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230 Recent Patents on CNS Drug Discovery, 2012, 7, 230-235

Immunocal® and Preservation of Glutathione as a Novel Neuroprotective

Strategy for Degenerative Disorders of the Nervous System

Erika K. Ross1, Josie J. Gray1, Aimee N. Winter1 and Daniel A. Linseman1,2,*

1
Department of Biological Sciences and Eleanor Roosevelt Institute, University of Denver, Denver, Colorado, USA;
2
Research Service, Veterans Affairs Medical Center, Denver, Colorado, USA

Received: February 28, 2012; Revised: May 23, 2012; Accepted: June 6, 2012

Abstract: Oxidative stress and glutathione (GSH) depletion are both recognized as significant contributors to the patho-
genesis of many devastating neurodegenerative diseases. In particular, mitochondrial dysfunction leads to the aberrant
production and accumulation of reactive oxygen species (ROS), which are capable of oxidizing key cellular proteins,
lipids, and DNA, ultimately triggering cell death. In addition to other roles that it plays in the cell, GSH functions as a
critical scavenger of these ROS. Therefore, GSH depletion exacerbates cell damage due to free radical generation. Strate-
gies that increase or preserve the levels of intracellular GSH have been shown to act in a neuroprotective manner, suggest-
ing that augmentation of the available GSH pool may be a promising therapeutic target for neurodegeneration. This
review discusses the capacity of a cystine-rich, whey protein supplement (Immunocal®) to enhance the de novo synthesis
of GSH in neurons, and highlights its potential as a novel therapeutic approach to mitigate the oxidative damage that
underlies the pathogenesis of various neurodegenerative diseases. Additionally, this review discusses various patents from
1993 to 2012 both with Immunocal® and other methods that modulate GSH in neurodegeneration.
Keywords: Antioxidant, cysteine, cystine, glutathione, Immunocal®, neuroprotection, neurodegeneration, oxidative stress,
whey protein.

OXIDATIVE STRESS AND NEURODEGENERATIVE
DISEASE
Oxidative stress due to aberrant ROS generation is a key
element involved in the pathogenesis of many debilitating
neurodegenerative diseases. These disorders include Alz-
heimer’s disease (AD), Parkinson’s disease (PD), Hunting-
ton’s disease, and amyotrophic lateral sclerosis (ALS), amongst
others [1]. Reactive oxygen species are generated in substan-
tial quantities during normal cell metabolism and in particu-
lar, at the level of mitochondria as a byproduct of electron
transport and ATP production. ROS play some essential
roles in cell signaling; however, when these free radicals are
produced in excess of antioxidant defense mechanisms, cell
damage and death will follow [2, 3]. The brain is especially
vulnerable to oxidative damage because of its high lipid con-
tent and high rate of oxygen consumption. Thus, region-
specific oxidative stress and the associated death of particu-
lar neuronal populations underlies the pathogenesis of each
of the above mentioned neurodegenerative diseases [4].
There is mounting evidence suggesting that deficiencies
in the electron transport chain (ETC) and subsequent oxida-
tive stress at the level of the mitochondria are integral factors
in a variety of neurodegenerative diseases [5, 6]. ETC defi-
cits lead to decreased ATP production and increases in ROS
[7]. More specifically, oxidative damage within the
*Address correspondence to this author at the University of Denver, De-
partment of Biological Sciences, 2199 S. University Blvd., Denver, CO,
80208; Tel: (303)-871-5654; Fax: (303)-871-3471:
E-mail: daniel.linseman@du.edu
mitochondria may target complex I of the ETC and further
increase ROS production, as is observed in PD [8]. Patho-
physiologically relevant complex I inhibitors such as rote-
none and N-methylpyridinium ion (MPP +), generate free
radicals and induce disease progression characteristic of PD
which is modeled in vitro and in vivo for studies of potential
therapeutics [9,10]. Methods aimed at bolstering endogenous
antioxidant defense mechanisms to enhance scavenging of
free radicals generated during disease progression hold
therapeutic potential for neurodegenerative diseases such as
PD.
In the context of AD, amyloid-
(A
) peptides generated
by the
-secretase-mediated cleavage of amyloid precursor
protein, have been shown to induce mitochondrial oxidative
stress (MOS) and cytochrome c release in vitro and in vivo
[11,12]. A
peptide accumulation and oligomerization has
been proposed to cause mitochondrial dysfunction and acti-
vation of an intrinsic apoptosis cascade [13]. Healthy and
functional mitochondria are imperative for defense against
A
peptide-induced oxidative stress; therefore, therapies
aimed at facilitating the activity of intrinsic antioxidants
have promise for AD [14]. In particular, strategies that pro-
tect cells from oxidative stress specifically at the level of the
mitochondria appear to have significant potential in the
treatment of a number of neurodegenerative diseases [15].
GSH IS A VITAL ENDOGENOUS ANTIOXIDANT
One of the crucial modulators of cell survival, under both
normal and pathological conditions, is the endogenous tri-
peptide glutathione (GSH, - L-glutamyl-L-cysteinyl-

2212-3954/12 $100.00+.00 © 2012 Bentham Science Publishers

Immunocal®: Neuroprotection for Neurodegenerative Disorders
glycine). GSH is involved in many essential cell processes
including DNA synthesis and cell metabolism, but its pri-
mary function is to act as an antioxidant by scavenging ROS
[16]. GSH exists in a reduced and active state, and is oxi-
dized to glutathione disulfide (GSSG) upon reducing unsta-
ble molecules, such as ROS. The reduction of ROS by GSH
is facilitated by the enzyme GSH peroxidase [17]. The en-
zyme, glutathione reductase then returns GSSG to its re-
duced form GSH [18].
Biosynthesis of GSH happens in two distinct steps. The
first occurs when  -glutamylcysteine ligase (GCL) combines
glutamate and cysteine together. This reaction is the rate-
limiting step in GSH synthesis. The second step in GSH syn-
thesis occurs when GSH synthetase adds glycine to the C-
terminus of the end product of the first step,  -
glutamylcysteine. Cysteine is the rate-limiting precursor re-
quired for GSH synthesis [18-20]. Because of its potent anti-
oxidant activity, and its neuroprotective capacity demon-
strated in many in vitro and in vivo systems (discussed be-
low), strategies that enhance GSH synthesis have drawn at-
tention as potentially novel therapies for neurodegenerative
diseases [reviewed in 21].
NEUROPROTECTIVE EFFECTS OF GSH AND GSH
PRECURSORS IN VITRO AND IN VIVO
Several strategies to increase levels of endogenous GSH
have previously shown to be effective in disease treatment in
vitro and in vivo. N-acetylcysteine (NAC) supplementation
provides cysteine in a cell-permeable and non-toxic form,
and has been studied for its efficacy in various neurodegen-
erative and progressive human diseases [22-24]. NAC sup-
plementation decreased ROS levels and increased cell anti-
oxidant potential against rotenone-induced apoptosis in vitro,
and in a rat model of PD in vivo. Neuronal survival was sig-
nificantly increased in NAC-treated cells and animals in
these models of PD, suggesting that NAC reduced toxicity
by sustaining the cellular GSH/GSSG ratio and decreasing
ROS [25]. NAC treatment in SH-SY5Y neuroblastoma cells
has also been shown to decrease levels of A
1-42 peptide,
and to decrease levels of phospho-tau protein, which are
pathological hallmarks of AD [26]. Finally, NAC has been
investigated in a model of ALS where it delayed clinical
onset and prolonged survival in G93A mutant SOD1 mice, a
model of familial ALS [27].
In addition to NAC, other means of increasing GSH lev-
els have been investigated for prevention and treatment of
various neurodegenerative diseases, though the research is
not as comprehensive as with NAC. Glutathione monoeth-
ylester (GSH-MEE) is a cell-permeable derivative of GSH.
Its efficacy has been investigated in several murine models
of neurodegeneration. GSH-MEE has been shown to prevent
mitochondrial GSH depletion and to provide neuroprotection
following transient focal cerebral ischemia [28, 29]. Addi-
tionally, GSH-MEE has been shown to improve recovery
and survival of spinal cord neurons in a model of spinal cord
injury in rats [30]. These approaches, among other methods
of increasing intracellular GSH, are unique and deserve fur-
ther investigation in both pre-clinical and clinical settings.
Recent Patents on CNS Drug Discovery, 2012, Vol. 7, No. 3 231
GSH DEPLETION PLAYS A CENTRAL ROLE IN ALS
PATHOGENESIS
ALS is the most common adult-onset motor neuron dis-
ease. It is characterized clinically by progressive muscle
weakness and atrophy, ultimately leading to respiratory fail-
ure and death typically within 1 to 5 years of diagnosis.
There is currently only one FDA approved drug for ALS,
riluzole, which is an anti-glutamatergic compound that
merely prolongs survival by 2 to 3 months and has little ef-
fect on quality of life for ALS patients [31, 32]. ALS is char-
acterized pathologically by a progressive loss of motor neu-
rons in the cortex, brainstem, and spinal cord [33]. Though
its underlying cause remains elusive, oxidative damage due
to aberrant production of ROS and associated mitochondrial
dysfunction appear to play key roles in motor neuron death
[34-36].
GSH depletion and a decline in antioxidant enzyme ac-
tivity have been observed in the erythrocytes of sporadic
ALS patients with active disease [37]. This decrease in anti-
oxidant defense enzyme activity and GSH metabolism has
been correlated directly with the rate of disease progression
[38]. Many characteristic pathological changes observed in
ALS may be induced by intracellular GSH depletion. For
example, the protein TAR DNA-binding protein-43 (TDP-
43) forms cytoplasmic inclusions, which is one of the hall-
mark pathologies seen in sporadic ALS patients. Remarka-
bly, these TDP-43 inclusions can be recapitulated in cultured
neurons subjected to GSH depletion [39]. Oxidative stress
also causes accumulation and aggregation of mutant Cu/Zn
superoxide dismutase (SOD1) in familial ALS, and these
inclusions have been shown to decrease GSH content which
in turn, leads to disruption of mitochondrial function [40,
41]. Similarly, SOD1 knockout causes oxidative stress and
subsequent distal motor axonopathy, as well as significant
GSH oxidation [42]. These cumulative data suggest a promi-
nent role for GSH depletion in ALS pathogenesis and there-
fore, there is great interest in developing a method to target
this aspect of the disease. Moreover, preservation of GSH
may also be beneficial in other neurodegenerative diseases,
as well as non-neuronal progressive human diseases such as
heart disease and cancer.
NUTRACEUTICAL MODULATION OF GSH: PRE-
CLINICAL AND CLINICAL STUDIES WITH IMMU-
NOCAL®
Immunocal® is a novel, undenatured whey protein sup-
plement designed to augment the available intracellular GSH
pool. Cellular GSH concentrations are highly dependent on
the availability of cysteine, which is the limiting precursor in
GSH synthesis [16, 43]. The cysteine precursor, cystine, oc-
curs in high levels in Immunocal® because the supplement is
rich in serum albumin, alpha-lactalbumin, and lactoferrin.
These proteins have a significant number of cystine residues
in this undenatured preparation. In addition, the direct GSH
precursor, glutamylcysteine, is also present in the serum al-
bumin fraction of this supplement. When cystine is provided
in this peptide form, it is readily cleaved and reduced to two
cysteine molecules within the target cell. This is significant,
as cysteine supplementation alone is cytotoxic [44]. Immu-
nocal® was initially developed as a nutritional supplement to
232 Recent Patents on CNS Drug Discovery, 2012, Vol. 7, No. 3
increase immune system function after dietary amino acids
were discovered to increase immune reactivity [45]. It has
been investigated in several human diseases, and it is one of
only a handful of nutritional supplements that are included in
the Physician’s Desk Reference (PDR). Immunocal® is com-
prised of natural food protein and is in the FDA category of
generally recognized as safe (GRAS) [46].
Immunocal® was initially studied in various animal mod-
els or human disorders of immune system deficiency and
cancer as an approach to increase the available GSH pool
and increase cellular antioxidant and immune system de-
fenses. Free radical generation is a key element of carcino-
genesis and diets rich in antioxidants are believed to decrease
one’s susceptibility to cancer [47, 48]. Consistent with this,
dietary whey protein decreased tumor burden in a mouse
model of dimethylhydrazine-induced colon cancer [49].
Similarly, Immunocal® was shown to increase apoptosis in
vitro in cultured human hepatoma cells by increasing the
cytotoxicity of specific chemotherapeutic agents such as
baicalein, a flavone used for its anti-inflammatory and anti-
proliferative properties in cancer cells [50]. In addition to its
anti-tumor effects, this undenatured whey protein was shown
to reverse weight loss in lung cancer patients receiving che-
motherapy or radiotherapy [51]. Thus, Immunocal® provides
anti-cancer activity while also preserving overall health, and
its method of action may be applicable in many other human
pathologies.
Immunocal® was also investigated as a potential antioxi-
dant therapy in patients infected with human immunodefi-
ciency virus (HIV) [52]. Oxidative stress is a known activa-
tor of HIV replication and based on this, Immunocal® was
tested as an approach to attenuate this stress by increasing
intracellular GSH levels. In a small pilot study, Immunocal®
administration over 3 months was shown to elevate blood
GSH by 70% in HIV-seropositive individuals back to normal
values [53]. Moreover, this restorative effect on blood GSH
was associated with parallel increases in bodyweight.
Oxidative stress plays a significant role in muscle weak-
ness. Immunocal® administration for 3 months was shown to
significantly increase muscle power and work capacity in
comparison to individuals that received placebo treatment.
Undenatured whey protein supplementation also signifi-
cantly increased lymphocyte GSH levels compared to pla-
cebo (36% +/- 11%) [54].
Oxidative stress is also central in the pathogenesis of
cystic fibrosis. It is well accepted that GSH is a major de-
fense in lung diseases, such as cystic fibrosis, and treatment
methods that augment the available pool of this antioxidant
are continually under investigation. In cystic fibrosis patients
receiving Immunocal® for 3 months, there was a 46.6% in-
crease in lymphocyte GSH levels compared to individuals
receiving a placebo [55].
This dietary whey protein supplement was also investi-
gated in aging. For instance, Immunocal® extended longevity
by 6.3 months in mice that received the supplement late in
life (specifically, over a 3 month period between 17 and 20
months of age). This lifespan extension in the mouse corre-
sponds to approximately 25 human years. Although there
was little investigation into the specific mechanism behind
Ross et al.
this effect, both heart and liver GSH levels were significantly
increased in Immunocal®-fed mice [56]. To date, there has
been minimal investigation into the efficacy of Immunocal®
treatment specifically in neurodegenerative diseases; how-
ever, with the wide array of human diseases that show prom-
ising results with this dietary supplement, it is encouraging
for other progressive human diseases. Immunocal® holds
potential to increase GSH levels in many relevant disorders
like neurodegeneration whose underlying pathology includes
oxidative stress.
IMMUNOCAL® AND RELATED PATENTS
Immunocal® has not been patented specifically for use in
neurodegenerative diseases; however, it has been investi-
gated in depth for several other human pathologies. Immu-
nocal® appears to show beneficial effects in these diseases
because of its capacity to act as a cysteine-donor, resulting in
an increase of GSH levels in treated individuals. Initial stud-
ies with this undenatured whey protein isolate were con-
ducted to investigate its efficacy in immune system defi-
ciency. Immunocal® is highly effective in bolstering immune
function, and was patented because of its ability to improve
active systemic humoral immune response [57]. More re-
cently, it was patented for use in HIV-seropositive individu-
als, in which it induces a decrease in viral load and an in-
crease in immune function in affected individuals [58]. Fi-
nally, Immunocal® was patented as an anti-cancer therapy
because of its ability to increase the efficacy of certain che-
motherapy agents, to decrease the oxidative stress compo-
nent of cancer, and to increase the body weight of patients
who were receiving chemotherapy back to healthy levels
[59]. This dietary supplement provides a unique approach to
increase intracellular GSH levels, and there is much promise
for Immunocal® in the treatment and prevention of neurode-
generative diseases (discussed below).
In addition to Immunocal® and its ability to augment the
available GSH pool with cystine supplementation, there have
been several other strategies that aim to increase intracellular
GSH. Liposome-encapsulated glutathione was recently pat-
ented for use in disease states related to GSH deficiency
[60]. Additionally, therapeutic compositions containing key
GSH precursors including glutamic acid, cystine, glycine,
and a selenium precursor have been patented for their use in
chronic disease [61]. Medical foods containing GSH precur-
sors have also been patented for their use in chronic disease
and neuropsychiatric disorders [62].
THERAPEUTIC APPROACHES TO MODULATE
GSH IN NEURODEGENERATIVE DISEASE
Based on extensive evidence that oxidative stress is a
fundamental aspect of neurodegeneration, multiple strategies
have been proposed to limit free radical damage to the nerv-
ous system. In particular, methods designed to augment in-
tracellular GSH levels are of great interest due to the evolv-
ing pool of literature indicating a key role of GSH depletion
and in particular, mitochondrial GSH depletion, in neurode-
generative disease [reviewed in 60]. GSH does not penetrate
the blood brain barrier (BBB) and therefore, therapies aimed
at indirectly modulating GSH levels have been investigated,
including the introduction of GSH precursors, analogs, and
Immunocal®: Neuroprotection for Neurodegenerative Disorders
mimetics [63, 64]. Other strategies, such as bolstering the
expression of enzymes responsible for increases in GSH syn-
thesis for example, are also gaining interest.
The NF-E2-related factor 2 (Nrf2) transcription factor
regulates the expression of several prominent antioxidant
defense proteins. Cellular GSH levels are dependent upon
Nrf2 translocation into the nucleus to induce the expression
of key GSH synthesis enzymes including the catalytic and
regulatory subunits of
-glutamylcysteine synthase (GCS)
[65, 66]. Nrf2 activation is therefore an interesting target to
modulate GSH in an effort to decrease neuronal cell death in
relevant neurodegenerative diseases. Several compounds that
stimulate Nrf2 nuclear translocation and subsequent in-
creases in GSH synthesis have been investigated and have
proven to be neuroprotective in vitro [67, 68]. Consistent
with these in vitro studies, Nrf2 overexpression in glial cells
and subsequent GSH secretion from astrocytes increased
survival and delayed disease onset in a familial mouse model
of ALS [69]. In a similar manner, stimulation of Nrf2-
dependent GSH synthesis by synthetic triterpenoid com-
pounds also delayed disease onset and enhanced survival in
the mutant SOD1 mouse model of ALS [70]. These studies
suggest that Nrf2 activation represents a promising approach
to increase GSH levels in the nervous system.
Glutamate transporter-associated protein 3-18 (GTRAP3-
18) interacts with excitatory amino acid carrier 1 (EAAC1)
at the plasma membrane. EAAC1 is a neuronal gluta-
mate/cysteine transporter, responsible for regulating intracel-
lular cysteine levels. It is suggested that GTRAP3-18 plays a
dominant negative role in cysteine transport when it interacts
with EAAC1 [71]. Accordingly, GTRAP3-18 deficient mice
show increased intracellular GSH levels, and these mice
have demonstrated a decreased sensitivity to oxidative stress
and increased cognitive function [72]. Thus, inhibitors of
GTRAP3-18 may be effective inducers of GSH accumula-
tion in the nervous system.
Another technique to boost cellular GSH that has re-
ceived attention is direct N-acetyl-L-cysteine (NAC) sup-
plementation. This has been shown to increase the available
cysteine pool and to decrease oxidative damage [73]. For
instance, wobbler mice that display lower motor neuron de-
generation have shown reductions in neurodegeneration and
retention of motor function when administered NAC. They
also display increased GSH levels and increased muscle
mass [74].
PRESERVATION OF GSH WITH IMMUNOCAL® AS
A NEUROPROTECTIVE AND THERAPEUTIC
STRATEGY FOR ALS
Recently, we have found that ad libitum supplementation
with Immunocal® in the hSOD1G93A familial mouse model of
ALS significantly delays disease onset, and that this protec-
tive effect is correlated with preservation of whole blood and
spinal cord GSH levels [75]. On the other hand, decreasing
available GSH levels by knocking out the glutamate-cysteine
ligase modifier subunit (GCLM) has recently been shown to
markedly accelerate motor neuron pathology in this mouse
model of ALS [76]. Nutritional supplementation with a die-
tary whey protein has been shown to preserve weight and
body mass index in patients with ALS [77], but the
Recent Patents on CNS Drug Discovery, 2012, Vol. 7, No. 3 233
effects of an undenatured cystine-rich whey protein like
Immunocal® on GSH levels in ALS patients remains to be
seen. These results suggest that Immunocal® may have clini-
cal relevance in ALS patients and warrants further investiga-
tion in this and other neurodegenerative diseases where GSH
depletion is believed to play a causal role. Regulation of
GSH synthesis and metabolism by pharmacologically or
nutritionally modulating cysteine holds therapeutic potential
in neurodegenerative disorders such as ALS.
CURRENT & FUTURE DEVELOPMENTS
Mitochondrial dysfunction and oxidative stress play key
roles in the pathogenesis of multiple neurodegenerative dis-
eases. With the accumulating data supporting GSH depletion
as an important contributor to mitochondrial oxidative stress
and dysfunction, strategies to augment the cellular GSH pool
are gaining attention as possible therapeutic interventions in
neurodegeneration. Immunocal® supplementation has yet to
be tested in the clinic in any human neurodegenerative dis-
ease. Based on its capacity to enhance GSH in various dis-
ease states, Immunocal® shows promise for ameliorating
oxidative stress and neuronal death. We are currently inves-
tigating Immunocal® and its efficacy in a familial model of
ALS, and plan to use it in tandem with FDA approved phar-
maceuticals to examine its potential to act synergistically in
a disease state. Immunocal® and other approaches that aug-
ment cellular GSH warrant further investigation in neurode-
generative diseases, such as ALS, in which oxidative stress
and GSH depletion are key pathogenic mechanisms.
CONFLICT OF INTEREST
The authors have received funding from Immunotec, Inc.
(Quebec, Canada) to support research on the neuroprotective
effects of Immunocal®.
ACKNOWLEDGEMENTS
Supported by a Merit Review grant from the Department
of Veterans Affairs and NIH R01 NS062766 to DAL.
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