Transcript Further characterization of the lipoic acid enantiomers

Further characterization of the lipoic
acid enantiomers provide new
research opportunities.
Stereochemistry, Pharmacokinetics & Metabolism:
Toward a Comprehensive Mechanism of Action
David A. Carlson
Director of R&D
GeroNova Research, Inc
www.geronova.com
R-(+)-Lipoic Acid & S-(-)-Lipoic
Acid are not Bioequivalent
 "R- and S- enantiomers of the
physiological compound alpha-lipoic
acid have been synthesized. The Senantiomer is not a naturally occurring
compound. This part of the racemate,
which is present as about a 50%
impurity, needs to be eliminated.“
(Zimmer et al 1995)
Structures of R-(+)-lipoic acid, S-(-)-lipoic acid, R(-)-dihydrolipoic acid & S-(+)-dihydrolipoic acid
O
S
H
S
OH
OH
*
S
S
R-(+)-ALA
H
[1200-22-2]
Oxidized
S-(-)-ALA
[1077-27-6]
O
Reduced
SH
O
SH
H
OH
OH
*
SH
HS
R-(-)-DHLA
[119365-69-4]
H
S-(+)-DHLA
[98441-85-1]
O
Number & Percentage of studies dealing
with racemic-LA versus the LA enantiomers
as of March, 2008.
 Rac-LA alone
 All enantiomer
 RLA alone
 SLA alone
 RLA vs. SLA
 RLA vs. rac-LA
 RLA vs. rac-LA vs. SLA
2466
109
46
0
31
2
30
4.3%
1.8%
0%
1.2%
0.08%
1.2%
Contribution of Stereochemistry
to the Pharmacodynamics
 Eutomer: the pharmacologically
preferred enantiomer
 Distomer: the pharmacologically non-
preferred enantiomer; isomeric ballast
vs. anti-metabolite/competitive
inhibitor
Select cases of SLA functioning as
an inhibitor of RLA.
 RLA is more actively transported than
rac-LA in a HUVEC.
(May et al 2006)
 RLA increased aortic blood flow in
working rat heart, whereas SLA and
rac-LA decreased it at 2 nmol/g.
(Zimmer et al 1995)
S-(-)-Lipoic Acid is toxic
in thiamine deficient rats
 20mg/kg (IP) rac-LA in B-1 deficient rats
lethal. (Gal 1960)
 20mg/kg SLA selective toxicity. (Gal 1965)
 20mg/kg RLA was not lethal.
 The presence of RLA as a 50% component of
rac-LA did not offset the toxicity of the
unnatural enantiomer.
[Cmax=72uM, AUC=2.9 ug hr/mL, Tmax =6 min, T
½=335 min]
Estimated yearly usage of LA
 800 MT rac-LA
 36 MT RLA
 <0.01 MT SLA
SLA is used to make
racemic-LA & RLA
 Most of the world’s supply of SLA is
used in our lab to develop processes
for racemizing it to racemic-LA for reresolution or
 Chiral inversion to RLA
Suna (13 yrs) and Willow (3 yrs)
Out of the lab & into our lives
 Suna & Willow are my yellow Labrador
retrievers. They have the same body
mass, the same blood markers &
activity levels. Suna is continuing to
learn new tricks & has been begging
for 22.5mg/kg/day RLA for the last 7
years. Suna is Willow’s aunt although
they frequently pass as siblings.
Rac-LA salts benefit old rats or increase
“oxidative damage” in old rats
 100 mg/kg per oral solution potassium
LA  normalizes markers of aging to
youthful levels. (Panneerselvam)
 100 mg/kg IP sodium LA  oxidation of
plasma proteins & signs of oxidative
stress. (Cakatay)
Beneficial effects of rac-LA & L-carnitine on
age-related changes in geriatric animals
(100 mg/kg PO as potassium-lipoate)
 improve GSH redox system.
(Kumaran et al 2004 a)
 improve skeletal muscle mitochondrial
respiration.
(Kumaran et al 2005)
 ameliorate decline in mitochondrial
enzymes.
(Savitha et al 2005)
R-(+)-Lipoic Acid
reverses age-related decline
 RLA & ALCAR improved memory by
reversed oxidative damage to nucleic
acids & improving mitochondrial
function. (Liu et al 2002)
 RLA increases Nrf2 translocation from
the cytosol and accumulation in the
nucleus. SLA is not effective.
(Petersen-Shay et al 2008)
R-(+)-Lipoic Acid
reverses age-related decline
 RLA reversed age-associated increase in
susceptibility of hepatocytes to tertbutylhydroperoxide both in vitro and in vivo. SLA
was ineffective. (Hagen et al 2000)
 RLA reversed the age-associated effects on ascorbic
acid concentration, recycling and biosynthesis after
oxidative stress. (Lykkesfeldt et al 2000)
 RLA improved mitochondrial metabolism in aging
rat heart. (Hagen et al 2002)
R-(+)-Lipoic Acid
reverses age-related decline
 RLA restores transcriptional activity of
Nrf2 age-related loss of glutathione
synthesis. (Suh et al 2004)
 RLA reverses the age-related
accumulation of iron and depletion of
antioxidants in the rat cerebral cortex.
(Suh et al 2005)
R-(+)-Lipoic Acid
reverses age-related decline
 RLA reverses the age-related loss in GSH
redox status in post-mitotic tissues:
evidence for increased cysteine
requirement for GSH synthesis. (Suh et al 2004)
 RLA reduces oxidative stress in the aging
rat heart. (Suh et al 2001)
Is lipoic acid useful in treating or
reversing age-related diseases or
markers of aging in humans?
• At the National Institute of Aging
conference in Oakland 2005 when the
group of experts was asked whether they
thought LA might be useful in treating or
reversing age-related deficits only 2
people out of the group answered yes.
RLA, Aging & Redox Status
• When I asked Aubrey de Grey about his
opinion on RLA & cellular redox control
in aging, he dismissed it saying it would
only buy us 10-15 years, max. While
Aubrey doubts that RLA will win us the
Methuselah Mouse award, I think
considering the options most of us would
be quite happy to add that time to our
“health span” or life-span.
Unsolved problems
in LA research
• Is the current state of the art sufficient to
explain the in vivo mechanism(s) of action of
LA? (Pershadsingh 2007)
• What is the contribution of stereochemistry
toward the in vivo mechanism(s) of action? (Carlson
et al 2008)
• What is the contribution of LA metabolites to
the overall mechanism of action of LA?
(Carlson et al 2008)
Lipoic Acid binds to the insulin receptor
 Enhanced Insulin signaling through
PI3K/Akt via increased
phosphorylation of the insulin receptor
and IRS-1. (Kiemer & Diesel 2008, Diesel et al 2007, Yaworsky et al
2000)
 Inhibition of phosphatases such as
PTB1B. (Petersen-Shay et al 2008, Cho et al 2003)
R-(+)-Lipoic Acid binding to
the insulin receptor
Diesel et al 2007, Yaworsky et al 2000
Used with permission of Diesel & Kiemer & ACS
a-lipoic acid (LA)
Kiemer & Diesel 2008
used with permission
of authors & Taylor &
Francis Publishing
insulin receptor
activation
activation of
PI3-K/Akt pathway
anti-inflammatory
• NF-kB↓
• CAMs ↓
• cytokines ↓
• chemokines ↓
• iNOS ↓
anti-apoptotic
• Bad ↓
• Bcl-2 ↑
• Bax ↓
• caspase activation
↓
anti-atherosclerotic
• eNOS ↑
• CAMs ↓
• insulin sensitizing (AMPK, PPARs)
Is the primary mechanism of LA
an inducible stress response?
 What is the nature of the stress response(s)?
 Primarily oxidative, reductive or both?
(Han et al
2008, Konrad 2005, Dicter et al 2002, Cho et al 2003)
 Metabolic stress response?
(Kim et al 2004, Lee et al 2005)
 A convergence of both redox & metabolic
effects?
Evidence for the primary mechanism of action
of LA being an inducible stress response
 Pharmacokinetics & Pharmacodynamics
are not correlated. (Krone 2002)
 40 mg/kg IP RLA (28.8 µg/mL[144 µM] &
5.8 µg hr/mL) increased nuclear levels of
Nrf2 & ARE transcriptional factor binding
activity at 12 h and remained elevated for
up to 48 h. (Suh et al 2004)
Evidence for the primary mechanism of action
of LA being an inducible stress response
 100 µM RLA increased Nrf2 nuclear
localization (24 hrs) whereas SLA was
NOT effective. (Petersen-Shay et al 2008)
 75 mg/kg IP rac-LA increased AMPK
within 1.5 hrs. (Lee et al 2004)
Evidence for the primary mechanism of action
of LA being an inducible stress response
 1 month feeding 0.25% wt/wt chow
increased AMPK.
(Lee et al 2004)
 40 µg/mL(200 µM) rac-LA (24hrs)
increased expression of PGC-1α.
(Kim et al 2007)
Evidence for the primary mechanism of action
of LA being an inducible stress response
 1% rac-LA increased hepatic phospho-
AMPK, 6 x and PGC-1a 4x. (Lee et al 2006)
 RLA activates PI3K [200 µM].
( Kiemer & Diesel
2008, Diesel et al 2007)
 rac-LA activates PI3K & PKG, ERK-1/2.
(Abdul & Butterfield 2007)
 Increases HO-1, HSPs, P38.
Oksala et al 2006, 2007)
(Ogborne et al 2005,
“Metabolic-Hormesis”
• What is the nature of the metabolic stress
activating AMPK?
• Is it possible that both the redox &
metabolic stress induced by LA is
mediated through a single transcription
factor such as PGC-1a?
“Metabolic-Hormesis”
• Can the redox stress from oxidative
activation of Nrf2 and transient dip in
NADP(H)/NADP+ from reduction of LA
& metabolites activate AMPK?
• Is AMPK activation stereospecific?
Comparison of pharmacokinetic profiles of 100
mg/kg R-lipoic acid and racemic lipoic acid in
male Wistar rats (PO solution)
20
19
18
17
Trivedi
16
Krone
15
Concentration (ug/mL)
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
0
1
2
3
4
Time (h)
5
6
7
8
adapted from Trivedi et al 2004, Krone 2002
Pharmacokinetic Values for R-Lipoic Acid tris salt
as oral solution administered to Wistar rats (Krone 2002)
6000
5000
10 mg
30 mg
100 mg
Concentration (ng/mL)
4000
3000
2000
1000
0
0
1
2
3
4
5
6
Time (h)
7
8
Used with Permission
from Dorothee Krone, Ph.D.
What is the origin of baseline levels of
R-(+)-Lipoic Acid in human plasma?
• Food: little increase in plasma or urinary levels of
RLA after eating.
• Endogenous synthesis. Is the lipoate activating
system functional in mammals? (Gunther et al 2005, Morikawa et al
2001, Dupre et al 1980)
• Lipoate salvaging: mitochondrial turnover and
lysosomal degradation of HSA.
• “Free”- RLA (non-lipoylated) levels found bound to
HSA in levels from ND to 200 ng/mL (~1 uM).
94
3
8
2
6
9
5
0
7
se
L
9
3
7
m
) (
m a
i
[ n R
)
-
L
A
]
(
+
Human
ofPharmacokine
R-(+)-Lipoic acid
600mgPharmacokinetics
Oral Dose Na
R-LA
tics
(
as Sodium R-(+)-Lipoate 600 mg
d
i
c
A
)
L
c
m
i
/
o
g

p
i
L
Plasma [R-LA] (µg/mL)
3
4
2
i .22
5/ D
0
m
l1
5 ma . 0
2
6
9
4
0
8
7
3
a
m
s
a
l
P
All Subjects (n=12)
18
18
mean
range
16
16
Cmax
14.2 µg/mL
10.6 - 33.8 µg/mL
14
14
Tmax
15 min
10-20 min
12
12
AUC
7.36 µg*hr/mL
3.93-13.4 µg*hr/mL
10
10
88
66
44
22
0
0
0
10
10
20
20
30
30
40
40
50
50
60
60 70
70 80
80
Time Time
(min)(min)
90
90 100
100 110
110 120
120
R-Lipoic Acid from baseline to Cmax
16000
Baseline RLA= Not Detected to 200 ng/mL
14000
1000mg RLA (free-acid form) to1000 ng/mL
600mg rac-LA=5000 ng/mL
Concentration ng/mL
12000
600mg NaRLA =15000 ng/mL
10000
8000
6000
4000
2000
0
©GeroNova Research
Plasma Redox effects of rac-lipoic acid
Plasma protein thiol levels
control
LA
(not labeled) control 15m
Method
LA
2h
Protein-SH + maleimide-biotin
Protein-S-maleimide-biotin
albumin
Western blot against
biotinylated proteins
This gel shows there are not much change to plasma
protein thiols levels. Small changes may be occurring but
another method is needed to detect these small changes.
Liver redox effects of rac-lipoic acid in vivo
Method
Liver protein disulfide levels
control
LA
15m
LA
2h
Protein-SH +
NEM
S S
Protein-S-NEM
S S
+ DTT
Protein-S-NEM
SH SH
+ maleimide-biotin
Protein-S-NEM
S
This gel shows no dramatic changes.
S
maleimide-biotin
maleimide-biotin
Western blot
Cmax comparing 600 mg of rac-LA from various
studies versus 600 mg Na-RLA & RLA
Tromethamine salt
18
Na-RALA (Carlson et al 2007)
16
RLA tromethamine salt (Krone 2002)
Contolled Release LA (Evans et al 2002)
14
rac-LA (Evans et al 2002)
rac-LA (Teichert et al 2003)
rac-LA (Krone 2002)
12
rac-LA (Breithaupt-Grogler et al 1999)
Plasma LA (µg/mL)
rac-LA (Gleiter et al 1996)
rac-LA (Rosak et al 1996)
10
rac-LA (Preiss et al 1996)
rac-LA (Chen et al 2005)
8
6
4
2
0
AUC comparing 600 mg of rac-LA from various
studies versus 600 mg of Controlled Release LA,
600 mg Na-RALA & RLA Tromethamine salt
8
Na-RALA (Carlson et al 2007)
7
RLA tromethamine salt (Krone 2002)
Controlled Release LA (Evans et al 2002)
rac-LA (Evans et al 2002)
rac-LA (Teichert et al 2003)
6
rac-LA (Krone 2002)
rac-LA (Gleiter et al 1996)
Mean AUC (µg x hr/mL)
rac-LA (Rosak et al 1996)
5
4
3
2
1
0
rac-LA (Preiss et al 1996)
rac-LA (Chen et al 2005)
2008 OCC Study: Comparison PK
profiles: rac-LA, RLA & SLA in 3
Plasma Lipoic Acid (g/mL)
subjects (600 mg)
RLA
SLA
rac-LA
1
8
16
14
12
10
8
6
4
2
0
10
20
30
40
50
60
70
80
90
Time (min)
3-way crossover in humans indicates possible stereospecific transport
This is the first human PK data with SLA
Plasma Lipoic Acid (µg/mL)
Single Subject
3 x 600mg Na R-(+)-Lipoate
Cmax 21.9 µg/mL
25
Tmax 45.0 min
20
AUC 17.5 µg*min/mL
15
10
5
0
10
20
30
40
50
60 70 80
Time (min)
90 100 110 120 130
Comparison of Pharmacokinetic values for
racemic-LA IV (20 min infusion; 30mg/min) and
NaRLA (3x600 mg) PO doses
Dose mg
Cmax
AUC
µg/mL µg hr/mL
Tmax
min
T1/2
min
NaRLA
3 x 600 PO
21.9
17.5
45.0
17.4
Rac-LA
1 x 600 IV
28.57
12.3
18.6
32.8
Carlson et al 2007, Teichert & Preiss 2008.
Extrapolation of in vitro & animal
pharmacokinetic (PK) models to
humans?
 80-90% of radioactive label is recovered in
24 hr. urine of rats. (Schupke et al 2001)
 Only 12 % LA and total metabolites is
recovered in 24 hr. urine. (Teichert & Preiss 2003)
 Do humans have a larger capacity to
accumulate LA than animals?
Unidentified Metabolites
 Humans administered rac-LA- 35 S
excreted 90-99% of the radioactivity
in 24 hr urine. (Takenouchi et al 1963)
This indicates unknown in vivo
desulfurization reactions, and
degradation of the dithiolane ring.
Is R-(-)-Dihydrolipoic Acid an in vivo
metabolite or pro-drug of R-(+)-Lipoic Acid?
 R-DHLA is plasma stable using acidic citrate
anti-coagulant but not EDTA or Heparin
tubes. (Carlson et al 2008)
 R-DHLA is a minor plasma metabolite.
(Carlson et al 2008)
 R-DHLA is also likely a minor intracellular
metabolite due to the increased toxicity of the
free sulfhydryls. (Kis et al 1997, Kulhanek-Heinz 2004)
Is Dihydrolipoic Acid the more
potent form of LA in vivo?
 In vitro studies have indicated DHLA is
a better scavenger of a variety of
radicals than LA but the disulfide
radical cation reacts faster. (Bucher et al 2005)
 Neither is likely to have much direct
impact on the redox status by
scavenging of radicals due to transient
presence. (Smith et al 2004)
Is Dihydrolipoic Acid the more
potent form of LA in vivo?
 Any therapeutic potential of DHLA
must be reconciled with its short ½ life
due to rapid bis-methylation. (Carlson et al 2008)
Lipoic Acid enantiomers disproportionate in plasma
after administration of rac-LA; RLA:SLA ~1.6-2:1 at Cmax
 Hepatic stereoselective 1st pass preferring SLA.
(Hermann & Niebch 1997)
 Hepatocytes never “see” a true racemic mixture but
“see” SLA with enantiomeric excess of ~60%.
 Renal stereoselective 1st pass preferring SLA. SLA
2:1 over RLA in urine
(Gal & Razevska 1960).
 Possible stereoselective transport from brush-border
or basal-lateral membrane.
(Carlson et al 2008)
R-Lipoic Acid Metabolites
O
O
OH
OH
R-BLAS
S
S
S
S
O
B-oxidation
O
O
microsomes
P450
NADPH oxidases
O
R-BLAS
B-oxidation
O
O
OH
OH
R-BLA
S
S
O
B-oxidation
R-BLA
S
S
microsomes
P450
NADPH oxidases
O
O
B-oxidation
OH
S
S
2-fold
B-oxidation
RLA
1-fold
B-oxidation
GSH Red
TRX Red
LipD
O
S
OH
S
H3 C
R-BNLA
O
GSH Red
TRX Red
LipD
GSH Red
TRX Red
LipD
TMT
CH3
S
OH
O
GSH Red
TRX Red
LipD
TMT
H3 C
S
CH3
S
H3C
S
O
TMT
CH3
S
OH
R-BMBA
S
HS
SH
R-TNLA
S
R-DHLA
OH
S
O
R-BMOA
OH
OH
R-BMHA
O
Human Plasma Metabolites
 DHLA (Carlson et al 2008, Haj Yehia et al 2000)
 BMOA: 6,8-bis(methylthio)octanoic acid
 BNLA: bisnorLA or 3-(1,2-dithiolan-3-yl) propanoic acid
 BMHA: 4,6-bis(methylthio)hexanoic acid
 TNLA: tetranorlipoic acid
 BMBA: 2,4-bis(methylthio)butanoic acid
(Teichert & Preiss 2008, Krone 2002, Schupke et al 2001)
Racemic-Lipoic Acid in rat hepatocytes
 Microsomal oxidation of RLA & SLA via
endoplasmic reticulum, P450s, lysosomes is
non-stereospecific and preferentially oxidizes
the dithiolane ring to BLA. (Lang 1992)
 SLA is more extensively B-oxidized than RLA
despite the fact that RLA reacts with
CoA/Acyl CoA synthetase. (Lang 1992)
LA enantiomer metabolism in rat liver fractions
SLA
Enantiomeric ratios
RLA
Homogenate
Mitochondria
Supernatant
Cytosol
E.C.6.2.1.3.
Microsome
Racemic lipoic acid &
racemic-tetranorlipoic acid
in rat hepatocytes
• 50 µM LA perfused into rat liver yields
almost equimolar amounts of LA and
TNLA. (Muller 2002)
• rac-LA =38.6 +/-7.9 nmol/g (7.96 µg/g)
• rac-TNLA= 36.9 +/-8.9 nmol/g (5.5 µg/g)
Do Lipoic Acid Metabolites contribute
to the mechanisms of action?
 Best evidence so far is the 3 fold
volume of distribution of
radioactivity relative to the RLA.
(Krone 2002)
 Dithiolane ring intact. (Krone 2002)
Do Lipoic Acid Metabolites contribute
to the mechanisms of action?
 rac-LA & rac-TNLA equimolar in
liver but 3-5:1 LA: TNLA in plasma.
(Krone 2002, Schupke et al 2001)
Do Lipoic Acid Metabolites contribute
to the mechanisms of action?
 Covalently bound LA & metabolites
(lipoyl-Protein mixed disulfides, lipoyl
CoA, and lipoyl glucuronides)
intracellularly, in blood & urine.
 Preliminary observation that the longer
MRT in intestine yields more BMHA in
plasma. (Carlson et al 2008, unpublished)
Enantioselective metabolism
 SLA is reduced faster by cytosolic
enzymes and B-oxidized more
extensively than RLA. (Lang 1992)
 RLA favors formation of R-BNLA & R-
TNLA in plasma after RLA
administration. (Krone 2002, Biewenga et al 1997)
Enantioselective metabolism
 The reduced forms are instantly (bis)-
methylated in vivo and released to
plasma, rac-BNLA and rac-TNLA levels are
low. (Teichert & Preiss 2008)
 The alleged contribution of DHLA to the
antioxidant effect of LA must be
reconciled the rapid appearance of BMHA
and with its rapid bis-methylation to
BMOA. (Carlson et al 2008)
LA intracellular metabolite
questions & considerations
• Bis-methylation (in vitro models seem to
have lower thiol methyl transferase
activity than in vivo).
• S-oxidation although B-lipoic is oxidized
in vitro, no evidence in plasma,
thiosulfinates and thiosulfonates formed
subsequent to reduction.
Mechanistic Hierarchy shows Stereochemical
Preference for RLA at top
R-lipoic acid
Ca/ Calmodulin
KK Pathway
AMPK
anti-inflammatory
• NF-kB↓
• CAMs ↓
• cytokines ↓
• chemokines ↓
• iNOS ↓
insulin receptor
activation
activation of
PI3-K/Akt pathway
anti-atherosclerotic
• eNOS ↑
• CAMs ↓
• insulin sensitizing (AMPK,
PPARs)
Nr-f2-Keap-1
anti-apoptotic
• Bad ↓
• Bcl-2 ↑
• Bax ↓
• caspases ↓
Metabolism of rac-lipoic acid in vivo
control
LA
15m
LA
2h
phospho-Akt
Akt
actin
(loading control)
Effect of LA on Akt phosphorylation. LA
feeding has a striking effect on Akt
phosphorylation (the active form). This
suggests that LA has anti-insulin effects,
since insulin works through activating Akt
by phosphorylation.
State III respiration
12
10
8
6
4
2
0
control
LA 15m
LA 2h
Effect of LA on mitochondria respiration.
Mitochondria respiration was performed in
the presents of succinate (complex II
substrate) and ADP. There is a trend of
decrease mitochondria respiration 15
minutes following LA treatment.
Na-R-(+)-Lipoate Clinical Trials
 Oregon Health Science University
1200 mg rac-LA, RLA (free-acid) vs.
NaRLA in MS patients. matrix
metalloproteinase-9 (MMP-9) soluble
intercellular adhesion molecule-1
(sICAM-1) (Yadav et al 2008; study in progress)
 Mayo Clinic 1800 mg NaRLA study.
(Bharucha et al 2008; study to begin March 2008)
Summary & Future studies
 All future work should consider the
quantitative and qualitative similarities
& differences between RLA, SLA &
racemic-LA by testing all three.
 Due to disproportionation of the
enantiomers in vivo, the cells never
“see” a true racemic mixture.
Summary & Future studies
 Evidence to date indicates R-(+)-Lipoic
Acid is the eutomer of Lipoic Acid.
 Even with non-stereospecific reactions
it is easier achieve effective
concentrations of NaRLA with 25% the
dose of the racemate.
Future Studies
 in vitro experiments should use
concentrations between 1 and 250 uM.
 Time of exposure corresponding to Tmax of
15 min-2 hrs as well as down stream effects
after 3-24 hrs in human cell lines.
 Rat models should use PO doses 20-100
mg/kg PO to correlate to human PK.
Future Studies
 in vitro time exposures should be
brief.
 look for downstream effects for hours
to days.
 Chronic feeding experiments should
use salt forms of RLA, SLA and rac-LA.
Possible Stereoselective Targets
AMPK
Leptin & adiponectin
Calcium channels
ATP ases
C-reactive protein (CRP )
cell surface thiols
dimethylarginine dimethylaminohydrolase (DDAH)
Plasma membrane redox system (PMRS)
Nucleus: histone R-lipoylation or acetylation or
alteration of transcription factor at promoters
 PI3K downstream targets



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Possible Stereoselective Targets
Protein tyrosine phosphatases
protein kinase C (PKC-delta)
PGC-1 alpha & Beta
Cyclooxygenase-2 ( COX-2)
5 & 15-lipoxygenase (LOX)
Flavine-disulfide oxidoreductases (FDRs)
Protein Disulfide Isomerase (PDI)
PPAR a, λ
 PPAR-B/δ
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How far we’ve come in 57 years:
A triumph of technology
 In March 1951 Lester Reed first isolated 30 mg RLA
from ten tons of beef liver.
 In 1984 Heinz Ulrich paid thousands of Deutsche
Marks for 0.5 g RLA.
 In 2008, RLA is produced in 100 kg batches, with
greater than 99.0 % chemical purity & enantiomeric
excess with less than 2% residual polymer.
 Crystalline RLA is now isolated from water to
eliminate the risk of residual organic solvents. This
high quality material is now readily affordable and
available as a nutraceutical on the metric ton scale.
Collaborators + co-workers
Sarah J. Fischer
Karyn L. Young
Anthony R. Smith, Ph.D.
Derick Han, Ph.D.
Gerald Muench, Ph.D.
Vijayshree Yadav, M.D.
Adil Bharucha, M.D.
Heinz Ulrich, M.D.
Lester Packer, Ph.D.