U.S. patent application number 12/966904 was filed with the patent office on 2011-05-05 for assays for diagnosing and evaluating treatment options for fabry disease.
This patent application is currently assigned to AMICUS THERAPEUTICS, INC.. Invention is credited to Jeff Castelli, Christine Kaneski, David Lockhart, Karin Ludwig, Gary Murray, Sang-Hoon Shin.
Application Number | 20110104727 12/966904 |
Document ID | / |
Family ID | 38723987 |
Filed Date | 2011-05-05 |
United States Patent
Application |
20110104727 |
Kind Code |
A1 |
Kaneski; Christine ; et
al. |
May 5, 2011 |
ASSAYS FOR DIAGNOSING AND EVALUATING TREATMENT OPTIONS FOR FABRY
DISEASE
Abstract
Provided are in vitro and in vivo methods for determining
whether a patient with Fabry disease will respond to treatment with
a specific pharmacological chaperone.
Inventors: |
Kaneski; Christine; (Dallas,
TX) ; Shin; Sang-Hoon; (Suwon, KR) ; Murray;
Gary; (Bethesda, MD) ; Ludwig; Karin; (Dana
Point, CA) ; Lockhart; David; (Del Mar, CA) ;
Castelli; Jeff; (New Hope, PA) |
Assignee: |
AMICUS THERAPEUTICS, INC.
Cranbury
NJ
|
Family ID: |
38723987 |
Appl. No.: |
12/966904 |
Filed: |
December 13, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11749512 |
May 16, 2007 |
7851143 |
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12966904 |
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60853631 |
Oct 23, 2006 |
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60801089 |
May 16, 2006 |
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Current U.S.
Class: |
435/18 ;
435/29 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 9/0053 20130101; A61P 43/00 20180101; A61K 31/445 20130101;
A61K 9/48 20130101; G01N 2800/52 20130101; A61P 9/00 20180101; A61P
9/10 20180101; A61K 31/45 20130101; G01N 2333/94 20130101; G01N
2333/924 20130101; G01N 2800/04 20130101; A61P 13/12 20180101; G01N
33/5094 20130101; A61P 3/00 20180101; C07D 211/46 20130101; A61K
31/445 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
435/18 ;
435/29 |
International
Class: |
C12Q 1/34 20060101
C12Q001/34; C12Q 1/02 20060101 C12Q001/02 |
Claims
1. A method for determining whether a patient having a deficiency
in activity of a protein will respond to treatment with a specific
pharmacological chaperone for the protein, which method comprises
a. contacting cells from a patient an individual with a
pharmacological chaperone specific for the protein; and b.
comparing protein activity in cells not contacted with the specific
pharmacological chaperone, with protein activity in cells contacted
with the specific pharmacological chaperone wherein an increase in
protein activity in cells contacted with the specific
pharmacological chaperone over activity in cells not contacted with
the specific pharmacological chaperone indicates that the patient
will respond to treatment with the specific pharmacological
chaperone.
2. The method of claim 1, wherein the deficiency of activity is
caused by a missense mutation in a gene encoding the protein.
3. The method of claim 1, wherein the protein is an enzyme.
4. The method of claim 3, wherein the enzyme is a lysosomal
enzyme.
5. The method of claim 4, wherein the patient has been diagnosed
with a lysosomal storage disorder.
6. The method of claim 5, wherein the lysosomal enzyme is
.alpha.-GAL and the lysosomal storage disorder is Fabry
disease.
7. The method of claim 5, wherein the specific pharmacological
chaperone is 1-deoxygalactonojirimycin.
8. The method of claim 7, wherein the cells are white blood cells
and the contact with the specific pharmacological chaperone occurs
in vivo.
9.-16. (canceled)
17. The method of claim 8, wherein the patient is administered
1-deoxygalactonojirimycin daily for about 2 weeks.
18. The method of claim 17, wherein the administration is oral.
19. The method of claim 17, wherein the 1-deoxygalactonojirimycin
is administered at a dose of about 50-500 mg/day.
20. The method of claim 19, wherein the dose is about 100-250
mg/day.
21. The method of claim 20, wherein the dose is about 150
mg/day.
22. The method of claim 19, wherein the 1-deoxygalactonojirimycin
is administered once a day.
23. The method of claim 17, further comprising collecting a blood
sample at the end of two weeks and separating the white blood
cells.
24. The method of claim 17 wherein .alpha.-GAL activity is
determined using a fluorometric assay that quantifies hydrolysis of
substrate in lysates from the white blood cells.
25. The method of claim 24 wherein the sufficient increase in
activity in the lysates in the presence of the
1-deoxygalactonojirimycin which indicates whether the patient will
respond is measured according to the following criteria: i) If
baseline activity is less than 1% of normal, the activity following
culture or following treatment with SPC must be at least 2% of
normal; ii) If baseline activity is between 1% but less than 5% of
normal then the activity following culture or treatment with SPC
must be at least 2 times the baseline level; iii) If baseline
activity is between 5% but less than 10% of normal, then the
activity following culture or treatment with SPC must be at least
5% of normal higher the baseline level of normal; iv) if baseline
activity is 10% of normal or more, then activity following culture
or treatment with SPC must be at least 1.5.times. the baseline
level.
26. A kit comprising: a. at least one T cell stimulatory agent; b.
a specific pharmacological chaperon c. a labeled substrate for the
chaperone; d. GalNAc; and e. instructions for performing a protein
enhancement assay.
27. The kit of claim 26, wherein the T-cell stimulatory agent is a
mitogen.
28. The kit of claim 27, wherein the mitogen is PHA.
29. The kit of claim 26, wherein the stimulatory agent is a
cytokine.
30. The kit of claim 29, wherein the cytokine is IL-2.
31. The kit of claim 26, wherein the pharmacological chaperone is
1-deoxygalactonojirimycin.
32. The kit of claim 26, further comprising one or more a blood
collection tubes, centrifuge tubes, and cryotubes.
33. The kit of claim 26, wherein the protein is an enzyme.
34. The kit of claim 33, wherein the enzyme is
.alpha.-galactosidase A.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. Ser.
No. 11/749,512, filed on May 16, 2007, which claims priority from
U.S. provisional patent application Ser. No. 60/801,089, filed on
May 16, 2006, and from U.S. provisional patent application Ser. No.
60/853,631, filed on Oct. 23, 2006, each of which is incorporated
by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention provides methods to determine whether
a patient with Fabry disease will benefit from treatment with a
specific pharmacological chaperone. The present invention
exemplifies two methods, one in vitro and one in vivo, for
determining .alpha.-galactosidase A responsiveness to a
pharmacological chaperone such as 1-deoxygalactonojirimycin in
patient cells. The invention also provides a method for diagnosing
Fabry disease in patients suspected of having Fabry disease.
BACKGROUND
[0003] Fabry disease is a glycosphingolipid (GSL) lysosomal storage
disorder resulting from an X-linked inherited deficiency of
lysosomal .alpha.-galactosidase A (.alpha.-GAL), an enzyme
responsible for the hydrolysis of terminal .alpha.-galactosyl
residues from glycosphingolipids (Brady et al. N Engl J Med. 1967;
276: 1163-7). A deficiency in the enzyme activity results in a
progressive deposition of neutral glycosphingolipids, predominantly
globotriaosylceramide (ceramide trihexoside, CTH, GL-3), in cell of
Fabry patients. Symptoms can be severe and debilitating, including
kidney failure and increased risk of heart attack and stroke.
Certain of the mutations cause changes in the amino acid sequence
of .alpha.-GAL that may result in the production of .alpha.-GAL
with reduced stability that does not fold into its correct
three-dimensional shape. Although .alpha.-GAL produced in patient
cells often retains the potential for some level of biological
activity, the cell's quality control mechanisms recognize and
retain misfolded .alpha.-GAL in the endoplasmic reticulum, or ER,
until it is ultimately moved to another part of the cell for
degradation and elimination. Consequently, little or no .alpha.-GAL
moves to the lysosome, where it normally hydrolyzes GL-3. This
leads to accumulation of GL-3 in cells, which is believed to be the
cause of the symptoms of Fabry disease. In addition, accumulation
of the misfolded .alpha.-GAL enzyme in the ER may lead to stress on
cells and inflammatory-like responses, which may contribute to
cellular dysfunction and disease.
[0004] Fabry disease is classified by clinical manifestations into
three groups: a classic form with generalized vasculopathy, an
atypical variant form with clinical manifestations limited to
cardiac tissue, and later-onset disease, which includes female
carriers with mild to severe forms of the disease.
[0005] The frequency of the classical form of disease is estimated
to be about 1:40,000 to 1:60,000 in males, and is reported
throughout the world within different ethnic groups. Classically
affected males have little or no detectable .alpha.-GAL levels and
are the most severely affected. The clinical manifestations include
angiokeratoma (small, raised reddish-purple blemishes on the skin),
acroparesthesias (burning in hands and feet), hypohidrosis
(decreased ability to sweat), and characteristic corneal and
lenticular opacities (The Metabolic and Molecular Bases of
Inherited Disease, 8th Edition 2001, Scriver et al., ed., pp.
3733-3774, McGraw-Hill, New York). Lipid storage may lead to
impaired arterial circulation and increased risk of heart attack or
stroke. The heart may also become enlarged and the kidneys may
become progressively involved. Other symptoms include fever and
gastrointestinal difficulties, particularly after eating. The
affected male's life expectancy is reduced, and death usually
occurs in the fourth or fifth decade as a result of vascular
disease of the heart, brain, and/or kidneys.
[0006] Individuals with later-onset Fabry disease can be male or
female. Late-onset Fabry disease presents as the atypical variant
form, and growing evidence indicates there may be a significant
number of "atypical variants" which are unaccounted for in the
world. Females, who inherit an X chromosome containing an
.alpha.-GAL mutation, may exhibit symptoms later in life,
significantly increasing the prevalence of this disease. These
patients typically first experience disease symptoms in adulthood,
and often have disease symptoms focused on a single organ. For
example, many males and females with later-onset Fabry disease have
enlargement of the left ventricle of the heart. Later-onset Fabry
disease may also present in the form of strokes of unknown cause.
As the patients advance in age, the cardiac complications of the
disease progress, and can lead to death.
[0007] In contrast, patients with the milder "cardiac variant" of
Fabry diseasenormally have 5-15% of normal .alpha.-GAL activity,
and present with left ventricular hypertrophy or a cardiomyopathy.
These cardiac variant patients remain essentially asymptomatic when
their classically affected counterparts are severely compromised.
Cardiac variants were found in 11% of adult male patients with
unexplained left ventricular hypertrophic cardiomyopathy,
suggesting that Fabry disease may be more frequent than previously
estimated (Nakao et al., N. Engl. J. Med. 1995; 333: 288-293).
[0008] The .alpha.-GAL gene has been mapped to Xq22 (Bishop et al.,
Am. J. Hum. Genet. 1985; 37: A144), and the full-length cDNA and
entire 12-kb genomic sequences encoding .alpha.-GAL have been
reported (Calhoun et al., Proc. Natl. Acad. Sci. USA. 1985; 82:
7364-7368; Bishop et al., Proc. Natl. Acad. Sci. USA. 1986; 83:
4859-4863; Tsuji et al., Eur. J. Biochem. 1987; 165: 275-280; and
Kornreich et al., Nucleic Acids Res. 1989; 17: 3301-3302). There is
a marked genetic heterogeneity of mutations that cause Fabry
disease (The Metabolic and Molecular Bases of Inherited Disease,
8th Edition 2001, Scriver et al., ed., pp. 3733-3774, McGraw-Hill,
New York; Eng et al., Am. J. Hum. Genet. 1993; 53: 1186-1197; Eng
et al., Mol. Med. 1997; 3: 174-182; and Davies et al., Eur. J. Hum.
Genet. 1996; 4: 219-224). To date, a variety of missense, nonsense,
and splicing mutations, in addition to small deletions and
insertions, and larger gene rearrangements, have been reported,
although the majority of mutations are missense mutations.
[0009] Fabry disease is heterogeneous and it is often difficult to
correlate genotype with phenotype. People with the same genotype
often exhibit different clinical symptoms and disease pathology.
However, there appears to be a correlation between residual enzyme
activity and disease severity, with the lower the .alpha.-GAL
activity resulting in the greatest severity of disease. Although
the vast majority of .alpha.-GAL mutations are missense mutations,
with most being outside the catalytic site, it difficult to predict
which mutations result in an unstable enzyme that could be
"rescued" by a specific pharmacological chaperone (SPC) which
stabilizes the enzyme, and which ones cannot be stabilized using a
SPC.
Diagnosis of Fabry Disease
[0010] Because Fabry disease is rare, involves multiple organs, has
a wide age range of onset, and is heterogeneous, proper diagnosis
is a challenge. Awareness is low among health care professionals
and misdiagnoses are frequent. Some examples of diagnoses seriously
considered in patients who were eventually diagnosed with Fabry's
disease include: mitral valve prolapse, glomerulonephritis,
idiopathic proteinuria, systemic lupus erythematosus, Whipple's
disease, acute abdomen, ulcerative colitis, acute intermittent
porphyrias, and occult malignancies. Thus, even for classically
affected males, diagnosis typically takes from about 5-7 years or
even longer. This is a concern because the longer a person has
Fabry disease, the more damage is likely to occur in the affected
organs and tissues and the more serious the person's condition may
become. Diagnosis of Fabry disease is most often confirmed on the
basis of decreased .alpha.-GAL activity in plasma or peripheral
leukocytes (WBCs) once a patient is symptomatic, coupled with
mutational analysis. In females, diagnosis is even more challenging
since the enzymatic identification of carrier females is less
reliable due to random X-chromosomal inactivation in some cells of
carriers. For example, some obligate carriers (daughters of
classically affected males) have .alpha.-GAL enzyme activities
ranging from normal to very low activities. Since carriers can have
normal .alpha.-GAL enzyme activity in leukocytes, only the
identification of an .alpha.-GAL mutation by genetic testing
provides precise carrier identification and/or diagnosis.
Treatment of Fabry Disease
[0011] The only approved therapy for treating Fabry disease is
enzyme replacement therapy, which typically involves intravenous,
infusion of a purified form of the corresponding wild-type protein
(Fabrazyme.RTM., Genzyme Corp.). One of the main complications with
protein replacement therapy is attainment and maintenance of
therapeutically effective amounts of protein in vivo due to rapid
degradation of the infused protein. The current approach to
overcome this problem is to perform numerous costly high dose
infusions.
[0012] Protein replacement therapy has several additional caveats,
such as difficulties with large-scale generation, purification, and
storage of properly folded protein; obtaining glycosylated native
protein; generation of an anti-protein immune response; and
inability of protein to cross the blood-brain barrier to mitigate
central nervous system pathologies (i.e., low bioavailability). In
addition, replacement enzyme cannot penetrate the heart or kidney
in sufficient amounts to reduce substrate accumulation in the renal
podocytes or cardiac myocytes, which figure prominently in Fabry
pathology.
[0013] Gene therapy using recombinant vectors containing nucleic
acid sequences that encode a functional protein, or using
genetically modified human cells that express a functional protein,
is also being evaluated to treat protein deficiencies and other
disorders that benefit from protein replacement. Although
promising, this approach is also limited by technical difficulties
such as the inability of vectors to infect or transduce dividing
cells, low expression of the target gene, and regulation of
expression once the gene is delivered.
[0014] A third, relatively recent approach to treating some enzyme
deficiencies involves the use of small molecule inhibitors to
reduce production of the natural substrate of deficient enzyme
proteins, thereby ameliorating the pathology. This "substrate
reduction" approach has been specifically described for a class of
about 40 related enzyme disorders called lysosomal storage
disorders that include glycosphingolipid storage disorders. The
small molecule inhibitors proposed for use as therapy are specific
for inhibiting the enzymes involved in synthesis of glycolipids,
reducing the amount of cellular glycolipid that needs to be broken
down by the deficient enzyme. This approach is also limited in that
glycolipids are necessary for biological function, especially
neurological function, and excessive deprivation may cause adverse
effects.
[0015] It has previously been shown that the binding of small
molecule inhibitors of enzymes associated with LSDs can increase
the stability of both mutant enzyme and the corresponding wild-type
enzyme (see U.S. Pat. Nos. 6,274,597; 6,583,158; 6,589,964;
6,599,919; 6,916,829, and 7,141,582 all incorporated herein by
reference). In particular, it was discovered that administration of
small molecule derivatives of glucose and galactose, which are
specific, selective competitive inhibitors for several target
lysosomal enzymes, effectively increased the stability of the
enzymes in cells in vitro and, thus, increased trafficking of the
enzymes to the lysosome. Thus, by increasing the amount of enzyme
in the lysosome, hydrolysis of the enzyme substrates is expected to
increase. The original theory behind this strategy was as follows:
since the mutant enzyme protein is unstable in the ER (Ishii et
al., Biochem. Biophys. Res. Comm. 1996; 220: 812-815), the enzyme
protein is retarded in the normal transport pathway
(ER.fwdarw.Golgi apparatus.fwdarw.endosomes.fwdarw.lysosome) and
prematurely degraded. Therefore, a compound which binds to and
increases the stability of a mutant enzyme, may serve as a
"chaperone" for the enzyme and increase the amount that can exit
the ER and move to the lysosomes. In addition, because the folding
and trafficking of some wild-type proteins is incomplete, with up
to 70% of some wild-type proteins being degraded in some instances
prior to reaching their final cellular location, the chaperones can
be used to stabilize wild-type enzymes and increase the amount of
enzyme which can exit the ER and be trafficked to lysosomes. This
strategy has been shown to increase several lysosomal enzymes in
vitro and in vivo, including .beta.-glucocerebrosidase and
.alpha.-glucosidase, deficiencies of which are associated with
Gaucher and Pompe disease, respectively.
[0016] However, as indicated above, successful candidates for SPC
therapy must have a mutation which results in the production of an
enzyme that has the potential to be stabilized and folded into a
conformation that permits trafficking out of the ER. Mutations
which severely truncate the enzyme, such as nonsense mutations, or
mutations in the catalytic domain which prevent binding of the
chaperone, will not likely be "rescuable" or "enhanceable" using
SPC therapy. While missense mutations outside the catalytic site
are more likely to be rescuable using SPCs, there is no guarantee,
necessitating screening for responsive mutations. This means that,
even when Fabry disease is diagnosed by detecting deficient
.alpha.-GAL activity in WBCs, it is very difficult, if not
impossible, to predict whether a particular Fabry patient will
respond to treatment with an SPC. Moreover, since WBCs only survive
for a short period of time in culture (in vitro), screening for SPC
enhancement of .alpha.-GAL is difficult.
[0017] In order to apply SPC therapy effectively, a broadly
applicable, fast and efficient method for screening patients for
responsiveness to SPC therapy needs to be adopted prior to
initiation of treatment. Thus, there remains in the art a need for
relatively non-invasive methods to rapidly assess enzyme
enhancement with potential therapies prior to making treatment
decisions, for both cost and emotional benefits to the patient.
SUMMARY OF THE INVENTION
[0018] The present invention provides two methods for determining
whether a patient will be a candidate for SPC therapy.
Specifically, the present invention provides in vitro and in vivo
assays to evaluate .alpha.-GAL activity in blood cells derived from
patients with Fabry disease in the presence or absence of an SPC.
The present invention also includes the basis for evaluation of SPC
as a treatment option for any number of other protein abnormalities
and/or enzyme deficiencies. The present invention also provides for
diagnostic kits containing the components required to perform the
assay. The present invention further provides an improved method of
diagnosing Fabry disease by determining .alpha.-GAL activity in T
cells from patients suspected of having Fabry disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1. Time course for enhancement for A97V. T-cells
bearing the A97V mutation in .alpha.-Gal A were cultured in the
absence (open circles) or presence (filled circles) of 20 .mu.M DGJ
for one to four days then assayed for .alpha.-GAL activity.
Changing media after 2 days and replacing with fresh media with
(open triangle) or without DGJ (filled triangle) had no effect on
the observed enzyme activity.
[0020] FIG. 2A-C. Concentration Dependence of DGJ in T-cells from
normal control and Fabry patients. T-cells from normal individuals
(2A) were incubated for 3 days with DGJ from 2 nM to 200 .mu.M then
assayed for .alpha.-GAL activity. The results of three experiments
on different days are shown. T cells from Fabry patients with the
A97V (2B), R112H (2C), or R301Q (2C) mutations, respectively, were
cultured with DGJ from 2 nM to 200 .mu.M then assayed for
.alpha.-GAL activity. Three independent sets of DGJ dosage
experiments, each of which were performed with triplicate sets are
shown.
[0021] FIG. 3. T-cells from various Fabry patients were cultured in
the absence of or in the presence of 20 .mu.M DGJ for three days
then assayed for .alpha.-GAL activity. Percent of the average
normal of specific activity of the .alpha.-GAL were graphed to show
the effect of DGJ rescue on the different genotypes of Fabry
patients. The lower panel shows the Western blot results for each
mutation, probed with polyclonal rabbit antibody specific for
.alpha.-GAL. This demonstrates increased protein stability for
enhanceable mutations A97V and R301Q and no increase in protein
amount for the mutations R356W, G132R and A143P.
[0022] FIG. 4. Graphical representation of in vivo enhancement of
.alpha.-GAL activity in WBC of Fabry patients following treatment
with DGJ.
[0023] FIG. 5. Graphical representation comparing in vitro and in
vivo enhancement of .alpha.-GAL activity for 10 genotypes.
DETAILED DESCRIPTION
[0024] The present invention provides two assays to allow the
accurate determination of whether an SPC enhances enzyme activity
from cells derived from patients with Fabry disease. These assays
permit a determination of whether the patient will be a candidate
for SPC therapy. The new in vitro assay is extremely sensitive and
can be performed on isolated T cells which do not need to be
extensively cultured and maintained in vivo, which speeds up the
time required to perform the assay (as compared to when fibroblasts
are used). This assay also can be used as a diagnostic assay for
patients suspected of having Fabry disease, especially females,
since it is more sensitive than the WBC assay typically used for
detecting .alpha.-GAL activity. The new in vivo assay is similarly
non-invasive and provides a very reliable method for determining
whether a SPC therapy will be effective in a particular patient. In
addition, in conjunction with genotyping, both assays provide a
method for determining whether newly discovered .alpha.-GAL
mutations (such as spontaneous mutations) cause the .alpha.-GAL to
misfold and, thus potentially would be "rescuable" using SPCs.
DEFINITIONS
[0025] The terms used in this specification generally have their
ordinary meanings in the art, within the context of this invention
and in the specific context where each term is used. Certain terms
are discussed below, or elsewhere in the specification, to provide
additional guidance to the practitioner in describing the
compositions and methods of the invention and how to make and use
them.
[0026] The term "Fabry disease" refers to an X-linked inborn error
of glycosphingolipid catabolism due to deficient lysosomal
.alpha.-galactosidase A activity. This defect causes accumulation
of globotriaosylceramide (ceramide trihexoside) and related
glycosphingolipids in vascular endothelial lysosomes of the heart,
kidneys, skin, and other tissues.
[0027] The term "atypical Fabry disease" refers to patients with
primarily cardiac manifestations of the .alpha.-GAL deficiency,
namely progressive globotriaosylceramide (GL-3) accumulation in
myocardial cells that leads to significant enlargement of the
heart, particularly the left ventricle.
[0028] A "carrier" is a female who has one X chromosome with a
defective .alpha.-GAL gene and one X chromosome with the normal
gene and in whom X chromosome inactivation of the normal allele is
present in one or more cell types. A carrier is often afflicted
with Fabry disease.
[0029] A "patient" refers to a subject who has been diagnosed with
a particular disease. The patient may be human or animal. A "Fabry
disease patient" refers to an individual who has been diagnosed
with Fabry disease and has a mutated .alpha.-GAL as defined further
below. Characteristic markers of Fabry disease can occur in male
hemizygotes and female carriers with the same prevalence, although
females typically are less severely affected.
[0030] Human .alpha.-galactosidase A (.alpha.-GAL) refers to an
enzyme encoded by the human Gla gene. The human .alpha.-GAL enzyme
consists of 429 amino acids and is in GenBank Accession No.
U78027.
[0031] As used herein in one embodiment, the term "mutant
.alpha.-GAL" includes an .alpha.-GAL which has a mutation in the
gene encoding .alpha.-GAL which results in the inability of the
enzyme to achieve a stable conformation under the conditions
normally present in the ER. The failure to achieve a stable
conformation results in a substantial amount of the enzyme being
degraded, rather than being transported to the lysosome. Such a
mutation is sometimes called a "conformational mutant."
[0032] Non-limiting, exemplary .alpha.-GAL mutations associated
with Fabry disease which result in unstable .alpha.-GAL include
L32P; N34S; T41I; M51K; E59K; E66Q; I91T; A97V; R100K; R112C;
R112H; F113L; T141L; A143T; G144V; S148N; A156V; L166V; D170V;
C172Y; G183D; P205T; Y207C; Y207S; N215S; A228P; S235C; D244N;
P259R; N263S; N264A; G272S; S276G; Q279E; Q279K; Q279H; M284T;
W287C; I289F; M296I; M296V; L300P; R301Q; V316E; N320Y; G325D;
G328A; R342Q; E358A; E358K; R363C; R363H; G370S; and P409A.
[0033] As used herein, the term "specific pharmacological
chaperone" ("SPC") or "pharmacological chaperone" refers to any
molecule including a small molecule, protein, peptide, nucleic
acid, carbohydrate, etc. that specifically binds to a protein and
has one or more of the following effects: (i) enhances the
formation of a stable molecular conformation of the protein; (ii)
induces trafficking of the protein from the ER to another cellular
location, preferably a native cellular location, i.e., prevents
ER-associated degradation of the protein; (iii) prevents
aggregation of misfolded proteins; and/or (iv) restores or enhances
at least partial wild-type function and/or activity to the protein.
A compound that specifically binds to e.g., .alpha.-GAL, means that
it binds to and exerts a chaperone effect on .alpha.-GAL and not a
generic group of related or unrelated enzymes. More specifically,
this term does not refer to endogenous chaperones, such as BiP, or
to non-specific agents which have demonstrated non-specific
chaperone activity against various proteins, such as glycerol, DMSO
or deuterated water, i.e., chemical chaperones (see Welch et al.,
Cell Stress and Chaperones 1996; 1(2):109-115; Welch et al.,
Journal of Bioenergetics and Biomembranes 1997; 29(5):491-502; U.S.
Pat. No. 5,900,360; U.S. Pat. No. 6,270,954; and U.S. Pat. No.
6,541,195). In the present invention, the SPC may be a reversible
competitive inhibitor.
[0034] A "competitive inhibitor" of an enzyme can refer to a
compound which structurally resembles the chemical structure and
molecular geometry of the enzyme substrate to bind the enzyme in
approximately the same location as the substrate. Thus, the
inhibitor competes for the same active site as the substrate
molecule, thus increasing the Km. Competitive inhibition is usually
reversible if sufficient substrate molecules are available to
displace the inhibitor, i.e., competitive inhibitors can bind
reversibly. Therefore, the amount of enzyme inhibition depends upon
the inhibitor concentration, substrate concentration, and the
relative affinities of the inhibitor and substrate for the active
site.
[0035] Following is a description of some specific pharmacological
chaperones contemplated by this invention:
[0036] 1-deoxygalactonojirimycin refers to a compound having the
following structures:
##STR00001##
[0037] This term includes both the free base and any salt forms.
The hydrochloride salt of DGJ is known as migalastat hydrochloride
(Migalastat).
[0038] Still other SPCs for .alpha.-GAL are described in U.S. Pat.
Nos. 6,274,597, 6,774,135, and 6,599,919 to Fan et al., and include
.alpha.-3,4-di-epi-homonojirimycin, 4-epi-fagomine, and
.alpha.-allo-homonojirimycin, N-methyl-deoxygalactonojirimycin,
.beta.-1-C-butyl-deoxygalactonojirimycin, and
.alpha.-galacto-homonojirimycin, calystegine A.sub.3, calystegine
B.sub.2, calystegine B.sub.3, N-methyl-calystegine A.sub.3,
N-methyl-calystegine B.sub.2 and N-methyl-calystegine B.sub.3.
[0039] As used herein, the term "specifically binds" refers to the
interaction of a pharmacological chaperone with a protein such as
.alpha.-GAL, specifically, an interaction with amino acid residues
of the protein that directly participate in contacting the
pharmacological chaperone. A pharmacological chaperone specifically
binds a target protein, e.g., .alpha.-GAL, to exert a chaperone
effect on .alpha.-GAL and not a generic group of related or
unrelated proteins. The amino acid residues of a protein that
interact with any given pharmacological chaperone may or may not be
within the protein's "active site." Specific binding can be
evaluated through routine binding assays or through structural
studies, e.g., co-crystallization, NMR, and the like. The active
site for .alpha.-GAL is the substrate binding site.
[0040] "Deficient .alpha.-GAL activity" refers to .alpha.-GAL
activity in cells from a patient which is below the normal range as
compared (using the same methods) to the activity in normal
individuals not having or suspected of having Fabry or any other
disease (especially a blood disease).
[0041] As used herein, the terms "enhance .alpha.-GAL activity" or
"increase .alpha.-GAL activity" refer to increasing the amount of
.alpha.-GAL that adopts a stable conformation in a cell contacted
with a pharmacological chaperone specific for the .alpha.-GAL,
relative to the amount in a cell (preferably of the same cell-type
or the same cell, e.g., at an earlier time) not contacted with the
pharmacological chaperone specific for the .alpha.-GAL. This term
also refers to increasing the trafficking of .alpha.-GAL to the
lysosome in a cell contacted with a pharmacological chaperone
specific for the .alpha.-GAL, relative to the trafficking of
.alpha.-GAL not contacted with the pharmacological chaperone
specific for the protein. These terms refer to both wild-type and
mutant .alpha.-GAL. In one embodiment, the increase in the amount
of .alpha.-GAL in the cell is measured by measuring the hydrolysis
of an artificial substrate in lysates from cells that have been
treated with the SPC. An increase in hydrolysis is indicative of
increased .alpha.-GAL activity.
[0042] The term ".alpha.-GAL activity" refers to the normal
physiological function of a wild-type .alpha.-GAL in a cell. For
example, .alpha.-GAL activity includes hydrolysis of GL-3.
[0043] A "responder" is an individual (diagnosed with or suspected
of having Fabry disease) whose cells exhibit sufficiently increased
.alpha.-GAL activity, and/or amelioration of symptoms or
improvement in surrogate markers, in response to contact with an
SPC. Non-limiting examples of improvements in surrogate markers for
Fabry disease include increases in .alpha.-GAL levels or activity
in cells (e.g., fibroblasts) and tissue; reductions in of GL-3
accumulation; decreased plasma concentrations of homocysteine and
vascular cell adhesion molecule-1 (VCAM-1); decreased GL-3
accumulation within myocardial cells and valvular fibrocytes;
reduction in cardiac hypertrophy (especially of the left
ventricle), amelioration of valvular insufficiency, and
arrhythmias; amelioration of proteinuria; decreased urinary
concentrations of lipids such as CTH, lactosylceramide, ceramide,
and increased urinary concentrations of glucosylceramide and
sphingomyelin (Fuller et al., Clinical Chemistry. 2005; 51:
688-694); the absence of laminated inclusion bodies (Zebra bodies)
in glomerular epithelial cells; improvements in renal function;
mitigation of hypohidrosis; the absence of angiokeratomas; and
improvements hearing abnormalities such as high frequency
sensorineural hearing loss progressive hearing loss, sudden
deafness, or tinnitus. Improvements in neurological symptoms
include prevention of transient ischemic attack (TIA) or stroke;
and amelioration of neuropathic pain manifesting itself as
acroparaesthesia (burning or tingling in extremities).
[0044] The dose that achieves one or more of the aforementioned
responses is a "therapeutically effective dose."
[0045] The phrase "pharmaceutically acceptable" refers to molecular
entities and compositions that are physiologically tolerable and do
not typically produce untoward reactions when administered to a
human. Preferably, as used herein, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopoeia or other
generally recognized pharmacopoeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the compound is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils. Water or aqueous solution saline solutions
and aqueous dextrose and glycerol solutions are preferably employed
as carriers, particularly for injectable solutions. Suitable
pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin, 18th Edition, or other
editions.
[0046] As used herein, the term "isolated" means that the
referenced material is removed from the environment in which it is
normally found. Thus, an isolated biological material can be free
of cellular components, i.e., components of the cells in which the
material is found or produced. In the case of nucleic acid
molecules, an isolated nucleic acid includes a PCR product, an mRNA
band on a gel, a cDNA, or a restriction fragment. In another
embodiment, an isolated nucleic acid is preferably excised from the
chromosome in which it may be found, and more preferably is no
longer joined to non-regulatory, non-coding regions, or to other
genes, located upstream or downstream of the gene contained by the
isolated nucleic acid molecule when found in the chromosome. In yet
another embodiment, the isolated nucleic acid lacks one or more
introns. Isolated nucleic acids include sequences inserted into
plasmids, cosmids, artificial chromosomes, and the like. Thus, in a
specific embodiment, a recombinant nucleic acid is an isolated
nucleic acid. An isolated protein may be associated with other
proteins or nucleic acids, or both, with which it associates in the
cell, or with cellular membranes if it is a membrane-associated
protein. An isolated organelle, cell, or tissue is removed from the
anatomical site in which it is found in an organism. An isolated
material may be, but need not be, purified.
[0047] The terms "about" and "approximately" shall generally mean
an acceptable degree of error for the quantity measured given the
nature or precision of the measurements. Typical, exemplary degrees
of error are within 20 percent (%), preferably within 10%, and more
preferably within 5% of a given value or range of values.
Alternatively, and particularly in biological systems, the terms
"about" and "approximately" may mean values that are within an
order of magnitude, preferably within 10- or 5-fold, and more
preferably within 2-fold of a given value. Numerical quantities
given herein are approximate unless stated otherwise, meaning that
the term "about" or "approximately" can be inferred when not
expressly stated.
Method
[0048] To easily determine whether SPC therapy will be a viable
treatment for Fabry patients, including female carriers, simple,
non-invasive DGJ rescue assay of .alpha.-GAL activity in WBCs, or
subsets of WBCs, from Fabry patients was developed.
I. In Vitro Assay
[0049] In one embodiment, the diagnostic method of the present
invention involves purifying T cells and establishing T cell
cultures from blood specimens from Fabry patients (or patients
suspected of having Fabry disease). T cell cultures are then
treated with or without an SPC, e.g., DGJ, for a sufficient time
period to demonstrate enhancement (i.e., increase) of .alpha.-GAL
activity. The T cells are then lysed, and the lysate is used in an
assay to determine enzyme activity. A sufficient increase in
.alpha.-GAL activity in the lysates from cells treated with the SPC
over the activity in the lysates from untreated cells indicates
that the patient will likely respond to SPC therapy i.e., the
patient will be a "responder").
[0050] This embodiment can be carried out as follows.
White Blood Cell Separation
[0051] The WBCs are prepared using standard techniques, e.g.,
collection, centrifugation, separation, and washing. More
specifically, they can be prepared according to the following
steps: [0052] 1. A blood sample is drawn from a Fabry patient. In
specific embodiments, approximately 8 to 10 mL are drawn into an
appropriate container such as a CPT tube from Becton-Dickenson
(containing an anti-coagulant and a separation medium). [0053] 2.
The blood sample is centrifuged to separate red blood cells from
white blood cells and plasma. Typically, this step can be performed
at room temperature (18-25.degree. C.) at about 1800.times.g with a
tabletop centrifuge for about 20-30 minutes, or until the red blood
cells are separated from plasma and white blood cells (WBCs).
However, other known white blood cell separation techniques may
also be used, e.g., Ficoll-Hypaque, Percoll or other similar
density gradients. In an alternative embodiment, T cells are
enriched from WBCs using antibody-mediated or magnetic separation
using negative selection to remove other cell types in order to
obtain unbound T cells. Any known technique for enriching for T
cells can be used, although the more expedient, least expensive
methods are preferred. [0054] 3. Half of the plasma layer is
discarded (without disturbing the white blood cell layer) and
remaining fluid containing white blood cells is transferred to a
centrifuge tube. [0055] 4. The WBCs are then pelleted and washed
for two or more times by re-suspending the pelleted cells in an
appropriate isotonic buffer, e.g., PBS, followed by centrifugation
for about 15-20 minutes at about 320.times.g. [0056] 5. The pellet
is then re-suspended with a small volume of appropriate isotonic
buffer, e.g., PBS. Half of the pellet is transferred to a labeled
cryovial for freezing. The other half is used for establishing T
cell cultures as described below. The sample that is to be frozen
is centrifuged and then resuspended in a small volume of
appropriate isotonic buffer, e.g., RPMI 1640 plus DMSO, prior to
freezing.
T-Cell Cultures
[0057] In one embodiment, T-cell cultures are established by
stimulating the T cells present in the WBC preparation, for
example, according to the following procedure. [0058] 1. The washed
cells from above are re-suspended in an appropriate cell culture
medium, such as RPMI supplemented with T cell stimulatory cytokines
and/or mitogens. Suggested stimulatory cytokines include IL-2,
IL-12, IL-15 phytohemagglutinin (PHA), concanavalin A (con A), and
pokeweed mitogen. In a particular embodiment, the WBCs are
re-suspended in an appropriate volume of RPMI 1640 medium
supplemented with FBS, IL-2 and a stimulatory concentration of PHA.
They can then be transferred to an appropriate culture vessel and
incubated for sufficient time to expand, e.g., about 2-3 days.
[0059] 2. After the T cells are expanded, they may be frozen, e.g.,
at about 3.times.10.sup.6 cells/vial using RPMI 1640 medium
supplemented for cryopreservation, e.g., containing FCS and DMSO.
This is sufficient to thaw 5 mL of culture at 5.times.10.sup.5
viable cells/mL.
[0060] It is noted that one of ordinary skill in the art will be
able to ascertain appropriate amounts of T cell stimulatory
cytokines or mitogens, although typically such agents are added at
amounts from between about 1 ng/ml and about 25 ng/ml (or about 100
U/ml) for cytokines. For mitogens, concentrations range from about
10 ng/ml to about 10 .mu.g/ml for mitogens with most being
effective in the low .mu.g/ml range.
Enzyme Activity/Enhancement Assay
[0061] Typically, T cells isolated above (e.g., approximately
2.5.times.10.sup.6) are grown in culture medium (preceded by
thawing if they are frozen), in an appropriate culture vessel in
the absence or presence of the SPC, e.g., DGJ, for enough time to
evaluate the change in .alpha.-GAL activity, e.g., 2 or 3 days.
Doses of DGJ expected to enhance .alpha.-GAL are in a range from
about 2 nM to about 150 .mu.M, preferably about 1 .mu.M to 100
.mu.M, and more preferably about 5 .mu.M to 50 .mu.M. In one
specific embodiment, DGJ is added at about 20 .mu.M. Cells can be
harvested by centrifugation and washed twice with PBS. Pellets can
be stored frozen at -80.degree. C. until assayed for enzyme
activity.
[0062] Cells are then lysed by the addition of lysis buffer (or
deionized water) and physical disruption (pipetting, vortexing
and/or agitation, and/or sonication) at room temperature or on ice,
followed by pooling of the lysates on ice, then splitting the
pooled lysate into small aliquots and freezing.
[0063] The lysates can be thawed immediately prior to the assay and
should be suspended by use of a vortex mixer and sonicated prior to
addition to appropriate wells e.g., in a microplate.
N-acetylgalactosamine (GalNAc) is then added to each well (to
inhibit .alpha.-galactosidase B), followed by a short incubation.
4-methylumbelliferyl-.alpha.-D-galactopyranoside (4-MU Gal), or
other appropriate labeled DGJ substrate, is then added and the
plate is gently mixed for a brief period of time, covered, and
incubated at 37.degree. C. for a sufficient time for substrate
hydrolysis, usually about 1 hour. To stop the reaction,
NaOH-glycine buffer, pH 10.7, is added to each well and the plate
is read on a fluorescent plate reader (e.g. Wallac 1420 Victor3.TM.
or similar instrument). Excitation and emission wavelengths were
customarily set at 355 nm and 460 nm, respectively. One unit of
enzyme activity is defined as the amount of enzyme that catalyzes
the hydrolysis of 1 nmole of 4-methylumbelliferone per hour. For
each patient sample at least three normal samples should be tested
concurrently.
[0064] Various modifications of this assay will be readily
ascertainable to one of ordinary skill in the art. Examples of
artificial substrates that can be used to detect .alpha.-GAL
activity include but are not limited to
p-nitrophenyl-.alpha.-D-galactopyranoside and 4-MU GAL. Obviously,
only substrates that can be cleaved by human .alpha.-GAL are
suitable for use. It is noted that while use of a fluorogenic
substrate is preferred, other methods of determining .alpha.-GAL
activity are contemplated for use in the method, including using
chromogenic substrates or immunoquantification techniques.
[0065] Diagnosis and Prognosis. The T cell assay can be easily
modified for use as a diagnostic assay to diagnose Fabry disease by
simply eliminating the step of culturing the T cells in the
presence of DGJ prior to performing the enhancement assay. The
activity of .alpha.-GAL in T cells established from an individual
suspected of having Fabry disease can instead be quantitated using
T cells from a normal individual as a control. Moreover, both
.alpha.-GAL activity and SPC enhancement assays can be performed
almost simultaneously using the same T cells derived from one
patient sample. Since T cells may express more .alpha.-GAL
(.alpha.-GAL in normal T cells as compared with WBCs is much
higher), it will be easier to confirm with more certainty whether a
patient has .alpha.-GAL activity below the normal range because the
margin of error will be smaller. Accordingly, use of the T cell
assay could potentially prevent misdiagnoses.
[0066] In addition, the modified assay also can be used to
periodically monitor the progress of patients in whom SPC therapy
was initiated to confirm that .alpha.-GAL activity remains
increased relative to prior to treatment initiation.
II. In Vivo Assay
[0067] In a second embodiment, WBCs are evaluated for .alpha.-GAL
enhancement by an SPC in vivo. In this embodiment, .alpha.-GAL
activity in WBCs derived from patients is assessed prior to SPC
administration, in order to obtain a baseline value. Patients are
then administered DGJ daily (e.g., 150 mg/day) for a sufficient
time period, e.g., about 10 days to about 2 weeks, followed by
extraction of blood and determination of changes in .alpha.-GAL
activity from the baseline value. Culturing the cells either prior
to or following administration is not required.
[0068] The dose and dosing regimen of DGJ administration during the
in vivo evaluation period may vary depending on the patient since
there is so much heterogeneity among mutations, and depending on
the patient's residual .alpha.-GAL activity. As non-limiting
examples, the following doses and regimens are expected to be
sufficient to increase .alpha.-GAL in most "rescuable" individuals:
25 mg b.i.d; 50 mg once a day; 50 mg b.i.d.; 50 mg once every other
day, 75 mg once a day; 75 mg b.i.d.; 100 mg once a day; 100 mg
b.i.d.; 150 mg once a day; 150 mg b.i.d., 150 mg once every other
day; 250 mg once a day; 250 mg b.i.d. and 250 mg once every other
day. In specific embodiments, the doses are 50 mg once a day; 50 mg
once every other day; 150 mg once a day; 150 mg once every other
day.
[0069] Administration of DGJ according to the present invention may
be in a formulation suitable for any route of administration, but
is preferably administered per os in an oral dosage form such as a
tablet, capsule or solution. As one example, the patient is orally
administered capsules each containing 25 mg, 50 mg, 75 mg or 100 mg
or combinations thereof. For this assay, in the case of oral
administration, it is preferred that the patient be administered
the DGJ without food (e.g., no food 2 hours before and for 2 hours
after dosing) since bioavailability may be lower if taken with
food, thereby risking inaccurate results.
[0070] Patients who have been on other therapies, such as ERT,
should cease treatment for at least about 28 days prior to the in
vivo assay to ensure the most accurate results.
White Blood Cell Separation
[0071] WBCs are isolated and separated as described above for the T
cell in vitro assay. However, no RPMI media or DMSO is to be added
to the pellets prior to freezing (as per step 5 above).
Enzyme Activity/Enhancement Assay
[0072] Pellets are thawed on ice and cells are then lysed by the
addition of lysis buffer and physical disruption (such as by use of
a vortex mixer and agitation, and/or sonication at room
temperature) for a sufficient time, followed by pooling of the
lysates in a polypropylene tube on ice, then splitting of the
pooled lysate into aliquots for freezing.
[0073] The WBC lysates are then thawed on ice and mixed (again, by
sonication and/or vortexing). Samples of each lysate, as well as
standards and negative controls, are then added to appropriate
wells in e.g., a 24 or 96 well microplate. Equal amounts of GalNAc
are added to each well in e.g., citrate/phosphate buffer, pH 4.6,
followed by addition of a labeled substrate, such as 4-MU Gal (also
in citrate/phosphate buffer, pH 4.6) to all wells, and incubation
for a short time at ambient temperature. The plate is then mixed
briefly and incubated at 37.degree. C. for a sufficient time period
to permit substrate hydrolysis, e.g., about 1 hour. After the
sufficient time period, the reaction is stopped by the addition of
stop buffer and the plate is read on a fluorescent plate reader
(e.g., Wallac 1420 Victor3.TM.) to determine enzyme activity per
well.
[0074] Various modifications of this assay will be readily
ascertainable to one of ordinary skill in the art. Examples of
artificial substrates that can be used to detect .alpha.-GAL
activity include but are not limited to
p-nitrophenyl-.alpha.-D-galactopyranoside and 4-MU Gal. Obviously,
only substrates that can be cleaved by human .alpha.-GAL are
suitable for use. It is noted that while use of a fluorogenic
substrate is preferred, other methods of determining .alpha.-GAL
activity are contemplated for use in the method, including using
chromogenic substrates or immunoquantification techniques.
Eligibility Determination Criteria
[0075] The criteria for determining eligibility for SPC therapy
depends on the patient's residual enzyme activity at baseline,
i.e., the activity determined in the untreated T cells in the in
vitro assay, or the activity in the WBCs prior to SPC
administration in the in vivo assay. The lower the residual
activity, the greater enhancement necessary in order for a patient
to be considered a likely responder to treatment.
[0076] In one embodiment, the criteria for determining eligibility
for the in vitro assay are as follows: [0077] If residual
.alpha.-Gal A activity in lymphocytes is less than 1% of normal,
then .alpha.-GAL activity after incubation with DGJ must be at
least 2% of normal; [0078] If residual .alpha.-GAL activity in
lymphocytes is between 1% of normal and <3% of normal, then
.alpha.-GAL activity after incubation with DGJ must be at least
2.times. the baseline level; [0079] If residual .alpha.-GAL
activity in lymphocytes is between 3% of normal and <10% of
normal, then .alpha.-GAL activity after incubation with DGJ must be
at least 3% of normal higher than the baseline level; and [0080] If
residual .alpha.-GAL activity in lymphocytes is 10% of normal or
more, then .alpha.-GAL activity after incubation with DGJ must be
at least 1.3.times. the baseline level.
[0081] In an alternative embodiment, patients with Fabry disease
could be categorized as eligible for SPC therapy if their
.alpha.-GAL activity in T cells in the presence of an SPC such as
DGJ is at least about 35-40 nmol/hr/mg protein, which is about 58%
of normal. According to the present invention, the average specific
was too variable to report as a global mean. Accordingly patient
T-cell samples were compared in activity to at least three normal
controls collected within 48 h of the collection date for the
patient specimen and grown under identical conditions (see Example
1). As a comparison, .alpha.-GAL activity in T cells from Fabry
patients with the A97V, R301Q, and R111H at baseline was 8
nmole/hr/mg protein, 4 nmol/hr/mg and 1.8 nmol/hr/mg. T cells
express higher levels of .alpha.-GAL compared with other WBCs, so
it is expected that .alpha.-GAL activity in a culture enriched for
T cells will be significantly higher than what is considered normal
in total WBCs (21 nmol/h/mg of protein to about 50 nmol/h/mg of
protein; Desnick et al., The Metabolic and Molecular Bases of
Inherited Diseases. 8th Edition 2001, Scriver et al., ed., pp.
3733-3774, McGraw-Hill, New York). For a comparison, three Fabry
patients having the mutations R220X, R356W, and G132R had WBC
.alpha.-GAL activity of 0.22, 0.18, and 0.26 nmol/hr/mg protein,
respectively.
[0082] In one embodiment, for the in vivo assay, the following
criteria are used to determine eligibility criteria: [0083] If
baseline .alpha.-GAL is less than 1% of normal, then Day 15
.alpha.-GAL activity after treatment with DGJ must be at least 2%
of normal; [0084] If baseline .alpha.-GAL is between 1% of normal
and <5% of normal, then .alpha.-GAL activity must be at least
2.times. the baseline level following the treatment period; [0085]
If baseline .alpha.-GAL is between 5% of normal and <10% of
normal, then .alpha.-GAL activity must be at least 5% of normal
higher than the baseline level following the treatment period; and
[0086] If baseline .alpha.-GAL is 10% of normal or more, then
.alpha.-GAL activity must be at least 1.5.times. the baseline level
following the treatment period.
[0087] In an alternative embodiment, an increase in activity of at
least about 20% in the cells cultured with SPC over the activity in
the cells not cultured with SPC, in either the in vitro or in vivo
assay, may be indicative that the patient will have a clinically
relevant (therapeutically effective) response to SPC therapy.
[0088] This discovery provides a method for improving the diagnosis
of and facilitating clinical treatment decisions for Fabry disease
in particular, and lysosomal storage disease in general. Moreover,
this method can be extended to a wide range of genetically defined
diseases in appropriate cell types. This class of disease includes
the other lysosomal storage disorders, Cystic Fibrosis (CFTR)
(respiratory or sweat gland epithelial cells), familial
hypercholesterolemia (LDL receptor; LPL-adipocytes or vascular
endothelial cells), cancer (p53; PTEN-tumor cells), and amyloidoses
(transthyretin) among others.
Kits
[0089] The present invention also provides for a commercial
diagnostic test kit in order to make therapeutic treatment
decisions. The kit provides all materials discussed above and more
particularly in the Examples below, for preparing and running each
assay in one convenient package, with the obvious exception of
patient blood, optionally including instructions and an analytic
guide.
[0090] As one non-limiting example, a kit for evaluating
.alpha.-GAL activity may contain, at a minimum: [0091] a. at least
one T cell stimulatory agent; [0092] b. a specific pharmacological
chaperone; [0093] c. a chromogenic or fluorogenic substrate for the
enzyme assay (including an appropriate standard); and [0094] d.
GalNAc. The kit may also contain instructions for optimally
performing the protein enhancement assay. In another embodiment,
the kit will contain the appropriate tubes, buffers (e.g., lysis
buffer), and microplates.
[0095] In one embodiment, the SPC is supplied in dry form, and will
be re-constituted prior to addition.
[0096] In another embodiment, the invention provides a kit for the
diagnosis of Fabry disease. In this embodiment, the SPC is not
included in the kit and the instructions are tailored specifically
to diagnosis.
[0097] Patients that test positive for enzyme enhancement with an
SPC can then be treated with that agent, whereas patients who do
not display enzyme enhancement with a specific agent can avoid
treatment which will save money and prevent the emotional toll of
not responding to a treatment modality.
EXAMPLES
[0098] The present invention is further described by means of the
examples, presented below. The use of such examples is illustrative
only and in no way limits the scope and meaning of the invention or
of any exemplified term. Likewise, the invention is not limited to
any particular preferred embodiments described herein. Indeed, many
modifications and variations of the invention will be apparent to
those skilled in the art upon reading this specification. The
invention is therefore to be limited only by the terms of the
appended claims along with the full scope of equivalents to which
the claims are entitled.
Example 1
In Vitro Method for Evaluating Effects of an SPC on .alpha.-GAL
Activity
[0099] The present Example provides the in vitro diagnostic assay
to determine a Fabry patient's responsiveness to a specific
pharmacological chaperone.
A. Preparation of Human WBC Pellets for Growth of T Lymphocytes
[0100] 1. Materials: [0101] CPT tube: Becton-Dickenson (BD
Vacutainer.RTM. CPT.TM. Cell Preparation Tube with Sodium Citrate,
cat #362761). [0102] Human IL-2 (recombinant), PreProTECH, cat
#200-02 [0103] Phytohemagglutinin (M Form) (PHA), liquid,
Invitrogen, cat #10576-015 [0104] RPMI-1640 medium, Mediatech Inc.,
cat #10-040-CV [0105] Fetal Bovine Serum, Mediatech Inc., cat
#35-010-CV [0106] Citric acid, monohydrate, ACS, Mallinckrodt, cat
#0627 [0107] Sodium phosphate dibasic (Na.sub.2HPO.sub.4), ACS,
Mallinckrodt cat #7917 [0108] Sodium hydroxide, volumetric solution
ION, Mallinckrodt cat # H385 [0109] Phosphoric acid, ACS,
Mallinckrodt cat # PX0995-3 [0110] 4-MU .alpha.-D-galactopyranoside
(4-MU-Gal), Sigma cat # M-7633 [0111] N-Acetyl-D-galactosamine
(GalNAc), Sigma cat # A-2795 [0112] 4-methylumbelliferone (4-MU),
Sigma cat # M-1381 [0113] Glycine, tissue culture grade, Fisher cat
# BP381 [0114] Double deionized water [0115] Dulbecco's Phosphate
Buffered Saline, PBS, (without Ca, without Mg), Mediatech Inc. cat
#21-031-CV [0116] Micro BCA Protein Assay Kit, Pierce cat #23235
[0117] 96-well microtiter plates, Costar black polystyrene 96 well
round bottom, cat #3792 [0118] Costar 24-well tissue culture
treated microplates, Corning Life Sciences, cat #3526 [0119] 15 mL
polypropylene Falcon tube, Becton Dickinson, cat #352097 [0120]
Sterile Cryovials [0121] Humidified 5% CO.sub.2, 37.degree. C.
incubator [0122] 37.degree. C. water bath [0123] Fluorescence plate
reader
[0124] 2. WBC Separation: [0125] Patient blood was drawn into an 8
mL CPT tube, which has been stored at 18-25.degree. C. [0126]
Immediately after collecting blood, it was mixed by inverting the
tube 8-10 times. [0127] The tube was centrifuged at room
temperature (18-25.degree. C.) for 30 minutes at 1800.times.g using
a tabletop centrifuge equipped with swinging buckets. Universal
precautions for handling blood specimens were taken, including the
use of a closed canister type bucket for centrifugation. [0128]
Following centrifugation, several layers of the blood composition
become distinguishable which represented separation of the red
blood cells from the plasma and white cells. If this does not
occur, warm in hands for 5 minutes and centrifuge again.
[0129] 3. Washing of WBC's [0130] Half of the plasma layer was
aspirated by vacuum and discarded without disturbing the white cell
layer. All of the remaining fluid, including the cell layer, was
transferred with a Pasteur pipette to a 15 mL conical screw-cap
Falcon centrifuge tube. [0131] PBS was added to bring the volume up
to 14 mL and the tube was mixed by inversion. [0132] The tube was
centrifuged at room temperature for 20-30 minutes at 1300 rpm
(approximately 320.times.g). [0133] Immediately after
centrifugation, as much supernatant as possible was aspirated by
vacuum and discarded without disturbing the cell pellet.
[0134] 4. Optional Wash [0135] The cell pellet was re-suspended in
the remaining liquid by tapping against the bottom of the tube.
[0136] 10 mL of PBS was added to the re-suspended cells, and
centrifuged at room temperature for 20 minutes at 1300 rpm. [0137]
Immediately after centrifugation, as much supernatant as possible
was aspirated by vacuum and discarded without disturbing the cell
pellet.
[0138] 5. Optional: Freezing WBC Pellet [0139] The cell pellet was
mixed in the remaining liquid by tapping a finger against the
bottom of the tube. [0140] 0.5 to 1 mL of PBS was added to the
re-suspended cells and one half of the pellet was transferred
(using a sterile tip on a micropipette) to a labeled 1.8 mL
cryovial. [0141] The cryovial was centrifuged at room temperature
for 5 minutes at 5000 rpm (approximately 2250 g) in a
microcentrifuge. [0142] All of the supernatant liquid was discarded
using a Pasteur pipette without disturbing the cell pellet. [0143]
0.5 to 1 ml of RPMI 1640 containing 10% FBS and 5% DMSO was then
added to the tube and mixed a pipette and frozen overnight at -80 C
prior to transferring to a liquid nitrogen cell storage freezer. B.
Establishment of T-cell Cultures from Blood Specimens [0144] 1. The
washed cells were re-suspended in 3.0 ml of RPMI 1640 medium with
10% Cosmic Calf Serum (CCS, Hyclone Laboratories, Logan, Utah),
about 25 ng/ml IL-2 (PreProTECH, Rocky Hill, N.J.) and the
manufacturer's recommended concentration of PHA (Life Technology,
Gaithersburg, Md.). The cells were then transferred to an upright
an upright 25 cm.sup.3 culture flask and incubated for 3-4 days at
37.degree. C., 5% CO.sub.2. [0145] 2. The cell culture was diluted
to 5 ml with growth medium (RPMI-1640, 10% FBS, 25 ng/ml IL-2). The
cell concentration was then adjusted to about 5.times.10.sup.5
cells/ml in the flask. [0146] 3. The growth of the cells was
monitored daily. Cells were maintained between 5.times.10.sup.5 and
1.5.times.10.sup.6 cells in an upright flask. The depth of the
medium in the flask did not exceed 1 cm (about 7 mLs in a T25 and
20 mLs in a T75). Cultures can be maintained for approximately 21
days with a doubling time of about 24 hrs. Senescence of the
culture will be apparent by a dramatic reduction in growth rate.
Culture time may possibly be extended by re-stimulation with PHA.
[0147] 4. Optional-Freezing T-lymphocytes: T-lymphocytes may be
frozen at 3.times.10.sup.6 cells/vial using RPMI1640 medium
containing 20% FCS and 7.5% DMSO. On day 5, 6, or 7 cryopreserve as
many vials as possible at 3.times.10.sup.6 cells/vial. This is
sufficient to thaw 5 mLs of culture at 5.times.10.sup.5 viable
cells/ml. When establishing T-cell cultures, the following should
be noted. [0148] Fresh blood specimens should be collected in
heparinized tubes (or tubes containing an appropriate
anti-coagulant) and used the same day. ACD tubes should be used if
specimens cannot be processed within 24 hours. (Clin Chem 1988
January; 34(1):110-3; Clin Diagn Lab Immunol. 1998 November;
5(6):804-7.). [0149] Eight-10 mLs of blood is usually sufficient to
establish 20 million cells by day 5. [0150] T lymphocytes are the
specific targets of the HIV virus. Use extreme care if the HIV
status of the patient is unknown. [0151] Each new lot of IL-2
should be tested to determine the optimal concentration. The lot
from PreProTECH used for these experiments was been found to be
optimal at 25 ng/ml with only a slight reduction in cell growth at
concentrations up to 50 ng/ml. [0152] Each lot of mitogen, e.g.,
phytohemagglutinin A (PHA), is assayed by the supplier (Invitrogen)
and should be used at the recommended dilution. [0153] All cultures
are maintained in a water saturated atmosphere at 37 C, 5%
CO.sub.2. [0154] Mononuclear cells and lymphocytes may also be
collected using either (lymphocyte separation medium
(Ficoll-Hypaque) or Lymphoprep tubes following the manufacturer's
standard procedure.
[0155] When analyzed by fluorescent activated cell sorting, the
regimen of IL-2 and PHA stimulation results in 99% CD3-positive
cells (which stains all T cell subsets), with equal numbers of
CD4-positive and CD4-negative cells (data not shown).
C. Chaperone Treatment
[0156] The density of the T cells was adjusted to 1.times.10.sup.6
per 3 ml of culture medium (RPMI-1640, 10% FBS, 25 ng/ml IL-2). 3
ml (.about.1.times.10.sup.6 cells) are then pipetted into each of 6
wells of a labeled 6-well culture plate and incubated overnight at
37.degree. C., 5% CO.sub.2. 3 ml of additional medium was then
added to 3 wells to give a final volume of 6 ml/well. To the three
remaining wells, 3 ml of medium containing DGJ (Cambridge Major
Laboratories, Inc., Germantown, Wis.) at a concentration of about
40 .mu.M (2.times.; final concentration is 20 .mu.M), for 4-5 days.
Cells were harvested by centrifugation (400.times.g for about 10
minutes) and washed 1.times. in 10 ml PBS. The resulting pellets
were re-suspended in 1 ml PBS and transferred to a 1.7 ml microfuge
tube and centrifuged in a refrigerated microfuge at 3000 rpm for 5
minutes. The supernatant was aspirated and the pellets were stored
frozen at -80.degree. C. until assayed for enzyme activity.
[0157] Note that prior to conducting the enhancement assay, the
optimum concentration of DGJ was determined using a range from 2
nM-200 .mu.M. It was determined that 20 .mu.M was optimal.
D. Preparation of Fibroblasts
[0158] For a comparison, fibroblast cultures were prepared as
described previously (e.g., U.S. Pat. No. 6,274,597). Briefly,
fibroblast cultures were derived from skin biopsies of patients and
grown in DMEM with 10% FBS until established (3-4 weeks).
E. Activity Assay
[0159] Prior to assay, the T cells were thawed on ice and sonicated
for 2 minutes, and all other assay reagents were thawed at room
temperature. Fluorometric assay of .alpha.-GAL activity was
performed essentially as described previously (Kusiak et al., J
Biol Chem. 1978; 253(1), 184-190). The cells were lysed in 0.2 ml
deionized water combined with vigorous pipetting and vortexing. The
supernatant obtained after centrifugation at 13000 rpm for 2 min at
4.degree. C. was put into a fresh tube and used as the source of
.alpha.-GAL. .alpha.-GAL activity was determined by incubating 50
.mu.l aliquots of the supernatant (containing comparable quantities
of protein as determined using 20 .mu.l in a standard protein
quantitation assay) in a 24-well microplate at 37.degree. C. with
3.75 mM 4-methylumbelliferyl-.alpha.-D-galactopyranoside (4-MU Gal)
(Research Products International, Mount Prospect, Ill.) in the
citric acid/phosphate buffer (27 mM citrate/46 mM phosphate buffer
pH 4.6) without taurocholate and with BSA (3 mg/ml). The percentage
of .alpha.-GAL was determined by comparing total activity with
activity observed in the presence of 117 mM N-acetylgalactosamine
(GalNAc) Sigma Chemical Co., St. Louis, Mo.), a specific inhibitor
of N-acetylgalactosaminidase. A Wallac 1420 Victor3.TM.
Fluorescence detection reader (Perkin Elmer, CA) was used to
measure the released 4-MU at excitation and emission wavelengths of
355 nm and 460 nm, respectively. Appropriate wells for fluorescent
standards, and negative (no substrate or no lysate) also were
employed. For each patient sample at least three normal samples
were tested concurrently.
[0160] Incubations were typically 30 minute duration but longer or
shorter periods may be employed with similar results.
[0161] Enzyme activity (nmol/hr/mg of protein) was calculated
according to the following:
Fluorescence of sample Flourescence of Standard * 60 mins
Incubation time ( mins ) * 1000 L Volume assayed ( L ) * 1 Protein
value ( mg / mL ) ##EQU00001##
One unit of enzyme activity is defined as the amount of enzyme that
catalyzes the hydrolysis of 1 nmole of 4-methylumbelliferone per
hour. The baseline "noise" in the fluorescence output was obtained
by evaluating the average of blank six times. If the activity
following SPC treatment was at least 2 standard deviations above
the baseline, it was considered responsive and not noise.
[0162] For the comparative fibroblast enhancement assay,
fibroblasts (.about.1.5.times.10.sup.6) were grown in 12 ml culture
medium in a T75 tissue culture flask in the absence or presence of
DGJ at 20 .mu.M for 3 days. At the end of the incubation period,
cells were removed from the flask by treatment with trypsin-EDTA
solution, collected by centrifugation and washed 3 times with
phosphate-buffered saline. Cell pellets were frozen at -80 C until
assayed for .alpha.-GAL activity. All steps for processing the cell
pellet for assay, including the extraction buffer, the time of
sonication and the volumes used are the same as used for the
T-cells assayed above.
F. Western Blots
[0163] The level of .alpha.-GAL protein measured by Western blot.
Protein was determined using a Micro BCA Protein Assay kit (Pierce,
Rockford, Ill.) with bovine serum albumin (BSA) as a standard.
Absorbance at 562 nm was measured using the Molecular Devices
VersaMax absorbance reader in a 96-well format. For gel
electrophoresis prior to western blotting, proteins were separated
using Novex Tris-glycine native or SDS-PAGE in 8-16% gradient gels
(Invitrogen). Western blots were developed using rabbit polyclonal
antibody against .alpha.-GAL was performed as described previously
(Park et al., Proc Natl Acad Sci USA. 2003; 100: 3450-54).
Results
[0164] This method described above using T cells is fast and
effective when compared with fibroblast-based .alpha.-GAL assays
conducted substantially similarly to the T cell assay (except that
about 1.5.times.10.sup.6 fibroblasts were plated in each well
instead of 2.5.times.10.sup.6 T cells).
[0165] Using this method, T cells from Fabry patients were
incubated without and with 20 .mu.M of DGJ for 1, 2, and 4 days,
respectively and the .alpha.-GAL activity was measured in cell
homogenates and compared to normal control values. When the media
was refreshed after 2 days and the cells incubated for 2 additional
days, .alpha.-GAL activity of A97V was 13% of the normal control
(FIG. 1, open circles). However, when 20 .mu.M DGJ was added to the
medium of the T cells, the .alpha.-GAL activity increased to about
40% of normal after only 1 day of incubation and continued to 80%
of normal after 4 days of incubation (FIG. 1, filled circles).
Addition of fresh DGJ and media after 2 days and incubation for an
additional 2 days did not result in any change in the profile from
that observed with a single addition of DGJ. The observed increase
in activity after 3 days to a level clearly distinguishable from
the .alpha.-GAL activity without DGJ led to the adoption of a
standard time of measurement after 3 days of incubation with 20
.mu.M of DGJ in subsequent experiments. The use of a three day time
course avoids the necessity to provide fresh media and/or splitting
the cells after 3 days in culture.
[0166] To determine the dosage effect of DGJ in T-cells from normal
controls, .alpha.-GAL activity was measured in patient cells using
a range of DGJ from 2 nM to 200 .mu.M (FIG. 2A) and compared to
untreated normal control values assayed on the same day.
.alpha.-GAL activity increased between 2 nM and 20 .mu.M of DGJ. At
200 .mu.M, DGJ inhibited normal .alpha.-GAL activity to
approximately 40% of the average of untreated normal controls. The
optimal enhancement of mutated .alpha.-GAL activity within this
same concentration range of DGJ was determined for the A97V
mutation and compared with normal controls (FIG. 2B). Three
separate experiments were carried out for the dosage effects on
A97V. DGJ in concentrations from 2 nM to 20 .mu.M increased the
.alpha.-GAL activity of A97V in a dose-dependent manner. However,
at DGJ concentrations of 200 .mu.M, there was a decrease in
.alpha.-GAL activity when compared to its highest level when cells
were grown in 20 .mu.M DGJ. In all three experiments the optimal
enhancement of activity of the A97V mutation was observed at 20
.mu.M DGJ with slightly lower activity at 2 and 200 .mu.M. When the
mutations R112H and R301Q were tested in the same concentration
range, a similar pattern emerged with the highest level of enhanced
activity observed at 20 .mu.M DGJ (FIG. 2C). The results showed
various mutations had similar dosage response profiles but
different levels of enhancements. Among three .alpha.-GAL mutant
genotypes tested for the dosage effects, the 20 .mu.M DGJ resulted
in an increase in .alpha.-GAL to at least 50% of the normal
control.
[0167] The rescue effect of mutant .alpha.-GAL from patients with
Fabry disease with at least 11 distinct genotypes has been observed
using a pharmacological chaperone using this T cell-based
.alpha.-GAL assay. Results, presented in Table 1, below, showed
that DGJ enhanced the activity of at least five distinct mutant
forms of .alpha.-GAL in T cells (T) and fibroblasts (F). However,
the pharmacological chaperone did not enhance activity of four
distinct mutant .alpha.-GAL forms. One classical Fabry patient's
.alpha.-GAL activity was enhanced by DGJ at the intermediate level.
The importance of this assay lies in the fact that it can be used
to screen for patients who might benefit from pharmacological
chaperone administration, thus avoiding the expense and frustration
of unnecessary therapy and tissue biopsies.
TABLE-US-00001 TABLE 1 Number of Activity Specimen Patient/
Replicates (% Normal) Enhancement Number Normal Sex Mutation (n=)
(-DGJ) (+DGJ) ratio Group 1 PT M T41I 3 48 147 3 E 2 PT M T41I 4 61
175 2.9 E 3 PT M M51K 2 6 29 4.6 E 4 PT M A97V 3 14 75 5.5 E 5 PT M
R112C 1 10 36 3.8 E 6 PT M R112C 3 8 49 6.3 E 7 PT M R112H 2 3 51
15.9 E 8 PT M R112H 2 8 73 9.4 E 9 PT M R112H 3 3 60 20 E 10 PT M
A143T 4 31 69 2.2 E 11 PT M A143T 5 49 62 1.3 E 12 PT M S201F 1 9
82 9.6 E 13 PT M P205T 1 37 108 2.9 E 14 PT M N215S 2 15 79 5.2 E
15 PT M P259R 1 9 297 32.6 E 16 PT M P259R 3 3 138 47.4 E 17 PT M
F295C 3 1 29 32.3 E 18 PT M L300P 5 2 72 36.1 E 19 PT M R301Q 1 7
91 12.3 E 20 PT M R301Q 3 22 204 9.1 E 21 PT M R301Q 4 7 80 12.1 E
22 PT M G328A 4 2 54 24.6 E 23 PT M R49C 9 3 11 4.3 I 24 PT M Y207S
3 4 15 3.5 I 25 PT M S276G 1 0 9 I 26 PT M S276G 3 1 12 9.8 I 27 PT
M C94S 2 2 2 NE NE 28 PT M G128E 1 2 4 NE NE 29 PT M G128E 5 2 2 NE
NE 30 PT M G132R 3 1 2 NE NE 31 PT M A143P 2 2 1 NE NE 32 PT M
A143P 2 1 1 NE NE 33 PT M R220X 3 0 1 NE NE 34 PT M R227Q 3 4 3 NE
NE 35 PT M W236R 3 1 2 NE NE 36 PT M G261D 1 0 1 NE NE 37 PT M
G271C 1 2 3 NE NE 38 PT M G271C 3 1 2 NE NE 39 PT M N272K 1 0 0 NE
NE 40 PT M W287C 1 1 1 NE NE 41 PT M W287C 1 1 2 NE NE 42 PT M
R356W 1 1 1 NE NE 43 PT M R356W 4 0 0 NE NE 44 PT M dE358 3 2 4 NE
NE 45 PT M L415P 1 0 3 NE NE 46 PT M unknown 1 1 2 NE NE 47 PT M
unknown 2 0 1 NE ME 48 PT M 1042insG 3 1 1 NE NE 49 PT M 256del1 1
0 0 NE NE 50 PT M 30delG 6 2 2 NE NE 51 PT M 82insG 2 1 0 NE NE 52
PT M del26bp21 1 0 1 NE NE 53 PT M del26bp21 1 0 0 NE ME 54 PT M
ivs4-1g/a 2 0 0 NE NE 55 PT M Q119X 1 2 1 NE NE 56 PT M R220X 3 0 1
NE NE 57 PT M R301X 4 0 7 NE NE All NL NL M or F 162 101 128 1.3
All NL NL M 115 101 124 1.2 Male All NL NL F 47 84 117 1.4 Female
58 NL F 8 120 161 1.3 59 NL F 3 88 100 1.1 60 NL F 1 95 120 1.3 61
NL F 9 85 104 1.2 62 NL F 8 103 150 1.5 63 NL F 3 107 165 1.5 64 NL
F 15 106 145 1.4 65 NL M 8 100 97 1 66 NL M 4 80 141 1.8 67 NL M 1
172 198 1.1 68 NL M 1 271 315 1.2 69 NL M 1 110 140 1.3 70 NL M 1
101 145 1.4 71 NL M 19 106 138 1.3 72 NL M 11 90 131 1.4 73 NL M 4
75 99 1.3 74 NL M 10 105 126 1.2 75 NL M 3 97 100 1 76 NL M 2 93
108 1.2 77 NL M 3 85 116 1.4 78 NL M 3 89 118 1.3 79 NL M 1 127 107
0.8 80 NL M 42 100 117 1.2 81 NL M 1 102 111 1.1 Abbreviations
used: PT: patient; NL: normal individual; E: enhanceable; I:
enhanceable (intermediate); NE: not enhanceable;
[0168] It was determined the optimal enhancement of mutated
.alpha.-GAL activity in the in vitro assay was achieved using about
20 .mu.M DGJ. Among three .alpha.-GAL mutant genotypes tested for
the dosage effects, the 20 .mu.M DGJ resulted in an increase in
.alpha.-GAL to at least 50% of the normal control for each (FIG.
3).
[0169] In Table 1, the enhanceable group included patients whose
.alpha.-GAL activity was at least 50% of normal controls when
cultured in the presence of DGJ (e.g., R112H was enhanced to 60% of
normal control activity). Included in the enhanceable group are the
mutations A97V, R301Q, R112H and L300P. For example, the activity
of the A97V mutation increased from 14 to 75% of normal in the
presence of DGJ, a 6-fold increase. Similarly R301Q increased from
7 to 80% of normal with 12-fold, and R112H increased from 3 to 60%,
nearly a 20-fold change. In addition, activity in the L300P
mutation was increased from 2% to 72% of normal, a 37-fold
increase, which was the highest among the enhanceable mutations
examined. L300P was unusual in that some of the activity without
DGJ was below the minimum threshold for detection. These results
demonstrate mutation-dependent enhancement levels and ratios.
[0170] The Western blots showed that the band intensity was
considerably increased by treatment with DGJ in normal control
cells and those with the A97V and the R301Q mutation, while no
increase was seen for R356W, G132R, and A143P (FIG. 3). The protein
appears to have shifted to a lower apparent molecular weight
indicating maturation of the enzyme by passage from the endoplasmic
reticulum, through the Golgi apparatus to the lysosome. The Western
blots show that enhancement of .alpha.-GAL activity by DGJ is
correlated with an increased amount of .alpha.-GAL protein. An
instance where increased protein levels as measured by Western
blots did not result in higher enzyme activity has not yet been
observed.
Discussion
[0171] The use of T cells in a test system for enhancement of
enzymes by SPCs offers significant advantages in the speed of assay
and convenience over other culture systems. A critical step in
determining which patients may benefit from SPC therapy was the
development of a rapid and reliable method for screening of
patient-derived cells for enhancement of .alpha.-GAL activity by
DGJ. The results demonstrate a method for quickly generating a
short-lived cell culture that permits the testing of the
enhancement and also provides a useful system for future studies on
the mechanism of action or for screening of additional chaperone
molecules. Leukocytes traditionally used for the diagnosis of
affected and carrier status do not survive long enough to permit
repeat assays if necessary.
[0172] Although Epstein-Barr virus transformed B lymphoblasts (Fan
et al., Nat Med. 1999; 5(1), 112-115) and primary fibroblast
cultures (Fan, supra; Mayes et al., Clin Chim Acta. 1981; 112(2),
247-251) have been tested, these are not convenient to use on a
large scale for screening of patients for clinical studies. Primary
fibroblast cultures require an invasive skin punch biopsy and
generally take at least three to four weeks to grow enough cells
for the assay. B cell lymphoblasts require Epstein Barr viral
transfection and selection process, which is time and labor
consuming, in addition to having unknown effects on enzymatic
activity.
[0173] The present invention provides a method for establishing T
cell cultures from fresh blood of normal control individuals and
patients with Fabry disease. These cultures can be grown for use in
an enhancement assay for .alpha.-GAL in 7 to 10 days. These data
also show that the effectiveness of DGJ enhancement was evident
after about 3 days in the T cell growth media. The data generated
are a reproducible measure of the degree of enhanced enzyme
activity by a SPC for a specific genotype.
[0174] This method can be used for other SPC-based enhancement
assays of other genetic diseases including glycosphingolipidoses,
mucopolysaccharidoses, and glycogen storage disease (Pompe) and can
be extended as a research and clinical protocol in a wide range of
genetically defined diseases, such as Cystic Fibrosis (CFTR) and
cancer (p53, PTEN), and others.
Example 2
In Vivo Method for Evaluating Effects of an SPC on .alpha.-GAL
Activity
[0175] This example describes results from an open label Phase II
study of DGJ in Fabry patients (n=11) with 10 different .alpha.-GAL
mutations and supports the use of the in vivo assay. The patients
were selected for the Phase II study based on the increase in
.alpha.-GAL activity in the T-cell assay described above. The
genotypes were as follows: T41L (2 patients); A143T; A97V; M51K;
S276G; L300P; G328A; P205T; N215S; and L415P.
[0176] Some patients (8) were administered DGJ according to the
following dosing schedule: 25 mg b.i.d. two weeks; 100 mg b.i.d.
weeks 2-4; 250 mg b.i.d. weeks 4-6; and 25 mg b.i.d. weeks 6-12.
Three patients received 150 mg of DGJ every other day throughout
the entire study. Blood was draw into an 8 mL Vacutainer CPT tube
at the end of each dosing period and treated as described
below.
A. Preparation of Human WBC Pellets for Assay
[0177] WBCs were prepared substantially as described in Example 1,
with the exception that no FBS/DMSO is added to the pellet prior to
freezing.
[0178] The preliminary data is summarized in the following
table.
B. Preparation of Human WBC Lysates for Assay
[0179] To the microtubes containing the WBC pellet, 0.6 ml of lysis
buffer (26 mM citrate/46 mM phosphate, pH 5.5) were added [0180]
Tubes were vortexed until the cells were re-suspended [0181] Tubes
were incubated at room temperature for about 15 minutes, but
agitate the suspension by vortexing every couple of minutes [0182]
Tubes were sonicated for 2 minutes, then vortexed for about 10
seconds [0183] Lysates were incubated on ice until chilled, and
then pooled into a pre-chilled polyproylene container (on ice)
[0184] Container was vortexed and pooled lysates were divided into
0.100 mL aliquots in pre-chilled labeled 0.5 mL screw-cap
polypropylene microcentrifuge tubes. Pooled lysates were mixed
while aliquoting by vortexing between every 10-20 aliquots. [0185]
Aliquots were stored at -80.degree. C. until use.
C. Human WBC Assay
[0185] [0186] Each tube containing lysate was thawed on ice,
sonicated for 2 minutes, then vortexed for 1 minute. [0187] 50
.mu.l of each standard, control, or clinical sample was added into
appropriate wells of a black polystyrene microplate (use 50 .mu.l
of 0.5% BSA in WBC lysis buffer for a standard) [0188] 50 .mu.l of
117 mM GalNAc was added to all wells, and the plate was incubated
for 5 minutes at ambient temperature; [0189] 50 .mu.l of 5 mM 4-MU
Gal substrate was added to all wells and the wells were mixed on a
plate shaker for 30 seconds [0190] The plate was covered and
incubated for about 1 hour at 37.degree. C. [0191] 100 .mu.l of
0.2M NaOH/Glycine buffer, pH 10.7 was added to each well to stop
the reaction [0192] The plate was read using a fluorescent plate
reader as described in Example 1
Results
[0193] The available data from the first eleven patients treated
with DGJ for at least 12 weeks suggest that treatment with DGJ
leads to an increase in the activity of the enzyme deficient in
Fabry disease in 10 of the 11 patients (FIG. 4). The patient with
the L415P genotype did not show an increase following DGJ (at 6
weeks) (FIG. 5). For purposes of calculating the percentage of
normal in the table, the level of .alpha.-GAL that is normal was
derived by using the average of the levels of .alpha.-GAL in white
blood cells of 15 healthy volunteers from the multiple-dose Phase I
study. The 11 patients represented 10 different genetic mutations
and had baseline levels of .alpha.-GAL that ranged from zero to 30%
of normal.
[0194] The data show an average 2-fold increase in .alpha.-GAL
activity, and up to 10-fold and 15-fold in some patients as
measured in white blood cells. Activity remained elevated from 6-24
weeks and counting when the dose was reduced back to 25 mg b.i.d
(data not shown).
Discussion
[0195] Based on these results, it appeared possible to screen
candidate patients for eligiblity for chaperone using an in vivo
assay or an in vitro assay using T cells as described in Example 1,
since 10 out of 11 patients who demonstrated a significant increase
in .alpha.-GAL activity in the T cell assay demonstrated an
increase following a 2 week treatment with DGJ. Performing an in
vivo screen may allow for a more accurate evaluation of the in vivo
response to DGJ and other chaperone treatment, since the in vitro
assay may not be fully predictive of an in vivo response due to
systemic interactions, and may be especially useful to determine
appropriate dosing. This dramatic increase in activity appeared
after 2 weeks at a low dose of only 50 mg/day (25 mg b.i.d.)
(although different dosing regimens are contemplated by the present
invention).
[0196] These methods can be used for chaperone based enhancement
assays for other genetic diseases including glycosphingolipidoses,
mucopolysaccharidoses and other lysosomal storage disorders in
addition to other genetically based diseases, such as cystic
fibrosis where maturation of the protein occurs in the ER.
[0197] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
[0198] Patents, patent applications, publications, product
descriptions, GenBank Accession Numbers, and protocols are cited
throughout this application, the disclosures of which are
incorporated herein by reference in their entireties for all
purpose.
* * * * *