Novel cell-penetrating α-keto-amide calpain inhibitors as potential treatment for muscular dystrophy

Article abstract of DOI:10.1016/j.bmcl.2005.08.064

Dipeptide-derived α-keto-amide compounds with potent calpain inhibitory activity have been identified. These reversible covalent inhibitors have IC₅₀ values down to 25 nM and exhibit greatly improved activity in muscle cells compared to the reference compound MDL28170. Several novel calpain inhibitors have shown positive effects on histological parameters in an animal model of Duchenne muscular dystrophy demonstrating their potential as a treatment option for this fatal disease.

Full text of DOI:10.1016/j.bmcl.2005.08.064

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5176–5181
Novel cell-penetrating a-keto-amide calpain inhibitors as
potential treatment for muscular dystrophy
a
a
a
a
Cyrille Lescop,a, Holger Herzner, Herve Siendt, Reto Bolliger, Marco Hennebo¨hle,
Philipp Weyermann,a Alexandre Briguet,b Isabelle Courdier-Fruh,b Michael Erb,b
Mark Foster,b Thomas Meier,b Josef P. Magyarb and Andreas von Sprechera
*
´
aMedicinal Chemistry Department, Santhera Pharmaceuticals, Hammerstrasse 25, CH-4410 Liestal, Switzerland
bBiology Department, Santhera Pharmaceuticals, Hammerstrasse 25, CH-4410 Liestal, Switzerland
Received 7 July 2005; revised 17 August 2005; accepted 22 August 2005
Available online 26 September 2005
Abstract—Dipeptide-derived a-keto-amide compounds with potent calpain inhibitory activity have been identified. These reversible
covalent inhibitors have IC50 values down to 25 nM and exhibit greatly improved activity in muscle cells compared to the reference
compound MDL28170. Several novel calpain inhibitors have shown positive effects on histological parameters in an animal model
of Duchenne muscular dystrophy demonstrating their potential as a treatment option for this fatal disease.
Ó 2005 Elsevier Ltd. All rights reserved.
Calpains are calcium-activated neutral proteases belong-
ing to the papain superfamily of cysteine proteases; the
best characterized are calpains I and II. Calpains are
widely distributed in mammalian cells and have been
implicated in a variety of diseases such as stroke, Alzhei-
merÕs disease, spinal cord injury, cardiac ischemia, mus-
cular dystrophy, and cataract.1 Thus, in recent years,
calpain inhibition has become an important pharmaco-
logical strategy to develop novel therapies.
Treatment options are at present very limited and in-
clude artificial respiration, orthopedic surgery, and the
short-term use of glucocorticoids primarily to limit the
disease-associated inflammation.
A pharmacological approach using enzyme inhibitors is
currently regarded as a possible therapeutic strategy for
the treatment of DMD. Absence of functional dystro-
phin protein in DMD muscle leads to impaired muscle
cell membrane integrity during cycles of contraction
and relaxation. Calcium influx through membrane le-
sions causes abnormal calpain activation and proteolysis
and, therefore, contributes to muscle cell deterioration.
Subsequent protein degradation by the proteasome
pathway further promotes muscle fiber damage and
muscle weakness. Therefore, activation of the cytosolic
Ca2+-dependent cysteine proteases calpain I and II is be-
lieved to be a critical step in the pathogenesis of DMD.
Consequently, it is considered that inhibition of calpains
holds the potential to slow down or stop disease
progression by preventing muscle degeneration and
necrosis.
Our drug discovery program for novel calpain inhibitors
emerged from our interest in finding a treatment for
Duchenne muscular dystrophy (DMD).2 Muscular dys-
trophies are a group of neuromuscular diseases charac-
terized by progressive weakness and degeneration of
body musculature. The most prevalent form is DMD,
an X-chromosome-linked inherited disease with an inci-
dence rate of 1 in 3500 newborn boys worldwide affect-
ing about 40,000 male patients in Europe and North
America. This fatal disease manifests between the age
of two and six, confines affected teenage children to
wheelchairs, and ultimately leads to death at early age.
A number of calpain inhibitors have been reported in
the literature. Most of these are modified peptides con-
taining reactive functional groups that interact with
the active-site cysteine thiol of calpain. These com-
pounds can be classified as either irreversible or revers-
ible inhibitors. Irreversible inhibitors include peptidyl
Keywords: Calpain inhibitor; Keto-amide; Duchenne muscular
dystrophy.
*
Corresponding author. Tel.: +41 61 906 89 85; fax: +41 61 906 89
88; e-mail: cyrille.lescop@santhera.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.08.064

C. Lescop et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5176–5181
5177
halo-ketones,3 diazo-ketones,4 epoxy-succinyls,5 and
Y
Y
other derivatives. They contain reactive functional
groups which can be nucleophilically displaced by the
cysteine thiol to form a sulfide, thereby permanently
inactivating calpain. Reversible inhibitors include pepti-
dyl aldehydes6 like the natural product leupeptin 1
(Fig. 1) and activated ketones7 like a-keto-esters, a-ke-
to-acids, and a-keto-amides. They inactivate the enzyme
in a transient manner by forming a reversible covalent
bond (hemithioacetal or -ketal) with the cysteine thiol.
However, most of these inhibitors have unsatisfactory
pharmacological properties regarding selectivity, solu-
bility, stability, or cellular penetration.1b
Passerini reaction
Acyl migration
d
R
HN
Boc
X
N
H
Boc
N
H
O
O
O
c
O
X = OH, 7
a
Boc
X = N(OMe)Me, 8
X = H, 9
N
H
R2
b
10
Y
Y
O
O
O
O
Starting from known calpain inhibitor lead compounds
such as MDL28170 2 (Fig. 1),8 we initiated a lead opti-
mization program to develop reversible calpain inhibi-
tors 3–6 which possess a chemically and metabolically
stable warhead and which show improved uptake
into muscle cells.9 We have focused on the synthesis
and evaluation of dipeptide-derived a-keto-carbonyl
compounds.
H
N
e,f
R
R3
H2N
R
N
H
N
N
H
N
H
H
O
R2
O
R2
OH
11
3-6
Scheme 1. Reagents and conditions: (a) NHMe(OMe), EDCI, NMM,
CH2Cl2, 0 °C to rt (ꢀ90%); (b) LiAlH4, Et2O, 0 °C (60–95%); (c) RNC,
Boc–AA2–CO2H, CH2Cl2, rt; (d) 1—TFA/CH2Cl2 (1/3), 0 °C to rt; 2—
NEt3, CH2Cl2, rt (40–55%, three steps); (e) R3–COOH, HBTU,
DIPEA, DMF, 0 °C to rt (80–95%); (f) Dess–Martin periodinane,
DMSO, CH2Cl2, 0 °C to rt, (25–85%).
Depending on the structure of the target compounds
and the availability of the starting materials, two syn-
thetic routes were used. The first method (method A) in-
volved the Passerini reaction as the key step to generate
the precursor 11 of the required keto-amide functional-
ity.10 As displayed in Scheme 1, aldehydes 9 were syn-
thesized from acids 7 under known conditions7c and
reacted with isocyanides and Boc-protected amino-acids
using Passerini conditions to yield 10. a-Acyloxyamides
10 underwent an acyl migration after Boc removal and
treatment with triethylamine to give the a-hydroxy-
amides 11. Subsequent coupling with different acids un-
der standard conditions (HBTU or EDCI/HOBt)
followed by Dess–Martin oxidation provided the
a-keto-amides 3–6.
The alternative protocol (method B) utilized the Wasser-
man acyl cyanophosphorane oxidative cleavage (Scheme
2).11 Coupling of the acids 7 with cyanomethyl-triphe-
nylphosphonium ylide afforded phosphoranes 12. Sub-
sequent ozonolysis of the phosphorus–carbon double
bond at low temperature resulted in the generation of
reactive acyl nitrile that was then displaced by the
appropriate nucleophiles to yield the a-keto-carbonyl
intermediates 13. Deprotection in acidic medium and
coupling of the left moieties provided the final com-
pounds 3–6 as a 1:1 mixture of diasteromers at P1.
H
Y
N
NH2
NH
Y
Ozonolysis
O
O
O
PPh3
CN
H
N
H
Boc
OH
Boc
1, leupeptin
N
N
H
N
H
N
H
a
H
b
O
O
O
O
O
O
12
7
Y
Y
H
N
2, MDL28170
O
H
N
H
O
O
O
H
N
Boc
R'
R3
R'
N
H
W
N
H
W
c,d
O
R2
O
O
P1
R1
13
P3
3-6
O
O
H
3-6
N
Scheme 2. Reagents and conditions: (a) Ph3P@CHCN, EDCI,
DMAP, CH2Cl2, 0 °C to rt, 14 h (48–83%); (b) 1—O3, CH2Cl2,
ꢁ78 °C, 45 min; 2—Ar flushing, ꢁ78 °C, 5 min; 3—+R0WH, ꢁ78 °C
for 1 h, then to rt, 14 h (esters: 24–45%; amides: 40–85%); (c) HCl
(3 M) in dioxane, 0 °C, 3 h (esters: 35–65%; amides: quant.); (d) R3–
COOH, EDCI, HOBt, NMM, CH2Cl2, 0 °C to rt, 14 h (esters: 25–
45%; amides: 55–85%).
R3
N
X
H
R2
P2
O
Figure 1. Leupeptin 1, MDL28170 2, and general structure of the
dipeptide-derived a-keto-carbonyl calpain inhibitors 3–6.

5178
C. Lescop et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5176–5181
Table 1. Inhibition of calpain I by P3 analogs 3a–m
According to numerous publications, the P1 and P2 res-
idues are quite well defined to achieve the optimal in vi-
tro activity: preferred P1 residues are large hydrophobic
groups like phenylalanine and preferred P2 residues are
valine and leucine.1 Therefore, we decided to focus first
on the P3 moiety (Fig. 1) by replacing the benzyloxycar-
bonyl group (Cbz) of MDL28170 with a residue which
would improve the uptake of the compounds into mus-
cle cells. Calpain I inhibition was initially determined in
a cell-free assay by measuring the time course of the
hydrolysis of a fluorogenic substrate Suc-Leu-Tyr-
AMC by purified porcine calpain I (increase in fluores-
cence over 30 min, kex = 360 nm, kem = 440 nm).12 Sub-
sequently, the inhibitory activity within muscle cells
was determined in a cell-based assay using cultured
C2C12 myoblasts which were loaded with the fluorogen-
ic substrate Suc-Leu-Tyr-AMC prior to the experiment.
Cellular calpains were activated by incubation with cal-
cium ionophore 4-Br-A23187 (10 lM) in the presence of
increasing concentrations of the inhibitor.
Cl
O
O
H
N
R3
N
H
N
H
O
Compound R3
Calpain I
inhibition
cell-freeb
Calpain I
inhibition
myoblastc
IC50 [lM]a IC50 [lM]a
1
Leupeptin
0.08
0 % at
10 lM
10
2
MDL28170
0.02
0.09
O
O
3a
15
S
S
3b
3c
3d
3e
3f
0.06
0.10
0.12
0.10
0.03
0.04
0.10
0.08
0.35
0.40
2.0
3.2
1.0
30
O
O
Using the Passerini strategy, we prepared a series of
inhibitors with ethyl a-keto-amide as warhead to cir-
cumvent the chemical and metabolic instability of the
aldehyde. We investigated different residues known to
be carried by biological transporters into the human
cells. A non-polar lipophilic residue, lipoyl 3b, was iden-
tified to mediate uptake into muscle cells as measured by
improved intracellular activity of the compound. Next,
we evaluated the influence of small changes on this type
of moiety as summarized in Table 1 with focus on a ser-
ies 3a–m characterized by valine in P2 and p-Cl-phenyl-
alanine in P1, respectively.
S
S
O
S
O
O
H
N
S
S
S
O
O
H
N
3g
3h
3i
S
S
O
H
N
S
S
20
O
O
Removal of one sulfur atom resulted in another potent
inhibitor 3c with a significantly improved intracellular
activity compared to the compound 3a bearing a Cbz
group. Interestingly, shortening of the chain length by
one (3d) or two carbons (3e) led to a loss of potency
in the cellular assay (5–8-fold decrease) while keeping
the activity against calpain I in the cell-free assay. Addi-
tion of a spacer between the lipoyl residue and the P2
moiety (3f–h) also resulted in a reduced intracellular
potency while the inhibitory activity as measured in a
cell-free assay remained in the same range. More polar
analogs like sulfoxide 3i and biotin 3j derivatives were
investigated as well. The sulfoxide derivatives were iso-
lated in some cases as a side product of the final oxida-
tion step. These compounds usually kept the inhibitory
potency against the enzyme but exhibited poor intracel-
lular activity (14–17-fold decrease). Replacement of the
sulfur ring by a phenyl (3k) or attachment of more lipo-
philic residues like 4-n-butylbenzoyl (3l) or lauroyl (3m)
resulted in weaker cell permeability or overall potency,
once again demonstrating the superiority of the lipoyl
residue over other lipophilic moieties.
6.0
S
O
O
O
HN
NH
H
3j
0.10
5.0
H
S
O
3k
3l
0.06
0.15
1.5
25
O
O
3m
>1.0
n.d.
O
a Values are means of triplicates (n.d. = not determined).
b Hydrolysis of fluorogenic substrate Suc-Leu-Tyr-AMC by purified
porcine calpain I.
c Activation of cellular calpain with Ca2+-ionophore Br-A-23187 and
hydrolysis of fluorogenic substrate Suc-Leu-Tyr-AMC by cellular
calpain.
erate only small lipophilic residues. Leucine and valine
are the preferred residues as illustrated in 3b and 4d
which are the most potent compounds in both enzymat-
ic and cellular assays. Surprisingly, tert-butylglycine,
reported to be a potent P2 moiety,6b resulted in the inac-
tive compound 4b. Large groups like cyclohexyl (4c)
proved to be detrimental. In another series, replacement
of leucine by its polar isoster threonine led to poor
Having identified the lipoyl group as the most potent P3
residue for improving the permeability into the muscle
cells, a study to confirm the best P2 residues was under-
taken (Table 2). With respect to the P2 position, our
results generally are in agreement with previous observa-
tions:1 the S2 pocket of calpain is quite tight and can tol-

C. Lescop et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5176–5181
Table 2. Inhibition of calpain I by P2 analogs 4a–e Table 3. Inhibition of calpain I by P1 analogs 5a–s
R1
5179
Y
O
O
H
S
N
N
H
N
H
S
O
O
O
O
H
N
S
Compound
R1
Calpain I
inhibition,
cell-free
Calpain I
inhibition,
myoblast
S
N
N
H
H
O
R2
O
IC50 [lM]a
IC50 [lM]a
Compound
R2
Y
Calpain I
inhibition,
cell-free
Calpain I
inhibition,
myoblast
IC50 [lM]a
3b
5a
5b
5c
5d
5e
5f
4-Cl-PhCH2–
PhCH2–
0.06
0.08
0.10
0.06
0.12
0.07
0.20
0.40
0.12
0.15
0.10
0.10
0.08
0.10
0.10
0.25
0.50
0.40
>1.0
>1.0
0.35
3.5
IC50 [lM]a
4-F-PhCH2–
4-Br-PhCH2–
2.5
0.20
0.22
0.35
1.1
3b
4a
4b
Cl
Br
Cl
0.06
0.18
0.35
9.0
4-MeO-PhCH2–
4-Me-PhCH2–
4-CF3-PhCH2–
4-t-Bu-PhCH2–
4-CN-PhCH2–
4-OH-PhCH2–
3-CF3-PhCH2–
2,4-Di-Cl-PhCH2–
3,4-Di-Cl-PhCH2–
3,4-Di-F-PhCH2–
2-Thienyl-CH2–
c-Hexyl-CH2–
i-Bu–
5g
5h
5i
1.8
5.0
>1.0
n.d.
2.5
4c
Br
>1.0
n.d.
5j
15
40
5k
5l
10
4d
4e
Cl
Cl
0.02
0.10
0.39
3.5
5m
5n
5o
5p
5q
5r
5s
0.70
6.0
3.0
0.25
2.0
a Values are means of triplicates (n.d. = not determined).
Ph–
Ph-CH2-CH2–
(Ph)2CH–
>10
n.d.
a Values are means of triplicates (n.d. = not determined).
inhibitors as well (data not shown) providing evidence
that the S2 pocket cannot accommodate polar groups.
As illustrated in Table 3, we also initiated a SAR study
to identify more potent P1 residues. We found that the
use of substituted phenylalanines as the P1 moiety affor-
ded inhibitors with a better binding to the enzyme usu-
ally with IC50 values below 0.15 lM. Calpain can
accommodate different substitution patterns on the
phenyl ring but the position of the substituent has a sig-
nificant influence on the intracellular activity. Substitu-
tion at the 4-position with hydrophobic groups
provided compounds with improved uptake into the
muscle cells (up to 17-fold improvement compared to
the unsubstituted analog 5a). Chloro- (3b), bromo-
(5c), methoxy- (5d), and methyl- (5e) were among the
best substituents. Di-substitution (5k–m) as well as the
use of polar substituents (5i) was detrimental to the cel-
lular activity. Replacement of the phenyl ring by a thie-
nyl (5n) or a cyclohexyl ring (5o) or by an isopropyl
group (5p) did not improve inhibitory activity. Interest-
ingly, the use of phenylglycine (5q) maintained moderate
activity, whereas increase of the chain length by one car-
bon resulted in a loss of activity (5r). Bulky a-branched
phenylalanines led to inactive compounds like 5s.
further efforts on the a-keto-amide series. N,N-disubsti-
tuted a-keto-amide analogs as exemplified with 6d were
much less potent than N-monosubstituted a-keto-
amides. This observation is consistent with previous
work7b and could indicate that the NH of the a-keto-
amide functional group forms a hydrogen bond with
an amino-acid residue of the active-site of calpain. Pri-
mary amide (6e) and other N-alkyl or N-alkylaryl amide
derivatives were also examined (data not shown). They
usually exhibited similar potency as 6a, but the a,a-di-
substituted N-alkyl residues like 6g were detrimental
for the binding to the enzyme. In order to prepare more
water soluble compounds, polar moieties were also eval-
uated. Intracellular potency of weakly basic residues on
the alkyl chain like pyridyl (6h) or morpholinyl (6i) was
comparable to the one of non-polar moieties, whereas
strongly basic substituents like piperidine led to a signif-
icant decrease of the intracellular activity with IC50 val-
ues above 5 lM (data not shown).
As depicted in Table 5, biochemical profiling against
other biologically relevant proteases demonstrated good
selectivity versus caspase 3 and thrombin. In the case of
cathepsin B, limited selectivity was observed. Some com-
pounds (5c) proved to be moderate 20S-proteasome
inhibitors. This property could be beneficial since both
calpain- and proteasome-mediated proteolytic pathways
are involved in muscular dystrophies.
We finally evaluated the effect of changes in the warhead
group on inhibitory potency as summarized in Table 4.
In the lipoyl-Leu-4-BrPhe series, changing the ethyl a-
keto-amide (6a) to the a-keto-acid (6b) resulted in a sim-
ilar potency against calpain but in a diminution of the
intracellular activity likely due to its polarity preventing
a good membrane permeability. The corresponding eth-
yl a-keto-ester 6c proved to be inactive against the en-
zyme (IC50 > 10 lM). Therefore, we decided to focus
Finally, we examined the influence of the chiral center of
the lipoyl residue by synthesizing the two isomers 6a-(R)
and 6a-(S) starting from (R)-lipoic acid and (S)-lipoic

5180
C. Lescop et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5176–5181
Table 4. Inhibition of calpain I by P0 analogs 6a–i
Br
O
O
H
N
S
S
N
X
H
O
O
Compound
X
Method
Calpain I inhibition cell-free IC50 [lM]a
Calpain I inhibition myoblast IC50 [lM]a
6a
6b
6c
6d
6e
6f
–NHEt
–OH
A
B
B
B
B
B
A
B
B
0.02
0.05
>10
0.5
5.0
–OEt
–N(CH2CH2)2-O
–NH2
4.5
>1.0
0.02
0.07
>1.0
0.23
0.12
n.d.
0.25
0.28
n.d.
0.8
–NH-c-Hex
–NH-t-Bu
6g
6h
6i
–NH(CH2)2-2-pyridyl
–NH(CH2)3-morpholinyl
1.0
a Values are means of triplicates (n.d. = not determined).
Table 5. Inhibitory activity profiling
Compound CalpI IC50 [lM]a CathB IC50 [lM]a 20S-Proteasome IC50 [lM]a Casp3 % inhib. at 10 lMa Thrombin % inhib.at 10 lMa
2
0.02
0.06
0.02
0.06
0.02
0.10
0.10
0.12
0.04
0.65
>1.0
0.50
0
17
0
0
36
20
0
3b
4d
5c
6a
0.52
0.18
0.62
0
0
30
a Values are means of triplicates.
acid.13 The inhibitory activity of 6a-(R) and 6a-(S) was
similar to that of the racemate 6a with IC50 value of
0.039 and 0.024 lM, respectively, in the cell-free assay
and IC50 value of 0.4 lM for both isomers in the myo-
blast assay. Therefore, compounds were used as race-
mates in the animal model.
diameters indicating elevated muscle fiber turnover. This
variability in muscle fiber diameters was quantified by
calculating the variance coefficient of the minimal Fer-
etÕs diameter of all muscle fibers in a given muscle.14
Several inhibitors showed a normalized histological
appearance, as illustrated in Figure 2, and improved
variance coefficient of the muscle fiber diameters. As
summarized in Table 6, between 25% and 30% improve-
ment was observed in diaphragm muscle upon applica-
tion of the keto-amides 3b, 5c, and 6a. Similar
histopathological recovery was achieved by overexpres-
sion of calpastatin (a specific endogenous calpain inhib-
itor) in mdx mice.15
The best inhibitors were tested in vivo in mdx mice, a
well-established animal model for DMD (ip treatment,
every second day between the third and seventh week
of age, 20 mg/kg, PEG200/saline (1:1) vehicle). A well-
documented hallmark of dystrophic muscles seen in
mdx mice is the increased variability of muscle fiber size
Figure 2. Representative images showing the normalization of the fiber size distribution in diaphragm muscle of mdx mice upon four weeks
application of 6a.

C. Lescop et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5176–5181
5181
Table 6. Variance coefficient of muscle fiber size in diaphragm muscle
D.; Josef, K.; Wells, G.; Tripathy, R.; Iqbal, M.; Bihov-
sky, R.; Senadhi, S. E.; Mallya, S.; OÕKane, T. M.;
McKenna, B. A.; Siman, R.; Mallamo, J. P. J. Med.
Chem. 1997, 40, 3820.
450
400
350
300
4. Crawford, C.; Mason, R. W.; Wikstrom, P.; Shaw, E.
Biochem. J. 1988, 253, 751.
5. Barrett, A. J.; Kembhavi, A. A.; Brown, M. A.; Kirschke,
H.; Knight, C. G.; Tamai, M.; Hanada, K. Biochem. J.
1982, 201, 189.
250
200
6. (a) Sasaki, T.; Kishi, M.; Saito, M.; Tanaka, T.; Higushi,
N.; Kominami, E.; Katunama, N.; Murachi, T. J. Enzyme
Inhib. 1990, 3, 195; (b) Iqbal, M.; Messina, P. A.; Freed,
B.; Das, M.; Chatterjee, S.; Tripathy, R.; Tao, M.; Josef,
K. A.; Dembofsky, B.; Dunn, D.; Griffith, E.; Siman, R.;
Senadhi, S. E.; Biazzo, W.; Bozyczko-Coyne, D.; Meyer,
S. L.; Ator, M. A.; Bihovsky, R. Bioorg. Med. Chem. Lett.
1997, 7, 539; (c) Chatterjee, S.; Gu, Z.-Q.; Dunn, D.; Tao,
M.; Josef, K.; Tripathy, R.; Bihovsky, R.; Senadhi, S. E.;
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Chem. 1998, 41, 2663; (d) Inoue, J.; Nakamura, M.; Cui,
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Wild type mdx
3bc
5cc
6ac
Meana
% Red.b
Mice
231.2
12
401.6
351.5
29.4
5
0.013f
354.7
27.5
5
0.008f
359.7
24.6
13
0.001f
27
< 0.001e
p-valued
a Variance coefficient.14
b % Reduction in the variance coefficient of muscle fiber sizes as
compared to untreated mice (= 100%).
c mdx treated with compound.
d p-value unpaired StudentÕs t-test.
e Comparison against wild type.
f Comparison against untreated mdx.
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