MX1013, a dipeptide caspase inhibitor with potent in vivo antiapoptotic activity

Article abstract of DOI:10.1038/sj.bjp.0705450

1. Caspases play a critical role in apoptosis, and are considered to be key targets for the design of cytoprotective drugs. As part of our antiapoptotic drug-discovery effort, we have synthesized and characterized Z-VD-fmk, MX1013, as a potent, irreversible dipeptide caspase inhibitor. 2. MX1013 inhibits caspases 1, 3, 6, 7, 8, and 9, with IC₅₀ values ranging from 5 to 20nM. MX1013 is selective for caspases, and is a poor inhibitor of noncaspase proteases, such as cathepsin B, calpain I, or Factor Xa (IC₅₀ values > 10 μM). 3. In several cell culture models of apoptosis, including caspase 3 processing, PARP cleavage, and DNA fragmentation, MX1013 is more active than tetrapeptide- and tripeptide-based caspase inhibitors, and blocked apoptosis at concentrations as low as 0.5 μM. 4. MX1013 is more aqueous soluble than tripeptide-based caspase inhibitors such as Z-VAD-fmk. 5. At a dose of 1 mgkg^-1 i.v., MX1013 prevented liver damage and the lethality caused by Fas death receptor activation in the anti-Fas mouse-liver apoptosis model, a widely used model of liver failure. 6. At a dose of 20 mgkg^-1 (i.v. bolus) followed by i.v. infusion for 6 or 12 h, MX1013 reduced cortical damage by approximately 50% in a model of brain ischemia/reperfusion injury. 7. At a dose of 20 mgkg^-1 (i.v. bolus) followed by i.v. infusion for 12 h, MX1013 reduced heart damage by approximately 50% in a model of acute myocardial infarction. 8. Based on these studies, we conclude that MX1013, a dipeptide pan-caspase inhibitor, has a good combination of in vitro and in vivo properties. It has the ability to protect cells from a variety of apoptotic insults, and is systemically active in three animal models of apoptosis, including brain ischemia.

Full text of DOI:10.1038/sj.bjp.0705450

British Journal of Pharmacology (2003) 140, 402–412
&
2003 Nature Publishing Group All rights reserved 0007–1188/03
$25.00
www.nature.com/bjp
MX1013, a dipeptide caspase inhibitor with potent in vivo
antiapoptotic activity
1
2
1
1
2
1
1Wu Yang, John Guastella, Jin-Cheng Huang, Yan Wang, Li Zhang, Dong Xue, Minhtam
Tran, Richard Woodward, Shailaja Kasibhatla, Ben Tseng, John Drewe & *,1Sui Xiong Cai
2
1
1
1
1Cytovia, Inc. (a subsidiary of Maxim Pharmaceuticals, Inc.), 6650 Nancy Ridge Drive, San Diego, CA 92121, U.S.A. and
2CoCensys, Inc., 213 Technology Drive, Irvine, CA 92718, U.S.A.
1
Caspases play a critical role in apoptosis, and are considered to be key targets for the design of
cytoprotective drugs. As part of our antiapoptotic drug-discovery effort, we have synthesized and
characterized Z-VD-fmk, MX1013, as a potent, irreversible dipeptide caspase inhibitor.
2
MX1013 inhibits caspases 1, 3, 6, 7, 8, and 9, with IC50 values ranging from 5 to 20 nm. MX1013 is
selective for caspases, and is a poor inhibitor of noncaspase proteases, such as cathepsin B, calpain I,
or Factor Xa (IC50 values 410 mm).
3 In several cell culture models of apoptosis, including caspase 3 processing, PARP cleavage, and
DNA fragmentation, MX1013 is more active than tetrapeptide- and tripeptide-based caspase
inhibitors, and blocked apoptosis at concentrations as low as 0.5 mm.
4
MX1013 is more aqueous soluble than tripeptide-based caspase inhibitors such as Z-VAD-fmk.
At a dose of 1 mg kgꢀ1 i.v., MX1013 prevented liver damage and the lethality caused by Fas death
5
receptor activation in the anti-Fas mouse-liver apoptosis model, a widely used model of liver failure.
At a dose of 20 mg kgꢀ1 (i.v. bolus) followed by i.v. infusion for 6 or 12 h, MX1013 reduced cortical
damage by approximately 50% in a model of brain ischemia/reperfusion injury.
At a dose of 20 mg kgꢀ1 (i.v. bolus) followed by i.v. infusion for 12 h, MX1013 reduced heart
damage by approximately 50% in a model of acute myocardial infarction.
Based on these studies, we conclude that MX1013, a dipeptide pan-caspase inhibitor, has a good
6
7
8
combination of in vitro and in vivo properties. It has the ability to protect cells from a variety of
apoptotic insults, and is systemically active in three animal models of apoptosis, including brain
ischemia.
British Journal of Pharmacology (2003) 140, 402–412. doi:10.1038/sj.bjp.0705450
Keywords: Apoptosis; brain ischemia/stroke; caspase inhibitor; cytoprotective; liver failure; myocardial infarction
Abbreviations: Ac-DEVD-AMC, acetyl-Asp-Glu-Val-Asp-7-amino-4-methyl-coumarin; Ac-DEVD-CHO, acetyl-Asp-Glu-
Val-Asp-carbaldehyde; Boc-D(OMe)-fmk, t-butyloxycarbonyl-Asp(OMe)-fluoromethylketone; CCA, common
carotid arteries; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; EDTA, ethylenediami-
netetraacetic acid; FACS, fluorescence-activated cell sorting; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesul-
fonic acid, sodium salt; HRP, horseradish peroxidase; LDCA, left descending coronary artery; MCA, middle
cerebral artery; MCAO, middle cerebral artery occlusion; MX1013, Z-VD-fmk, benzyloxycarbonyl-Val-Asp-
fluoromethylketone; PARP, poly(ADP)ribose polymerase; PIPES, 1,4-piperazinebis(ethanesulfonic acid, sodium
salt); PVDF, polyvinylidene fluoride; SGPT, serum glutamic pyruvic transaminase; SGOT, serum glutamic
oxaloacetic transaminase; suc-Leu-Tyr-AMC, succinyl-Leu-Tyr-7-amino-4-methyl-coumarin; TTC, 2,3,5-
triphenyltetrazolium chloride; Z-D(OMe)E(OMe)VD(OMe)-fmk, benzyloxycarbonyl-Asp(OMe)-Glu
(OMe)-Val-Asp(OMe)-fluoromethylketone; Z-E(OMe)VD(OMe)-fmk, benzyloxycarbonyl-Glu(OMe)-Val-Asp
(OMe)-fluoromethylketone; Z-VAD(OMe)-fmk, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone; Z-
VD(OMe)-fmk, benzyloxycarbonyl-Val-Asp(OMe)-fluoromethylketone
Introduction
Apoptosis is
a
highly regulated process involving the
embryonic and post-embryonic development, and also plays
an important role in the remodeling of adult tissues. However,
as with any complex physiological pathway, defects in
apoptosis signaling, including inappropriate apoptosis induc-
tion or failure of apoptosis induction when required for
normal growth, can cause disease (Thompson, 1995).
systematic disassembly and death of cells. The ability to
undergo apoptosis appears to be a general property of
metazoan cells, and the essential features of the process are
conserved from worms to man (Aravind et al., 1999).
Apoptosis is a normal part of tissue homeostasis during
The basic molecular framework underlying apoptosis is
reasonably well established. It comprises a complex system of
signal transduction pathways (Zimmermann & Green, 2001)
consisting of cell surface and intracellular death receptors and
*Author for correspondence; E-mail: scai@maxim.com
Advance online publication: 26 August 2003

Wu Yang et al
MX1013, an in vivo active dipeptide caspase inhibitor
403
their ligands; a network of protein–protein interactions
extending from the plasma membrane to the nucleus; the
participation of organelles, such as mitochondria, which act as
both intracellular stress sensors and a source of amplifying
apoptotic signals; and the activation of caspases, a group of
cysteine proteases. The caspases are a family of structurally
related cysteine proteases, which mediate an array of death-
promoting proteolytic reactions within the cell and are
considered to play a pivotal role in the apoptotic program
(Thornberry & Lazebnik, 1998). These enzymes are respon-
sible for both the induction of apoptosis signaling (carried out
by initiator caspases, such as caspase 8), as well as the myriad
phenotypic changes that characterize cell death (carried out by
effector caspases such as caspase 3). More importantly, the
activation of the caspase proenzymes is largely controlled by
the caspases themselves, either by the proteolytic action of one
family member on another or through autocatalytic activation
via an induced proximity mechanism (Salvesen & Dixit, 1999).
The result is a caspase-mediated cascade of molecular events
that maintains and amplifies the original apoptotic stimulus.
Since the caspases play such an important role in initiating,
regulating, and carrying out apoptosis, as well as in their own
biochemical activation, they represent a key molecular target
for the discovery and development of antiapoptotic drugs
(Talanian et al., 2000).
studies provide a comprehensive analysis of this broad-
spectrum caspase inhibitor, and suggest that MX1013 may
be useful in treating human apoptosis-related disorders, which
fulfills the need for cell death inhibitors that show efficacy in
whole-cell models of apoptosis and are active in animal models
of apoptosis (Thornberry, 1998).
Methods
Materials
MX1013 (Z-VD-fmk; Figure 1) was prepared by coupling
Z-Val-CO2H with t-butyl 3-amino-5-fluoro-4-hydroxy-pen-
tanoate (Revesz et al., 1994). The coupling product was
oxidized by Dess-Martin reagent to give the t-butyl ester of
MX1013, which was then cleaved by trifluoroacetic acid to
yield MX1013 as the free acid. The structure of MX1013 was
confirmed by 1H-NMR, mass spectrometry, and elemental
analysis. Other caspase inhibitors, including Z-D(OMe)E(O-
Me)VD(OMe)-fmk, Z-E(OMe)VD(OMe)-fmk, Z-VD(OMe)-
fmk, Boc-D(OMe)-fmk, and Z-VAD(OMe)-fmk, were
obtained from Enzyme System Products (Livermore, CA,
U.S.A.). The Animal Care Committee of CoCensys and
Maxim approved all procedures for in vivo experiments.
There is now a large body of data that show that caspase-
mediated apoptosis accounts for at least part of the cell death
associated with a variety of diseases, such as ischemic stroke
(Schulz et al., 1999; Han et al., 2002), myocardial infarction
(Haunstetter & Izumo, 1998), and neurodegenerative disorders
(e.g., Huntington’s disease, Alzheimer’s disease, and amyo-
trophic lateral sclerosis) (Wellington & Hayden, 2000). More-
over, evidence from several laboratories shows that genetic or
pharmacological inhibition of caspases can reduce or even
prevent the cell death caused by endogenous and exogenous
apoptosis stimuli (Yaoita et al., 1998; Braun et al., 1999;
Cursio et al., 1999; Grobmyer et al., 1999; Schulz et al., 1999;
Lee et al., 2000; Mocanu et al., 2000). Taken together, these
studies suggest that drugs that block apoptosis may be able to
halt or reduce disease progression.
Protease inhibition assays
The ability of MX1013 to inhibit the activity of human
recombinant caspases was determined using
a standard
fluorometric microplate assay (Thornberry, 1994). Briefly,
caspase enzyme (Pharmingen, San Diego, CA, U.S.A.) was
incubated at 371C with 5 mm of the fluorogenic caspase 3
substrate Ac-DEVD-AMC (BioMol, Plymouth Meeting, PA,
U.S.A.) in a buffer containing 10 mm of either HEPES or
PIPES (pH 7.4), with 100 mm NaCl, 10% sucrose, 1 mm
EDTA, 0.1% CHAPS, and 10 mm DTT. At the end of the
incubation period, the amount of cleaved AMC was measured
on a fluorescent microplate reader, using an excitation
wavelength of 355 nm and an emission wavelength of
460 nm. IC50 values were calculated from 12-point inhibition
curves using drug concentrations ranging from 1 ꢁ 10ꢀ5 to
5 ꢁ 10ꢀ11 m. Values for kinactivation were determined as described
(Morrison and Walsh, 1988; Thornberry et al., 1994). The
ability of MX1013 to inhibit the activity of the noncaspase
proteases was conducted with the following proteases (Cal-
biochem) and substrates (Bachem or Molecular Probes):
Cathepsin B and Z-Phe-Arg-AMC (Tchoupe et al., 1991),
In this paper, we describe our search for a peptide-based
caspase inhibitor with good in vitro and in vivo activities and
our discovery of MX1013 as a potent and broad-spectrum
caspase inhibitor containing a dipeptide scaffold and a
fluoromethyl ketone warhead. Despite being 10–100-fold less
potent in caspase enzyme inhibition assays than caspase
inhibitors with tripeptide or tetrapeptide scaffolds, MX1013
has unexpectedly strong activity as a cytoprotectant. MX1013
also is more water-soluble than the commonly used caspase
inhibitor Z-VAD(OMe)-fmk. Previously, we showed that
MX1013 (CV1013) was effective in blocking apoptosis and
death in a rodent model of endotoxemia (Jaeschke et al., 2000).
In the current report, we present data showing that the
dipeptide scaffold of MX1013 provides a good combination of
in vitro and in vivo activities for a caspase inhibitor. We show
that MX1013 has antiapoptotic activity in three cell culture
models of apoptosis, where it prevents the appearance of the
main biochemical markers of apoptosis and blocks cell death,
and that it is efficacious by intravenous (i.v.) administration in
three rodent models of apoptosis: anti-Fas-induced liver
failure, transient focal brain ischemia/reperfusion, and myo-
cardial ischemia (MCI)/reperfusion. These in vitro and in vivo
calpain
cathepsin D and BODIPY FL casein (Molecular Probes),
renin and suc-Arg-Pro-Phe-His-Leu-Leu-Val-Tyr-AMC
(Murakami et al., 1981), Factor Xa and Boc-Ile-Glu-Gly
I and suc-Leu-Tyr-AMC (Meyer et al., 1996),
O
O
N
F
O
N
O
OH
O
Figure 1 Structure of MX1013.
British Journal of Pharmacology vol 140 (2)

404
Wu Yang et al
MX1013, an in vivo active dipeptide caspase inhibitor
Arg-AMC (Morita et al., 1977), and thrombin and Boc-
Asp(OBzl)-Pro-Arg-AMC (Laura et al., 1980). Substrate
cleavage was measured in the presence and absence of drug,
using the fluorogenic substrates and assay conditions, as
described in the references.
and 8 ꢁ 105 cells mlꢀ1. Cells (1 ꢁ 105 in 0.1 ml of media) were
placed into a well of a 96-well plate, and were treated with
doxorubicin (1 mm) at 371C for 18 h. MX1013 (10 mm) in
DMSO was added to the appropriate samples at the time of
compound addition. Control samples were treated with the
same volume of solvent (DMSO). After 18 h, cells were
observed by phase-contrast microscopy to visualize morpho-
logic changes induced by the apoptosis stimuli.
HeLa cell protection assays
HeLa S3 cells (ATCC-American Type Culture Collection,
Manassas, VA, U.S.A.) were grown in minimal essential
medium with 2 mm glutamine and 10% fetal bovine serum. For
cell protection assays, the cells were seeded in multiwell plates
and allowed to grow for 24 h. The cells were then preincubated
with inhibitors for 2 h and incubated with 25 ng mlꢀ1 TNF-a
(Roche Molecular Biochemicals, Indianapolis, IN, U.S.A.)
and 30 mg mlꢀ1 cycloheximide (CHX) (Sigma, St Louise,
MO, U.S.A.) for 18–24 h. All incubations were performed
at 371C in a CO2 incubator. The viability of the cells was
assessed by phase-contrast microscopy, or quantified by
uptake of calcein AM (Molecular Probes, Eugene, OR,
U.S.A.). For the latter assay, the cultures were washed twice
with PBS to remove nonadherent cells, and incubated at room
temperature for 30–45 min with 4 mm calcein AM in serum-free
and phenol red-free culture medium. The amount of calcein
AM fluorescence in each well, which gives a quantitative
measure of the number of remaining cells, was then measured
using an excitation wavelength of 485 nm, and an emission
wavelength of 525 nm using a Wallac fluorescence microplate
reader. Data were expressed as percent control, with
control values taken from cultures incubated with CHX, but
without TNF-a.
Mouse liver apoptosis model
In vivo apoptosis protection assays, using the mouse liver
failure model, were run as described (Rodriguez et al., 1996).
Female ND4 Swiss Webster mice (16.5–21 g, Harlan Spra-
gue–Dawley, San Diego, CA, U.S.A.) were injected i.v. with
8 mg of anti-Fas monoclonal antibody (clone JO-2; Pharmin-
gen), followed 5 min later by i.v. administration of various
doses of MX1013 formulated in an aqueous vehicle containing
50 mm Tris-HCl, pH 8.0. Control animals received anti-Fas
antibody, followed by vehicle. The number of surviving
animals in each treatment group was determined at 1, 3, 24 h
and 5 days postinjection, and was expressed as a percentage of
the total number of animals in each group. To assess the
prevention of liver failure by MX1013 quantitatively, a
separate experiment was run, where serum levels of two liver
enzymes, SGPT (serum glutamic pyruvic transaminase) and
SGOT (serum glutamic oxaloacetic transaminase), were
measured using an Ames Seralyzer.
Rat transient focal brain ischemia model
Male Fischer-344 rats (200–240 g, Harlan Sprague–Dawley,
San Diego, CA, U.S.A.) were anesthetized with halothane and
implanted with two catheters: one through the left femoral
vein to the inferior vena cava for drug administration, and the
other in the left femoral artery for monitoring blood pressure
and blood gasses. In addition, a PhysioTel Transmitter was
implanted in the peritoneal cavity to remotely monitor the
body temperature. Following preparative surgery, a ventral
midline cervical incision was made to expose both common
carotid arteries (CCA). The right CCA was permanently
ligated with 4-O silk suture, and the left CCA was clamped
with an atraumatic aneurysm clip. In order to gain access to
the right middle cerebral artery (MCA), a 1-cm incision was
made between the lateral canthus of the right eye and the
external auditory canal, and the underlying temporalis muscle
was excised and retracted. A 2 mm burr hole was drilled
2–3 mm rostral to the fusion of the zygomatic arch and
the squamosal bone, and the dura was cut and retracted to
expose the MCA proximal to the rhinal fissure. The right
MCA was occluded using a Codman microaneurysm clip
(No. 1), and interruption of blood flow was verified visually
using a dissecting microscope. The incisions were then
closed with surgical clips, anesthesia was discontinued, and
the animals returned to their cages. After 2.5 h, the animals
were reanesthetized, the surgical clips removed, and the
aneurysm clips occluding the right MCA and left CCA
were removed. Restoration of blood flow was confirmed
visually. The incisions were closed and the animals returned
to their cages for 24 h. MX1013, formulated in the aqueous
with 50 mm Tris-HCl, pH 8.0, was tested in two different
dosing regimens: one in which the drug was administered
Western blots
Jurkat cells (ATCC) were seeded in 2 ml of RPMI 1640 media
with 10% FCS in 35 mm dishes, at a density of 0.5 ꢁ 106 –
1 ꢁ 106 per culture. The cells were preincubated at 371C in a
CO2 incubator for 2 h with inhibitor or vehicle (DMSO),
followed by incubation for an additional 4 h with 500 ng mlꢀ1
anti-Fas monoclonal antibody (clone CH1) (PanVera, Madi-
son, WI, U.S.A.). At the end of the incubation period, the cells
were harvested by centrifugation, washed with PBS and lysed
in a buffer containing 50 mm Tris-Cl, pH 7.4, 150 mm NaCl,
1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and a
cocktail of protease inhibitors (Complete Protease Inhibitor
tablets, Roche Molecular Systems). The amount of protein in
each sample was measured by the method of Bradford, using a
BioRad Protein Assay Kit. Samples of lysate containing
equivalent amounts of protein were separated by
SDSFPAGE, and transferred to a PVDF membrane. The
membranes were probed with either a monoclonal anticaspase
3 antibody coupled to HRP (Transduction Technologies) or a
polyclonal antibody to poly(ADP)ribose polymerase (PARP;
Enzyme Systems Products), followed by goat-anti-rabbit HRP
second antibody. Antibody binding was visualized by chemi-
luminescence (Pierce SuperSignal).
Jurkat cell protection assay with doxorubicin as activator
of apoptosis
Jurkat cells were grown in RPMI 1640 media with 10% FCS at
371C in a CO2 incubator maintained at a cell density between 4
British Journal of Pharmacology vol 140 (2)

Wu Yang et al
MX1013, an in vivo active dipeptide caspase inhibitor
405
as a 20 mg kgꢀ1 i.v. bolus 10 min after the onset of ischemia,
followed by a continuous i.v. infusion of 5 mg kgꢀ1 hꢀ1 for
6 h, and the other in which the infusion time was extended
to 12 h. For determination of infarct volumes, animals were
killed by overanesthetization and the brains removed.
Then, 2 mm coronal sections of the right hemispheres
were prepared and stained with 2,3,5-triphenyltetrazolium
chloride (TTC). Infarct areas were measured in each section
using image analysis software, and the infarct volume
calculated by summing the areas in adjacent sections.
Statistical analyses were conducted using Sigmastat software
(Jandel Scientific Software, San Rafael, CA, U.S.A.). Stu-
dent’s t-tests were used for unpaired data, and ANOVA for
multiple comparisons. A P-value of o0.05 was considered
significant.
et al., 2002). We examined the efficacy of different length
peptides in a cell assay where cytoprotective activity against
TNF-a-induced apoptosis of HeLa cells was measured.
Methyl ester forms of the acidic amino acids were used,
since this modification had been proposed to enhance the
cell-permeation properties of peptides with acidic amino
acids. Enzyme-inhibitory activity, though, depends upon
removal of the methyl groups (Rotonda et al., 1996) by
ubiquitous cellular esterases. Using HeLa cells and a TNF-a-
induced apoptosis model, we compared the potencies of
the following four methyl ester peptide inhibitors whose
peptide scaffolds were designed based on the caspase 3
recognition site present in PARP (Nicholson et al., 1995): (1)
Z-D(OMe)E(OMe)VD(OMe)-fmk, a tetrapeptide which con-
tains the P1 –P4 amino acids of the PARP site; (2) Z-
E(OMe)VD(OMe)-fmk, a methyl ester tripeptide compound
which contains the P1 –P3 amino acids; (3) Z-VD(OMe)-fmk, a
dipeptide which contains the P1 and P2 amino acids; and, (4)
Boc-D(OMe)-fmk, a monopeptide which contains only the P1
amino acid.
Rat myocardial infarction model
Male Fischer-344 rats (200–250 g, Harlan Sprague–Dawley,
Indianapolis, IN, U.S.A.) were intubated by insertion of an
endotracheal tube, and ventilated with a rodent respirator.
A left thoracotomy was performed, and the proximal branch
of the left descending coronary artery (LDCA) was exposed,
following procedures described previously (Anversa et al.,
1986). An atraumatic balloon device (prepared with a length of
5 mm silastic tubing connected to prolonged teleflex tubing)
was introduced to allow occlusion and reperfusion of the
LDCA while the animal was conscious. Two rounds of
suture loop were wound loosely around the LDCA to allow
enclosure of both the balloon and the artery. The volume of
balloon inflation necessary for interruption of blood was
determined, and the teleflex tubing was exteriorized through
the back of the animal. Mechanical ventilation was discon-
tinued, and the animals regained spontaneous respiration
within seconds. Using this procedure, MCI was effected for
1 h by inflating the implanted balloon to the predetermined
volume with sterile saline injected through the teleflex
tubing. Reperfusion was achieved by removing the saline and
deflating the implanted balloon. MX1013, formulated in the
aqueous with 50 mm Tris-HCl, pH 8.0, or its vehicle, was
administered as a single i.v. bolus of 20 mg kgꢀ1 upon initiation
of ischemia, followed by continuous infusion at 5 mg kgꢀ1 hꢀ1
for 12 h. Infarcts were assessed 24 h after surgery by TTC
staining. Briefly, the heart tissue was sliced into sections,
stained with TTC, and the extent of ischemic injury deter-
mined for the volume of the infarcted area. The infarct was
computed as the ratio of injured/total volume of the left
ventricle. Data are expressed as percent tissue injury.
Statistical differences between groups were determined using
Student’s t-test.
At a concentration of 50 mm, the methyl ester mono, di, and
tripeptides effectively protected HeLa cells from TNF-a, while
the methyl ester tetrapeptide was almost completely ineffective
(Figure 2), as assessed by the round floating or apoptotic cells.
At a concentration of 5 mm, the methyl ester mono and
tripeptides were only marginally effective, and they were
completely ineffective at 0.5 mm (Figure 2). In contrast, the
methyl ester dipeptide retained substantial cytoprotective
activity at both 5 and 0.5 mm. We also compared the methyl
ester dipeptide to the tripeptide pan-caspase inhibitor Z-
VAD(OMe)-fmk, a compound that is widely used in cell
culture apoptosis experiments and which has been found to be
an effective cytoprotectant in a variety of animal models of
apoptosis (Yaoita et al., 1998; Braun et al., 1999; Cursio et al.,
1999; Mocanu et al., 2000). Z-VAD(OMe)-fmk was an
effective cytoprotectant at 50 mm, but like the methyl
ester tetra, tri, and monopeptides, it had little activity
at 0.5 mm (Figure 3), whereas the dipeptide was effective.
Based on these results, we selected the dipeptide as a pre-
ferred scaffold for optimal caspase inhibition in cells. We
next examined the difference between the free acid and
methyl ester derivative of the dipeptide Z-VD-fmk on
cytoprotection. We found that the IC50’s of both com-
pounds were very similar, approximately 0.1 mm, indicating
that the monocharged Z-VD-fmk has good cell-permeation
properties, compared to tetra- and tripeptide-based inhibitors.
Therefore, we chose the free acid dipeptide Z-VD-fmk
(MX1013) (Figure 1) as a lead compound for further in vitro
and in vivo studies.
Caspase inhibition studies
In order to characterize the basic in vitro properties of
MX1013, we examined its ability to inhibit the activity of
recombinant human caspases, particularly the principal
effector caspase caspase 3. MX1013 inhibited recombinant
human caspase 3 with an IC50 of 30 nm (Figure 4a) and
Results
Z-VD-fmk (MX1013) is a potent cytoprotectant
Previous studies of caspase 1 inhibitors of different peptide
lengths indicated that mono and dipeptides showed equivalent
potencies, whereas a tripeptide had approximately 50 ꢁ
greater potency, which suggested that the longer peptides
with the appropriate recognition sequences were preferred
due to their increased potency (Prasad et al., 1995; Linton
K
inactivation of 18 700 mꢀ1 ꢀ1. Progress curves, run in the absence
S
and presence of compound, were used to determine the nature
of inhibition. In the absence of MX1013, substrate cleavage
increased linearly with time, and the reaction rate increased as
the substrate concentration was shifted from 5 mm (1Km) to
British Journal of Pharmacology vol 140 (2)

406
Wu Yang et al
MX1013, an in vivo active dipeptide caspase inhibitor
0.5 µM
5 µM
50 µM
a
BOC-D(OMe)-fmk
Z-VD(OMe)-fmk
Z-E(OMe)VD(OMe)-fmk
Z-D(OMe)E(OMe)VD(OMe)-fmk
b
DMSO
Control
TNF-α/CHX
Figure 2 Effect of caspase inhibitors containing different peptide lengths on TNF-a/CHX-induced apoptosis of HeLa cells. HeLa
cells were preincubated for 2 h with inhibitors as indicated, followed by treatment for 20 h with 25 ng mlꢀ1 TNF-a and 30 mg mlꢀ1
cycloheximide to induce apoptosis. Cells were photographed using phase-contrast optics at ꢁ 10 magnification. Protected cells can
be seen as a normal adherent monolayer, whereas apoptotic cells can be seen as rounded, phase-bright, floating cells in the medium.
The results shown are representative examples of at least two experiments. (a) Apoptotic cells treated with different concentration of
inhibitors. (b) Control panel including control cells, DMSO-treated control cells, and apoptotic cells without treatment by
inhibitors.
500 mm (100Km) (Figure 4b). By contrast, in the presence of
MX1013, enzyme activity rapidly tailed off, approaching a
plateau starting at about 15 min. Incubation of the enzyme
with an excess (100Km) of substrate prior to addition of
MX1013 protected it from inhibition, indicating that the drug
is competitive for substrate. However, the addition of excess
substrate 15 min after MX1013 addition did not result in an
increase in activity, indicating that MX1013 acts irreversibly.
These findings are consistent with the known characteristics of
peptide-halomethylketone inhibitors, and show that decreas-
ing the size of the peptide scaffold from tetra to dipeptide does
not change the basic characteristics of the inhibitor.
100
75
50
25
0
0
0.5
µM
5
50
0
0.5
µM
5
50
We extended these enzyme-inhibition studies by testing the
ability of MX1013 to block other members of the caspase
family, as well as six noncaspase proteases. MX1013 was
roughly equipotent against caspases 1 (IC50 ¼ 20 nm) and 3
(IC50 ¼ 30 nm) and caspases 6–9 (IC50 ¼ 5–18 nm). In contrast,
the compound was a poor inhibitor of calpain I, cathepsin B,
cathepsin D, and renin (IC50410 mm), as well as thrombin and
Factor Xa (IC504100 mm). These results indicate that MX1013
is a broad-spectrum caspase inhibitor, but not a general
protease inhibitor.
MX1013
Z-VAD(OMe)-
fmk
Figure 3 Comparison of the cytoprotective effects of MX1013 and
Z-VAD(OMe)-fmk. HeLa cells were treated with inhibitors at the
concentrations indicated, followed by TNF-a/cycloheximide, as
described in Figure 2. Plates were washed and processed for calcein
AM assays as described in Methods. The values measured at the
different concentrations of MX1013 or Z-VAD(OMe)-fmk tested in
triplicate were expressed as a percentage of the control cells, which
were not treated with apoptosis inducer. The results are shown as the
mean7s.e.m.
British Journal of Pharmacology vol 140 (2)

Wu Yang et al
MX1013, an in vivo active dipeptide caspase inhibitor
407
processing and PARP cleavage, and these apoptotic events
were readily detectable at lower concentrations (0.25, 0.1, and
0.05 mm) of Z-VAD(OMe)-fmk. An analysis of DNA fragmen-
tation confirmed these results: concentrations of MX1013 as
low as 0.5 mm prevented the formation of DNA ladders
(Figure 5b).
a
100
80
60
40
20
0
MX1013 protects cells from doxorubicin-induced
apoptosis
-11
-10
-9
-8
-7
-6
-5
-4
To determine if MX1013 can protect cells against another
activator of apoptosis, Jurkat cells were treated with doxor-
ubicin in the absence or presence of MX1013. Apoptosis was
monitored for membrane blebbing by microscopy. Membrane
blebbing, a characteristic effect of apoptosis, was blocked by
addition of MX1013 along with doxorubicin (data not shown).
log [MX1013]
(M)
1 X Km, Control
b
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1 X Km with MX1013
100 X Km, Control
100 X Km with MX1013
MX1013 is an effective antiapoptotic agent in vivo
Added substrate at 100 X K
m
To test the cytoprotective activity of MX1013 in vivo, we
utilized three rodent disease models involving apoptosis: anti-
Fas-induced liver failure, transient focal brain ischemia/
reperfusion, and MCI/reperfusion. The liver failure model is
a
straightforward method for determining the in vivo
antiapoptotic efficacy of caspase inhibitors (Rodriguez et al.,
1996), and is based on the fact that hepatocytes express high
levels of Fas, a cell death receptor, which can be activated by
agonistic antibodies. To measure drug efficacy in this model,
the number of surviving animals as a function of time after
challenge with anti-Fas antibody was determined. When mice
were challenged with 8 mg of anti-Fas antibody followed by
vehicle, all the mice died by 3 h time point (Table 1). In
contrast, the lowest dose of MX1013 (0.25 mg kgꢀ1, i.v.
injection) protected 66% (4/6) of the mice from the lethal
effects of anti-Fas antibody at the 3 h time point, and 1 and
10 mg kgꢀ1 dose of MX1013 protected 100% of the mice at the
3 h time point. Moreover, animals that were protected by
MX1013 at the 3 h time point were all alive at 24 h post-
antibody challenge, and were alive after 5 days at which time
the experiment was terminated, indicating that MX1013 truly
protected the mice from the initial injury cause by anti-Fas
antibody. These results are similar to what have been reported
for another irreversible caspase inhibitor (IDN-1965), which at
a dose of 1 mg kgꢀ1 i.p. completely blocked lethality measured
up to 7 days after anti-Fas antibody administration (Hoglen
et al., 2001). These results show that the cytoprotective activity
of MX1013 observed in cell culture studies extends to
apoptosis in vivo, and that MX1013 not only inhibits local
tissue apoptosis, but also can protect animals against its lethal
effects. In separate experiments, the levels of SGOT and SPGT
activity in the serum (markers for liver toxicity) were also
drastically reduced when treated with drug (data not shown),
in accord with the survival data.
0
5
10
15
20
25
30
Time
(min)
Figure 4 Effect of MX1013 on recombinant human caspase 3
activity. (a) A representative dose–response curve of caspase 3
inhibition by MX1013. Each concentration of drug was tested in
triplicate in a standard caspase 3 enzyme fluorometric assay, using
Ac-DEVD-AMC as the substrate. The percent inhibition was
calculated using control samples in which activity was measured in
the absence of drug. Each data point represents the mean, and the
error bars represent the s.e.m.; in some cases the error bars are too
small to be seen. (b) Progress curves from competition experiments
with MX1013. Standard fluorometric caspase 3 enzyme assays were
run in the presence or absence of MX1013 (30 nm), and the activity
was determined as a function of assay time. Substrate was added at
5 mm (1Km) or 500 mm (100Km). In the experiment with 5 mm
substrate, an excess of substrate (500 mm) was added 15 min after
the assay was initiated, in order to determine whether the inhibition
was reversible. The figure shown is a representative example of three
experiments.
MX1013 inhibits three key markers of apoptosis
Apoptosis is characterized by a series of biochemical events,
which ultimately lead to cell death. We tested the ability of
MX1013 to block three of these events: the proteolytic
maturation of caspase 3, the caspase-mediated cleavage of
PARP, and the fragmentation of genomic DNA. During
apoptosis, caspase 3 is proteolytically processed from a single-
chain zymogen to its mature subunits, which consist of a
17 kDa large subunit and a 12 kDa small subunit (Nicholson
et al., 1995). In addition, PARP, one of the key substrates for
caspase 3 (Lazebnik et al., 1994), is cleaved to form 25 and
85 kDa fragments. Both proteolytic events can be monitored
by Western blotting. In anti-Fas-treated Jurkat cells preincu-
bated with 0.5 mm MX1013, neither caspase 3 processing, nor
PARP cleavage, could be detected (Figure 5a). At concentra-
tions of MX1013 as low as 0.05 mm, caspase 3 processing and
PARP cleavage were still markedly reduced. In contrast,
0.5 mm Z-VAD(OMe)-fmk only partially inhibited caspase 3
The rat transient middle cerebral artery occlusion (MCAO)
model has been used to evaluate the efficacy of caspase
inhibitors as anti-stroke agents. This model involves the
transient induction of brain ischemia by MCAO, and an
estimate of the ability of a test agent to reduce the size of the
resulting infarct after reperfusion. We tested MX1013 in this
model in two separate experiments, one in which the drug was
administered as a 20 mg kgꢀ1 i.v. bolus 10 min after the onset
of ischemia, followed by a continuous i.v. infusion of
British Journal of Pharmacology vol 140 (2)

408
Wu Yang et al
MX1013, an in vivo active dipeptide caspase inhibitor
a
anti-Fas
−
anti-Fas
+
0
+
+
+
+
−
+
0
+
+
+
+
Z-VAD(OMe)fmk
(µM)
MX1013
(µM)
0
0.05 0.1 0.25 0.5
0
0.05 0.1 0.25 0.5
Pro
Caspase-3
PARP
LS
FL
Cl
b
anti-Fas
−
+
0
+
+
5
MX1013
(µM)
St
0
0.5
Figure 5 The effect of MX1013 on three biochemical markers of apoptosis. Jurkat T-lymphocytes were treated with MX1013 or
Z-VAD(OMe)-fmk, at the indicated concentrations, followed by an agonistic anti-Fas antibody, to initiate apoptosis. (a) The cells
were processed for Western blotting in order to visualize the processing of caspase 3 (top panels) or PARP (bottom panels). Pro
indicates the migration position of caspase 3 proenzyme, LS the caspase 3 large subunit, FL the full-length PARP, and Cl indicates
the position of cleaved PARP. (b) Samples were processed for agarose gel electrophoresis, and stained with ethidium bromide in
order to visualize DNA laddering. St lane indicates DNA size standards and the other lanes indicate the concentration of MX1013
inhibitor. The results shown are representative examples of at least three experiments.
Table 1 Survival of mice with MX1013 treatment
after induction of apoptosis by anti-Fas antibody
The rat model of acute MCI is a widely used model for
studying the effects of cytoprotective drugs on cardiac
ischemia/reperfusion injury. Similar in principle to the rat
brain ischemia model, it involves interruption of the blood
supply to the heart by occlusion of the LDCA, followed by
restoration of blood flow (reperfusion) and determination of
the size of the resultant infarct. When administered as a single
i.v. bolus of 20 mg kgꢀ1 at the time of ischemia, followed by
continuous infusion at 5 mg kgꢀ1 hꢀ1 for 12 h, MX1013
treatment resulted in a reduction of infarct size (by 52%,
Po0.05) (Figure 7). In comparison, Z-VAD(OMe)-fmk was
reported to be active in the MCI model by i.v. administration
and produced 21% reduction of infarct volume (Yaoita et al.,
1998), which is less efficacious than that of MX1013.
% Survival (n ¼ 6)
Dose of MX1013 (mg kgꢀ1
)
1 h
3 h
24 h
5 days
0
0.25
1
100
100
100
100
0
0
66
100
100
0
66
100
100
66
100
100
10
Mice were injected intravenously (i.v.) with 8 mg of anti-Fas
antibody, followed 5 min later with i.v. injection of MX1013
at the indicated doses or by vehicle. Six mice were treated in
each group, and the number surviving was monitored for up
to 5 days.
Taken together, these results show that MX1013 is an
effective antiapoptotic agent in vivo. It can reduce the tissue
damage and lethality associated with activation of death
receptor pathways, as well as the amount of cellular damage
found in two different models of ischemia/reperfusion injury.
5 mg kgꢀ1 hꢀ1 for 6 h (Figure 6a), and the other in which the
infusion was extended to 12 h (Figure 6b). MX1013 signifi-
cantly reduced cortical infarct size in both cases (by 46 and
57%, respectively; Po0.05). The drug did not cause any
changes in blood pressure, blood gasses, or body temperature
(data not shown). The efficacy of MX1013 administered by i.v.
injection in the MCAO model is comparable with Z-
VAD(OMe)-fmk administered by i.c.v. injection in a MCAO
model, which was reported to reduce infarct volume by
approximately 40–50% (Hara et al., 1997).
Discussion
Our studies of MX1013 show that it is an effective
cytoprotective agent, in several tissue culture models of
apoptosis, in a mouse liver apoptosis model, and in two
British Journal of Pharmacology vol 140 (2)

Wu Yang et al
MX1013, an in vivo active dipeptide caspase inhibitor
409
30
a
200
150
100
50
20
*
*
10
0
VEHICLE
MX1013
* p < 0.05
0
VEHICLE
MX1013
Figure 7 Effects of MX1013 treatment in an acute myocardial
infarction and reperfusion model in rats. Ischemia was effected by
occlusion of the left anterior descending artery of rat hearts for 1 h,
followed by reperfusion for 23 h. Drug was administered i.v. at the
time of occlusion as a bolus (20 mg kgꢀ1), followed by i.v. infusion
(5 mg kgꢀ1 hꢀ1) for 12 h. At the end of the reperfusion, the hearts
were sectioned, stained, and analyzed for infarct volume, as
described in Methods. The average of 12 animals (1073.5) in the
MX1013-treated group and 10 animals (2172.4) in the vehicle
control group is shown as the mean7s.e.m. The statistical
significance value is indicated.
* p < 0.05
b
200
150
100
50
*
poor cytoprotectant, possibly due to the requirement to
remove the methyl esters.
Despite containing a peptide scaffold with only two amino
acids, MX1013 still has reasonable potency in cell-free enzyme
assays, which is probably due to the fact that the compound
retains the essential features of a caspase-recognition sequence
(an aspartic acid residue at the P1 position and a small
hydrophobic residue in the P2 position) and utilizes an
irreversible warhead. The crystal structures of caspases 1, 3,
7, and 8 bound to the peptide aldehyde inhibitor Ac-DEVD-
CHO reveal that the P1 aspartic acid forms a hydrogen bond
network with the side chains of Arg341, Arg179, and Gln283 of
the enzyme (Wei et al., 2000). Moreover, the P1 amide NH
group forms hydrogen bonds with the backbone carbonyl of
Ser339. The crystal structures also show that the P2 valine of
Ac-DEVD-CHO lies against a hydrophobic portion of the
binding pocket, and its backbone carbonyl forms a hydrogen
bond with Arg341. The P1 and P2 amino acids of MX1013
would be expected to participate in all of these interactions.
The methyl ester version of MX1013 was found to be 470-
fold less active than MX1013 in the caspase-3 enzyme assay,
supporting the notion that the carboxylic acid in P1 is critical
for interaction with caspases. It is also likely that the
fluoromethylketone-leaving group of MX1013, which is
known to bind irreversibly to the active site cysteine of
caspases (Mittl et al., 1997), plays a major role in the
compound’s high potency. The reversible aldehyde form of
MX1013 has an IC50 of 16 mm against caspase 3. Thus, the loss
of potency resulting from truncation of the peptide scaffold
can apparently be compensated for by use of an irreversible
warhead, but this truncation does not decrease its specificity
against caspases.
0
VEHICLE
MX1013
* p < 0.05
Figure 6 Effects of MX1013 treatment in the cortical infarct
volume of animals in a transient MCAO. The MCA of rats was
occluded for 2.5 h before release of the occlusion. MX1013 was
administered i.v. with a bolus of 20 mg kgꢀ1 10 min after the onset of
occlusion, followed by an i.v. infusion of 5 mg kgꢀ1 hꢀ1 for 6 h
(Figure 6a, 162726, n ¼ 16 for vehicle, and 86718, n ¼ 15 for
MX1013 treated) or 12 h (Figure 6b, 143718, n ¼ 12 for vehicle, and
62720, n ¼ 10 for MX1013 treated) by the jugular vein. At 24 h after
occlusion, the brain was sectioned and stained with TTC. Sections
were made and the infarct area was determined, and the volume of
the infarct calculated. The average for the MX1013-treated and the
control vehicle-treated group is shown as the mean7standard error
of mean (s.e.m.). The statistical significance value is indicated.
different animal models of ischemia/reperfusion. These results
suggest that, although tetrapeptide-based inhibitors may be
more potent inhibitors of caspases under cell-free conditions,
dipeptide inhibitors have superior in vivo properties. It is likely
that the strong in vivo activity of MX1013 is due to its ability to
permeate the plasma membrane and reach the caspases, which
are intracellular enzymes (Reed, 2002). This enhanced
permeation may be a function of both the size and structure
of MX1013: it contains only two amino acids, only one of
which is charged. In contrast, caspase inhibitors containing
more than one charged or polar amino acid (such as Z-DEVD-
fmk, which has three free carboxy acids) probably do not
readily pass through the plasma membrane. Based on the fact
that charged molecules penetrate cell membranes poorly,
conversion of the free carboxy acids of acidic amino acids to
methyl esters has been used to enhance cell permeation. We
have not observed that the methyl ester version of Z-DEVD-
fmk demonstrates such an enhancement, as it is still a relatively
The P4 amino acid in tetrapeptide-based caspase inhibitors
appears to be a key determinant of specificity between caspases
(Thornberry et al., 1997). Charged or polar residues in this
position are preferred by caspases 3 and 7, while large
hydrophobic residues are preferred by caspases 1 and 4
(Thornberry et al., 1997). In MX1013, the absence of the P4
British Journal of Pharmacology vol 140 (2)

410
Wu Yang et al
MX1013, an in vivo active dipeptide caspase inhibitor
and P3 amino acids would be expected to reduce the selectivity
of the compound, allowing it to inhibit most, if not all,
caspases. Our data, which show an IC50 value in the low to
mid-nanomolar range for inhibition of caspases 1, 3, and 6 to
9, support this prediction. Since apoptosis signal transduction
involves the activation of multiple caspases, it can be argued
that, in order to achieve optimal inhibition of apoptosis, the
ability to inhibit most or all of the caspases is a desirable
feature in a cytoprotective drug. However, it is important that
a pan-caspase inhibitor does not inhibit all proteases. MX1013
is not a general protease inhibitor, as shown by its relatively
low potency as an inhibitor of calpain I, cathepsin B, cathepsin
D, renin, thrombin, and Factor Xa. MX 1013 is not a general
protease inhibitor, as shown by its relatively low potency as an
inhibitor of calpain I, cathepsin B, cathepsin D, renin
thrombin and Factor Xa. The selectivity of MX 1013 for
caspases vs other proteases is probably due to the free
carboxylic acid group of the P1 Asp.
The ability of MX1013 to reduce the size of ischemic infarcts
in the brain and heart is in agreement with the known role that
apoptosis plays in ischemia/reperfusion injury (Gottlieb and
Engler, 1999; Schulz et al., 1999). In both organs, ischemia/
reperfusion triggers the activation of caspases and DNA
fragmentation, two key biochemical markers of apoptosis.
The development of these markers, as well as the resulting
cell death, can be reduced by Z-VAD(OMe)-fmk and the
tetrapeptide caspase inhibitors, as well as a peptidomimetic
caspase inhibitor (Hara et al., 1997; Mocanu et al., 2000;
Deckwerth et al., 2001). It is important to note that with
MX1013 we were able to achieve large, statistically significant
reductions in brain infarct size with i.v. administration,
suggesting that MX1013 can pass the blood–brain barrier.
In contrast, neuroprotection by tripeptide, tetrapeptide in-
hibitors, and a selective caspase 3 inhibitor appears to require
direct injection into the cerebral ventricles (i.c.v.) (Loddick
et al., 1996; Hara et al., 1997; Endres et al., 1998; Han et al.,
2002). In general, these peptide-based inhibitors have low
brain penetration, and they are given by i.c.v. injection to
overcome this limitation (Robertson et al., 2000; Loetscher
et al., 2001). For clinical application, i.c.v. administration is
impractical, and systemic administration such as i.v. is
preferred. However, there are a few caspase inhibitors that
have been reported to be active by the i.v. route in the brain
ischemia model (Deckwerth et al., 2001), and it has been
suggested that clinical studies of caspase inhibitors for cerebral
ischemia have not yet been performed due to the lack of
synthetic caspase inhibitors that cross the blood–brain barrier
(Schulz et al., 1999). Therefore, the ability of MX1013 to
provide neuroprotection by i.v. administration, suggesting that
it has improved brain penetration properties vs the earlier
tetrapeptide and tripeptide based caspase inhibitors, is a
critical advantage for any drug proposed for use in treating
human stroke patients.
Due to its relatively small size and the free carboxylic acid
group, MX1013 is more water soluble than the commonly used
caspase inhibitor Z-VAD(OMe)-fmk. For in vivo studies,
MX1013 can be formulated in a slightly basic pH 8.0 aqueous
solution with 50 mm Tris-HCl, at
a concentration of
410 mg mlꢀ1 for i.v. administration. In comparison, Z-
VAD(OMe)-fmk has low water solubility, and generally has
been formulated in DMSO/water at a concentration of
1 mg mlꢀ1 (Farber et al., 1999; Huang et al., 2000).
Our studies of MX1013 in rodent models of cell death show
that the drug is effective in preventing anti-Fas-induced liver
failure and the ensuing lethality, and also in reducing the size
of brain and myocardial ischemic infarcts. In the mouse anti-
Fas liver apoptosis model, MX1013 reduced the levels of two
biochemical markers of liver damage 3 h after anti-Fas
challenge, and reduced the number of deaths in a dose-
dependent manner up to 5 days after antibody challenge. The
efficacy of MX1013 was comparable to that of Z-VAD(OMe)-
fmk in previously reported studies, although in those experi-
ments Z-VAD(OMe)-fmk was administered in multiple doses
(Rodriguez et al., 1996), while MX1013 was effective when
given as a single dose. The mouse liver damage model has been
widely used to test the in vivo efficacy of caspase inhibitors
(Hoglen et al., 2001), and has been proposed as a model for
human fulminant hepatic failure arising from viral hepatitis
(Kondo et al., 1997). The cytoprotective activity of MX1013 in
this model suggests that it may be useful in humans in the
prevention of acute liver failure mediated by caspase-depen-
dent mechanisms.
Based on these studies, it appears that the dipeptide
structure of MX1013 provides a good combination of in vitro
and in vivo activities for a caspase inhibitor, which are useful
for the design of novel caspase inhibitors. MX1013 is effective
in inhibiting the deleterious effects of apoptosis in a number of
animal models, and is a leading candidate for additional
preclinical studies.
We would like to thank Professor John Keana and Dr Eckard Weber
for contributory discussions. We thank Lisa D’Arcy for her excellent
technical assistance.
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(Received March 31, 2003
Revised May 29, 2003
Accepted July 14, 2003)
British Journal of Pharmacology vol 140 (2)