Pharmacokinetic-Based Design of New CNS-Active Analogs of

Valproic Acid Derivatives with Improved Potency and Low-

Toxicity

Jakob Avi ShimshoniJakob Avi Shimshoni

Supervisors: Prof. Meir Bialer & Prof. Boris YagenSupervisors: Prof. Meir Bialer & Prof. Boris Yagen

OutlineOutline

Common features of epilepsy and bipolar disorder

Part I

Epilepsy and antiepileptic drugsEpilepsy and antiepileptic drugs

Valproic acid and its major side-effectsValproic acid and its major side-effects

Study objectives and resultsStudy objectives and results

Part II

Bipolar disorder and drug treatment thereof Bipolar disorder and drug treatment thereof

Molecular targets of mood-stabilizers and their Molecular targets of mood-stabilizers and their effect on growth cone behavioreffect on growth cone behavior

Study objectives and resultsStudy objectives and results

Common Features of Epilepsy & Bipolar Disorder

Both disorders respond to several identical drugs :

Gradual progression in intensity and frequency of epileptic seizures and manic-depressive episodes

20-40% of bipolar and epileptic patients are resistant to current drug-therapy

Amann et al, Epilepsia, 2005

N

C NH2O

Valproic AcidCarbamazepine Lamotrigine

Common neurological condition occurring in about 1% of the global population

Characterized by periodic and unpredictable occurrence of seizures, due to disordered, synchronous firing of a population of brain neurons

Classification into partial seizure and generalized seizure

Part I: Epilepsy

Antiepileptic Drugs (AEDs)

Old DrugsOld Drugs New DrugsNew Drugs

CarbamazepineCarbamazepine

PhenobarbitalPhenobarbital

PhenytoinPhenytoin

Valproic AcidValproic Acid

FelbamateFelbamate

GabapentinGabapentin

LamotrigineLamotrigine

LevetiracetamLevetiracetam

OxcarbazepineOxcarbazepine

PregabalinPregabalin

RufinamideRufinamide

StiripentolStiripentol

TiagabineTiagabine

TopiramateTopiramate

VigabatrinVigabatrin

ZonisamideZonisamide

Valproic Acid (VPA)

COOH

Efficient in many types of epilepsy

Migraine prophylaxis

Bipolar disorder

Major rare side effects:

hepatotoxicity & teratogenicity

VPA-Induced Fatal HepatotoxicityVPA-Induced Fatal Hepatotoxicity Worldwide till 1999:Worldwide till 1999:

179 cases of fatal 179 cases of fatal hepatotoxicityhepatotoxicity

Risk Factors of Fatal Hepatic FailureRisk Factors of Fatal Hepatic Failure

Children under the age of two

Polytherapy

Metabolic disturbances and

liver disease

Chang et al, Drug Metab Rev, 2006Chang et al, Drug Metab Rev, 2006

COOH COOHCYP2A6CYP2A6CYP2C9CYP2C9

-oxidation-oxidation -oxidation-oxidation

COOHCOOH

VPAVPA

2-ene 2-ene VPAVPA

4-ene VPA4-ene VPA

2,4-diene 2,4-diene VPAVPA

CYP450CYP450

ffmm= 0.5-1%= 0.5-1%

COOH COOH

VPAVPA

2,2,3,3-tetramethylcyclopropane- carboxylic acid (TMCA)

Quaternary Carbons & Acylurea containing compounds & Hepatotoxicity

CONHCONH2

3,3-dimethylbutanoylurea (DBU)

VPA-Induced Teratogenicity

Finnell et al, Epilepsia, 2003; Sankar, Acta Neurol Scan, 2007Finnell et al, Epilepsia, 2003; Sankar, Acta Neurol Scan, 2007

Major malformations associated VPA therapy:

abnormalities of the skeleton, CNS,

cardiovascular and urogenital system

Neural Tube Defects (NTD):

10-20 fold increased risk

Mechanism of VPA-Induced Teratogenicity

Inhibition of folate metabolism

HDAC-inhibition

Inhibition of neuroepithelial proliferation

Formation of cytotoxic, teratogenic metabolites:

2,4-diene-VPA, 4-ene-VPA

VPA Constitutional Isomers, Amide derivatives and Teratogenicity

Radatz et alRadatz et al, Epilepsy Res, 1998; Isoherranen et alIsoherranen et al, Epilepsia, 2002

VPA

ValpromideValpromide

COOHCOOH

Valnoctic Acid

CONH2COOH

Propylisopropyl acetic Acid

AEDs Containing Urea Moiety in Their Structure

NH

HN OO

H3C

O

NH

HN

O

O

N

CO NH2

CH2CONHCONH2 CHCONHCONH2

C2H5

PhenobarbitalPhenobarbital PhenytoinPhenytoin CarbamazepineCarbamazepine

PhenacemidePhenacemide PheneturidePheneturide

CONHCONH2

TMCUTMCU

Study Objectives: Part I

Design and synthesis of urea derivatives of VPA constitutional isomers and homologs

Evaluation of anticonvulsant activity, neurotoxicity and teratogenicity of the above compounds

PK study of the most potent and safe candidate among the urea derivatives

Maximal Electroshock Seizures (MES)

Animal Models of Epilepsy

sc Metrazole (scMet)

Identifies drugs effective against generalized seizures; seizure spread inhibition

Identifies drugs effective against absence seizures; increase in seizure threshold

Mice : Rotated rod Rats : Positional sense test

6Hz psychomotor seizure test

Identifies drugs effective against therapy- resistant epilepsy

Neurotoxicity

Synthesis of Urea Derivatives of VPA Constitutional Isomers

R1 C

O

OHLDA, THF

R1 C

O-

O-

2Li+

R1 C

O

OH

R2

R1 C

O

Cl

R2

UREA, ACN R1 C

O

NHCONH2

R2

R-I

SOCl2

CONHCONH2

VPU

CONHCONH2

VCU

CONHCONH2

DIU

CONHCONH2

PIU

CONHCONH2

R-PIU

CONHCONH2

S-PIU

CONHCONH2

OCU

Shimshoni et al et al, J Med Chem, 2007

*

Shimshoni et alShimshoni et al, J Med Chem, 2007

Anticonvulsant Activity and Toxicity

CONHCONH2

PIU

CONHCONH2

VCU

CONHCONH2

DIU

Significantly different from the corresponding amide (p<0.05); $ significantly different from the corresponding

enantiomer (p<0.05)

CompoundMES-ED50

( mg/kg)

scMet-ED50

(mg/kg)

Neurotoxicity-TD50

(mg/kg)

PI(MES)

PI(scMet)

VPA485646 7841.61.2

VPU 5477 2324.33.0

VCU 2414*9746.9

DIU 33*16*561.73.5

(R,S)-PIU 16*45 955.92.1

(R)-PIU36$22$1243.45.6

(S)-PIU 18*371186.53.2

OCU >300>300>300--

*CONH2

CONH2 CONH2

CompoundED50 at 32mA

(mg/kg)

ED50at 44mA

(mg/kg)

VPA126 (PI=3.2) 310 (PI=1.3)

Levetiracetam191089

VPU 58 (PI<1.7)105 (PI<0)

VCU 21* (PI<4.7)48* (PI<2.1)

DIU 43*(PI<2.3)49* (PI<2)

(R,S)-PIU 42 (PI<2.4)71 (PI<1.4)

(R)-PIU 43 (PI=2.7)56# (PI=2.1)

(S)-PIU 46* (PI<6.5)75 (PI<4)

Anticonvulsant Activity in Mice 6Hz Model

Shimshoni et alShimshoni et al, J Med Chem, 2007

Significantly different from the corresponding amide (p<0.05); $ significantly different from the corresponding enantiomer (p<0.05)

CONHCONH2

VCU

CONHCONH2

DIU

CONHCONH2

S-PIU

CONH2 CONH2

CONH2

DrugMES-ED50

(mg/kg)

scMet- ED50

(mg/kg)

Neurotoxicity-TD50

(mg/kg)

PI(MES)

PI(scMet)

VPA4856467841.61.2

PVU69302283.37.6

IVU>25083300<TD50<500-3.6<PI<6

DBU64261432.25.5

Anticonvulsant Activity and Toxicity of VPU Homologs

Shimshoni et alShimshoni et al, submitted to Epilepsia, 2008

CONHCONH2

CONHCONH2

CONHCONH2

COOH

TestDBU’s ED50 or TD50

(mg/kg)

PI (DBU)VPA’s ED50 or TD50

(mg/kg)

PI (VPA)

6Hz (32mA) 803126 3.2

6Hz (44mA)1331.8310 1.3

Toxicity244-398-

Treatment GroupaDosemg/kg

(mmol/kg)

No. Litters No. of Live Fetuses

Exencephaly%

Control25% CEL151880

VPA 452 (2.7)1314129.1*

VPA301 (1,8)121331.5

VPU261(1.8)108013.7*

VPU167(0.9)101150.9

VCU205 (1.1)10752.7

(R)-PIU335 (1.8)95215.4*#

(R)-PIU205 (1.1)91040

(S)-PIU335 (1.8)101235.7*

(S)-PIU205 (1.1)101240.8

Teratogenicity of Acylurea Isomers of VPU in SWV Mice

Shimshoni et alShimshoni et al, J Med Chem, 2007

Significantly different from control (p<0.05); # significantly different from the corresponding enantiomer (p<0.05)

Treatment GroupDosemg/kg

(mmol/kg)

No. of Litters

No. of Live Fetuses

Exencephaly%

Control25% CEL151880

VPA452 (2.7)1314129.1*

IVU520 (3.6)121350

PVU520 (3.6)131800.6

DBU671 (3.6)10981

DBU502 (2.7)121560.6

Shimshoni et alShimshoni et al, submitted to Epilepsia, 2008

Teratogenicity of Acylurea Homologs of VPU in SWV Mice

Significantly different from control (p<0.05)

PK Profile of DBU in Rats (10mg/kg, i.v.)

CL (L/h•kg) 0.12 0.24 0.36

Vss (L/kg) 0.78 0.87 0.86

t1/2 (h) 4.5 4.0 1.6

MRT (h) 6.5 3.5 2.5

fe (%) 2.4 1.8 6.3

DBU VPA TMCU

Shimshoni et alShimshoni et al, submitted to Epilepsia, 2008

0.0

5.0

10.0

15.0

20.0

25.0

0 5 10 15 20 25

Time (h)

Pla

sm

a C

on

c. (

mg

\L) CONHCONH2

Conclusions: Part I

1. Urea derivatives of VPA constitutional isomers exhibited potent and broad anticonvulsant activity

2. PIU exhibited enantioselective activity (S-PIU was more potent in the MES, whereas R-PIU was more potent in the scMet and 6Hz tests)

3. Even at doses 3 times larger than their ED50 values, PIU, VCU and VPU were non teratogenic

4. PIU enantiomers demonstrated enantioselective teratogenicity (R-PIU was more teratogenic than S-PIU)

5 .Homologs of VPU with 2-3 carbon atoms less, but containing quaternary carbon (DBU and PVU) retain high anticonvulsant activity and are non-teratogenic

6 .Based on the pharmacokinetic study in rats, DBU’s half-life was in a similar magnitude as VPA

7 .VCU, PIU and DBU have the potential to become antiepileptic drugs second generation to VPA

Conclusions: Part I

Part II: Bipolar Disorder (BD)

BD is common, affecting approximately 1-2% of the population

BD is characterized by unpredictable swings in mood from mania to depression

Manic episodes emerge gradually and last as long as several months to years when untreated

Frequent age of onset: 20-30 years

Belmaker, N Engl J Med, 2004

Clinical Features

ManiaDepression

Moodelevated, labiledepressed, miserable

Talkfast, flight of ideasslow, monotonous

Energyexcessive, increased psychomotor activity

lacking energy, apathy

Ideationgrandiose, self confidentguilt, unworthiness

Physicalinsomnia, weight lossfatigue

Behaviordisinhibition, excessive spending

retardation or agitation, poverty of movements

Belmaker, N Engl J Med, 2004

FDA Approved Drug Treatments for BD

ManiaMania MaintenanceMaintenance

AntipsychoticsAntipsychotics

LithiumLithium

CarbamazepineCarbamazepine

Valproic AcidValproic Acid

LithiumLithium

LamotrigineLamotrigine

OlanzapineOlanzapine

AripiprazoleAripiprazole

((atypical antipsychotic)

DepressionDepression

Olanzapine (3mg) Olanzapine (3mg) +

Fluoxetine (25mg) Fluoxetine (25mg)

(Symbyax®)(Symbyax®)

Ketter et al, Psychopharm Bull, 2006

Berridge et al, Cell, 1989; Silverstone et al, Bipolar Disorder, 2005

diacylglycerol

PLC

Inositol

IP IP2

Inositol synthaseIno-1

Glucose-6-phosphate

IMPase

BD & the Inositol Depletion Theory

Mania:

Inositol

Depression:

Inositol

VPA

Li+

Neurites elongate by growth at their distal end called the growth cone

Growth cones integrates external signals and translate them into changes in the rate and direction of growth

The Effect of Mood Stabilizers on Growth Cone Behavior

Harwood et al, Clin Neuroscience Res, 2004

The Effect of Mood Stabilizers on Growth Cone Behavior

VPA, CBZ & Li+

Common mechanism :growth cone spreading via inositol depletion

Antipsychotics and Antidepressant have no growth cone spreading effect

The spread of growth cones provides a cell-based assay that may be utilized as a screening approach for mood stabilizing properties

Williams et al, Nature, 2002

Inositol

Study Objectives: Part II

Evaluate the effect of VPA constitutional isomers, cyclopropyl analogs for mood stabilizing properties

Evaluate the inositol depleting activity of the aforementioned compounds

VPA Constitutional Isomers, Cyclopropyl Analogs & their Corresponding Amide

Shimshoni et al, Mol Pharmacol, 2007

COOH

COOH

COOH COOH COOH

COOH CONH2 CONHCH3 CONHCONH2

COOH

CONH2 CONH2 CONH2 CONH2

VPA

4-ene-VPA 4-yne-VPA

PIA DIA VCA

VPD PID DID VCD

TMCA TMCD MTMCD TMCU

Growth Cone-Based Assay: Dorsal Root Ganglion (DRG)

DRG are composed of several thousand cell bodies of somatosensory neurons

Rat DRG showing neuron outgrowth (x20)

a. Control

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Spread (um2) Spread (um2) Spread (um2)

Shimshoni et al, Mol Pharmacol, 2007

Growth Cone Spreading Effect of Aliphatic VPA Constitutional Isomers

%

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b. 3mM TMCD

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Growth Cone Spreading Effect of VPD Constitutional Isomers, Cyclic VPA Analogs &

their Amides

Shimshoni et al, Mol Pharmacol, 2007

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Shimshoni et al, Mol Pharmacol, 2007

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0

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trol

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InsP

3 l

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* * *

Conclusions : Part II

1. PIA and DIA, the constitutional isomers of VPA, as well as the amide of cyclic VPA analog, MTMCD are more effective than VPA in increasing growth cone spreading via inositol depletion mechanism

2. Anticonvulsant potency and growth cone spreading effect of the above compounds are poorly correlated, suggesting a different mechanisms of their action

3. PIA, DIA and MTMCD have a potential as new antibipolar drugs

1. Shimshoni JA, Dalton EM, Jenkins A, Eyal S, Ewan K, Williams RSB, Pessah N, Yagen B, Harwood AJ, Bialer M. The effects of CNS-active valproic acid constitutional isomers, cyclopropyl analogues and amide derivatives on neuronal growth cone behaviour. Mol Pharmacol, 2007, 71: 884-92

2. Shimshoni JA, Bialer M, Wlodarczyk B, Finnell RH, Yagen B. Potent anticonvulsant urea derivatives of constitutional isomers of valproic acid. J Med Chem, 2007, 50: 6419-6427

3. Shimshoni JA, Bialer M, Yagen B. Synthesis and anticonvulsant activity of aromatic tetramethylcyclopropanecarboxamide aromatic derivatives. Submitted to Bioorg & Med Chem, 2008

4. Shimshoni JA, Yagen B, Pessah N, Wlodarczyk, Finnell, Bialer M. Anticonvulsant profile and teratogenicity of 3,3-dimethylbutanoylurea: a potential for a second generation drug to valproic acid. Submitted to Epilepsia, 2008

List of Publications

Acknowledgements

Prof. Meir Bialer & Prof. Boris YagenProf. Meir Bialer & Prof. Boris Yagen

Prof. Adrian HarwoodProf. Adrian Harwood

Dr Ken EwanDr Ken Ewan

Dr Robin WilliamsDr Robin Williams

Miss Emma DaltonMiss Emma Dalton

Dan KaufmannDan Kaufmann

Dorit MimrodDorit Mimrod

Neta PessahNeta Pessah

Naama HenNaama Hen

Idit AchachIdit Achach

Cardiff University Texas University

Prof. Richard H. FinnellProf. Richard H. Finnell

Dr Bogdan WlodarczykDr Bogdan Wlodarczyk

Lab Members