Amyotrophic Lateral Sclerosis.

2009; 10: 85
94

ORIGINAL ARTICLE

Combined riluzole and sodium phenylbutyrate therapy

in transgenic amyotrophic lateral sclerosis mice

STEVEN J. DEL SIGNORE1, DANIEL J. AMANTE1, JINHO KIM1,4,

EDWARD C. STACK1,4, SARAH GOODRICH1, KERRY CORMIER4, KAREN SMITH1,4,
MERIT E. CUDKOWICZ5 & ROBERT J. FERRANTE1
4

Departments of 1Neurology, 2Laboratory Medicine and Pathology, 3Psychiatry, Boston University School of Medicine,
Boston, 4Geriatric Research Education Clinical Center, New England Veterans Administration, Bedford, and 5Neurology
Clinical Trials Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA

Abstract
Recent evidence suggests that transcriptional dysregulation may play a role in the pathogenesis of amyotrophic lateral
sclerosis (ALS). The histone deacetylase inhibitor, sodium phenylbutyrate (NaPB), is neuroprotective and corrects aberrant
gene transcription in ALS mice and has recently been shown to be safe and tolerable in ALS patients while improving
hypoacetylation. Since many patients are already on riluzole, it is important to ensure that any proposed therapy does not
result in negative synergy with riluzole. The combined treatment of riluzole and NaPB significantly extended survival and
improved both the clinical and neuropathological phenotypes in G93A transgenic ALS mice beyond either agent alone.
Combination therapy increased survival by 21.5%, compared to the separate administration of riluzole (7.5%) and NaPB
(12.8%), while improving both body weight loss and grip strength. The data show that the combined treatment was
synergistic. In addition, riluzole/NaPB treatment ameliorated gross lumbar and ventral horn atrophy, attenuated lumbar
ventral horn neuronal cell death, and decreased reactive astrogliosis. Riluzole/NaPB administration increased acetylation at
H4 and increased NF-kB p50 translocation to the nucleus in G93A mice, consistent with a therapeutic effect. These data
suggest that NaPB may not interfere with the pharmacologic action of riluzole in ALS patients.

Key words: Amyotrophic lateral sclerosis, riluzole, histone deacetylase inhibition, sodium phenylbutyrate, therapy

Introduction those observed in human ALS (4). Genetic mouse

Amyotrophic lateral sclerosis (ALS) is a rapidly
developing disease characterized by degeneration of
upper and lower motor neurons, progressive muscle
weakness and atrophy, and morbidity associated
with failure of respiratory muscles (1). The inci-
dence of ALS is 1
2 per 100,000 per year and may
be rising. Death usually occurs within 2
5 years of
diagnosis. Although the root cause of disease is
unclear, genetic mutations have been discovered to
underlie 10% of cases, termed familial ALS (FALS).
Mutations in the enzyme Cu/Zn superoxide dismu-
tase (SOD1) cause about 25% of FALS cases and
their clinical and pathological features are indistin-
guishable from those in sporadic ALS (2,3). Over-
expression of mutant SOD1 in animal models is
sufficient to cause motor neuron degeneration re-
sulting in clinical and pathological features similar to
models of ALS have provided a greater understand-
ing of disease pathogenesis and have helped to
develop potential therapeutic strategies for human
clinical trials (5,6). Glutamate mediated excitotoxi-
city, transcriptional dysregulation, oxidative stress,
and bioenergetic dysfunction have all been proposed
as pathophysiological mechanisms of disease in ALS,
and each has been the focus of successful preclinical
trials in animal models with mutations in the SOD1
gene (7
10). Pharmacotherapies that can address
multiple disease pathways may provide a promising
route to slowing disease progression.
Since the first conceptualization of glutamate
excitotoxicity (11), there has been strong evidence to
support its importance in the pathology of ALS (12).
The only drug currently approved to treat ALS is
riluzole, a drug with anti-excitotoxic properties
(13,14). The neuroprotective effect of riluzole may

Correspondence: R. J. Ferrante, GRECC Unit 182B, Bedford VA Medical Center, 200 Springs Road, Bedford, MA 01730, USA. Fax: 781 687 3515.
E-mail: rjferr@bu.edu

(Received 28 March 2008; accepted 20 May 2008)

ISSN 1748-2968 print/ISSN 1471-180X online # 2009 Informa UK Ltd. (Informa Healthcare, Taylor & Francis AS)
DOI: 10.1080/17482960802226148
86 S. J. Del Signore et al.
stem from a number of pharmacological actions
(15). Riluzole has been shown to inhibit glutamate
release, possibly through inhibition of presynaptic
calcium and sodium channels (16,17) and to mod-
ulate depolarizing currents at alpha-amino-3-
hydroxy-5-methyl-4-isoxazolepropionic acid and
N-methyl-D-aspartic acid receptors (18,19). While
several experiments in animal models have replicated
the therapeutic effect observed with riluzole in ALS
patients (7,20), there have also been a number of
ALS rodent trials demonstrating an enhanced ther-
apeutic effect by combining one or more other drugs
with riluzole (21
23). In addition, a clinical trial
investigating the combination of riluzole and high
doses of vitamin E has been reported (24).
In addition to excitotoxicity, transcriptional dys-
regulation has also been shown to play a role in the
neurodegeneration observed in ALS (25
28). Tran-
scription is regulated by complex interactions be-
tween many proteins. Among the most important of
these proteins are transcription factors and histones,
which affect the actions of RNA-Polymerase II on
individual genes. A well-recognized mechanism of
transcriptional regulation is histone modification.
Acetylation of lysine 9 and 13 at histone H4 is
associated with an enhanced transcriptional state.
Histone deacetylase (HDAC) inhibitors increase
acetylation of histones, promoting transcriptional
activation. Of the five classes of HDAC inhibitors,
the butyrates are the most developed for clinical use
because they are able to penetrate the blood-brain-
barrier (29
31). Sodium phenylbutyrate (NaPB) has
been shown to increase acetylation at H4, and
improve the clinical, neuropathological, and mole-
cular phenotype in the G93A mouse model of ALS
(8). In addition, we recently reported that NaPB was
safe and tolerable in ALS patients and was ther-
apeutically effective in improving hypoacetylation
levels found in this patient population (32).
Given the multifactorial nature of disease patho-
genesis of ALS, combination therapies that address
multiple disease pathways hold great promise for
achieving maximal therapeutic effect. As riluzole is
the only FDA-approved drug currently available to
patients, it is especially important to develop com-
binatorial treatments that are safe and effective, that
do not negatively interfere with riluzole. We investi-
gated the effects of the combined administration of
riluzole and NaPB on the pathological phenotype
observed in the G93A mouse model of ALS.
Materials and methods
Experimental animals
Transgenic mice overexpressing human SOD1 with
the G93A mutation (TgSOD1 (G93AGUR) F1/J,
Jackson Labs, Bar Harbor, ME) were bred with
females of similar background (B6/SJL) from an
established colony maintained at the Bedford Veter-
ans Affairs Medical Center and used for the experi-
ments. PCR assay of tail DNA was used to genotype
the offspring. Animals in each experiment were from
the same ‘f’ generation, born within four days of one
another. Only male mice were used in the treatment
studies because we have observed gender differences
in survival in the G93A transgenic ALS mouse
model, with extended survival and greater variability
in time of death in female G93A mice (33). All
animal experiments were performed in accordance
with the NIH Guide for the Care and Use of
Laboratory Animals and were approved by the
Veterans Administration Animal Care Committee.
Treatment
Animals were randomly distributed into one of four
treatment groups (n 10) at four weeks of age:
untreated, NaPB-treated (300 mg/kg), riluzole-
treated (16 mg/kg), or riluzole/NaPB-treated using
the above doses. NaPB (Triple Crown USA, Inc.,
Perkasie, PA) was dissolved in physiological saline at
a concentration of 75 mg/ml. Fresh solutions were
made daily and administered via intraperitoneal
injection. Riluzole was obtained as RilutekTM, 50-
mg tablets and dissolved in drinking water at a
concentration of 100 ug/ml. Solution was made fresh
weekly and stored at 48C. Fresh solution was
provided to the animals twice per week. Treatments
were started at 30 days. The untreated G93A mice
were injected with saline (vehicle). Separate groups
(n 10) of treated and untreated mice were used for
histological analyses.
Survival
Animals were assessed twice daily (early morning
and late afternoon) for morbidity and mortality by
two independent observers who were blinded to the
assigned treatment (RJF and KS). Motor perfor-
mance and ability to feed was closely monitored and
used to determine when to euthanize the mice.
Animals were euthanized when they were unable to
right themselves after being placed on their back for
30 s. Some deaths occurred overnight and were
recorded the following morning. Body weights
were recorded weekly on the same day and time.
Behavior analysis
Neuromuscular function was assessed using the Grip
Strength apparatus (Columbus Instruments, Co-
lumbus, OH). This instrument measures the peak
amount of force an animal applies in grasping
specially designed pull-bar forelimb and hindlimb
assemblies. Briefly, mice were held by the scruff of
their back and drawn along in a straight line along
the sensor assemblies to allow the mice to grasp the
 Combined riluzole and sodium phenylbutyrate therapy 87

forelimb pull-bar assembly, recording the force
applied by the forelimbs of the mouse in Newtons.
In a continued sweep, the mouse was drawn along
until the hindlimbs grasped and released the hin-
dlimb assembly, again recording the force applied by
the hindlimbs of the mouse. Forelimb and hindlimb
strength were measured by the same investigator
(SJD) three times and averaged during each weekly
assessment. It is critically vital that a single investi-
gator performs the grip strength measurements due
to inter-rater differences in tensile strength.
Histology
Additional groups of 10 animals from each
treatment paradigm were deeply anesthetized and
transcardially perfused with 4% buffered parafor-
maldehyde at 120 days of age. Forty mice were used
for neuropathological analysis. Spinal columns were
removed and placed in fixative overnight. Cords
were dissected and cryoprotected in 10%, then 20%,
glycerol in DMSO/TBS-T. The lumbar section of
each cord was then blocked and cut on a freezing
sledge microtome at 50 mm. Sections were stored in
phosphate buffer with 0.01% sodium azide at 48C
and subsequently stained for Nissl substance, and
immunostained for glial fibrillary acidic protein
(GFAP) (1:100; Boehringer-Mannheim, Indianapo-
lis, IN), acetylated histone H4 (rabbit polyclonal
antibody, 1: 1000; Upstate Biotechnology, Lake
Placid, NY, USA), and nuclear factor-kappa B
(NF-kB) p50 (rabbit polyclonal antibody, 1: 500;
Cell Signaling Technology, Inc.).
Neuronal quantitation
Serial lumbar spinal cord tissue sections (n 20)
from L3
L5 spinal cord segments were used for
gross spinal cord areas and neuronal analysis. Gross
areas of the spinal cord sections were quantified
from each experimental cohort using NIH Image.
From the same sections, the ventral horn was
delineated by a line from the central canal laterally
and circumscribing the belly of gray matter. Abso-
lute neuronal counts of Nissl-positive neurons were
performed in the ventral horns in the lumbar spinal
cord. Only those neurons with nuclei were counted.
All counts were performed with the experimenter
(SJD) blinded to treatment conditions. Counts were
performed using Image J (NIH) and manually

Figure 1. Effects of riluzole and NaPB on survival, body weight, and grip strength in G93A transgenic ALS mice.
Untreated G93A mice have a median survival of 126 days (A). Treatment with either riluzole (18 mg/kg) or NaPB (300 mg/kg) resulted in a
significant survival extension of 7.5% and 12.8%, respectively. Combined riluzole/NaPB treatment resulted in the greatest survival
extension, 21.5% over untreated mice. There is a significant improvement in body weight in all treated groups, compared to untreated
control mice (B). Mice treated with combined riluzole and NaPB show the greatest improvement, with body weight significantly elevated
above all other groups. G93A mice treated with combined riluzole and NaPB resulted in a marked improvement in forelimb grip strength
compared to untreated mice (C). Starting at 91 days of age the combined therapy resulted in improved grip strength that persisted
throughout testing. In contrast, an analysis of hind limb grip strength revealed no improvement in combined riluzole and NaPB treated
G93A mice compared to untreated mice (D).
88 S. J. Del Signore et al.

verified and the data represent the average neuronal improvement in combined riluzole/NaPB treated
number from the sections used. G93A mice, compared to untreated mice (Figure
1D). It should be noted that a differential paralysis
occurs early and most severely in the hindlimbs, with
Statistical analysis less and later involvement in the forelimbs. This
Data are expressed as the mean9SEM. Statistical early and more pronounced involvement of the
comparisons of grip strength, weight data and hindlimbs may be too great for riluzole-NaPB
histological data were made by ANOVA or re- therapeutic treatment to overcome.
peated-measures ANOVA. Survival data were ana- Neuropathological analysis at 120 days revealed
lyzed using Kaplan-Meier survival curves. All other marked gross spinal cord atrophy, with increased
statistical analyses were performed using Student’s astrogliosis and neuronal loss in the ventral horns
t-test. from the lumbar spinal cord in untreated G93A
mice. These pathological findings were significantly
reduced by riluzole/NaPB administration. There
Results was significant gross atrophy of the lumbar spinal
cord in untreated G93A mice, compared to wt
Both riluzole (16 mg/kg) and NaPB (300 mg/kg) littermate control mice (wt littermate control:
treatment alone significantly extended survival in the 3.0890.18 106 mm3; untreated G93A mice:
G93A ALS mice by 7.5% and 12.8%, respectively, 2.3190.95 106 mm3, p B0.0001) with significant
compared to untreated G93A mice (Figure 1A). amelioration of gross spinal cord atrophy in riluzole/
These data are consistent with previously reported NaPB treated mice (riluzole/NaPB treated G93A
findings. The combined administration of riluzole/ mice: 2.6790.43 106 mm3, p B0.01) (Figure 2A
NaPB also significantly extended survival in the and 3A
C). The gross atrophy was largely asso-
G93A mice by 21.5%, suggesting that the efficacy ciated with the ventral horn of the lumbar spinal
was synergistic without having a negative impact on cord (wt littermate control: 7.2190.45 105;
survival, compared to drug treatments alone (un-
treated G93A mice: 126.192.7 days; riluzole 16 mg/
kg per day: 135.594.4 days, p B0.05; NaPB 300
mg/kg per day: 142.295.4 days, p B0.01; riluzole/
NaPB 16 mg/kg and 300 mg/kg, respectively, per
day: 153.299.1 days, p B0.001).
There was a significant improvement in body
weight in all the treatment groups, compared to
untreated control mice (p B0.01) (Figure 1B). The
riluzole treatment arm significantly ameliorated
body weight loss, compared to untreated controls,
starting at 115 days (p B0.05). The NaPB treatment
improved body weight starting at 101 days and
continued through the end of the trial (p B0.01).
G93A mice treated with combined riluzole and
NaPB showed the greatest improvement, with body
weight significantly elevated above the untreated
G93A mice at 91 days (p B0.01) and above the
other treatment groups starting at 115 days, remain-
ing consistent through the completion of the trial
(p B0.01). Forelimb and hindlimb grip strength
were assessed as a measure of neuromuscular
performance and motor function. Others and we
have previously reported that motor performance is
improved by riluzole or NaPB treatment alone (7,8).
As such, it is of import to demonstrate that G93A
mice treated with combined riluzole and NaPB also Figure 2. Combined riluzole and NaPB treatment improves gross
area of lumbar spinal cord and ventral horn areas in G93A
improve this outcome measure. There was a marked transgenic ALS mice.
improvement in forelimb grip strength in riluzole/ Analysis of gross lumbar spinal cord area reveals a marked
NaPB treated G93A mice, compared to untreated decrease in untreated G93A mice compared to wild-type mice.
littermate G93A mice. At 91 days of age, the Treatment with riluzole and NaPB results in a significant
combined therapy resulted in significantly improved improvement in lumbar spinal cord area (A). A similar analysis
of the ventral aspect of the lumbar spinal cord revealed a marked
grip strength (p B0.01) that persisted throughout reduction in untreated G93A mice relative to wild-type control
the trial (Figure 1C). In contrast, however, an mice (B). Treatment with riluzole and NaPB significantly
analysis of hindlimb grip strength revealed no improved ventral horn area in G93A ALS mice.
 Combined riluzole and sodium phenylbutyrate therapy 89

Figure 3. Neuroprotective effects of combined riluzole and NaPB in the lumbar spinal cord in G93A transgenic ALS mice. Nissl staining of
the lumbar spinal cord from (A) wild-type mice, (B) G93A mice treated with riluzole and NaPB, and (C) untreated G93A mice shows a
marked gross atrophy in untreated G93A mice. Combined treatment with riluzole and NaPB significantly improves the gross
neuropathological changes, as shown in Figure 2. Bar in A 200 mm and is the same for B and C. High magnification photomicrographs
of Nissl staining in the ventral horn from the same sections in A, B, and C of the lumbar spinal cord from (D) wild-type mice, (E) G93A
mice treated with riluzole and NaPB, and (F) untreated G93A mice demonstrates ventral neuron loss and atrophy in untreated G93A mice.
The combined treatment with riluzole and NaPB markedly improves these neuropathological changes. Bar in D50 mm and is the same for
E and F. Glial fibrillary acidic protein (GFAP) expression within the lumbar spinal cord in wild-type mice reveals low levels of reactive
astrogliosis (G). In contrast, there is marked astrogliosis, as indicated by GFAP immunostaining, in the lumbar spinal cord from untreated
G93A mice (I). Treatment with both riluzole and NaPB markedly attenuates reactive astrogliosis (H). Bar in G 50 mm and is the same in
H and I.
90 S. J. Del Signore et al.
untreated G93A mice: 5.7190.61 105, p B0.05
mm3) and was significantly improved by riluzole/
NaPB treatment (riluzole/NaPB treated G93A mice:
6.5990.37 105 mm3, p B0.01) (Figure 2B). Con-
sistent with the above findings, there was marked
ventral horn neuronal loss in the untreated G93A
mice, compared to the wt littermate control mice
(wt littermate control: 93.5914.5; untreated G93A
mice: 20.495.60, p B0.0001) (Figure 3D, F), with
significant reduction in neuronal loss as a conse-
quence of riluzole/NaPB administration (riluzole/
NaPB-treated G93A mice: 68.4917.0, p B0.01)
(Figure 3E). Reactive astrogliosis, a prominent
finding in untreated G93A mice spinal cord speci-
mens, was ameliorated by riluzole/NaPB treatment,
as shown by the marked reduction of glial fibrillary
acidic protein expression in the lumbar spinal cord
(Figure 3G
I).
We have previously reported that hypoacetylation
of histone activity is present in both G93A mice and
ALS patients and that histone acetylation is im-
proved by NaPB treatment (8,32). These findings
demonstrate a parallelism in pathophysiological
disease mechanism between mouse and human.
We confirmed the hypoacetylation of histone H4 in
lumbar spinal cord of untreated G93A mice, com-
pared to wt littermates, using immunocytochemical
analysis in tissue specimens and showed that rilu-
zole/NaPB treatment in G93A mice significantly
improves the hypoacetylation of H4 (Figure 4A

D). It has also been reported that NF-kB activation
through phosphorylation of IkB or directly via
butyrate administration increases NF-kB transloca-
tion to the nucleus, improving transcription and
inhibiting apoptosis (8,34,35). Immunoreactivity of
NF-kB p50 was significantly reduced in the spinal
cords of untreated G93A mice, when compared to
wt littermates (Figure 4 E
H). Riluzole/NaPB treat-
ment significantly increased the spinal cord tissue
expression of NF-kB in ventral horn neurons from
G93A mice (Figure 4G), resulting in nuclear trans-
location of NF-kB activity, consistent with that
observed in littermate control mice. It is of interest
to note that among members of the NF-kB family in
mammals, the p50 subunit has been shown to be
neuroprotective (36).
Discussion
A clear understanding of the molecular events
leading to motor neuron death in ALS remains to
be elucidated. While a number of pathways have
been incriminated, both glutamatergic excitotoxicity
and transcriptional dysregulation have been sug-
gested to play roles in the insidious neuronal death
observed in ALS (8,12,32). To date, riluzole is the
only FDA-approved ALS drug therapy that slows or
abrogates the disease process in ALS and is asso-
ciated with a 2
3 month prolongation of survival
(13,14). Riluzole acts upon a number of physiologi-
cal pathways that include the inhibition of both
glutamate release and post-synaptic glutamate re-
ceptor activation, as well as by inactivating voltage
sensitive sodium channels (15). It has also been
shown to have neurotrophic and anti-apoptotic
effects, and reduces mitochondrial swelling. Riluzole
is well-tolerated, although liver function monitoring
is necessary especially during the first year of
therapy. Since many ALS patients may well be
placed on riluzole, it is critically vital to make certain
that any prospective therapeutic agent does not
negatively impact the patient or the progression of
the disease.
An important advance in developing drug com-
pounds for clinical trials in ALS patients has been
the introduction of transgenic mice models of ALS.
Of the available ALS murine models, mice expres-
sing mutant forms of SOD1, particularly the G93A
transgenic mice, have been the most widely used in
studying pathophysiological pathways and in devel-
oping candidate ALS therapies (5,6). There is a
strong body of evidence suggesting that the pheno-
types from mouse models of neurological diseases
closely correlate with human diseases and may
validate known CNS drug targets in a therapeutically
relevant manner. The strengths of the ALS mouse
models are in their utility to provide parallel patho-
physiological targets that are present in ALS pa-
tients, in their potential as sensitive predictors for
therapeutic intervention, and their promise in the
development of novel drug agents (5). Although
drug trials in mice confirm therapeutic direction, the
challenge is in determining what dose might be of
value in patients since the pharmacokinetics of mice
and man is dissimilar. It is noteworthy that riluzole,
which extends life in humans with ALS, has a similar
survival benefit in ALS mice. While the predictive
value of therapeutic trials in the G93A ALS mice has
been established using riluzole, there are drug
therapies that are efficacious in ALS mouse models
that do not provide benefit to patients with the
disease (37). In contrast, several potential therapies
that have not shown efficacy in mouse models
have also been ineffective in human trials (6,38).
The latter scenarios may be the consequence of
insufficient fully-powered clinical trials in humans to
permit a comparison of therapeutic efficacy between
mouse and man. Alternatively, optimal therapeutic
dosing may be underestimated. Current thought
indicates that Human Equivalent Dose extrapolation
measurements derived from body surface area cri-
teria in animals may not accurately predict the
maximum-recommended safe dose in neurological
disorders. This is evident in human trials where
human equivalent dosing of bioenergetic agents
comparable to that given to mice, while considered
safe and tolerable and also effective in mice, have not
demonstrated significant efficacy in ALS patients
 Combined riluzole and sodium phenylbutyrate therapy 91

Figure 4. Effects of combined riluzole and NaPB treatment on acetylated H4 and NF-kB immunoreactivity in G93A mice. Compared to
wild-type mice (A), there is a significant hypoacetylation in the untreated G93A mice (B) in the ventral horn of the lumbar spinal cord,
consistent with reduced transcription. Combined riluzole and NaPB induced a marked increase in acetylated H4 (C). Quantitative analysis
confirms the H4 findings (D). There is also a marked reduction in NF-kB expression in the lumbar spinal cord in untreated G93A mice
with little or no nuclear expression (F), compared to wild-type control mice (E). Combined riluzole and NaPB treatment results in a
notable increase in NF-kB expression in G93A mice, with nuclear translocation (arrows). Quantitative analysis confirms the NF-kB
findings (H). Bar in A 50 mm and is the same for B and C. Bar in D25 mm and is the same for E and F.
92 S. J. Del Signore et al.
(39) and in Huntington’s disease patients (40).
Higher doses of potential therapeutic agents may
result in slowing disease progression in ALS. As
evidence, a double-blind, randomized, controlled
study trial in Parkinson’s disease patients, using
CoQ10 at 1200 mg/day, slowed the rate of deteriora-
tion in the UPDRS score, with higher tolerated
doses (41). Of great interest, doses up to 3000 mg/
day appear safe and well tolerated in ALS patients
(42).
Sodium phenyl butyrate (NaPB), a histone
deacetylase (HDAC) inhibitor, is a potential candi-
date for use in ALS patients (5,8). Histones are
important nuclear proteins that bind DNA in the
formation of nucleosomes and control transcription.
Chromatin remodeling and transcription is regulated
in part by histone acetylation, which is promoted by
the opposing activities of histone acetyltransferases
and HDACs. HDAC inhibitors affect histones as
well as transcription factors that are influenced by
acetylation. As altered nucleosome dynamics may
play a role in the pathogenesis of ALS, the recruit-
ment of HDACs to DNA alters the nucleosome
structure locally and inhibits transcription. HDAC
inhibitors can promote either transcription activa-
tion or suppression by relaxing DNA conformations.
Because HDAC inhibitors induce growth arrest in
cell proliferation models, HDAC inhibitors have also
been used as anti-cancer drugs (31). While other
compounds, such as trichostatin A and suberoylani-
lide hydroxamic acid, have been studied, the buty-
rates have been the best clinically studied
compounds and are known to readily reach the brain
(43).
HDAC inhibition has been shown to be neuro-
protective in mouse models of spinal muscular
atrophy and Huntington’s disease (44,45), and is
under investigation in these patient populations. We
have reported that NaPB dramatically improves the
behavioral, neuropathological, and molecular phe-
notype in G93A ALS mice, extending survival by
approximately 22% beyond untreated G93A mice
and restoring histone acetylation to near normal
levels (8). Of relevance is the finding that NaPB,
either directly or post-transcriptionally, corrected
gene transcription associated with apoptosis and
mitochondrial dysfunction in these mice, two addi-
tional pathophysiological mechanisms thought to
play a role in neuronal death in ALS. These findings
provided the rationale for a safety and tolerability
trial using NaPB in ALS patients. The results of this
work showed that NaPB was safe and tolerable (32).
Interestingly, a pharmacodynamic analysis showed
that histone acetylation was decreased by approxi-
mately 50% in blood buffy-coat specimens at screen-
ing from enrolled patients and was significantly
increased after four weeks of NaPB administration
and throughout the remainder of the 20-week trial.
These findings parallel those observed in the ALS
mice. Hypoacetylation, as a potential biomarker of
manifest disease in ALS, may improve the power and
cost-effectiveness of drug trials in this population.
The current findings show that combined rilu-
zole and NaPB treatment in G93A mice did not
adversely affect the impact of treatment and out-
come measures, compared to the efficacy of riluzole
and NaPB alone. Interestingly, the riluzole/NaPB
combination slowed the course of the disease,
evidenced by the altered gradual slope in the
Kaplan-Meier survival curve, compared to the un-
treated G93A mice. There was an additive improve-
ment in both survival and body weight, to a greater
degree than either compound alone, with a con-
comitant improvement in forelimb grip strength in
G93A mice receiving combined riluzole and NaPB.
Riluzole/NaPB also significantly ameliorated the
neuropathological sequela observed in the G93A
mice. There was reduced gross spinal cord atrophy
and ventral horn loss, attenuated ventral horn
neuronal death, markedly diminished reactive astro-
gliosis, and improved both histone H4 acetylation
and NF-kB expression in the lumbar spinal cord
from G93A mice.
It must be said, however, that the treatment was
started prior to manifest clinical symptoms in the
G93A mice and, as such, NaPB may not provide
efficacy if administered after diagnosis in patients
with sporadic ALS. While the G93A mice may not
be a perfect model of sporadic disease, starting
treatment in these mice with only a few weeks
remaining until their death makes any positive
treatment outcome very unlikely. In defense of the
present treatment paradigm, the disease begins very
early in life in G93A mice, with motor unit number
decline from 40 days, N-acetyl aspartate levels
decreased by 34 days, and mitochondrial swelling
and cytoplasmic microvacuoles occurring by two
weeks of age. It has been suggested that the clinical
and pathological features of familial ALS patients
are indistinguishable from those in sporadic ALS
(2,3). As such, pathophysiological mechanisms may
begin well before clinical symptoms, and the efficacy
in the G93A mice in this study may be proof of
concept. As stated above, NaPB administration in
ALS patients improved molecular pathological as-
pects of the disease (32).
ALS remains an untreatable and uniformly lethal
disease. HDAC inhibitors target multiple pathways
important in ALS pathogenesis. Our data strengthen
the hypothesis that transcription dysfunction may
play a role in the pathogenesis of ALS, and suggest
that therapies aimed at improving histone acetyla-
tion and transcription may provide a novel treatment
strategy that translates to clinical benefits in patients
with ALS. Most importantly, the combined treat-
ment using riluzole and NaPB shows no negative
impact, compared to either agent alone. NaPB has
been used clinically to treat patients with a number
 Combined riluzole and sodium phenylbutyrate therapy
of medical conditions and its pharmacokinetics and
side-effects are well defined in humans (46,47).
Thus, NaPB remains as a compelling candidate for
testing in humans with ALS.
Acknowledgements
This work was supported by a Merit Review Award
from the Veterans Administration (RJF and MEC).
Disclosure of interests: The authors have had no
involvements that might raise the question of bias in
the work reported or in the conclusions, implications
or opinions stated.
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