α-Tocotrienol quinone modulates oxidative stress response and the biochemistry of aging

Article abstract of DOI:10.1016/j.bmcl.2011.04.085

We report that α-tocotrienol quinone (ATQ3) is a metabolite of α-tocotrienol, and that ATQ3 is a potent cellular protectant against oxidative stress and aging. ATQ3 is orally bioavailable, crosses the blood-brain barrier, and has demonstrated clinical res

Full text of DOI:10.1016/j.bmcl.2011.04.085

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3693–3698
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
a-Tocotrienol quinone modulates oxidative stress response
and the biochemistry of aging
William D. Shrader a, , Akiko Amagata a, Adam Barnes a, Gregory M. Enns c, Andrew Hinman a,
⇑
Orion Jankowski a, Viktoria Kheifets a, Ryo Komatsuzaki a, Edgar Lee a, Paul Mollard a,
Katsuyuki Murase a, Alfredo A. Sadun d, Martin Thoolen a, Kieron Wesson a, Guy Miller a,b
a Edison Pharmaceuticals, Inc., 350 North Bernardo Avenue, Mountain View, CA 94043, USA
b Adjunct Clinical Instructor, Department of Anesthesiology, Critical Care Medicine, Stanford University, Stanford, CA 94305, USA
c Department of Pediatrics, Division of Medical Genetics, Lucile Packard Children’s Hospital, Stanford, CA 94305-5208, USA
d Doheny Eye Institute, USC-Keck School of Medicine, 1450 San Pablo St., Los Angeles, CA 90089, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We report that a-tocotrienol quinone (ATQ3) is a metabolite of a-tocotrienol, and that ATQ3 is a potent
Received 14 March 2011
Accepted 19 April 2011
Available online 24 April 2011
cellular protectant against oxidative stress and aging. ATQ3 is orally bioavailable, crosses the blood–brain
barrier, and has demonstrated clinical response in inherited mitochondrial disease in open label studies.
ATQ3 activity is dependent upon reversible 2e-redox-cycling. ATQ3 may represent a broader class of
unappreciated dietary-derived phytomolecular redox motifs that digitally encode biochemical data using
redox state as a means to sense and transfer information essential for cellular function.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Redox
Antioxidants
Quinone
Aging
Digital Biology
The precise relationship between antioxidants, aging, and met-
abolic control remains a puzzling biological problem.1–3 Many the-
ories have been put forth to explain how antioxidants may be
responsible for modulating the aging process.4,5 Initial theories
suggested antioxidants blocked free radicals, preventing oxidative
injury, and preserving cellular structure and function.6–8 While
there have been numerous cellular and animal studies that have
validated this concept,9,10 few have been recapitulated in human
studies of health and disease.11
have specifically focused on a subset of unambiguous inherited
mitochondrial diseases as models of oxidative stress and human
aging. In these diseases quinone based antioxidants have regis-
tered positive preclinical and clinical responses.22–29
The most widely studied quinone in this class is Coenzyme Q10
(CoQ10).30–32 In contrast to single-electron donor antioxidants,
CoQ10 undergoes reversible two-electron cycling.33 There are
numerous published and ongoing clinical trials demonstrating
34
the clinical relevance of CoQ10
,
but no systematic attempt has
Recent theories have attempted to link antioxidants contained
in certain food sources with the genetic control of caloric expendi-
ture and aging,12–14 but initial promising animal studies15–17 have
not been reproduced in controlled clinical trials.18 While a clear
explanation of how antioxidants may work remains elusive, it is
clear that individuals who eat diets high in naturally occurring
antioxidants generally enjoy an improved quality of life with a
lower incidence of diseases of aging: diabetes, obesity, and cardio-
vascular disease.19–21
To study how antioxidants may be beneficial, we have investi-
gated diseases where both the mitochondria and oxidative stress
have been implicated. While mitochondria have been implicated
in a broad range of neurodegenerative and metabolic diseases we
been made to identify why CoQ10 is active nor whether its mar-
ginal but clinically measurable activity can be improved upon.
CoQ10’s mechanism of action is typically explained by an
enhancement in mitochondrial electron transport function, neu-
tralization of free radicals, or both.35,36 Not generally considered
is whether CoQ10’s activity may be ascribed to other mechanisms
or targets,37,38 especially when administered at supra-physiologic
doses where off-target effects are common.
Given the known role of quinones as biochemical redox signal-
ing agents,39 it may be that CoQ10 is a weak version of a more po-
tent and unidentified quinone agonist. To test this hypothesis, we
set out to identify and characterize dietary-derived quinones in
search of potent quinone regulators of metabolic control and oxi-
dative stress.
Herein we report that quinones derived from the hydrolysis of
⇑
Corresponding author.
E-mail address: bshrader@edisonpharma.com (W.D. Shrader).
vitamin E—called vitamin E quinones40—are 1000–10,000 times
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.085

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W. D. Shrader et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3693–3698
more potent than CoQ10 in a battery of human cell assays of oxida-
tive stress. We explore the hypothesis that vitamin E quinones may
be members of a new class of native and essential quinones critical
to the control of metabolism and the aging process through redox
signaling and information transfer and sensing mechanisms
(Fig. 1).
(Scheme 1 insert). To investigate the relationship of biological ac-
tion with redox dependence, a redox silenced version, chemically
locked in a single state was prepared by the hydride reduction of
(R)-a-tocotrienol quinone to the dihydroquinone, with subsequent
acylation and trapping as its bis-pivaloyl ester (RS-ATQ3).
The pharmacokinetic parameters, oral bioavailability, and tissue
distribution of ATQ3 were obtained in multiple species (Table 1).
From these studies, ATQ3 was found to have good oral bioavailabil-
ity. To support these studies, a LC/MS/MS bioanalytical method
was developed in mice, rats, dogs and humans to detect ATQ3 in
its oxidized state. The reduced dihydroquinone of ATQ3 present
in fresh plasma or tissue samples was allowed to fully oxidize
ex vivo in air to the quinone before quantification. The pharmaco-
kinetic studies revealed that the presence of food increased the oral
absorption and absolute bioavailability of ATQ3 in the rat from <5%
to 89%. In the dog, the absolute bioavailability was 17% under fast-
To investigate
three-step synthesis was developed starting from an Elaeis
guineensis extract enriched in - and d-tocotrienol vitamin E
isoforms. This is the first synthesis and characterization reported
for the natural product, -tocotrienol quinone (Scheme 1). Step
one was an exhaustive aminomethylation of the - and d-tocotri-
a-tocotrienol quinone in detail, an efficient
c
a
c
enol isoforms (1, 2) contained within the E. guineensis extract.41
This was accomplished by treating the crude E. guineensis extract
with paraformaldehyde and 1-methylpiperazine to yield a mixture
of mono- and diaminomethylated tocotrienol derivatives (3, 4).
During work-up, the aminomethylated derivatives were separated
from the crude reaction mixture by protonation with formic acid,
and extraction of the resulting ammonium formate salt into the
acetonitrile layer of an organic/organic biphasic system with hep-
tanes. Step two converted the crude aminomethyl species (3 and 4)
ing conditions and the Cmax and AUC increased by 16% and 28%,
1
respectively, when administered with food. The elimination of
ATQ3 in both rat and dog, demonstrated a similar biphasic decline
in plasma levels with a terminal half-life of approximately 3.5 h
(po) to 9.6 h (iv) for rat and approximately 7.5 h for both routes
of administration for dog. The clearance was moderate in the rat
(716–2086 ml/h/kg) and low in the dog (574 ml/h/kg). In human
adults, a single po dose of 200 mg yielded peak plasma concentra-
tions at 2 h with a Cmax average of 167 ng/ml.
to a single compound a-tocotrienol (5) by a reductive deamination
with sodium cyanoborohydride in amyl alcohol at elevated tem-
peratures.42 Silica gel chromatography yielded high purity
a-toco-
trienol as a white waxy solid.43
The
a
-tocotrienol chroman (5) was converted to the
a
-tocotri-
The high volume of distribution (Vdss) observed in the initial
pharmacokinetic studies prompted further experiments to deter-
mine the tissue distribution of this particular metabolite of vitamin
E (ATQ3). A series of distribution studies revealed that ATQ3 read-
ily distributed into the brain, retina and heart. In mice, ATQ3 was
dosed IP at 25 mg/kg and monitored at each time point (n = 3 per
time point, t = 0.5, 4, 12 and 24 h) for ATQ3 levels in brain, heart,
and plasma. Peak brain concentrations were observed at t = 12 h,
with a brain-serum ratio of 1.83 (539 ng/g: 300 ng/ml). The heart
tissue–serum ratio was 0.71 at t = 0.5 h, with a heart concentration
of 848 ng/g and a plasma level of 1188 ng/ml. Distribution of ATQ3
into ocular tissues was measured at the conclusion of a 7-day dog
po repeat dose study (po, QD, 300 mg/kg/day). Levels of ATQ3 in
the retina were 11,200 and 755 ng/g in the cornea, with the asso-
ciated peak plasma concentrations of approximately 10,000 ng/
ml. ATQ3 concentrations were variable in vitreous humor ranging
from below quantifiable levels to 682 ng/mL with no ATQ3 de-
tected in the aqueous humor. This is in contrast to what has been
enol quinone (ATQ3) by an oxidation with ceric (IV) ammonium
nitrate in isopropyl acetate and water.45 Analytically pure
(R)-
gel chromatography, in an overall yield of 45% based on the start-
ing - and d-tocotrienol w/w content in the E. guineensis extract. To
confirm the structure of (R)- -tocotrienol quinone, a series of de-
tailed 1H, 13C, HSQC, and HMBC NMR experiments were performed
a-tocotrienol quinone was obtained as a yellow oil after silica
c
a
reported for vitamin E members with the phytyl tail, such as
a-
tocopherol, which do not enter the eye or cross the blood–brain
barrier.46–49
The conversion of the parent a-tocotrienol to ATQ3 metabolite
was monitored in vivo by the simultaneous detection of both the
parent chroman and the quinone metabolite using a modified LC/
MS/MS method. In this study,
a-tocotrienol was dosed either iv
(5 mg/kg) or po (25 mg/kg) in rat with parent chroman and metab-
olite (ATQ3) monitored at each time point (t = 0.1–24 h). In both
the iv and po study arms, the ATQ3 quinone metabolite concentra-
tion followed the parent a-tocotrienol chroman concentration at a
level approximately 10-fold lower: 7–11% of parent concentration
for iv, and 5–9% for po. This study demonstrated that the level of
the ATQ3 metabolite was directly correlated to the level of its par-
ent chroman—
trienol to -tocotrienol quinone could also be achieved in vitro
by incubation of -tocotrienol (20 M) with human and mouse li-
ver microsomes (25 g). These conditions yielded a 15% overall
conversion to ATQ3 relative to the initial levels of -tocotrienol,
a-tocotrienol. The conversion of the parent a-toco-
a
a
l
l
a
and were independent of the presence or absence of NAD(P)H.
ATQ3 was not mutagenic when evaluated in the in vitro
bacterial reverse mutation (Ames) assay and was negative in
the CHO micronucleus test. A toxicological evaluation of ATQ3
Figure 1. The conversion of a-tocotrienol to a-tocotrienol quinone.

W. D. Shrader et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3693–3698
3695
Scheme 1. Synthesis of a-tocotrienol quinone. Reagents and conditions: (a) 1-methylpiperazine, paraformaldehyde, 120 °C, 18 h; (b) formic acid, liquid–liquid partition with
acetonitrile and heptanes, aqueous base wash; (c) NaBH3CN, amyl alcohol, 120 °C, 6 h, silica chromatography; (d) Ceric (IV) ammonium nitrate, isopropyl acetate, 0 °C, 0.5 h,
silica chromatography; (e) NaBH4, pivaloyl chloride, THF, silica chromatography. Proton and carbon NMR assignments of human metabolite, ATQ3 (all shift values in ppm,
C6D6). ⁄Assignment inconclusive.
Table 1
A summary of the in vivo plasma pharmacokinetics of ATQ3 in rat, dog, and mouse44
Parameter
Species
Species
Rat
Rat
Rat
Rat
Dog
Dog
Mouse
Mouse
Human
Dose route
Dose level (mg/kg)
Compartment
Cmax (ng/ml)
tmax (h)
AUCt (h ng/ml)
AUC1 (h ng/ml)
t1/2 (h)
F (%)
Cl (ml/h/kg)
Vi (ml/kg)
Vss (ml/kg)
Vz (ml/kg)
iv
2
Plasma
—
—
5882 431
5954 411
3.5 1.0
—
716 50
117 14
222 81
3726 1137
po
25
Plasma
248
4.0
961
—
—
iv
2
Plasma
—
—
984 117
1069 134
9.6 0.7
—
2086 359
567 299
11497 1881
27392 3014
po
10
iv
2
Plasma
—
—
3408 589
3572 587
7.5 0.7
—
574 112
95 14
2535 893
6249 829
po
10
ip
25
ip
25
po
2b
Plasma
931 83
3.5 0.2
4728 349
4781 351
3.5 0.1
89(fed)
—
Plasma
442 294
2.8 1.0
2693 1656
2817 1704
7.6 5.1
17
—
—
—
—
Plasma
1188 601
Brain
Plasma
167 110
2
539 232a
0.5
4835
5862
7.6
—
—
—
—
—
12
7738a
10724a
12.4
—
—
—
—
—
—
—
—
—
4.1(fasted)
6
—
—
—
—
—
—
—
—
—
—
—
a
Tissue samples, units are ng/g and h ng/g.
Average mg/kg dose for a 50 kg adult.
b
demonstrated a high therapeutic index between no-observable-ad-
verse-effect level (NOAEL) and the levels required for pharmaco-
logic activity. A 28-day repeat-dose toxicity study in both dogs
and rats after oral (gavage) administration showed that the NOAEL
of ATQ3 was 100 mg/kg. In both species and sexes at P100 mg/kg/
day there were dose-dependent prolongations of prothrombin
time (PT) and activated partial thromboplastin time (APTT) relative
to controls. The dose-limiting effect is an increase in coagulation
times explained by weak vitamin K antagonism previously as-
cribed to this structural class of compounds.50,51 All alterations in
coagulation parameters had resolved by the end of the 4 week
recovery period in both species and sexes.
To determine if ATQ3 had similar cellular pharmacology to
other antioxidant quinones, CoQ10 and idebenone, we established

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W. D. Shrader et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3693–3698
an assay using stressed primary human fibroblasts. Utilizing the
inherent oxidative stress of fibroblasts from patients with mito-
chondrial disease, we further increased their oxidative burden by
depleting cellular glutathione with an inhibitor of glutathione syn-
thesis, L-buthionine-(S,R)-sulfoximine (BSO). As a result, while
fibroblasts from healthy individuals retained full viability, patient
fibroblast exhibited complete cell death within 48 hr of the BSO in-
sult (ꢀ100
lM). As an example, we show the results obtained with
primary fibroblasts of a Friedreich ataxia (FRDA) patient, a neuro-
degenerative disease resulting from a triplet expansion in the nu-
clear frataxin gene (Fig. 2a), as well fibroblasts from a patient
with Leigh syndrome caused by a compound heterozygous muta-
tion SURF1 mutation (Fig. 2b). Cellular viability was measured with
a redox independent dye Calcein-AM. The data presented herein
shows that ATQ3 is a potent cellular oxidative-stress protectant
with in vitro potency and efficacy of 102–104 times higher than
either structurally-related quinones, such as CoQ10 and idebenone,
or structurally dissimilar antioxidants such as polyphenolic
stilbenes, represented by resveratrol. The importance of ATQ3
redox-cycling for compound efficacy was tested via a redox silent
bis-pivoyl adduct of ATQ3, which showed no activity in any cell
line tested.
Figure 2b. Dose–response and redox dependence of ATQ3 for cellular activity in
Leigh syndrome (SURF1 mutation) primary human fibroblasts.
either human or mouse microsomes resulted in substantial conver-
In two ongoing FDA-approved, open-label human studies52,53 in
children and adults with inherited mitochondrial disease at doses
between 100 and 400 mg three times daily, no significant elevation
of clotting times or significant drug-related adverse events have
been reported in approximately 6250 patient dosing days. Initial
clinical data in subjects with inherited respiratory chain diseases
of the mitochondria suggest ATQ3 has an acceptable therapeutic
index and alters the natural course of disease. ATQ3 is being ad-
vanced into controlled clinical trials to verify these initial positive
open-label clinical findings.
sion of a-tocotrienol to ATQ3. While the precise mechanism of this
transformation is under investigation, the generalized possibility
that a vitamin E vitamer could be a ‘pro-drug’ (pro-vitamin) raises
new questions about the biochemistry of vitamin E deficiency. We
are actively investigating whether a vitamin E deficiency syndrome
in otherwise healthy subjects might be explained in part by a vita-
min E quinone deficiency and not a vitamin E (chroman) defi-
ciency. Furthermore, various diseases of aging may be examples
of the subclinical presentations of a vitamin E quinone deficiency
syndrome.
Because manifestations of inadequate levels of ATQ3 may be
both subclinical and pernicious, ATQ3 deficiency syndromes may
be hard to detect in otherwise healthy subjects. As children with
inherited mitochondrial disease present with clinical characteris-
tics similar to adult diseases of aging they may magnify the impor-
tance of ATQ3’s role in metabolic function. Thus inherited
mitochondrial disease may represent time-compressed ‘models’
of aging.
There is ample precedent for pro-vitamin biochemistry in, for
example, vitamins A and D. But the fact that an active form of vita-
min E may result from the conversion of a chroman to a quinone
that is critical for metabolic control and aging has not been previ-
ously described.
The lack of cellular efficacy with the covalent modification of
ATQ3 to its redox-silenced, bis-pivoyl adduct suggests that re-
dox-cycling is critical to the mode of action of ATQ3. It may be that
this structural modification also precludes target binding, though
other covalent modifications have resulted in similar loss of activ-
ity, suggesting that the redox-cycling is the key factor. Additional
experiments are underway to construct a periodic table of antiox-
idants correlating electronic (redox) properties with biological
activity.
The observed biological activity in both laboratory and clinical
settings raises the question of how to reconcile the relationship
of ATQ3 with its purported precursor,
a-tocotrienol, particularly
in the broader context of redox signaling, metabolic control, and
aging.
Preliminary studies suggest that ATQ3 may be derived from die-
tary sources of
a
-tocotrienol. Incubation of
a
-tocotrienol with
The observation that the biological activity of ATQ3 may have
an obligate dependence on its redox properties may be reconciled
through its involvement as a redox-signaling agent. Enzymes such
as NAD(P)H:Quinone oxidoreductase 1 (NQO1, DT-diaphorase)
that bind quinones have long been thought to detoxify such sub-
strates.54,55 But recent data now suggests these enzymes may be
regulated by quinones. Further studies now also link oxidoreduc-
tases with oxidative stress response, cell death, and aging.56–61
Compounds of the structural class of ATQ3 are also known NQO1
modulators.62–64 We believe ATQ3 may exert its beneficial effects
on metabolic control and aging through action at oxidoreduc-
tases—thus establishing the missing link between antioxidants
and aging.
Lastly, we note that physical chemistry and material science
investigators have exploited the binary nature of quinone redox
states to synthesize solid-phase quinone computing chips to en-
code digital data.65–69 We propose that dietary-derived quinones
such as ATQ3 may represent nature’s version of digital biol-
ogy,70–72 thereby explaining how select antioxidants may regulate
metabolism and aging. We term compounds with these select
Figure 2a. Dose–response of ATQ3 versus other antioxidants in Friedreich ataxia
(FRDA) primary human fibroblasts.

W. D. Shrader et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3693–3698
3697
Figure 3. Cyclic voltammograms of ATQ3 (Fig. 3a), resveratrol (Fig. 3b), and generalized D-BITS (Fig. 3c). Cyclic voltammograms were obtained for ATQ3 and resveratrol using
a BASi C3 electrochemical analyzer under standard reported conditions. Cyclic voltammograms were obtained using 2 mM analyte under the following conditions: anhyd
acetonitrile with 0.1 M (CH3CH2CH2CH2)4N+(BF4)ꢁ as the supporting electrolyte; working electrode Pt; reference electrode Ag/AgCl; sweep rate 100 mV/s. ATQ3 exhibits
reversible oxidation reduction properties with a reduction potential of ꢁ650 mV, as evidenced by an equivalent current flow in oxidation and reduction phases (Fig. 3a).
Resveratrol exhibits primarily irreversible oxidation with a single oxidation potential at 1050 mV (Fig. 3b). The generalized D-BITS case is presented that illustrates how
nature could deploy structural changes in ring substitution patterns (here shown as a quinone) to alter redox properties and thereby discretely encrypt biological data
(Fig. 3c).
8. Shigenaga, M. K.; Hagen, T. M.; Ames, B. N. Proc. Natl. Acad. Sci. U.S.A. 1994, 91,
functional properties D-BITS—Digital Biochemical Information
Transfer and Sensing compounds.
10771.
9. Behl, C.; Davis, J.; Cole, G. M.; Schubert, D. Biochem. Biophys. Res. Commun. 1992,
186, 944.
10. Forrest, V. J.; Kang, Y. H.; McClain, D. E.; Robinson, D. H.; Ramakrishnan, N. Free
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We would like to thank Professor Sidney Hecht, Dr. Pat Conf-
alone and Dr. Robert Ings, respectively, for their helpful discussions
on redox biochemistry, synthetic methods and the pharmacoki-
netic data and analysis presented herein.
154.
12. Lagouge, M.; Argmann, C.; Gerhart-Hines, Z.; Meziane, H.; Lerin, C.; Daussin, F.;
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