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 S enantiomer 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

H S * S R-(+)-ALA [1200-22-2] Oxidized O O S S OH H Reduced SH SH S-(-)-ALA [1077-27-6] * SH HS H R-(-)-DHLA [119365-69-4] OH H S-(+)-DHLA [98441-85-1] O O OH OH

Number & Percentage of studies dealing with racemic-LA versus the LA enantiomers as of March, 2008.

 Rac-LA alone 2466  All enantiomer 109 4.3%  RLA alone 46 1.8%  SLA alone 0 0%  RLA vs. SLA 31 1.2%  RLA vs. rac-LA 2 0.08%  RLA vs. rac-LA vs. SLA 30 1.2%

Contribution of Stereochemistry to the Pharmacodynamics

 Eutomer: the pharmacologically preferred enantiomer  Distomer: inhibitor the pharmacologically non preferred enantiomer; isomeric ballast vs. anti-metabolite/competitive

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 re resolution 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 tert butylhydroperoxide 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

Kiemer & Diesel 2008 used with permission of authors & Taylor & Francis Publishing

a-lipoic acid (LA) insulin receptor activation activation of PI3-K/Akt pathway anti-inflammatory

• NF-kB

↓

• CAMs

↓

• cytokines

↓

• chemokines

↓

• iNOS

↓ anti-atherosclerotic

• eNOS

↑

• CAMs

↓

• insulin sensitizing (AMPK, PPARs)

anti-apoptotic

• Bad

↓

• Bcl-2

↑

• Bax

↓ ↓

• caspase activation

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 5.8 µ g hr/mL) up to 48 h. (28.8 µ g/mL[144 µ M] & increased nuclear levels of Nrf2 & ARE transcriptional factor binding activity at 12 h and remained elevated for (Suh et al 2004)

Evidence for the primary mechanism of action of LA being an inducible stress response

 100 µ M 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 µ g/mL(200 µ M) rac-LA (24hrs) increased expression of PGC-1 α . (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. ( Ogborne et al 2005, Oksala et al 2006, 2007)

“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?

20 19 18 17 16 15 5 4 3 2 1 0 14 13 12 11 10 9 8 7 6 Comparison of pharmacokinetic profiles of 100 mg/kg R-lipoic acid and racemic lipoic acid in male Wistar rats (PO solution) Trivedi Krone 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 4000

10 mg 30 mg 100 mg

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).

6 1 i .

2 / a 0 L 9 m a

(

[ n R ) L A ] (

Human Pharmacokinetics of R-(+)-Lipoic acid as Sodium R-(+)-Lipoate 600 mg d i c A ) L i / c m o All Subjects (n=12) C

max

T

max

mean 14.2 µg/mL 15 min 10.6 33.8 µg/mL 10-20 min range

AUC

7.36 µg *hr/mL 3.93 13.4 µg *hr/mL i L a m s a l P 8 8 6 6 4 4 2 2 0

R-Lipoic Acid from baseline to Cmax 16000 14000 12000 Baseline RLA= Not Detected to 200 ng/mL 1000mg RLA (free-acid form) to1000 ng/mL 600mg rac-LA=5000 ng/mL 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 (not labeled) control LA 15m LA 2h

Method

Protein-SH + maleimide-biotin albumin Protein-S-maleimide-biotin 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 Liver protein disulfide levels

control LA 15m LA 2h

Method

Protein-SH +

S S

NEM Protein-S-NEM

S S

+ DTT Protein-S-NEM

SH SH

+ maleimide-biotin Protein-S-NEM

S S

maleimide-biotin maleimide-biotin This gel shows no dramatic changes. Western blot

Cmax comparing 600 mg of rac-LA from various studies versus 600 mg Na-RLA & RLA Tromethamine salt 18 16 14 12 10

Na-RALA (Carlson et al 2007) RLA tromethamine salt (Krone 2002) Contolled Release LA (Evans et al 2002) rac-LA (Evans et al 2002) rac-LA (Teichert et al 2003) rac-LA (Krone 2002) rac-LA (Breithaupt-Grogler et al 1999) rac-LA (Gleiter et al 1996) rac-LA (Rosak et al 1996) rac-LA (Preiss et al 1996) rac-LA (Chen et al 2005)

8 6 4 2 0

1 0 3 2 4 7 6 5 8 AUC comparing 600 mg of rac-LA from various studies versus 600 mg of Controlled Release LA, 600 mg Na-RALA & RLA Tromethamine salt

Na-RALA (Carlson et al 2007) RLA tromethamine salt (Krone 2002) Controlled Release LA (Evans et al 2002) rac-LA (Evans et al 2002) rac-LA (Teichert et al 2003) rac-LA (Krone 2002) rac-LA (Gleiter et al 1996) rac-LA (Rosak et al 1996) rac-LA (Preiss et al 1996) rac-LA (Chen et al 2005)

1 16 14 12 10 8 6 4 2 2008 OCC Study: Comparison PK profiles: rac-LA, RLA & SLA in 3 subjects (600 mg) RLA SLA rac-LA 0 10 20 30 40

Time (min)

50 60 70 80 90 3-way crossover in humans indicates possible stereospecific transport This is the first human PK data with SLA

Single Subject 3 x 600mg Na R-(+)-Lipoate 25 20 15 10 5 C T

max max AUC

21.9 µg/mL 45.0 min 17.5 µg*min/mL 0 10 20 30 40 50 60 70 80 90 100 110 120 130 Time (min)

Comparison of Pharmacokinetic values for racemic-LA IV (20 min infusion; 30mg/min) and NaRLA (3x600 mg) PO doses

Dose mg NaRLA 3 x 600 PO Rac-LA 1 x 600 IV Cmax µg/mL AUC µg hr/mL 21.9

17.5

Tmax min 45.0

28.57

12.3

18.6

T1/2 min 17.4 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 1 st (Hermann & Niebch 1997) pass preferring SLA.  Hepatocytes never “see” a true racemic mixture but “see” SLA with enantiomeric excess of ~60%.

 Renal stereoselective 1 st 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 B-oxidation O B-oxidation S S O R-BLA 2-fold S O S B-oxidation TMT S S R-BLAS R-TNLA O OH GSH Red TRX Red LipD H 3 C S S CH3 OH R-BMBA O microsomes P450 NADPH oxidases O OH microsomes P450 H 3 C S OH NADPH oxidases S RLA S SH HS GSH Red TRX Red LipD O OH R-DHLA GSH Red TRX Red LipD S CH3 R-BMOA S S O O O S O S OH TMT S R-BLAS R-BLA S 1-fold B-oxidation R-BNLA O OH GSH Red TRX Red LipD OH O H 3 C S S CH3 R-BMHA O OH OH O B-oxidation B-oxidation TMT OH

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

RLA SLA 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 low.

in vivo

(Teichert & Preiss 2008) and released to plasma, rac-BNLA and rac-TNLA levels are  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 insulin receptor activation AMPK anti-inflammatory

• NF- k

B ↓

• CAMs

↓

• cytokines

↓

• chemokines

↓

• iNOS

↓ activation of PI3-K/Akt pathway anti-atherosclerotic

• eNOS

↑

• CAMs

PPARs) ↓

• insulin sensitizing (AMPK,

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) 12 10 8 6 4 2 0

control LA 15m LA 2h

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.

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

brief. time exposures should be  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

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

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.