0022-3565/09/3283-723–731$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics
JPET 328:723–731, 2009Vol. 328, No. 3
149054/3444539
Printed in U.S.A.

Preclinical Efficacy and Safety of 1-Deoxygalactonojirimycinin Mice for Fabry Disease
Satoshi Ishii, Hui-Hwa Chang, Hidekatsu Yoshioka, Tatsuo Shimada, Kazuaki Mannen,
Yasunori Higuchi, Atsumi Taguchi, and Jian-Qiang Fan

Received November 21, 2008; accepted December 22, 2008

ABSTRACT
Fabry disease is an inborn error of glycosphingolipid metabolism caused by deficiency of ␣-galactosidase A (␣-Gal A) activity. It has been shown that protein misfolding is primarily
responsible for the enzyme deficiency in a large proportion of
mutations identified in Fabry patients with residual enzyme
activity, and 1-deoxygalactonojirimycin (DGJ) can effectively
increase the residual enzyme activity in cultured patient’s cells.
Herein, we demonstrate the preclinical efficacy and safety of
DGJ in transgenic mice that express human mutant ␣-Gal A
activity. ␣-Gal A activity in heart, kidney, spleen, and liver wasincreased dose- and time-dependently. The mutant ␣-Gal A
was increased in cardiomyocytes and distal convoluted tubulesof the transgenic mice in a null background after 2 weeks of
DGJ treatment. Globotriaosylceramide storage was remarkably

Fabry disease is an X-linked recessive disorder caused by
the deficient activity of ␣-galactosidase A (␣-Gal A; EC
3.2.1.22) (Brady et al., 1967). The enzymatic defection resultsin the accumulation of neutral glycosphingolipids with terminal ␣-galactosyl residues, predominantly globotriaosylceramide (Gb3), in body fluids and tissue lysosomes. Patientswith the early onset or classic phenotype typically have noresidual ␣-Gal A activity and often present symptoms such as
This work was supported in part by the Ministry of Education, Science andCulture of Japan; the Ministry of Health, Labor and Welfare of Japan; Mizutani Glycoscience Foundation; Irma T. Hirschl Foundation; and American
Heart Association.
S.I. and J.-Q.F. are coinventors of patents related to the active-site-specificchaperone technology, which is now licensed to Amicus Therapeutics, Inc., anddeclare competing financial interests.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.108.149054.

reduced in kidney of mice after a 4-week treatment at a dosageof approximately 3 mg/kg body weight/day. The half-life of DGJ
was less than 1 day in all major issues and that of the enzyme
synthesized during the DGJ treatment period was approximately 4 days. No abnormality of blood chemistry and pathological tissue damage was found in mice treated with DGJ at⬃30 mg/kg body weight/day for 9 weeks. Furthermore, nochange was observed in appearance, growth, fertility, and life
span in mice during a 2-year period of continuous administration of DGJ at the effective dosage. These preclinical results
indicate that DGJ is effective in restoring mutant enzyme activity in tissues and reversing substrate storage in kidney and iswell tolerated in mice.

angiokeratoma, acroparesthesias, hypohidrosis, and characteristic corneal and lenticular opacities. In contrast, the lateonset variants who present residual ␣-Gal A activity typically are mild, and clinical symptoms are limited to single
organs, either heart or kidney (Desnick et al., 2001). The
severity of the disease is well correlated with the level of theresidual enzyme activity. Enzyme replacement therapy has
been approved for the treatment of Fabry disease in the mostof the developed countries. Intravenous infusion of recombinant ␣-Gal A purified from Chinese hamster ovary cells or
fibroblasts has been shown to be effective for lowering theaccumulation of substrate in tissues, reducing pain in classically affected patients and resulting in improvement ofquality of life (Eng et al., 2001b; Schiffmann et al., 2001).
However, delivery of the macromolecular protein to certain
cell types, including cardiomyocytes, distal convoluted tubules, and glomerular podocytes, is rather ineffective. The

ABBREVIATIONS: ␣-Gal A, ␣-galactosidase A; Gb3, globotriaosylceramide; ER, endoplasmic reticulum; ERAD, ER-associated degradation;
ASSC, active-site-specific chaperone; DGJ, 1-deoxygalactonojirimycin; TgM mouse, transgenic mouse expressing human mutant ␣-Gal A(R301Q); TgM/KO mouse, transgenic mouse expressing human mutant R301Q ␣-Gal A in ␣-Gal A knockout background; Non-Tg, non-transgenic;
4MU-␣-Gal, 4-methylumbelliferyl ␣-D-galactopyranoside; PAGE, polyacrylamide gel electrophoresis; TLC, thin layer chromatography; HPTLC,
high-performance TLC; HE, hematoxylin and eosin; AST, aspartate aminotransferase; ALT, alanine aminotransferase; LDH, lactic dehydrogenase;
BUN, blood urea nitrogen.
723

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Department of Human Genetics, Mount Sinai School of Medicine, New York, New York (S.I., H.-H.C., J.-Q.F.);
Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan (S.I., A.T.); and Faculty of Medicine,
Oita University, Oita, Japan (H.Y., T.S., K.M., Y.H.)

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HO

OH
NH

HO

OH

Fig. 1. Structure of DGJ.

knockout mice (Ishii et al., 2004). These mice express exclusively the human mutant enzyme without endogenous ␣-Gal
A activity. Although they lack typical clinical symptoms for
Fabry disease, presumably caused by high enough levels of
␣-Gal A activity because of the human mutant enzyme intissues, they are an excellent biochemical animal model for
preclinical studies of DGJ. We report herein the dose- andtime-dependent increase in tissue ␣-Gal A activity, changes
of DGJ concentrations in tissues upon DGJ treatment, efficacy of DGJ treatment in reducing of Gb3 storage in kidney,and safety of DGJ in mice. These preclinical results provide
a rationale and guidance for clinical trials in man. DGJ(Migalastat) currently is under clinical phase II trials.

Materials and Methods
Animals. Transgenic mice expressing human mutant ␣-galactosidase A (TgM) were generated by injection of the DNA fragment
comprising chicken ␤-actin promoter and R301Q mutant ␣-Gal A
cDNA into the pronuclei of fertilized mouse ova taken from superovulated C57BL/6CrSlc female mice (Ishii et al., 1998). The TgM
mice were crossbred with ␣-Gal A-deficient mice (Ohshima et al.,1997) to provide transgenic mice that express human R301Q ␣-Gal A
in a null background (TgM/KO) (Ishii et al., 2004). Four to five mice
typically were housed together and fed with regular rodent pellets
and autoclaved drinking water with or without DGJ. Protocols foranimal studies were reviewed and approved by the corresponding
Institutional Animal Care and Use Committee.
Administration of DGJ. DGJ was purchased from Toronto Research Chemicals Inc. (North York, ON, Canada) and dissolved indrinking water at different concentrations without any other substances. Age-matched male homozygous TgM, TgM/KO, and nontransgenic (Non-Tg) C57BL/6 mice were supplied drinking watercontaining DGJ ad libitum. Daily water consumption was recorded,
and no difference was observed at a statistical significant level
among all groups. Body weight was monitored weekly during the
DGJ treatment period. Average daily DGJ dosage was estimatedbased on the consumption of drinking water. Mice fed drinking watercontaining DGJ at 0.05 mM typically received DGJ at approximately
3 mg/kg body weight/day.
Tissue Preparation. After DGJ treatment, animals were sacrificed for collecting tissues and blood samples. Blood was collected via
cardiac puncture and immediately transferred to heparinized tubes(Thermo Fisher Scientific, Waltham, MA) for hematological examination. Serum was separated and pooled for blood chemistry analyses. Tissues were quickly removed and rinsed with ice-cold phosphate-buffered saline. All samples were kept on ice and processed as
rapidly as possible. Tissue homogenates [⬃10% (w/v)] prepared inwater using a microhomogenizer (Physcotron; Niti-on, Inc., Chiba,
Japan) were used for enzyme assay and tissue DGJ determination as
described below. For histological examination, tissues were fixed
immediately in 10% formalin for 24 h.
Determination of ␣-Gal A Activity and Protein in Tissues.
Both 4-methylumbelliferyl ␣-D-galactopyranoside (4MU-␣-Gal) andN-acetyl-D-galactosamine were purchased from Sigma-Aldrich (St.
Louis, MO). ␣-Gal A activity was assayed with a mixture (50 ␮l) of
4MU-␣-Gal (5 mM) as substrate and N-acetyl-D-galactosamine (75mM) as inhibitor for ␣-Gal B in 0.1 M sodium citrate buffer, pH 4.6,
at 37°C for 30 min, as described previously (Ishii et al., 2004). The
reaction was stopped by the addition of 1.2 ml of 0.1 M glycine-NaOH
buffer, pH 10.7. The released 4-methylumbelliferone was determinedby fluorescence measurement at 360 and 450 nm as excitation andemission wavelengths, respectively. One unit of enzyme activity wasdefined as the amount of enzyme that releases 1 nmol/h 4-methylumbelliferone. The protein concentration was determined using a
DC Protein Assay kit (Bio-Rad, Hercules, CA) and bovine serum
albumin as a standard.

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treatment is expensive, with an annual cost at approximately$250,000 for each patient. Therefore, an effective and affordable alternative therapeutic strategy would be desirable fortreating a wide range of patients.
A substantial number of disease-causing mutations are
missense mutations in Fabry disease (Human Gene Mutation Database). Among them, many missense mutations intheir purified forms are known to be catalytically comparablewith their wild-type counterpart but are less thermo- andpH-stable (Ishii et al., 2007). Studies of trafficking and degradation of various missense ␣-Gal A mutants indicate thatthe mutant enzymes are retained in the endoplasmic reticulum (ER), and their cellular degradations are associated with
ER-associated degradation (ERAD), caused by their misfolded tertiary conformations (Ishii et al., 2007). This provides a rationale for a therapeutic intervention by small
molecules that are pharmacological chaperones or more specifically active-site-specific chaperones (ASSCs) (Fan andIshii, 2007; Fan, 2008). Effective ASSCs can act as a folding
template for those mutant proteins with a fragile conformation and shift the folding dynamics in favor of the native or
proper conformation, thereby avoiding their retardation in
the ER and further degradation in the ERAD. As a result,
ASSCs promote normal trafficking of mutant enzymes
through the secretory pathway of the ER and, ultimately,
increase enzyme activity in lysosomes (Hamanaka et al.,2008).
We have shown that cultivation of lymphoblasts established from Fabry patients with the R301Q or Q279E mutation with 1-deoxygalactonojirimycin (DGJ; Fig. 1) could effectively increase the residual enzyme activity (Fan et al.,1999) and proposed that reversible competitive inhibitors
could serve as effective ASSCs for rescuing misfolded mutant
enzyme that otherwise would be retarded and degradedwithin the ERAD (Fan, 2003, 2008). DGJ has been shown tobe effective in increase of residual enzyme activity in cultured cells of patients with a variety of disease-causing mutations (Ishii et al., 2007; Shin et al., 2007), suggesting that
DGJ could be an effective drug agent for treating certain
genotypes of Fabry disease. This strategy using competitive
inhibitors as ASSCs has been applied for the rescue of mutant enzymes in other lysosomal storage diseases, including
Gaucher disease, Pompe disease, GM1-gangliosidosis, TaySachs, and Sandhoff diseases, indicating that this approach
may be widely applicable for the treatment of metabolic
deficiencies caused by misfolding of mutant proteins (forrecent review, see Fan and Ishii, 2007; Fan, 2007; Tropak
and Mahuran, 2007; Yu et al., 2007).
To further evaluate DGJ for the clinical feasibility, we havedeveloped a mouse model by crossbreeding transgenic miceexpressing human mutant R301Q ␣-Gal A with ␣-Gal A

Preclinical Efficacy and Safety of 1-Deoxygalactonojirimycinries obtained by purified glucosylceramide, lactosylceramide (bothpurchased from Wako Pure Chemicals, Osaka, Japan), and Gb3(Nacalai Tesque, Kyoto, Japan).
Histological Examination. Tissues including heart, kidney,spleen, liver, and lung of mice treated with DGJ at 0.5 mM concentration for 9 weeks were fixed immediately in 10% formalin after
removal from the animal, followed by embedding in paraffin. The
paraffin section (4 ␮m) of each tissue was stained with hematoxylin
and eosin (HE) and examined by light microscopy to determine theextent of tissue damage. The HE staining and examination were
performed by the Center for Comparative Medicine and Surgery,
Clinical Pathology Laboratory, Mount Sinai School of Medicine.
Blood Chemistry Analyses and Hematological Profile. Bloodchemistry analyses including aspartate aminotransferase (AST),
alanine aminotransferase (ALT), and lactic dehydrogenase (LDH), as
parameters of acute cardiac and hepatic toxicity, blood urea nitrogen(BUN), and creatinine, as parameters of kidney functions, and hematological profile of the mice treated with DGJ were performed
using standard methods at the Center for Comparative Medicine andSurgery, Clinical Pathology Laboratory, Mount Sinai School of
Medicine.
Statistical Analysis. Conventional statistical methods were employed to calculate mean value and S.D. Differences between groups
were tested for significance using Student’s t test for unpaired observations. A value of p ⬍ 0.05 was considered statistical significant.

Results
Comparison of ␣-Gal A Activity in TgM and TgM/KO
Mice. TgM mice were generated to express human mutant
␣-Gal A activity under a ␤-actin promoter (Ishii et al., 1998).
The apparent enzyme activity determined in tissue homogenates is a combination of the endogenous mouse enzyme andthe transgenic human mutant enzyme. To rule out the possible interference between these two versions of the enzyme,
we examined tissue ␣-Gal A activity in age-matched TgM,
TgM/KO mice, and wild-type mice with the similar genetic
background (Non-Tg mice). The tissue enzyme activities in
TgM/KO mice seemed to be statistically equivalent to thecorresponded tissue enzyme activities in TgM mice after
subtraction of those of Non-Tg mice as presented under Net
in Table 1. This indicated that the expression of the endogenous ␣-Gal A activity and the transgenic human mutant
activity are independent of each other. Furthermore, we further examined the responses of these two enzymes to the
DGJ treatment in mice because DGJ was known to be effective in the increase of the wild-type enzyme activity in cultured cells, albeit at a lesser level (Fan et al., 1999). Afteradministration of DGJ to mice at 0.05 mM in drinking waterfor 1 week, tissue ␣-Gal A activity in Non-Tg mice increasedslightly (⬃1.4-fold). Upon subtracting the enzyme activity
found in Non-Tg mice from those of TgM mice, the net tissue
enzyme activities in heart, kidneys, spleen, and liver were
statistically equivalent to those of the TgM/KO mice, respectively. This suggested that results generated from both
TgM/KO mice and TgM mice are considered to be equivalentand interchangeable upon adapting necessary controls with
Non-Tg mice.
Time-Dependent Increase in ␣-Gal A Activity and
DGJ Concentrations in Tissues. Time course of DGJ
treatment was performed with TgM mice fed with drinking
water containing DGJ at 0.5 mM concentration, corresponding to a dosage of approximately 30 mg/kg body weight/day(Fig. 2). Tissue ␣-Gal A activity was time-dependently in-

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To distinguish the enzyme activity in TgM mice attributed to thetransgene from the endogenous GLA, a group of Non-Tg mice wasused as a paralleled control. The net increase in enzyme activity
attributed to the transgene was calculated by subtracting the ␣-Gal
A activity in tissues of Non-Tg mice from the total ␣-Gal A activity incorresponded tissues of TgM mice.
Determination of DGJ Concentration in Tissues. Tissue DGJ
concentration was determined based on inhibitory activity against
coffee bean ␣-galactosidase (Sigma-Aldrich), which is reportedly sensitive to DGJ with a Ki value at 3 nM (Asano et al., 2000). Before theaddition to the reaction mixture, tissue homogenates were boiled at
95°C for 2 min to terminate the residual ␣-Gal A activity and thencentrifuged at 10,000g for 5 min. Supernatant (40 ␮l) was added to areaction mixture consisting of 10 ␮l of 4MU-␣-Gal (6 mM) in 150 mM
2-(N-morpholino)ethanesulfonic acid-NaOH buffer, pH 6.5, and 10 ␮lof coffee bean ␣-galactosidase (0.1 mU) and incubated at 37°C for 10
min to determine the inhibitory potency. DGJ concentration in tissue
supernatant was calculated from a standard inhibition curve thatwas established under the same condition using control tissue homogenates supplemented by a series of DGJ with known concentrations.
Western Blot Analysis. Western blot analysis was performed
with a polyclonal anti-␣-Gal A antibody produced in rabbits (Ishii etal., 1994) and horseradish peroxidase-conjugated anti-rabbit IgG
antibody produced in goat (Pierce Chemical, Rockford, IL). Tissue
homogenate containing approximately 30 ␮g of protein was applied
to a 10% polyacrylamide gel. After SDS-polyacrylamide gel electrophoresis (PAGE), proteins were transferred electrophoretically to a
polyvinylidene difluoride (Immobilon P) membrane (Millipore Corporation, Billerica, MA) and visualized with SuperSignal Chemiluminescent Substrate (Pierce Chemical). Prestained SDS-PAGE standards, including bovine serum albumin (77 kDa), ovalbumin (50kDa), carbonic anhydrase (34.3 kDa), and soybean trypsin inhibitor(28.8 kDa), purchased from Bio-Rad, were used to determine molecular mass.
Immunohistological Examination of Tissue ␣-Gal A. Heartand kidney obtained from the TgM/KO mouse treated with DGJ at
0.5 mM concentration in drinking water for 2 weeks were fixed in 4%
paraformaldehyde/phosphate-buffered saline, embedded in paraffin,
and sectioned at 4-␮m thickness. The paraffin section was stained by
the immunogold silver technique described by Takita et al. (1990). In
brief, binding of the anti-␣-Gal A polyclonal antibody to sections was
detected using goat anti-rabbit IgG gold (particle size, 5 nm) (Janssen Pharmaceuticals, Antwerp, Belgium), followed by developer solutions (10% gum Arabic aqueous solution containing 0.1% silver
nitrate, 0.6% bromohydroquinone, and 1% citric acid). Sections werecounterstained with nuclear fast red solution.
Determination of Gb3 Storage in Kidney. Kidney Gb3 wasdetermined as described previously (Nakamura et al., 2008). Minced
tissues from female heterozygous TgM/KO mice were homogenizedusing a microhomogenizer (Physcotron) in H2O. After the determination of protein, cellular lipids were extracted from the homogenates containing 5 mg of protein with 20 volumes of chloroform/
methanol [2:1 (v/v)]. After filtration by 90-mm 5A filter papers (ToyoRoshi Kaisha, Ltd., Tokyo, Japan), crude extracts were dried andstored as a crude extract fraction. Crude extracts were further
treated with 1 ml of methanolic NaOH (0.2 M NaOH in methanol) at
40°C for 2 h. After neutralizing the solution with glacial acetic acid,glycosphingolipids were further subjected to the Folch’s partition[chloroform/methanol/H2O, 8:4:3 (v/v)]. Glycosphingolipids recoveredin the lower phase were pooled and dried under an air stream of dry
N2. Glycosphingolipids resuspended in 0.1 ml of chloroform/methanol [2:1 (v/v)] were quantitatively applied to a high-performance TLC(HPTLC)-Silica gel 60 plate (Merck, Whitehouse Station, NJ). TLC
analysis was performed using a solvent system of chloroform/methanol/water [60:35:8 (v/v)] and visualized by spraying orcinol/sulfuric
acid reagent. Band intensities were determined by the Scion Image
software (Scion Corporation, Frederick, MD). Gb3 concentrations
were calculated in comparison with glycosphingolipid standard se-

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TABLE 1Comparison of tissue ␣-Gal A activity in TgM and TgM/KO mice
Age matched non-Tg, TgM, and TgM/KO mice (male, n ⫽ 3) were used for this study. DGJ was dissolved in drinking water at 0.05 mM concentration, corresponding to a daily
dosage of 3 mg/kg body weight, and orally administered to each group for 1 week. After scarification, heart, kidney, spleen, and liver were harvested, and tissue ␣-Gal A
activity was determined in homogenates using 4MU-␣-Gal as substrate. Means and S.D. are shown.
␣-Gal A Activity
Non-TgTgM

Neta

TgM/KO

U/mg proteinab

3.2 ⫾ 0.3
11.2 ⫾ 1.2
49.7 ⫾ 3.9
36.3 ⫾ 2.3

59.6 ⫾ 4.9
34.7 ⫾ 11.9
80.2 ⫾ 5.9
104.0 ⫾ 11.2

56.3 ⫾ (4.6–5.1)
23.5 ⫾ (10.7–12.6)
30.5 ⫾ (2.1–9.4)
67.7 ⫾ (9.2–13.4)b53.1 ⫾ 7.4
18.3 ⫾ 8.3
29.6 ⫾ 2.7
44.4 ⫾ 8.6

3.5 ⫾ 0.1
15.8 ⫾ 0.5
54.0 ⫾ 6.6
47.0 ⫾ 10.1

300.4 ⫾ 35.3
61.7 ⫾ 14.1
137.4 ⫾ 12.5
153.6 ⫾ 11.1

296.9 ⫾ (35.2–35.3)
45.9 ⫾ (13.6–14.5)
83.4 ⫾ (6.2–18.4)
106.6 ⫾ (7.0–20.9)

298.6 ⫾ 49.7
43.2 ⫾ 4.4
80.8 ⫾ 10.0
96.6 ⫾ 4.9

Enzyme activities under Net were calculated by subtraction of tissue activity in non-Tg mice from those of TgM mice.
The statistical significance of the difference between Net and TgM/KO mice was determined by Student’s t test; p values were 0.033 to 0.064.

Fig. 2. Time-dependent change in ␣-Gal A activity and DGJconcentrations in TgM mice. DGJ was administered to TgM
mice and Non-Tg mice as a 0.5 mM solution available adlibitum in their drinking water for the indicated period.
␣-Gal A activity was the net activity that was determinedby subtracting tissue activity in Non-Tg mice from that ofTgM mice. DGJ concentration in tissues of TgM mice wasdetermined based on inhibitory activity against coffee bean
␣-galactosidase. ␣-Gal A activity (F) and DGJ concentration (}) were the mean values ⫾ S.D. of four of TgM and
Non-Tg mice per point. A, heart; B, kidney; C, spleen; D,liver.

creased for approximately 11 days and gradually reached aplateau. The ␣-Gal A activity attributed to the human mutant transgene in TgM mice after 7- and 18-day treatments
was estimated to be 807 U/mg protein (14-fold increase over
the activity observed on day 0) and 2091 U/mg (38-fold) inheart, 119 (6-fold) and 197 (9-fold) U/mg in kidney, 180(6-fold) and 232 (8-fold) U/mg in spleen, and 190 (3-fold) and
280 (4-fold) U/mg in liver, respectively. Tissue DGJ concentrations were increased with the increasing concentration ofadministered DGJ in kidney, spleen, and liver for 7 days andin heart for 11 days and eventually reached a plateau, indicating that DGJ did not accumulate in the tissues over thetime. Because the increase in ␣-Gal A activity in all major
tissues was substantial after 1 week of treatment, a 1-weektreatment of DGJ was considered to be sufficient for further
study of ␣-Gal A activity in mouse tissues.
Dose-Dependent Increase in ␣-Gal A Activity and
DGJ Concentrations in Tissues. DGJ was orally administered to TgM mice through drinking water at different
concentrations for 1 week, and ␣-Gal A activity in major

tissues including heart, kidney, spleen, and liver was determined at the end of the experiment. The tissue ␣-Gal A
activity derived from the human mutant transgene was increased in a dose-dependent manner within the DGJ concentrations of 0 to 1.0 mM in drinking water (Fig. 3). In particular, a remarkable increase was observed between DGJconcentrations at 0 to 0.5 mM, and enzyme activity graduallyreached a plateau at DGJ concentrations greater than 1.0
mM. The enzyme activity attributed to the transgene in the
TgM mice treated with DGJ at 0.05 mM was determined to
be 270 U/mg protein (corresponding to ⬃6-fold of day 0) inheart, 69 U/mg (⬃2-fold) in kidney, 80 U/mg (⬃3-fold) in
spleen, and 84 U/mg (⬃2-fold) in liver, respectively. This
clearly indicates a substantial increase in tissue enzyme
activity in mice treated with DGJ at a concentration even as
low as 0.05 mM. Based on the average daily water consumption, daily DGJ dosage in mice treated with DGJ at 0.05 mM
was calculated as approximately 3 mg/kg body weight.
Tissue DGJ concentrations in TgM mice treated with DGJ
dissolving in drinking water at various concentrations were

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Nontreatment
Heart
KidneySpleenLiver
DGJ-treatedHeart
KidneySpleenLiver

Preclinical Efficacy and Safety of 1-Deoxygalactonojirimycin
50
40

800

400

50

B

300

40

30 200

30

20

20
100

10
0

0

10
0

0

50
300

C

40

200

50

D

300

40

30 200

30

20

20

100

100
10

0

0
0.0

1.0

2.0

Tissue DGJ concentration (pmol/mg protein)A

Fig. 3. Dose-dependent change in ␣-Gal A activity and DGJconcentrations in TgM mice. TgM mice and Non-Tg mice
were fed with DGJ solution ad libitum as drinking water atindicated concentrations for 1 week. Tissue ␣-Gal A activity(F) was obtained by subtracting the enzyme activity in
Non-Tg mice from that of TgM mice. Each data point was
the average of four TgM mice and four age-matched Non-Tg
mice. DGJ concentration (}) in tissues of TgM mice wasdetermined based on inhibitory activity against coffee bean
␣-galactosidase. The data shown are the mean values ⫾S.D. A, heart; B, kidney; C, spleen; D, liver.

10
0

0

3.0

0.0

1.0

2.0

3.0

DGJ concentration (mM)

Remaining α-galactosidase A activity (%)

100

A

B

nanomolar concentrations, which are not expected to cause
substantial inhibition of the enzyme.
Post-Treatment Changes in ␣-Gal A Activity and
DGJ Concentration in Tissues. To investigate the depletion rate of ␣-Gal A activity increased by DGJ treatment andthe clearance rate of DGJ in tissues after termination of the
DGJ treatment, ␣-Gal A activity and DGJ concentration intissues were determined in TgM mice after the administration of DGJ in drinking water at 0.5 mM, a daily dosage
corresponding to approximately 30 mg/kg body weight. DGJ
was rapidly cleared from all tissues after termination of the
DGJ treatment (Fig. 4). DGJ concentrations in all tissues
examined were below 50% within 24 h after the terminationof the treatment. In particular, DGJ concentration was reduced more than 90% in kidney in 1 day, suggesting that
100

75

75

50

50

25

25

0

0

100

C

D

100

75

75

50

50

25

25

0

0
0

7

14

21

28

0

7

14

Days after DGJ treatment

21

28

Remaining DGJ concentration (%)

determined according to a method described under Materials
and Methods (Fig. 3). Tissue DGJ concentrations were increased with the increasing DGJ concentrations in drinking
water initially and eventually reached a plateau in heart,spleen, and liver after the administered DGJ concentrations
reached 1 mM. Compared with other tissues, DGJ concentration in kidney was significantly higher in all treated groups.
DGJ concentration in kidney of TgM mice treated with 2.5
mM DGJ increased 36% over those in mice treated with 1.0
mM DGJ. In mice treated with DGJ at 0.05 mM in drinking
water, DGJ concentrations in heart, kidney, spleen, and liver
were 1.5, 3.7, 0.5, and 1.8 pmol/mg protein, respectively.
Based on an assumption that tissue protein concentrationsare ⬃10 mg/ml, the tissue DGJ concentrations in micetreated with 0.05 mM DGJ would be expected to be at low

Fig. 4. Post-treatment changes in ␣-Gal A activity and DGJconcentration in TgM mice. DGJ was administered to TgM
mice as drinking water (0.5 mM DGJ) for 2 weeks adlibitum. The estimated DGJ dosage is approximately 30
mg/kg body weight/day. After the treatment, the mice werefed with fresh water for the indicated period. ␣-Gal A derived from the human mutant transgene was determinedby subtraction of the enzyme activity obtained from Non-Tg
mice from those of TgM mice. The data shown are the meanvalues ⫾ S.D. of four TgM mice and four Non-Tg mice perdata point. A, heart; B, kidney; C, spleen; D, liver. F, tissue
␣-Gal A activity; }, tissue DGJ concentration.

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α -Galactosidase A activity (U/mg protein)

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Fig. 5. Immunoblot analysis of ␣-Gal A in heart of TgM mice treated with
DGJ. The heart samples (approximately 20 ␮g of protein) from homogenates of untreated TgM mouse (lane 1) or from TgM mice treated with
0.05, 0.5, 1, and 2.5 mM DGJ corresponding to a daily dosage of 3, 30, 60,
and 150 mg/kg body weight for 1 week (lanes 2–5, respectively) wereapplied to a 10% SDS-PAGE gel. Immunoblot analysis was performed
with anti-␣-Gal A antibody as described under Materials and Methods.

Fig. 6. Immunohistochemistry of heart and kidney of TgM/KO micetreated with DGJ. A TgM/KO male mouse was treated with 0.5 mM DGJ
in drinking water ad libitum for 2 weeks. The estimated DGJ dosage wasapproximately 30 mg/kg body weight/day. ␣-Gal A activities in the heartfrom nontreated and DGJ-treated mice were 55 and 1240 U/mg protein,respectively, and those in kidney were 9.5 and 79.0 U/mg protein, respectively. The secondary antibody (goat anti-rabbit IgG gold) used for theheart samples was 5-fold diluted compared with those for the kidneysamples. A, glomeruli; B, distal convoluted tubule; C, proximal convoluted tubule.

in the kidney was determined to be 1.9 ␮g/mg protein, a 46%
reduction, despite no change being found in glucosylceramide
and lactosylceramide. This result clearly indicated that administration of DGJ at a dosage of approximately 3 mg/kg
body weight/day results in a reduction of Gb3 storage inkidney.
Preliminary Toxicology Examination of DGJ in
Mice. DGJ were administered to TgM/KO mice by drinking
water containing DGJ at 0.5 mM for 9 weeks. The estimateddaily dosage is approximate 30 mg/kg body weight. Because
the effective dosage for mice is considered to be 3 mg/kg bodyweight/day, this represents an approximately 10-fold excessof the effective dosage. The results are summarized in Table 2.
The average body weights at the beginning of the treatment were 22.0 and 21.2 g for the control and treated groups,
respectively. At the end of the experiment, the body weights
were 28.0 and 27.3 g for the respective groups, indicatingthat there was no significant difference between these twogroups. There was also no significant difference in the bodyweight between these two groups at any point over the entire
experiment period. The weights of heart, kidney, spleen, andliver were found to be essentially the same between these twogroups (0.40, 1.31, 0.33, and 4.69% body weight in control
group versus 0.43, 1.33, 0.31, and 4.64% body weight in the
DGJ-treated group, respectively) at the end of the study,
despite that ␣-Gal A activity was increased 49.6-, 3.8-, 2.5-,
and 2.0-fold in heart, kidney, spleen, and liver, respectively.
AST, ALT, and LDH in serum were used as markers forpotential acute cardiac and hepatic toxicity. BUN and creatinine levels were used as indicators for kidney function. All
these biochemical parameters in both groups were normal
and did not significantly fluctuate by the long-term DGJ
treatment. White blood cell counts were not significantly
different in both control and DGJ-treated groups, and the
profile of leukocytes did not change. Likewise, the concentrations of red blood cells and platelet were normal and were notsignificantly different between the two groups. In addition,
no histological abnormality in DGJ-treated mouse was revealed in heart, kidney, spleen, liver, and lung comparedwith those of the control mouse (Fig. 8), indicating that DGJ
does not cause any tissue damage at the dosage of 30 mg/kg
body weight/day.

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DGJ was cleared through renal filtration in mice. DGJ wascompletely cleared from kidney, spleen, and liver after 7 days
and reduced to less than 15% in heart. The slower clearance
rate in the heart is considered the result of deep penetrationof DGJ into cardiomyocytes during the treatment period.
Tissue ␣-Gal A activity also decreased upon the terminationof the treatment, however, at a slower rate compared withthe clearance rate of DGJ. Enzyme activity in all tissues
maintained at least 23% of the level at the termination for 1
week. In particular, the enzyme activity in liver was still
maintained over 24% after 2 weeks, indicating that the mutant enzyme synthesized during the DGJ treatment period
remained partially stable in tissues after the clearance of DGJ.
Immunohistochemical Examination of Tissues inMice Treated with DGJ. Heart and kidney are two major
tissues affected by Fabry disease. To confirm that the increase in ␣-Gal A activity in tissues was the direct result of
increase in the enzyme protein, Western blot analysis was
performed with the heart homogenate of the TgM micetreated with DGJ at a dosage of approximately 0 to 150
mg/kg body weight/day for 1 week. It was shown clearly
that the enzyme protein was increased dose-dependently(Fig. 5).
To examine whether DGJ treatment can increase ␣-Gal A
activity in specific cell types in addition to the overall increase in tissues, immunohistochemical examination was
performed with the TgM/KO mouse treated with DGJ in
drinking water at 0.5 mM for 2 weeks. Without the DGJ
treatment, no immunoreactive mutant ␣-Gal A could be detected in the heart of the TgM/KO mouse under the experimental condition. However, granular immunoresponses appeared in all cell matrixes of cardiomyocytes of the TgM/KO
mouse treated with DGJ (Fig. 6). In kidney, a remarkableincrease in intensity of ␣-Gal A was observed in distal convoluted tubules. A slight increase was also seen in proximal
convoluted tubules, but no marked change was observed inendothelial and epithelial cells of glomerulus and Bowman’s
capsule.
Effect of DGJ Treatment on Gb3 in Kidney of
TgM/KO Mice. Female heterozygous TgM/KO mice wereused to determine the effect of DGJ on Gb3 storage in thekidney because a small amount of accumulation of Gb3 could
be found in such mice. The average Gb3 amount was determined to be 3.5 ␮g/mg protein in the kidney of female heterozygous TgM/KO mice. As a comparison, Gb3 in the kidneyof ␣-Gal A-deficient mice and wild-type mice was determined
to be 19.9 and 0.4 ␮g/mg protein, respectively. Neutral glycosphingolipids were extracted from kidneys of three micetreated with 0.05 mM DGJ for 4 weeks and were subjected to
TLC analysis (Fig. 7). After the DGJ treatment, Gb3 amountPreclinical Efficacy and Safety of 1-Deoxygalactonojirimycin

729

Fig. 7. Effect of DGJ treatment on Gb3 storage inkidney of TgM/KO mice. Ten-week-old female heterozygous TgM/KO mice were treated with a DGJ
solution (0.05 mM) as drinking water (corresponding to a daily dosage of approximately 3 mg/kg bodyweight) for 4 weeks. A, HPTLC analysis of neutral
glycosphingolipids extracted from kidney of mice.
Lanes 1 to 3, untreated mice; lanes 4 to 6, DGJtreated mice. B, average concentrations of neutral
glycosphingolipids from kidney of mice were determined from the band intensity of HPTLC based onstandards. Light-colored bar, non-DGJ-treatedgroup; dark-colored bar, DGJ-treated group. GlcCer, glucosylceramide; LacCer, lactosylceramide.
The statistical significance of the difference wasdetermined by Student’s t test; ⴱ, p ⬍ 0.05.

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TABLE 2
Toxicological profiles of DGJ in mice
DGJ was administered to age-matched TgM/KO mice as a 0.5 mM solution available
ad libitum in a water tap for 9 weeks. Four male mice each were used in the
DGJ-treated and control groups. The DGJ dosage is estimated to be 30 mg/kg bodyweight/day. Values are means ⫾ S.E.M., and the significant difference between two
groups was determined by Student’s t test.

Body weight (g)Tissue weight (% of body weight)
Heart
KidneySpleenLiver
␣-Gal A activity (U/mg protein)Heart
KidneySpleenLiver
Biochemicals
AST (U/l)
ALT (U/l)
LDH (U/l)
BUN (mg/dl)
Creatinine (mg/dl)
Hematology profile
Leukocytes
White blood cells (K/␮l)
Neutrophils (K/␮l)
Lymphocytes (K/␮l)
Monocytes (K/␮l)
Eosinophils (K/␮l)
Basophils (K/␮l)
Erythrocytes
Red blood cells (M/␮l)
Hemoglobin (g/dl)
Hematocrit (%)
Mean corpuscular volume (fl)Mean corpuscular hemoglobin (pg)Mean corpuscular hemoglobin
concentration (g/dl)
Red cell distribution (%)
Thrombocytes
Platelet (K/␮l)Median platelet volume (fl)Control Group

Treated Group

28.0 ⫾ 1.5

27.3 ⫾ 1.1

0.400 ⫾ 0.031
1.313 ⫾ 0.105
0.331 ⫾ 0.024
4.687 ⫾ 0.316

0.427 ⫾ 0.011
1.328 ⫾ 0.025
0.306 ⫾ 0.011
4.636 ⫾ 0.030

57.3 ⫾ 2.2
67.1 ⫾ 3.7
122.8 ⫾ 2.8
132.1 ⫾ 13.9

2841.7 ⫾ 246.4*
257.0 ⫾ 28.3*
309.7 ⫾ 40.7†
267.7 ⫾ 22.8†

75.3 ⫾ 9.7
31.0 ⫾ 4.6
305.5 ⫾ 33.9
23.67 ⫾ 1.20
0.90 ⫾ 0.51

79.8 ⫾ 8.4
28.3 ⫾ 1.3
391.5 ⫾ 43.4
26.25 ⫾ 2.96
1.38 ⫾ 0.45

7.43 ⫾ 3.24
3.62 ⫾ 2.08
3.51 ⫾ 1.28
0.24 ⫾ 0.12
0.05 ⫾ 0.03
0.01 ⫾ 0.01

4.06 ⫾ 0.75
1.20 ⫾ 0.30
2.69 ⫾ 0.46
0.14 ⫾ 0.01
0.03 ⫾ 0.01
0.01 ⫾ 0.01

8.53 ⫾ 0.90
13.98 ⫾ 1.25
46.35 ⫾ 4.51
54.58 ⫾ 1.03
16.48 ⫾ 0.40
30.20 ⫾ 0.41

8.20 ⫾ 0.62
13.50 ⫾ 1.04
44.35 ⫾ 2.85
54.25 ⫾ 0.79
16.48 ⫾ 0.22
30.35 ⫾ 0.52

18.00 ⫾ 0.51

18.45 ⫾ 0.25

1038 ⫾ 212
7.28 ⫾ 2.541124 ⫾ 78
5.90 ⫾ 1.14

* p ⬍ 0.001 versus control group.
† p ⬍ 0.005 versus control group.

To determine potential long-term toxicity of DGJ, 4-weekold TgM mice were maintained and treated with or without
DGJ at 0.05 mM, corresponding to a dosage of 3 mg/kg bodyweight/day for 20 weeks. All mice in the DGJ-treated groupsurvived after the 20-week treatment. The body weight of themice in both groups was monitored weekly, and no significant
differences in body weight of both groups were observed (Fig.
9). At the end of the study, tissue weights of major tissues,
including heart, kidney, spleen, and lung, were essentiallythe same between the DGJ-treated and nontreated groups.

Fig. 8. Histochemistry of tissues of TgM/KO mice treated with DGJ. DGJ
in drinking water (0.5 mM) was administered to TgM/KO mice for 9
weeks. The tissues were removed and fixed in 10% formalin, followed byembedding with paraffin. The paraffin sections of each tissue were
stained with HE reagent and examined under optical microscopy (magnification, ⫻20). A, C, E, G, and I, tissues from nontreated TgM/KO
mouse; B, D, F, H, and I, tissues from DGJ-treated TgM/KO mouse. A andB, heart; C and D, liver; E and F, kidney; G and H, spleen; I and J, lung.

In addition, no abnormality in appearance, growth, and fertility was found in mice administered by DGJ at a daily
dosage of approximately 3 mg/kg body weight for 2 years. The

730

Ishii et al.

life span of the DGJ-treated group seemed to be the same as
the control group. These results indicated that DGJ is welltolerated in mice.

Discussion
After the evaluations of DGJ for efficacy and mechanistic
studies in vitro and in cell-based systems, we have developed
suitable mouse models for further evaluation of DGJ in vivo.
We initially generated a transgenic mouse model (TgM) that
expresses human mutant R301Q ␣-Gal A activity. Because
these mice contained both the endogenous ␣-Gal A gene andthe transgene, the ␣-Gal A activity measured in tissues was
the combination of two enzymes. Initial in vivo studies wereconducted with these TgM mice, using a set of Non-Tg micewith a similar genetic background to subtract the endogenous enzyme activity. Although these initial in vivo studies
were undertaken, we (Ishii et al., 2004) further developed amouse model (TgM/KO) exclusively expressing human mutant enzyme in tissues by crossbreeding the TgM mice with
␣-Gal A KO mice. Despite the transgene product being amutant form of human ␣-Gal A, the tissue ␣-Gal A activity inheterozygous TgM/KO mice was found to be comparable or
only slightly lower than that of wild-type Non-Tg mice, presumably because of the highly efficient ␤-actin promoter of
the transgene. In comparison of tissue ␣-Gal A activities in
TgM mice with those of TgM/KO mice, we found that theendogenous enzyme did not interfere the human mutant
enzyme in TgM mice and that the activity derived from thehuman mutant enzyme could be estimated by subtracting theendogenous enzyme activity determined in age-matched control Non-Tg mice from the total apparent enzyme activity(Table 1). Furthermore, response of the human mutant ␣-Gal
A to DGJ in TgM mice behaved in a similar manner of thatfound in TgM/KO mice. These results indicated that resultsof tissue ␣-Gal A activity generated from TgM mice after
correction are comparable with those of TgM/KO mice. Although TgM mice were equally useful to TgM/KO mice forstudies of tissue ␣-Gal A activity, TgM/KO mice had advantages to avoid use of a large number of Non-Tg mice, therefore reducing experimental animal size and consumption of
DGJ. In addition, because of the low level of ␣-Gal A activity
in kidney of heterozygous TgM/KO mice, a small amount ofaccumulation of Gb3 was found in the kidney of such mice.

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Fig. 9. DGJ effect on body weight of TgM mice. Five 4-week-old mice wereused for each group. DGJ was administered to the treated group as
dinking water at a concentration of 0.05 mM corresponding to a dosage of
3 mg/kg body weight/day. Body weight of both nontreated (ⴱ) and treated(E) groups was measured weekly, and average and S.D. are shown.

These mice were particularly useful as a pathological animalmodel for evaluating DGJ in vivo.
The responses of tissue ␣-Gal A activity to DGJ were evaluated in TgM mice using Non-Tg mice as controls. DGJ wasorally administered ad libitum to the mice in drinking water.
Upon administration of DGJ, ␣-Gal A activity responded tothe treatment quickly, and a substantial increase in the
enzyme activity could be observed in all major tissues within
3 days (Fig. 2). The DGJ effect reached a maximum between
11 and 14 days in major tissues and remained as a plateau
afterward. The increase of tissue ␣-Gal A activity was dosedependent in TgM mice treated with DGJ at 0.05 to 1 mM in
drinking water (corresponds to daily dosage at 3– 60 mg/kg
body weight) for 1 week (Fig. 3). Even when DGJ was administered at 0.05 mM in drinking water (approximately 3 mg/kg
body weight/day), ␣-Gal A activity increased more than 2-foldin major tissues, including heart and kidney, that are severally affected by the disease in patients. Because a 2-foldincrease in the enzyme activity in this mutation (R301Q) is
wildly expected to have a therapeutic effect in clinical, we
further administered DGJ at 0.05 mM in drinking water toheterozygous TgM/KO mice, which retain a small amount of
Gb3 accumulation in kidney. After 4 weeks of treatment, Gb3
was substantially reduced to 54% of untreated mice in kidney(Fig. 6). This clearly indicates that a continuous dose of DGJ
at approximately 3 mg/kg body weight/day cannot only increase tissue enzyme activity but also provide clinical benefit
in mice for this mutation. Therefore, we consider a daily
dosage of 3 mg/kg body weight is the effective dosage in mice.
DGJ is the most potent inhibitor for ␣-Gal A and the mosteffective ASSC for rescuing mutant ␣-Gal A (Asano et al.,2000). High doses of DGJ may extend the ASSC effect but
may also cause stronger inhibition for the enzyme activity invivo. We have shown that DGJ included in the culture medium at concentrations that were effective for ASSC function
did not inhibit the enzyme activity in cultured cells (Fan etal., 1999). However, it is a valid concern that overdose of DGJ
in vivo may well result in rescuing of mutant enzyme while
partially or totally inhibiting its activity if the tissue DGJ
concentration is higher than the effective inhibitory concentrations. Therefore, we determined tissue DGJ in TgM miceadministered with DGJ at various dosages (Fig. 3). The tissue DGJ concentrations were increased with the increasing
dose of DGJ in all major tissues. However, DGJ was notaccumulated in all tissues in mice that were continuously
administered for 2 weeks at a dosage of 30 mg/kg bodyweight, a 10-fold higher dose than the expected effective
dose. It is noticeable that DGJ concentrations in kidney weresignificantly higher compared with other tissues. This may
indicate that DGJ was concentrated and cleared through thekidney route in vivo. Upon termination of the DGJ treatment, clearance of DGJ in tissues was rapid, particularly inkidney, where more than 98% DGJ was cleared in 1 day (Fig.
4). This further indicated that urea excretion could be themain route for the clearance of DGJ in mice. No DGJ could
been detected in kidney, spleen, and liver after 1 week oftermination, although a small amount of DGJ was found in
the heart after 1 week.
To examine the half-life of the mutant ␣-Gal A synthesizedunder the DGJ treatment, tissue ␣-Gal A activities were
determined in TgM mice pretreated with DGJ for 2 weeks.
After ceasing the treatment, ␣-Gal A activity declined imme-

Preclinical Efficacy and Safety of 1-Deoxygalactonojirimycin

ing a variety of genetic disorders and provide the basis fordesign of further clinical trials of DGJ for Fabry diseasein man.
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Address correspondence to: Dr. Jian-Qiang Fan, Mount Sinai School of
Medicine, Department of Human Genetics, 5th Avenue at 100th Street, New
York, NY 10029. E-mail: jian-qiang.fan@mssm.edu

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diately in all tissues (Fig. 4). The half-life of the enzyme wasdetermined to be approximately 4 days in heart and spleen
and 7 days in kidney and liver, respectively, and was significantly longer compared with that of DGJ, which was lessthan 1 day in all tissues. This could be significant for providing a clinically effective window that permits the full functionality of the rescued enzyme in a relatively DGJ-free environment. Although DGJ in tissues of mice treated at adosage of 3 mg/kg body weight/day was not expected to cause
substantial inhibition of ␣-Gal A, an alternate dosing schedule may still be beneficial for maximizing the therapeutic
effect of DGJ treatment. Because the clearance of DGJ intissues was rapid, even higher doses of DGJ, which could
result in a greater increase of tissue ␣-Gal A, may havegreater clinical benefit when an alternate dosing strategy isapplied.
Heart and kidney are main organs affected by Fabry disease in patients. In the clinical trials of enzyme replacement
therapy, although vascular endothelial Gb3 was effectively
reduced in endomyocardial and kidney biopsies, Gb3 storage
remained unchanged in the cardiomyocytes, distal convoluted tubules, and glomerular podocytes, respectively (Eng etal., 2001a), presumably caused by the difficulty to deliver the
replacement enzyme to these cell types. To determine
whether DGJ treatment would affect these cell types, we
examined ␣-Gal A in the heart and kidney of TgM/KO micetreated with DGJ for 2 weeks for ␣-Gal A by immunohistochemical microscopic studies. Although no clear change wasfound in endothelial and epithelial cells of glomerulus, aremarkable increase of ␣-Gal A was observed in the matrix ofcardiomyocytes and distal convoluted tubules after the DGJ
treatment (Fig. 6). These results indicate that DGJ can bephysiologically effective in the increase of ␣-Gal A activity insuch cell types and might be clinically effective in reducing
Gb3 storage in these cell types.
To examine the safety of DGJ in mice, we have performed
blood biochemistry and hematological profiling and pathological examination of heart, kidney, spleen, lung, and liver in
TgM/KO mice treated with DGJ at approximately 30 mg/kg
body weight/day (a 10-fold higher dosage than the targeted
effective dosage) for 9 weeks. No abnormality in all tests andexaminations was found at the end of treatment. Furthermore, no abnormality in appearance, growth retardation,
fertility, and life span could be observed in mice treated with
DGJ at approximately 3 mg/kg body weight/day for 2 years.
In clinical phase I trials of DGJ in healthy volunteers,
no drug-related adverse event was reported (http://www.
amicustherapeutics.com), indicating that DGJ is also welltolerated in man.
In summary, DGJ has been shown to be effective in increase of ␣-Gal A in vivo and reduction of Gb3 storage in thekidney of TgM/KO mice. DGJ has a high safety profile in miceeven at a high dose. These in vivo efficacy and safety results
further indicate the potential of this novel therapeutic strategy of using orally active small molecules as ASSCs for treat-731