disease affects all groups without an ethnic
predilection (Martiniuk, F. et al., Amer. J. Med. Genet. 79:69-72
(1998); Ausems, M. G. E. M. et al., Eur. J. Hum. Genet. 7:713-716
(1999)).
[0003] Clinically, GSD-II encompasses a range of phenotypes
differing as to age of onset, organs involved and clinical
severity, generally correlating with the residual amount of GAA
activity. In its most severe presentation (infantile GSD-II, or
Pompe disease, in which less than 1% of normal GAA activity is
present), infants are affected by a hypertrophic cardiomyopathy,
generalized muscle weakness and hypotonia secondary to massive
glycogen accumulation in cardiac and skeletal muscles (for review,
see Hirschhorn, supra). The disease progresses rapidly, with death
from cardiac failure usually occurring by 1 year of age. Juvenile
(1-10% of normal GAA activity) and adult-onset (10-40% of normal
GAA activity) forms of the disease are characterized by lack of
severe cardiac involvement, later age of onset, and slower
progression, but eventual respiratory or limb muscle involvement
results in significant morbidity and mortality for the affected
individuals.
[0004] Drug treatment strategies, dietary manipulations, and bone
marrow transplantation have been employed as means for treatment
for GSD-II, without significant success (Hug, G. et al., Birth
Defects Org. Ser. 9:160-183 (1967); Slonim, A. E. et al., Neurology
33:34 (1983); Watson, J. G. et al., N. Engl. J. Med. 314:385
(1986)). Early attempts at enzyme replacement were also
unsuccessful (Hug, G. and Schubert, W. K., J. Clin. Invest. 46:1073
(1967); de Barsy, T. et al., Birth Defects Orig. Art. Ser. 9:84-190
(1973); Williams, J. C. and Murray, A. K., "Enzyme replacement in
Pompe disease with an alpha glucosidase low-density lipoprotein
complex", in Desnick, R. J. (ed), Enzyme Therapy in Genetic
Diseases: 2, New York, Alan R. Liss 1980; pp. 415-423)). A need
remains for effective treatment of GSD-II.
SUMMARY OF THE INVENTION
[0005] The present invention is drawn to methods of treating
glycogen storage disease type II (infantile, juvenile or
adult-onset) in an individual, by administering to the individual a
therapeutically effective amount of acid .alpha.-glucosidase (e.g.,
less than about 15 mg enzyme per kilogram of body weight,
preferably about 1-10 mg enzyme per kilogram of body weight, more
preferably about 10 enzyme per kilogram of body weight or about 5
mg enzyme per kilogram of body weight), at a regular interval
(e.g., monthly, bimonthly, weekly, twice weekly, daily). The acid
.alpha.-glucosidase is human acid .alpha.-glucosidase, preferably
recombinant human acid .alpha.-glucosidase, more preferably,
precursor form of human acid .alpha.-glucosidase, and even more
preferably precursor form of human acid .alpha.-glucosidase
produced in Chinese hamster ovary cells. The acid
.alpha.-glucosidase is administered periodically (e.g., monthly,
bimonthly, weekly, twice weekly, daily). In preferred embodiments,
the acid .alpha.-glucosidase is administered intravenously;
intramuscularly; intrathecally; or intraventricularly.
[0006] The methods of the invention provide the first effective
means to treat an individual with glycogen storage disease type
II.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1A-1C are a series of graphic representations depicting
longitudinal data (for the first 16 months of age) on motor
development as assessed by Alberta Infant Motor Scale (AIMS)
(closed diamonds), and titer of antibodies to recombinant human
acid .alpha.-glucosidase (rhGAA) (open diamonds) in three patients
(patient 1, FIG. 1A; patient 2, FIG. 1B; patient 3, FIG. 1C) with
infantile Pompe disease receiving enzyme replacement therapy. The
arrow indicates when the enzyme therapy was initiated. AIMS scores
in normal patients are plotted as dotted curves against age
(5.sup.th, 10.sup.th, 25.sup.th, 50.sup.th, 75.sup.th and 95th
percentile, from bottom to top).
[0008] FIG. 2A-2F are a series of graphic representations depicting
longitudinal two-dimensional echocardiographic measurements of left
ventricular volume (FIG. 2A-2C) and mass (FIG. 2D-2F) in the three
infantile Pompe disease patients receiving enzyme replacement
therapy (patient 1, FIGS. 2A and 2D; patient 2, FIGS. 2B and 2E;
patient 3, FIGS. 2C and 2F). Week 0 depicts the measurements at the
time of enzyme therapy initiation. Open diamonds, end-diastolic
volume measurement; closed diamonds, end-systolic volume
measurement.
DETAILED DESCRIPTION OF THE INVENTION
[0009] The present invention is drawn to methods of treating
glycogen storage disease type II (GSD-II) in an individual, by
administering the enzyme, acid .alpha.-glucosidase (GAA) to the
individual, as well as the use of the enzyme, GAA, in the
manufacture of a medicament for the treatment of glycogen storage
disease type II. As described herein, Applicants have successfully
treated infants suffering from GSD-II by administering GAA to the
infants on a regular basis; the infants demonstrated improvement of
cardiac status, pulmonary function, and neurodevelopment, as well
as reduction of glycogen levels in tissue.
[0010] As a result of these findings, it is now possible for the
first time to treat GSD-II, including infantile, juvenile and
adult-onset GSD-II. Although the results described herein discuss
individuals with the most severe form of GSD-II (infantile GSD-II),
it is expected that the methods will be equally effective in
individuals affected by juvenile or adult-onset GSD-II, and may, in
fact, be even more effective, as individuals with juvenile or
adult-onset GSD-II have higher levels of residual GAA activity
(1-10%, or 10-40%, respectively), and therefore are likely to be
more immunologically tolerant of the administered GAA (e.g., they
are generally cross-reactive immunoreactive material
(CRIM)-positive for endogenous GAA, so that their immune systems do
not perceive the GAA as a "foreign" protein, and they do not
develop anti-GAA antibodies). The enhanced efficacy in such
individuals can be seen in patient 3, who was CRIM-positive and did
not develop anti-GAA antibodies, and who demonstrated a normal
progression of developmental milestones, in contrast with the
variable course that was seen in CRIM-negative patients 1 and 2
(who did develop anti-GAA antibodies).
[0011] 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 cardiac status (e.g., increase of end-diastolic
and/or end-systolic volumes, or reduction, amelioration or
prevention of the progressive cardiomyopathy that is typically
found in GSD-II) or of pulmonary function (e.g., increase in crying
vital capacity over baseline capacity, and/or normalization of
oxygen desaturation during crying); improvement in neurodevelopment
and/or motor skills (e.g., increase in AIMS score); reduction of
glycogen levels in tissue of the individual affected by the
disease; or any combination of these effects. In one preferred
embodiment, treatment includes improvement of cardiac status,
particularly in reduction or prevention of GSD-II-associated
cardiomyopathy. The terms, "improve," "increase" 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 GSD-II (either
infantile, juvenile or adult-onset) 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).
[0012] The individual being treated is an individual (fetus, child,
adolescent, or adult human) having GSD-II (i.e., either infantile
GSD-II, juvenile GSD-II, or adult-onset GSD-II). The individual can
have residual GAA activity, or no measurable activity. For example,
the individual having GSD-II can have GAA activity that is less
than about 1% of normal GAA activity (infantile GSD-II), GAA
activity that is about 1-10% of normal GAA activity (juvenile
GSD-II), or GAA activity that is about 10-40% of normal GAA
activity (adult GSD-II). The individual can be CRIM-positive or
CRIM-negative for endogenous GAA. In a preferred embodiment, the
individual is CRIM-positive for endogenous GAA. 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 for to
minimize the effects of the disease and to maximize the benefits of
treatment.
[0013] In the methods of the invention, human acid
.alpha.-glucosidase (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., heart, muscle). In one
preferred embodiment, the human GAA is administered in its
precursor form, as the precursor contains motifs which 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, can be administered. In a particularly
preferred embodiment, the GAA is the precursor form of recombinant
human GAA.
[0014] GAA is obtainable from a variety of sources. In a
particularly preferred embodiment, recombinant human acid
.alpha.-glucosidase (rhGAA) has been 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); the entire teachings of
these references are incorporated herein by reference). Production
of GAA in CHO cells appears to yield a product having glycosylation
which allows significant and efficient uptake of GAA in the desired
tissues (heart and muscle); it is assumed that the glycosylation
differs from that of GAA that is produced in transgenic mouse and
rabbit milk (see, e.g., Bijvoet, A. G. A. et al., Hum. Mol. Genet.
7:1815-1824 (1998); Bijvoet, A. G. A. et al., Hum. Mol. Genet.
8:2145-2153 (1999)).
[0015] The GAA has a specific enzyme activity in the range of about
1.0-3.5 .mu.mol/min/mg protein, preferably in the range of about
2-3.5 .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 2.0 .mu.mol/min/mg protein; even more preferably, a specific
enzyme activity of at least about 2.5 .mu.mol/min/mg protein; and
still more preferably, a specific enzyme activity of at least about
2.75 .mu.mol/min/mg protein.
[0016] GAA can be administered alone, or in compositions or
medicaments comprising the GAA (e.g., in the manufacture of a
medicament for the treatment of the disease), 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.
[0017] 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.
[0018] 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.
[0019] 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 may 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.
[0020] 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.
[0021] 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), or nervous system (e.g.,
direct injection into the brain; intraventricularly;
intrathecally). More than one route can be used concurrently, if
desired.
[0022] 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. 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 preferred embodiment, if the
individual is CRIM-negative for endogenous GAA, 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. Such a regimen can also be used
in individuals who are CRIM-positive for endogenous GAA but who
have, or are at risk of having, 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.
[0023] GAA (or composition or medicament containing GAA) is
administered in a therapeutically effective amount (i.e., a dosage
amount that, when administered 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 which 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 may optionally be employed to help identify
optimal dosage ranges. The precise dose to be employed will 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 may
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 15 mg enzyme/kg
body weight of the individual, preferably in the range of about
1-10 mg enzyme/kg body weight, and even more preferably about 10 mg
enzyme/kg body weight or about 5 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.
[0024] The therapeutically effective amount of GAA (or composition
or medicament containing GAA) is 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.
[0025] In one preferred embodiment, a therapeutically effective
amount of 10 mg enzyme/kg body weight is administered weekly. In
another preferred embodiment, a therapeutically effective amount of
5 mg enzyme/kg body weight is administered twice weekly.
[0026] The invention additionally pertains to a pharmaceutical
composition comprising human acid .alpha.-glucosidase, 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 glycogen
storage disease type II, such as by the methods described
herein.
[0027] The invention will be further and more specifically
described by the following examples.
Exemplification: Phase I/II Trial of Use of Recombinant Human Acid
.alpha.-glucosidase
Materials and Methods
[0028] Patients: Inclusion criteria were infants affected with
infantile GSD-II having virtually absent GAA activity (<1% of
normal in skin fibroblasts and/or muscle biopsy) and less than one
year of age. Exclusion criteria included severe cardiorespiratory
failure at baseline and/or other medical conditions likely to
decrease survival. Because of the limited life expectancy of the
disease following diagnosis, no placebo control was used.
Historical control data indicated that virtually all patients died
before 1 year of age (Table 1).
TABLE-US-00001 TABLE 1 Historical control data of infantile
glycogen storage disease, type II Length of disease Onset (months)
Death (months) course (months) Duke University Medical Center (n =
30)* Mean .+-. SD 5.1 .+-. 1.8 8.6 .+-. 2.4 3.5 .+-. 2.3 Range
2.4-10.3 3.3-12.4 0.0-9.0 Slonim et al. (n = 10)** Mean .+-. SD 2.5
.+-. 1.0 7.2 .+-. 2.8 4.7 .+-. 2.4 Range 1.0-4.0 4.0-12.0 2.0-9.0
*Data from Duke University Pompe Disease Registry **Data from
Slonim et al., J. Pediatr. 137: 283-285 (2000).
[0029] Three infants affected with infantile GSD-II as evidenced by
reduced acid .alpha.-glucosidase activity to less than 1% of normal
in skin fibroblasts and/or muscle biopsy were enrolled in the
study. At the protein level, both patients 1 and 2 had no
detectable GAA protein while patient 3 had reduced levels of GAA
protein detected by immunoblot analysis. The baseline clinical data
before the initiation of the therapy are summarized in Table 2.
TABLE-US-00002 TABLE 2 Baseline Clinical Data on 3 Infantile Pompe
Disease Patients Patient Age Motor GAA Activity in Number/ Ethnic
rhGAA Development Skin Fibroblasts CRIM* Sex Background Started
Cardiac Status Pulmonary Function (AIMS Score) (% of normal) Status
Current Age Patient Caucasian 4 months Severe Borderline normal,
<<5.sup.th% 0.84% Negative 29 1/male cardiomyopathy; left
main bronchus months status post compression due to cardiac arrest
markedly enlarged heart, O.sub.2 desaturation Patient African- 3
months Moderate O.sub.2 desaturation during <5.sup.th% 0.57%
Negative 25 2/male American cardiomyopathy crying months Patient
Caucasian 21/2 months Borderline Normal <<5.sup.th% 0.69%
Positive 23 3/male cardiomyopathy months *CRIM = cross reactive
immunoreactive material
Patient 1 presented at 2 months of age with cardiac arrest during
elective surgical repair of an inguinal hernia. Subsequent
evaluation when he was 4 months of age demonstrated evidence of
severe hypotonia, with a motor development age estimated to be
equivalent to that of a 3 week old. He also had profound
cardiomyopathy and severe cardiomegaly with compression of the left
main bronchus resulting in partial atelectasis of the left lung,
and feeding difficulties and failure to thrive. Patients 2 and 3
were prenatally diagnosed with Pompe disease; importantly, each had
a previous sibling that had died of symptoms typically attributable
to the infantile GSD-II. Both patients had evidence of motor
delays; in addition patient 2 had feeding difficulty, failure to
thrive and severe cardiomyopathy.
[0030] Basic Design: The study was designed as a Phase I/II,
open-label, single-dose, safety and efficacy study of rhGAA
administered twice weekly in the 3 patients with infantile Pompe
disease. The study was approved by the institutional review board,
and parental written informed consent was obtained.
[0031] The study consisted of an initial Screening Phase, a 13-week
Treatment Phase, and a Follow-up Treatment Phase. During the
Screening Phase the initial clinical status of the patients was
assessed; in addition, GAA and glycogen levels were determined in
skeletal muscle biopsy samples. During the Treatment Phase,
patients received intravenous infusions of rhGAA (5 mg/kg) twice
weekly. Patients were closely monitored for any adverse responses
to the enzyme infusions, as well as for any impact the rhGAA
administrations had on the clinical progression of infantile
GSD-II. General clinical assessments included routine physical
examinations, supplemented by complete urine, hematological, and
clinical chemistry analyses (electrolytes, glucose, creatinine,
BUN, CO.sub.2, protein, albumin, ALT, AST, bilirubin, alkaline
phosphatase, CK and isozyme, uric acid). Exhaustive neurologic and
motor function evaluations included manual muscle strength testing,
Denver development testing, and AIMS (Alberta Infant Motor Scale;
see Piper, M. C. and Darrah, J., Motor Assessment of the Developing
Infant, WB Sanders Company, Philadelphia, 1994). Two-dimensional,
M-mode and Doppler echocardiography were used to assess left
ventricular mass, wall thickness and systolic as well as diastolic
functions. Additionally, a variety of pulmonary functions (crying
vital capacity, trend pulse-oximetry and end tidal carbon dioxide
measurement, as well as negative inspiratory force maneuver) were
monitored throughout the study. At the conclusion of the 13-week
treatment phase, GAA activity, glycogen levels and histopathology
of muscle biopsies obtained from the quadriceps muscles of the
contra-lateral thigh of the pre-treatment biopsies were determined.
The muscle biopsies were taken 3 days after the rhGAA infusion.
[0032] Enzyme source: rhGAA purified from the culture medium of
rhGAA secreting CHO cells (Van Hove, J. L. K. et al., Proc. Natl.
Acad. Sci. USA 93:65-70 (1996)) was provided as a GMP-grade,
sterile and colorless solution by Synpac (North Carolina), Inc., 99
Alexander Drive, Suite NW20, Research Triangle Park, N.C. 27709.
rhGAA was purified primarily as the 110-kD precursor protein with
specific enzyme activity of 2.77-3.02 .mu.mol/min/mg protein.
[0033] ELISA for anti-rhGAA antibodies: The ELISA for anti-rhGAA
antibodies was a standard sandwich assay performed by Phoenix
International Life Sciences, Inc. (Saint-Laurent, Quebec). Briefly,
microtiter plates were coated with rhGAA at 2.0 .mu.g/ml overnight
and then blocked with bovine IgG. Patient serum, diluted to 1:100
and then serially diluted at 1:2, was reacted with the rhGAA on the
plate. The amount of bound antibody was detected with a horseradish
peroxidase conjugated goat anti-human secondary antibody and
tetramethylbenzidine substrate by measuring the absorbances at 450
nm. Positive samples were defined as having an absorbance that was
higher than the negative cutoff. This was defined as twice the A450
value of the normal human serum negative control. Titer was defined
as the dilution of the serum that still had an A450 reading above
the negative cutoff value.
[0034] GAA activity, glycogen content and Western blot analysis:
GAA activity was assessed by measurement of
4-methyl-umbelliferyl-.alpha.-D-glucoside cleavage at pH 4.3 as
previously described (Reuser, A. J. J. et al., Am. J. Hum. Genet.
30:132-143 (1978)). As an internal standard,
acid-.beta.-galactosidase activity was similarly assayed with the
4-methyl-umbilliferyl derivative as the substrate (Wenger, D. A.
and Williams, C., "Screening for lysosomal disorders" in Hommes, F.
A. (ed.), Techniques in diagnostic human biochemical genetics: a
laboratory manual, Wiley-Liss, New York, 1991, pp. 587-617).
Glycogen content was determined by treatment of tissue extracts
with A. niger amyloglucosidase and measurement of glucose released
(Van Hove, J. L. K. et al., Proc. Natl. Acad. Sci. USA 93:65-70
(1996)). Western blot analysis was performed with antibody raised
in rabbits against purified placenta GAA (Van Hove, J. L. K. et
al., supra).
[0035] Histology: One specimen of muscle was mounted on a chuck
with gum tragacanth and quick-frozen in isopentane cooled by liquid
nitrogen. Five micron sections were obtained and stained with
hematoxylin and eosin, modified Gomori trichrome, ATPase at pH 4.35
and 9.4, nicotinamide dehydrogenase tetrazolium blue reductase, and
phosphorylase. A second specimen was clamped in situ and placed in
2.5% glutaraldehyde. The tissue was processed without en bloc
staining with uranyl acetate in order to avoid loss of glycogen.
Semithin sections (0.5 micron) were stained with toluidine blue and
thin sections stained with uranyl acetate and lead citrate and
mounted on a copper grid for electron microscopy.
Results
[0036] Patient Reaction to Treatment: The three patients with
infantile Pompe disease received twice weekly intravenous infusions
of rhGAA for 21-25 months. No serious allergic reactions occurred
during enzyme therapy. However, three episodes of skin rash,
accompanied by a mild fever and increased irritability occurred in
two of the patients (patient 1 two episodes, patient 2 single
episode). These symptoms resolved promptly after intravenous
administration of diphenhydramine. After a second episode of skin
rash, patient 1 was premedicated with oral diphenhydramine just
prior to all subsequent rhGAA infusions, without further episodes.
Patient 2 was similarly premedicated with oral diphenhydramine just
prior to all subsequent infusions, without further episodes.
Multiple hematological parameters, liver functions, renal
functions, and urinalyses have all been in the normal range
throughout the therapy period in all treated patients.
[0037] Anti-rhGAA antibodies of IgG class were detected in patients
1 and 2 as early as 3 weeks after the initiation of the enzyme
therapy (FIGS. 1A-1C). Anti-rhGAA antibody titers increased to
1:1600 by week 16 in patient 1 (FIG. 1A) and 1:6400-1:12,800
between weeks 11-19 in patient 2 (FIG. 1B). As anti-rhGAA antibody
titers increased, we noted that clinical improvements (noted early
during therapy--see below) were no longer advancing. Neither
untoward effects nor anti-rhGAA antibodies have been detected in
patient 3 (FIG. 1C).
[0038] Cardiac status: Prior to the initiation of the enzyme
therapy, patients 1 and 2 had severe hypertrophic cardiomyopathy
associated with an increased left ventricular (LV) mass, concentric
thickening of the ventricular wall and a decrease in size of the
ventricular cavity (FIG. 2B, the cavity in patient 2 was almost
obliterated at the end of systole). All of these features are
typically seen in the untreated patient with the infantile form of
Pompe disease. Additionally patient 2 was noted to have an
increased LV ejection fraction (shortening fraction, 84%)
reflective of a hyperdynamic shortening. None of the patients,
however, had any evidence of obstruction of the ventricular outflow
tract. The longitudinal echocardiographic data assessed in the
patients during the first 3 months of rhGAA therapy are shown in
FIG. 2A-2C. During the treatment period, in both patients 1 and 2,
the LV end-diastolic and end-systolic volumes (2-D measurements)
progressively increased, and up to almost 2-3 fold by the end of 3
months of therapy as compared to those measured during the
pre-treatment phase (FIGS. 2A and 2B, respectively). Similar
increases were noted by M-mode analysis (data not shown). The
two-dimensional LV mass measurements (FIG. 2D-2F) initially
increased as the LV volumes increased, but then steadily decreased
during therapy, to a value that was less than the pre-treatment LV
mass (reduced to 60-70% of the baseline pretreatment levels). The
initial increase in mass was most likely due to an increase in the
LV volume, without any changes in the LV wall thickness. These
overall improvements in cardiac parameters, were sustained through
the latest follow-up evaluation, although patient 1 required an
intensive daily enzyme infusion for 10 days when LV mass was
further increased and cardiac function compromised at the time of
viral pneumonia. Otherwise the ventricular function in both
patients had been normal and remained normal at the latest
follow-up. Thus, the progressive cardiac morbidity normally noted
in untreated infantile Pompe disease was clearly averted.
[0039] Patient 3 had a Lv mass of 64 g/.beta..sup.2 (upper normal
limits 65) but otherwise of normal baseline cardiac evaluation at
the initiation of therapy, and has continued to be normal (with LV
mass now of 33 g/.beta..sup.2) since 7 months post-therapy.
[0040] Pulmonary function: In the first 2 months of therapy,
improvement of pulmonary function was evident by increases in
crying vital capacity (improvements of greater than 28% and 70%, in
patients 1 and 2, respectively) over baseline capacities, and
normalization of O.sub.2 desaturation during crying (O.sub.2
saturation of 70% in patient 1 and 81% in patient 2 during maximal
crying). Decreased respiratory muscle strength was also evidenced
in patient 1 before the therapy by a negative inspiratory force
maneuver (NIFM) of -45 cm H.sub.2O. With treatment, the NIFM
increased to -55 cm H.sub.2O. The initial improvements noted in the
pulmonary functions of both patients, however, plateaued over the
next 2-3 months and declined subsequently, concomitant with the
rising anti-rhGAA antibodies. Both patients have subsequently
become ventilator dependent after episodes of viral pneumonia
precipitated respiratory insufficiency.
[0041] Patient 3 had a normal pulmonary function at initiation of
therapy and has continued to demonstrate normal pulmonary function
testing at the latest follow-up.
[0042] Neurodevelopment and motor assessment: Alberta Infant Motor
Scale (AIMS) was used to evaluate the motor development in these
infants. AIMS scores for all 3 patients started below the 5th
percentile for age (FIG. 1A-1C). Patient 1 remained below the 5th
percentile but showed increases within that range before beginning
to decline at week 13 of the therapy (FIG. 1A). Patient 2 rose to
the 25th percentile by week 5, dropped back to remain below the 5th
percentile after week 7 despite increasing skills, then showed a
rapid decline and loss of skills between weeks 13 and 17 (FIG. 1B).
The onset of clinical declines, again was concomitant with the
rising anti-rhGAA antibodies (FIG. 1A, 1B).
[0043] Concurrently administered neurologic and Denver
Developmental evaluations showed in patient 1, normal
personal-social, language, and fine motor developmental domains
with ongoing but improving gross motor delay until week 10 when a
plateau and subsequent regression became apparent. Importantly,
gross motor skills had shown significant progress until week 10 but
never reached normal. Patient 2 showed mild developmental delay in
the gross motor sphere only with attainment of normal developmental
skills in the fine motor, personal-social, and language domains
until weeks 14-16 when regression occurred. Currently, both
patients have normal personal-social development for age but delay
in all other domains.
[0044] Patient 3 showed a steady increase of AIMS score, rising
over the 10.sup.th percentile by week 11 of the therapy and rising
above the 25.sup.th percentile by week 20 (FIG. 1C), and 90.sup.th
percentile at latest follow-up. At age 9 months, he maintained
independent sitting, belly crawled reciprocally for mobility, and
maintained standing with hands held. Remarkably, he has been
walking independently since 12 months of age and has been able to
move between squatting and standing without hand use since 14
months of age. He currently also has normal for age neurologic and
Denver development evaluations in all domains.
[0045] Muscle GAA activity and glycogen content: Muscle biopsies
were performed at baseline 1 week prior to the start of the rhGAA
therapy except in patient 1 who had a biopsy done at the time of
diagnosis which was 2 months prior to initiation of rhGAA therapy.
After 4 months of rhGAA therapy, muscle biopsies were obtained from
the contra-lateral quadriceps 3 days after the enzyme infusion
(trough level). With rhGAA treatment GAA activity increased 2-3
fold over baseline pre-treatment levels in both patients 1 and 2,
and 18 fold in patient 3 (Table 3).
TABLE-US-00003 TABLE 3 Muscle Acid .alpha.-glucosidase Activity and
Glycogen Content in Infantile Pompe Disease Patients Treated with
rhGAA GAA Activity Glycogen Content nmole/hr/mg Protein % Wet
Weight Patient 1 Pre-therapy 0.41 5.90% Post-therapy 0.95 7.50%
Patient 2 Pre-therapy 0.67 5.68% Post-therapy 1.97 4.43% Patient 3
Pre-therapy 0.1 5.13% Post-therapy 1.84 1.43% Control 23.92 +/-
8.63 0.94 +/- 0.55% (upper normal limit; 1.5%)
The absolute level of GAA activity approached 8% of the GAA
activity seen in normal muscles. There were no appreciable changes
in the muscle glycogen content in patients 1 and 2, but glycogen
levels were reduced to within normal range in patient 3.
[0046] Histology: The pre-treatment biopsies of all the patients
showed marked vacuolization of the muscle fibers in the frozen
sections. Evaluation of the semithin sections demonstrated the
fibers to be expanded by glycogen with the formation of glycogen
lakes. In some fibers faint outlines of residual membranes could be
discerned. Electron microscopy confirmed the presence of glycogen
both in expanded lysosomes and lying free in the cytoplasm. The
biopsy from patient 3 had more glycogen remaining within lysosomes
than did the other two patients (data not shown).
[0047] The 4-month post-treatment biopsies of patients 1 and 2 were
similar to the pre-treatment biopsies in terms of glycogen
accumulation. The post-treatment biopsy of patient 3, however, had
a marked decrease in visible glycogen and essentially normal
histology in most of the muscle fibers. Electron microscopy showed
many remaining distended lysosomes were depleted of glycogen. Some
glycogen lakes and glycogen-rich lysosomes remained.
[0048] Western Blot Analysis
[0049] To investigate why anti-rhGAA antibodies developed in
patients 1 and 2, but not 3, we performed a Western blot analysis
specific for detection of expressed (but nonfunctional) GAA protein
in fibroblasts derived from each of the patients. No GAA protein
was detected in the fibroblasts of patients 1 and 2, whereas a
readily detectable precursor form of GAA protein (110 kD) was found
in patient 3. These patterns were previously seen in other patients
with infantile GSD-II (Van der Ploeg, A. T. et al., Am. J. Hum.
Genet. 44:787-793 (1989)). Normal fibroblasts as expected have GAA
protein predominantly of 95 kD and 76 kD.
Further Studies
[0050] Three more patients have been enrolled in an additional
study. All three are CRIM positive. After treatment (10 mg/kilogram
body weight, weekly intravenous infusions of rhGAA) for 3-6 weeks,
improvement of heart function, muscle strength, and motor
development have been seen.
[0051] The teachings of all publications cited herein are
incorporated herein by reference in their entirety.
[0052] While this invention has been particularly shown and
described with reference to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
spirit and scope of the invention as defined by the appended
claims.
* * * * *