paolo mainardi - · pdf filepaolo mainardi mincep, october 21, 2008 3 ... epilepsia. 1992...

Paolo MainardiMincep, October 21, 2008

History of AEDs in the US

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1850 1870 1890 1910 1930 1950 1970 1990 2010 2030

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N° of A

EDs

Bromides PhenobarbitalPhenytoin

Primidone

Ethosuximide Carbamazepine

Valproic acid

Gabapentin LamotrigineTopiramate Tiagabine

Levetiracetam Oxcarbazepine

Zonisamide

Clonazepam

Felbamate

The problem of refractory epilepsy has not been solved, despite the increasing number of new AEDs


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Novel mechanisms of action are needed to solve drug resistance

W. Löscher, I.E. Leppik . Critical re-evaluation of previous preclinical strategies for the discovery and the development ofnew antiepileptic drugs. Epilepsy Research 50 (2002) 17–20

“to solve this problem, ….a better hypothesis of the basic mec hanisms of pharmacoresistanceare needed”.

Anticonvulsive effect of Serotonin:

� 1956 Bonnycastle: DPH increases brain serotonin

� 1957 Bonnycastle: AEDs increases brain serotonin

� 1973 BogganWO et al. Antiepileptic effect of 5-HTP in animals

� 1973 Wenger GR et al. Decreasing in brain 5-HTP increases seizure in animals

� 1975 Maynert E et al. Decreasing in brain 5-HTP increases seizure in humans

� 1983 Lazarova M. Serotonin mediation of the protective effect of clonidine againstpentylenetetrazol-induced seizures in rats.


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� 1975: Truscot TC et al. Anticonvulsant effect of Fluoxetine in mice. Pharmacol Biochem Behav 3, 909 1975.

� 1992: Leander JD. Fluoxetine, a selective serotonin-uptake inhibitor, enhances theanticonvulsant effects of phenytoin, carbamazepine, and ameltolide. Epilepsia. 1992May-Jun;33(3):573-6.

� 1992: Pasini A, Tortorella A, Gale K. Anticonvulsant effect of intragranial fluoxetine.Brain Res. 1992 Oct 16;593(2):287-90.

� 1993: Prendiville S, Gale K. Anticonvulsant effect of fluoxetine on focally evoked limbicmotor seizures in rats. Epilepsia. 1993 Mar-Apr;34(2):381-4.

Anticonvulsive effect of SSRIs:


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Favale E, …, Mainardi P, …, Albano C. Anticonvulsant effect of fluoxetine in humans. Neurology. 1995Oct;45(10):1926-7

Favale E,…, Albano C. The anticonvulsant effect of citalopram as an indirect evidence of serotonergic impairment inhuman epileptogenesis. Seizure. 2003 Jul;12(5):316-8.

Cupello A, …, Albano C. Decrease of serotonin transporters in blood platelets after epileptic seizures. NeurochemRes. 2005 Apr;30(4):425-8.

Albano C, et al. Successful treatment of epilepsy with serotonin reuptake inhibitors: proposed mechanism.Neurochem Res. 2006 Apr;31(4):509-14. Epub 2006 May 13

Cupello A, …., Albano C. Epileptic seizures but not pseudoseizures are associated with decreased density of theserotonin transporter in blood platelet membranes. Neurochem Res. 2008 Nov;33(11):2263-8. Epub 2008 Apr 22.

In 1994 our group started clinical trials by adding SSRIs to AED therapy in drug-resistant patients.

As SSRIs we used both• fluoxetine • citalopram

Our data showed the anticonvulsant effect of SSRIs:

potentiation of the serotoninergic system helpsto control epileptic seizures.

Jobe, 2005: high doses of SSRIs are pro-convulsants, low doses are anti-convulsants


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Tryptophan, the only precursor of serotonin

� An essential amino acid� A large neutral amino acid (LNAA)� All LNAAs compete to the same carrier to cross BBB

The rate of Trp brain uptake depends on Trp/LNAAs plasmatic ratio

R=Trp/LNAAs

LNAAs: Tyr, Val, Met, Ile, Leu, Phe, Trp

Cughani, by PET, confirmedthat brain serotonin synthesis depends on brain Trp uptake

**

***


6 Low Trp levels in blood of epileptic patients:

• Marion JL, Bigot JC, Goas JY . Presse Med 1985;14(12):681–3• Ko FJ, Chiang CH, et al.. Ko Hsiung I Hsueh Ko Hsueh Tsa Chih 1993;9(3):131–42

Our own data:

� Lunardi G, Mainardi P, et al. Tryptophan and epilepsy. In: Recent Advances in Tryptophan Research (Eds G. Allegri Filippini and V. L. Costa) New York and London, Plenum Press, 1995: pp. 101–102

� Lunardi G, Mainardi P, et al.. Tryptophan and epilepsy. Adv Exp Med Biol. 1996;398

R=Trp/LNAAs

1/3 reduction in brain influx rate!

To increase plasmaticR=Trp/LNAAs

To increase brain Trp uptake

To increase brain serotonin synthesis

To increase seizure control

A new idea:Paolo Mainardi

Mincep, October 21, 20087

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In the late 80’s 14 million people were assuming oral tryptophan to:

Clinical studies on oral Trp administration in more severe diseases, i.e. epilepsy and depression, carried out controversial results.


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• DBA/2J,• Phe increases seizures,• Trp decreases seizures

• O'Grady mice, • 5-HTP decreases seizures

• electrical stimulation of thedorsal hippocampus of rats.

• systemic injections of 5-HTP• a great reduction in the electrographic seizure activity

• GEPR.• varied doses of 5-HTP ip + fluox. (12.5, 25, 50 + 15 mg/kg)• the severity of audiogenic seizures was decreased dose-dependently

Trp in experimental models

• GEPR-9s• 15 mg/kg fluox. reduced seizures in 33% of rats• 12.5 mg/Kg 5-HTP no anticonvulsant effect• Together decrease seizures in 83% of rats

I.p. or i.v. Trp administration is effective, while oral administration, is not.

Conclusion:


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� Free amino acids are not fully absorbed� In the GI system LNAAs compete for the same carrier as in BBB

Free amino acids vs protein intestinal absorption

� Peptides arising from protein demolition are freely adsorbed by GI system� Enzymatic proteolysis continue in the blood � Whey proteins produce a higher postprandial level of plasma amino acids compared to casein (Hall WL,et al. Br J Nutr 2003;89:239-248)

Frenhani PB, Burini RC. Mechanism of action and control in the digestion of proteins and peptides in humans.Arq Gastroenterol. 1999 Jul-Sep;36(3):139-47

• Whey proteins do not coagulate under acidic conditions

• They are considered to be “fast proteins”

• They reach the jejunum rapidly after ingestion


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• Alpha-lactalbumin (ALAC) is a whey protein• naturally occurring in human milk (2 g/l)• high Trp/LNAAs ratio: contains 4.8% trp.• fully adsorbed by gastro-intestinal system as short peptides• increases the Trp/LNAAs ratio up to 48% compared to casein

produced by Davisco Foods Intl as ultrapure protein


Serplus (Giofarma Srl) is a food supplementcontaining 0.75 g/tablet of ultra pure ALAC

ALAC is supplied as pure protein by Davisco Foods

12 Paolo MainardiMincep, October 21, 2008

SERPLUS TRIAL pt 1: sex F, age 41, PC II gen

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SERPLUS TRIAL pt 2: sex M, a ge 39, PC II gen

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SERPLUS TRIAL pt 3: sex F, a ge 51, PC II gen

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SERPLUS TRIAL pt 9: sex M, a ge 53, Mioclonic

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Single oral administration

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Results obtained by De Sarro, Confidential data

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Audiogenic mice: GEPR-9s

N=5 animals per dose and time group

ALAC on experimental models

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5 days chronic administration

12 days chronic administration


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ALAC in DBA/2 mice

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N=10 animals per dose and time group

MES test

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MES test:5 days repeated ALAC administrations


ALAC on experimental modelsPaolo Mainardi


Animal groupDose (mg/kg)

Pilocarpine (mg/kg)250 270 300

Control 10/10 10/10 10/10

CBZ 20 10/10 10/10 10/10

CBZ 40 10/10 10/10 10/10

CBZ 80 10/10 10/10 10/10

ALAC 125 4/10 6/10 9/10

ALAC 250 0/10 3/10 9/10

ALAC 500 0/10 8/10 10/10

ALAC on seizures induced by Pilocarpine

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ALAC 15 days

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Acute seizuresChronic seizures




Chronic administration of ALAC increases Trp/LNAAs plasmatic ratio.

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R trp/LNAAs

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ALACCAS

Our own data Fuertè et al.

Our initial hypothesis has been confirmed: an increase in Trp/LNAAs ratio permits to improve the seizure control.



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• Only 1% Trp is uptaked in the brain• Cortisol increases the liver catabolism of Trp• Cortisol levels are elevated in epileptic and depressed pts

Markus CR et al. The bovine protein alpha-lactalbumin increases the plasma ratio of tryptophan to the other large neutral amino acids, and in vulnerable subjects aises brain serotonin activity, reduces cortisol concentration, and improves mood understress.Am J Clin Nutr. 2000 Jun;71(6):1536-44.

• Trp (i.v.) decreases other LNAAs, increasing protein synthesis

Is the anticonvulsant effect due toTrp/LNAAs increase only?

• ALAC can decrease cortisol levels


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Diet and epilepsy: Ketogenic diet

Is the antiepileptic effect of the ketogenic diet d ue to ketones?Mainardi P, Albano C. Med Hypotheses. 2008;70(3):536 -9.

APR: Acute phase reactionStig Bengmark , Acute and ‘‘chronic’’ phase reaction - a mother of disease. Clinical Nutrition (2004) 23, 1256–1266

NEIntestinal stimuli:

• Starvation….... (Biblical times)

• Fats…….. (Ketogenic diet)

• Medium Chain Triglycerids… (MCT diet)

• Tryptophan and or Serotonin…. (SSRIs, AEDs, ALAC )

Brain synthesis:• Leptin• NPY “endogenous anticonvulsant”• Norepinephrine

All of them are reported to be anticonvulsant when injected in the brain


NE

Starvation

Fats

MC

Ts

Tryptophan

Serotonin

Intestinal stimuli

NPY “endogenous anticonvulsant” Leptin Norepinephrine

•El Bahh B et al. The anti-epileptic actions of neuropeptide Y in the hippocampus are mediated by Y and not Y receptors. Eur J Neurosci. 2005 Sep;22(6):1417-30.•Richichi C. et al. Anticonvulsant and antiepileptogenic effects mediated by adeno-associated virus vector neuropeptide Y expression in the rat hippocampus. Neurosci. 2004 Mar 24;24(12):3051-9.•Woldbye DP et al. Neuropeptide Y inhibits in vitro epileptiform activity in the entorhinal cortex of mice. Neurosci Lett. 2002 Nov 22;333(2):127-30.•Sabrina Diano1,2 and Tamas L. Horvath1,2,3 Anticonvulsant effects of leptin in epilepsy J. Clin. Invest. 118:26–28 (2008).•Lin Xu, et al Leptin inhibits 4-aminopyridine– and pentylenetetrazole-induced seizures and AMPAR-mediated synaptic transmission in rodents. J. Clin. Invest.. 118:272

Norepinephrine, Leptin and NPY are reported to be anticonvulsant when injected in the brainBrain synthesis


Jun YAMADA, et al. Involvement of Leptin in Hypophagia Induced by theSerotonin Precursor 5-Hydroxytryptophan (5-HTP) in Mice. Biol. Pharm. Bull.29(3) 557—559 (2006)

1) 5-HTP, i.p. injected, increases serum leptin

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2) Leptin increases Insulin secretion

Ceddia RB, et al. Modulation of insulin secretion by leptin, General PharmacologyVolume 32, Issue 2, , February 1999, Pages 233-237.

3) Fats (KD) increases insulin secretion

Valls E, et al. Modifications of insulin and growth hormone after medium chain triglycerides ingestion An Esp Pediatr. 1978 Oct;11(10):675-82

C. Cruciani-Guglielmacci, et al. Early changes in insulin secretion and action induced by high-fat diet are related to a decreased sympathetic tone Am J Physiol Endocrinol Metab 288

4) MCT diet increases insulin secretion


Insulin levels

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ALAC, orally administered, has the same effect as Trp (i.p. or I.v. injected)


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Martillotti J, Weinshenker D, Liles LC, Eagles DA. A ketogenic diet and knockout of the norepinephrine transporter both reduce seizure severity in mice. Epilepsy Res. 2006 Mar;68(3):207-11.

• Augmentation of norepinephrine increases anticonvulsant effect of KD

Ahern TH, Javors MA, Eagles DA, Martillotti J, Mitchell HA, Liles LC, WeinshenkerD. The effects of chronic norepinephrine transporter inactivation on seizuresusceptibility in mice. Neuropsychopharmacology. 2006 Apr;31(4):730-8.

• Norepinephrine has both anticonvulsant and antidepressive effects

Fischer W, Müller M. Pharmacological modulation of central monoaminergic systems and influence on the anticonvulsant effectiveness of standard antiepileptics in maximal electroshock seizure. Biomed Biochim Acta. 1988;47(7):631-45.

• Pharmacological stimulation of noradrenergic system increases anticonvulsant activity of drugs. Conversely pharmacological suppression reduces the activity of AEDs.

The role of catecholamine in epilepsy

In agreement with anticonvulsive and antidepressive effect of SSNRIs


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Trp/LNAAs plasmatic ratio was found lower in:epilepsy, depression, MS, ALS,…

Leptin was found lower in: epilepsy, depression, SM,….

Further studies are needed to better understand the neuroendocrine response of ALAC

Nayanah Siva. Can ketogenic diet slow progression of ALS? The Lancet Neurology, Volume 5, Issue 6, June 2006, Page 476Samuel T. Henderson, Ingrid Van der Auwera, Stefaan Wera and Fred Van Leuven. A ketogenic diet reduces amyloid beta 40 and 42 in a mouse model of Alzheimer’s disease. Alzheimer's and Dementia, Volume 2, Issue 3, Supplement 1, July 2006, Pages S637-S638Patricia Murphy, Sergei Likhodii, Kirk Nylen and W.M. Burnham. The antidepressant properties of the ketogenic diet. Biological Psychiatry, Volume 56, Issue 12, 15 December 2004, Pages 981-983Gasior M, Rogawski MA, Hartman AL. Neuroprotective and disease-modifying effects of the ketogenic diet. Behav Pharmacol. 2006 Sep;17(5-6):431-9

Ketogenic diet has been tested in other neurological diseases, asdepression, ALS, MS, Alzheimer, …



Possible future perspective and co-operation:

• ALAC controlled clinical trials• Studies on ALAC neuroendocrine response• …. other proposals are welcome!

Keep in touch !

[email protected]

Thank you for your patienceand your kind attention ☺

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paolo mainardi - · pdf filepaolo mainardi mincep, october 21, 2008 3 ... epilepsia. 1992...

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