Alternative models to study Cannabidiol and Dravet Syndrome

Prof. Fábio Klamt, PhDLaboratory of Cellular Biochemistry/UFRGS

Doencas Neurodegenerativas (# 46698/2014-8)

MJFF – RRIA 2014

Everett’s story

Everett was born on January 13, 2012. He left the hospital a normal baby boy.Everett continued to develop normally and had no medical issues for approximatelythe first five months of his life. On June 7, 2012, Everett had a seizure in hismother’s arms.

He was diagnosed with a febrile seizure and we were sent home with antibiotics foran ear infection. Later that night, Everett had another seizure lasting 30 minutesbefore being stopped at the emergency room by emergency anti epilepticmedication.

Unfortunately, this was not an isolated incident and Everett’s seizures continued.From June 2012 until November 2012, Everett had four hospital stays, fiveemergency room visits, underwent anesthesia four times and was placed on antiepileptic medication that intensified his daily seizures.

In November 2012, Everett experienced a status seizure lasting over one hour. Hewas unable to breathe on his own and was intubated and placed in ICU for anumber of days and received the Dravet syndrome diagnosis.

We began to deal with the reality that our son was always going to live withseizures.

Shortly after his diagnosis, Everett was placed on clobazam, valproic acid andstiripentol. Since being placed on these drugs in November 2012, Everett has gonefrom having daily convulsive seizures to having rare convulsive seizures.

Today Everett attends an Early Learning Program four days per week andabsolutely loves his teachers, therapists, classmates and nurse.

Dravet Syndrome

- Devastating infantile-onset epilepsy syndrome

- Incidence: 1:22.000 / 10-20% mortality

- Seizures begin at 6 months classically with recurrent febrile status epilepticus

- Development is normal until aged 1–2 years, despite frequent, prolonged seizures

- 80% of patients have mutations of the sodiumchannel α-1 subunit gene, SCN1A, whichencodes the voltage-gated channel Nav1.1,predominantly expressed in GABAergicInterneurons, influencing directly the actionpotential and excitatory control over pyramidal neurons

- Most of patients present treatment-resistant epilepsy (TRE)

- There are currently no U.S. Food and Drug Administration (FDA)-approved therapies for TRE

- Cannabis-based therapies have beenused to treat epilepsy for millennia

- Only in the last few years we havebegun to collect data adequatelypowered by placebo-controlledrandomized trials

Dr. Raphael Mechoulam

Cannabidiol (CBD)

Evaluation of cannabidiol from a toxicological point of view…

Morphologies of SH-SY5Y cells; the proliferative and differentiated phenotypes.

Immunohistochemistry images of neural markers in SH-SY5Y cells.

β‐actin

NSE

NeuN

TH

Nestin

0          4          7          10     Days of differentiation

Mr(K)

42‐

177‐62‐

45‐

46‐

Lopes et al. Brain Research 1337 (2010) 85 ‐94

Neuronal Differentiation protocol

NSE

NeuN

TH

nestin

proliferative RA‐differentiated

SH‐SY5Y cells

92%

95%

85%

0%

RA-DifferentiatedRA-DifferentiatedProliferativesProliferatives

Cell model: human neuroblastoma SH-SY5Y

ORIGINAL ARTICLE

RA Differentiation Enhances Dopaminergic Features, ChangesRedox Parameters, and Increases Dopamine TransporterDependency in 6-Hydroxydopamine-Induced Neurotoxicityin SH-SY5Y Cells

Fernanda M. Lopes1,2 &Leonardo L isbôa da Motta1 &Marco A. De Bastiani 1 &Bianca Pfaffenseller 1 &Bianca W. Aguiar 1 &Luiz F. de Souza3 &Geancar lo Zanatta1,4 &Daiani M. Vargas1 &Patrícia Schönhofen1 &Giovana F. Londero1 &L iana M. de Medeiros1 &Valder N. Freire4 &Alcir L. Dafre3 &Mauro A. A. Castro5 &Richard B. Parsons2 &Fabio Klamt 1

Neurotox Res (2017) 31:545–559DOI 10.1007/s12640-016-9699-0

Dr. Fernanda LopesUndiff. RA-Diff.

Patrícia Schönhofen

Patrícia Schönhofen

Patrícia Schönhofen

Patrícia Schönhofen

Researching the potentialadverse effect of CBDadministration with iPSCs fromDravet Syndrome (DS) patient

iPS from DS (DRVT-2):5 years old male with nonsense mutationc.1837 C>T on exon 11 of SCN1A geneand mutation at splicing site IVS4-91 G>A

First mutation already reported to causesevere myoclonic epilepsy and secondmutation cause febrile convulsions

Dr. Raphael Mechoulam

Cannabidiol (CBD)

4’-F-CBD (HU101)

CBD (HU556)

(-)-5’-DMH-CBD

4’-F-CBD (HU101)

Evaluation of cannabidiol from a toxicological point of view…using iPSCs!!!

Ongoing Collaborations

Prof. José Alexandre CrippaNeuroscience DepartmentFMRP/USP

Prof. Stevens K. RehenIDOR/UFRJProfa Marilia Z.P. GuimarãesUFRJDr. Marcelo ReisDr. Jarek SochackiDra. Sylvie DevalleGabriela VitoriaRoberson Girão

Prof. Geancarlo ZanattaPhysics DepartmentUFC

Doencas Neurodegenerativas (# 46698/2014-8)

MJFF – RRIA 2014