cannabidiol, a broad-spectrum therapeutic … · 2018-11-28 · • isolated from cannabis plant in...

CANNABIDIOL, A BROAD-SPECTRUM

THERAPEUTIC CANNABINOID:

EMPHASIS ON ITS NEUROPROTECTIVE

PROPERTIES

Javier Fernández-Ruiz

Departamento de Bioquímica y Biología Molecular, Facultad de

Medicina, Universidad Complutense, Madrid, Spain

Disclosures:

• Member of the Scientific Advisory External Committee

of VivaCell Biotechnology-Spain

• Member of the Editorial Board of the British Journal of

Pharmacology

• Current research contracts or collaborations with GW

Pharmaceuticals, Emerald Health Pharmaceuticals,

VivaCell Biotechnology-Spain and Symrise

• Isolated from Cannabis plant in 1940 (Roger Adams)

The phytocannabinoid cannabidiol (CBD)

• Its structure elucidated in 1963 (Raphael Mechoulam)

Cannabidiol

• Devoid of psychoactivity of 9-THC because it does

not activate CB1 receptors (nor CB2)

• Broad-spectrum therapeutic profile

Although the range of effects of CBD in CNS disorders is impressive,

however, to date, the molecular mechanisms through which CBD exerts its

action remain elusive (adapted from Iuvone et al., 2009)

Cannabidiol as a therapeutic agent

• Antiinflammatory

• Neuroprotective

• Antidepressant

• Antipsychotic

• Anxiolytic

• Antiemetic

• Anticonvulsant

Inflammatory disorders,

neurodegeneration, psychiatric

disorders, vomiting and nausea,

epilepsy…

Reference

Thomas et al., 2007

Thomas et al., 2007

Pertwee et al., 2010†

Yang et al., 2010

De Petrocellis et al., 2011



Ross et al., 2008





Ahrens et al., 2009





Zhu et al. 2006

Pertwee et al., (2008)†

Cannabidiol

concentration

<1 µM

1 to 10 µM

>10 µM

<1 µM

1 to 10 µM

>10 µM

Table 1. A selection of receptors, ion channels, enzymes and cellular uptake processes that CBD has

been reported to activate, antagonize or inhibit in vitro (Fernández-Ruiz et al., BJCP 2013).

*apparent allosteric modulation; (+). activation; (-), inhibition or antagonism; †review article

Pharmacological target and effect

Receptors & channels

CB1 receptor (-)

CB2 receptor (-)

GPR55 (-)

5-HT3A ligand-gated channel (-)*

TRPM8 cation channel (-)

TRPA1 cation channel (+)

PPARγ nuclear receptor (+)

CaV3 T-type Ca2+ channels (-)

TRPV1 cation channel (+)

TRPV2 cation channel (+)

5-HT1A receptor (+)

µ & δ opioid receptors (-)*

α1 & α1β glycine ligand-gated channels (+)*

Transporters and cellular uptake

adenosine uptake by cultured microglia &

macrophages (-)

calcium uptake by synaptosomes (-)

NE, DA, 5-HT & GABA uptake by synaptosomes (-)

anandamide and palmitoylethanolamide cellular

uptake (-) P-glycoprotein (drug efflux transporter) (-)

choline uptake by rat hippocampal homogenates (-)

Pharmacological target and effect

Enzymes

CYP1A1 (-)

CYP1A2 & CYP1B1 (-)

CYP2B6 (-)

CYP2D6 (-)

CYP3A5 (-)

Mg2+-ATPase (-)

arylalkylamine N-acetyltransferase (-)

indoleamine-2,3-dioxygenase (-)

15-lipoxygenase (-)

phospholipase A2 (+)

glutathione peroxidase (+)

glutathione reductase (+)

CYP2A6 (-)

CYP3A4 & CYP3A7 (-)

fatty acid amide hydrolase (-)

5-lipoxygenase (-)

superoxide dismutase (-)

catalase (-)

NAD(P)H-quinone reductase (-)

progesterone 17α-hydroxylase (-)

testosterone 6β-hydroxylase (-)

testosterone 16α- hydroxylase (-)

Reference

Yamaori et al., 2010

Yamaori et al., 2010

Yamaori et al., 2011b

Yamaori et al., 2011c

Yamaori et al., 2011a


Koch et al., 2006

Jenny et al., 2009

Takeda et al., 2009

Pertwee et al., (2008)†

Massi et al., 2006; Usami et al., 2008

Massi et al., 2006; Usami et al., 2008

Yamaori et al., 2011b

Yamaori et al., 2011a


Takeda et al., 2009

Usami et al., 2008

Usami et al., 2008

Usami et al., 2008

Watanabe et al., 2005; Funahashi et

al., 2005 Watanabe et al., 2005

Watanabe et al., 2005

Cannabidiol

concentration

<1 µM

1 to 10 µM

>10 µM

Table 1. A selection of receptors, ion channels, enzymes and cellular uptake processes that CBD has

been reported to activate, antagonize or inhibit in vitro (Fernández-Ruiz et al., BJCP 2013).

*apparent allosteric modulation; (+). activation; (-), inhibition or antagonism; †review article

Therefore, to identify the key mechanism(s) supporting its therapeutic

properties remains as the major challenge

Cannabidiol as a therapeutic agent

The synthesis of CBD derivatives has provided information on these

mechanism(s) of action (enhancing/reducing/modifying therapeutic

properties):

• Classic analogs (desoxy-, 11-hydroxy-, acid…)

• Abnormal CBD GPR18

• CBD quinones (more toxic) and derivatives (PPAR- activators)

• Fluorinated CBD (more potent)

ab

CB1

∆9-THC ∆9-THCV

ab

CB2

Normalization of

glutamate homeostasis

Inflammatory mediators

prosurvival factors

Control of glial activation and toxicity

Glutamate toxicity

Neurons Glial cells

Cannabidiol as a neuroprotective agent

Again, to identify the key mechanism(s) supporting its neuroprotective

profile remains as the major challenge

Induction of

antioxidant enzymes

ROS

Improvement of

antioxidant defenses

Nrf-2?

Cannabidiol

ROS

Recovery of

antioxidant defenses

abCB

NFB

COX-2, iNOS

Inflammatory

mediators

Unknown targets:

CBX?, PPARs?

A2a receptor

5HT1a receptor

Activation of CB2

receptors (Martínez-

Orgado’s lab)

Blockade of CB2

receptors or acting as

NAM (Pertwee’s lab)

Immature brain

In vitro data

FAAH enzyme

Phytocannabinoids

Huntington’s

disease

Parkinson’s

disease

Alzheimer’s

disease

Neonatal ischemia

Stroke/Brain

trauma

Amyotrophic lateral

sclerosis

CBD + Δ9-THC

Δ9-THCV + CBDCBD

Δ9-THC

CBD + Δ9-THC

CBD + Δ9-THC

Δ9-THCV

CBN

CBD

CBD

CBD

Huntington’s

disease:

Rationale for using a broad-spectrum cannabinoid-based therapy in HD

pathogenesis

- Early down-regulation of CB1 receptors

- Up-regulation of CB2 receptors in glial cells

- Changes in endocannabinoid levels

- Alterations in FAAH and other enzymes

infl

am

mati

on

Selective CB2

receptor agonists:

- HU308

Oxid

ativ

e d

am

ag

e

Antioxidant

cannabinoids:

- 9-THC

- CBD

excitotoxicity

Selective CB1 and

CB2 receptor

agonists

• Genetic origin (CAG)>36

in htt gene

• Striatal and cortical

degeneration

• Choreic movements and

dementia

• No useful treatments

Potential of cannabinoids

as a disease-modifying

agent in HD

Animal models of Huntington’s disease

• Transgenic mice (R6/1, R6/2 y HD94); knock-in mice?

• Neurotoxin-based models:

- excitotoxins (quinolinate)

- mitochondrial toxins (inhibitors of complex II such as 3NP or malonate)

• Cellular models:

- STHdh(Q7/Q7) versusSTHdh(Q111/Q111)

Control

HD94

control

3-NP

PENK-mRNA

control 3NP 3NP + Sativex

*

control 3NP 3NP-Sativex0

50

100

150

Nu

mb

er

of

sta

ined

cells

(% o

f co

ntr

ol g

rou

p)

Nissl staining

3NP-lesioned rats Oxidative stress

Calpain activation Antioxidant cannabinoids (CBD, CBG) were

neuroprotective

Fluoro-Jade staining

Controls 3NP 3NP + Sativex

controls 3NP 3NP + Sativex0

100

200

300 ***##

*

Nu

mb

er o

f d

egen

erat

ing

cel

ls

(% o

ver

co

ntr

ols

)Sagredo et al., J Neurosci Res 2011

GFAP immunostaining

Controls 3NP 3NP + Sativex

OX-42-immunostained cells

control 3NPcontrol 3NP 3NP + Sativex


Calpain activation

Antioxidant cannabinoids (CBD, CBG) were

neuroprotective

Sagredo et al., J Neurosci Res 2011

*

#

*

IGF-1 gene expression

control 3NP 3NP + Sativex0

50

100

150

mR

NA

leve

ls (

%)

*

CB1 receptor gene expression

control 3NP 3NP + Sativex0

50

100

150

mR

NA

leve

ls (

%)

*

Calpain gene expression


50

100

150

200

mR

NA

leve

ls (

%)

SOD-1 gene expression


50

100

150

mR

NA

leve

ls (

%)


Calpain activation


neuroprotective


*

##

#


control 3NP 3NP 3NP + SR1 3NP + AM6300

50

100

150

200

Sativex

mR

NA

le

ve

ls (

%)


Calpain activation


neuroprotective


SOD-1 gene expression

control 3NP 1:1 1:2 2:10

50

100

150

mR

NA

le

ve

ls (

%)


control 3NP 1:1 1:2 2:10

50

100

150

200

250

mR

NA

levels

(%

)

*

#

1:1 Δ9-THC-CBD

1:2 Δ9-THC-CBD

2:1 Δ9-THC-CBDtotal cannabinoid = 4.63 mg/kg (equivalent to 3 mg/kg of pure compound)


Calpain activation


neuroprotective


in vivo NMR imaging:

+ SativexMALONATECONTROLS

sham malonate malonate+sativex0.00

0.02

0.04

0.06

0.08

ed

em

a vo

lum

e (c

m3)

malonate-lesioned rats Inflammation

Activation of apoptosis Neuroprotection was afforded by selective CB2

receptor agonists but CB1 receptor blockade

aggravated striatal damage

Valdeolivas et al., ACS Chem. Neurosci. 2012

Nissl stainingControls Malonate Malonate + Sativex

Controls Malonate Malonate + Sativex

Fluoro-Jade staining

Controls Malonate Malonate + Sativex

GFAP immunostaining





Are Sativex effects in this model mediated by CB1 and/or CB2 receptors?


Iba-1 immunostainingControls Malonate Malonate + Sativex





Both CB1 and CB2 receptors appear to be involved in Sativex effects in this model

Sham

+ Sativex + Sativex + SR1 + AM630

Malonate

Nissl staining

* ** *

control malonate Sativex SR141716 AM630 both0

40

80

120

Nu

mb

er

of

Nis

sl-

stain

ed

cells

(% o

ver

co

ntr

ols

)


4 5 6 7 8 9 10 110

20

40

60

80

100

120

140Wild-type R6/2 R6/2 + Sativex

Age (weeks)

Tim

e i

n r

ota

rod

(% o

ve

r b

as

al

va

lue

s a

t 4

we

ek

s)

* ** **** **

Clasping behavior at 10 weeks

Wild-type R6/2 R6/2 + Sativex0.0

0.5

1.0

1.5

2.0

2.5

3.0

Cla

sp

ing

(sco

re) ***

*

##

R6/2 mice Htt aggregation

Neurological deficit

Neuronal death

Neuroprotection was afforded with agonists of

both CB1 and CB2 receptors

Valdeolivas et al., IJMS 2017

caudate-putamen

wild-type R6/2 R6/2 + Sativex0

5

10

15

20

*

SU

V

globus pallidus


5

10

15

20

*

SU

V

whole brain


5

10

15

20

*

#

SU

VR6/2 mice Htt aggregation


Neuronal death




Lac/NAA


0.5

1.0

1.5

2.0

2.5

Rati

o

****

Tau/NAA


0.5

1.0

1.5

2.0

2.5

Rati

o

***

*###

Glu/NAA


0.5

1.0

1.5

Rati

o

*** ***

Tau/Cre


0.5

1.0

1.5

Rati

o

*

GSH/Cre


0.4

0.8

1.2

Rati

o

NAA/Cho


0.25

0.50

0.75

1.00

1.25

Rati

o **

##

R6/2 mice Htt aggregation


Neuronal death




Pilot clinical trial with Sativex in early symptomatic HD patients

Recruited from Hospital Ramón y Cajal, Madrid, Spain

Coordinated by Justo García de Yébenes and including the laboratories

of Manuel Guzmán, Julián Romero and Javier Fernández-Ruiz

In collaboration with GW Pharmaceuticals Ltd.

- 24 patients (12 treated with Sativex and 12 with placebo)

- Patients were crossed at 12 weeks leaving a period of wash-out of 4-6 weeks

- Then patients were treated again with Sativex (previously treated with placebo) or

placebo (previously treated with Sativex) for another 12 weeks

- Primary endpoints were safety of Sativex in patients

- Secondary endpoints were to get any evidence of slower HD progression with Sativex

- Measures included neurological analysis with the UHDRS scale, in vivo imaging,

analysis of biomarkers (BDNF, cytokines, endocannabinoid receptors and enzymes)

in CSF, plasma, lymphocytes or cultured fibroblasts from skin biopsies), and others

López-Sendón et al., J Neurol 2016

placebo

Sativex

Week 1 Week 12 Week 16 Week 28

Skin

biopsy

CSF

sampling

wash-out

CSF

sampling

Skin

biopsy

control visit


Total, N = 25Group A (Sativex®-

Placebo) N = 12

Group B (Placebo-

Sativex®) N = 13

Age (years) 47.6 ± 12.4 49.2 ± 11.4 46.1 ± 13.6

Male:female 14:11 7:5 7:6

Age at onset (years) 41.0 ± 10.5 40.7 ± 9.4 41.1 ± 11.8

Caucasian:Latino 24:1 12:0 12:1

Disease duration (years) 6.6 ± 4.3 8.4 ± 4.7 4.8 ± 3.1

CAG repeats 45.7 ± 3.7 44.9 ± 3.4 46.4 ± 3.9

BMI (kg/m2) 22.0 ± 3.4 21.9 ± 2.9 22.2 ± 4.1

Other medication

Dopamine blockers 6 2 4

Tetrabenazine 6 4 2

Amantadine 1 1 0

Antidepressants 5 3 2

Benzodiacepines 15 8 7

Table 1. Patient demographic and baseline characteristics


Sativex Placebo P value

CSF protein concentrations

aβ-42 (ρg/mL) 757.1 (318.9) 655.3 (275.9) 0.258

Tau (ρg/mL) 206.4 (91.3) 202.4 (76.0) 0.876

p-Tau (ρg/mL) 47.9 (14.9) 50.2 (15.6) 0.627

CSF monoamine levels

MHPG (ηg/mL) 14.5 (3.7) 14.5 (3.9) 0.992

DOPAC × 100 (ηg/mL) 0.3 (0.1) × 100 0.3 (0.2) × 100 0.876

Trp/10 (ηg/mL/10) 588.6 (186.7)/10 621.0 (233.2)/10 0.618

5HIAA (ηg/mL) 27.1 (9.3) 27.8 (9.3) 0.804

HVA (ηg/mL) 51.0 (19.1) 53.1 (22.1) 0.732

Plasma miR-34b levels 14.5 (1.0) 14.3 (0.91) 0.595

CSF endocannabinoid levels

AEA (pmol/mL) <0.15 <0.15 –

PEA (pmol/mL) 2.5 (0.6) 2.8 (0.9) 0.374

2-AG (pmol/mL) <1 <1 –

CSF BDNF levels 27.7 (8.1) 30.2 (9.5) 0.322

Endocannabinoid gene expression in lymphocytes

CB2 (2−ΔΔCt) 0.00046 (0.00006) 0.00071 (0.00009) <0.005

FAAH (2−ΔΔCt) 0.00169 (0.00023) 0.00227 (0.00049) n.s.

MAGL (2−ΔΔCt) 0.00047 (0.00011) 0.00061 (0.00017) n.s.

ACO2 (2−ΔΔCt) 0.00821 (0.00140) 0.00641 (0.00112) n.s.

UCP2 (2−ΔΔCt) 0.126 (0.018) 0.108 (0.013) n.s.

Table 2. Biomarker determinations in patients after Sativex and placebo treatments


Sativex, N = 25 Placebo, N = 24No treatment

N = 25p value

Anxiety 6 (24) 3 (12.5) 5 (20) 0.479

Sleepiness 5 (20) 1 (4.17) 3 (12) 0.102

Dizziness 4 (16) 0 2 (8) 0.045*

Disturbance in

attention4 (16) 0 1 (0) 0.045*

Insomnia 2 (8) 1 (4.17) 3 (12) 0.564

Behavioral

changes1 (4) 3 (12.5) 3 (12) 0.317

Fever 1 (4) 2 (8.33) 2 (8) 0.564

Local infection 4 (16) 1 (4.17) 1 (4) 0.317

Upper respiratory

infection7 (28) 8 (33) 9 (36) 0.317

Diarrhea 6 (24) 3 (12.5) 1 (4) 0.414

Vomiting 3 (12) 0 3 (12) 0.083

Headache 4 (16) 4 (16.67) 9 (36) 1

Muscular pain 2 (8) 2 (18.33) 5 (20) 1

Table 4. Reported adverse events


Changes during

Sativex®

Changes during

Placebop value

Motor scores

Eye movements 0 (1.9) 0.4 (1.3) 0.238

Maximal dystonia −0.4 (1.6) −0.4 (1.2) 1.000

Maximal chorea 0 (2.2) 0.6 (2.3) 0.405

Remaining items −0.2 (3.4) 1.4 (2.6) 0.678

TMS −0.6 (6.4) 2.0 (5.0) 0.286

Cognitive scores

Verbal fluency 0.6 (5.6) −0.7 (6.6) 0.405

Categorical fluency 11.7 (4.8) 11.6 (5.1) 0.824

Stroop interference 1.6 (6.2) 0.7 (5.5) 0.824

Behavioral scores

bUHDRS −2.6 (5.6) −0.1 (8.0) 1.000

HADS −2 (3.5) −0.2 (5.1) 0.405

NPI −2.6 (5.6) −0.1 (7.9) 0.134

Functional scores

fUHDRS 2.8 (11.6) 0.3 (1.2) 0.581

Table 5. Changes in outcomes between treatment allocations


• Beyond to be a nonpsychotropic cannabis component, CBD is a broad-

spectrum therapeutic compound serving as antiinflammatory, anticonvulsant,

antiemetic, antidepressant, anxiolytic, antipsychotic and neuroprotective agent

Conclusions….

• Its mechanisms of action are diverse (and not completely identified), involving

targets within the endocannabinoid system but, in particular, outside this

system, which may explain why its effects are also diverse

• Preclinical work in Huntington’s disease with a Sativex-like combination of Δ9-

THC- and CBD-botanical extracts demonstrated that this combination may be

a promising disease-modifying agent in this disorder

• Preliminary clinical testing demonstrated that this combination is safe in

Huntington’s disease, but its disease-modifying potential could not be

demonstrated. A new clinical trial with a different design (longer periods with

the active treatment) will be developed soon

• Its neuroprotective profile is relevant and is being studied, alone or in

combination with other phytocannabinoids, in numerous neurodegenerative

disorders including ischemia, Alzheimer’s disease and Parkinson’s disease

Departamento de Bioquímica y

Biología Molecular, Facultad de

Medicina, Universidad Complutense

Javier Fernández-Ruiz (Full Professor)

José A. Ramos (Emeritus Full Professor)

Mariluz Hernández (Associate Professor)

Eva de Lago (Associate Professor)

María Gómez Ruiz (Associate Professor)

Onintza Sagredo (Associate Professor)

Concepción García (Assistant Professor)

Carmen Rodríguez (Postdoctoral researcher)

María Gómez Cañas (Postdoctoral researcher)

María Ceprián (Predoctoral researcher)

Laura García Toscano (Predoctoral researcher)

Irene Santos-García (Predoctoral researcher)

Eva Luna (Predoctoral researcher)

Cristina Alonso (Predoctoral researcher)

Sonia Burgaz (Predoctoral researcher)

Claudia Gonzalo (Predoctoral researcher)

Marta Gómez Almería (Master student)

Yolanda García Movellán (Assistant)

Anke Witting (Ulm University)

Mauro Maccarrone (University of Rome)

Vincenzo Di Marzo (ICMIB)

Gabriele Murineddu (University of Sassari)

Patricia Maciel (University of Porto)

Cecilia Hillard (Medical College of Wisconsin)

Peter McCormick (University of Surrey)

Grupo Cannabinoides-BBM3



Medicina, Universidad

Complutense

Collaborators:

Manuel Guzmán (UCM)

Felipe Ortega-María Teresa Miras (UCM)

Pilar Goya-Nadine Jagerovic (IQM-CSIC)

Julián Romero (HUFA)

José Martínez Orgado (HCSC-UCM)

Carmen Guaza (I.Cajal-CSIC)

Adolfo López de Munain (Biodonostia)

Ana Martínez (CIB-CSIC)

Eduardo Muñoz (UCO-VivaCell)

Rafael Franco (UB)

GW Pharmaceuticals

VivaCell Biotechnology Spain

Emerald Health Pharmaceuticals

Grupo Cannabinoides-BBM3



Medicina, Universidad

Complutense

Current funding:

CIBERNED (CB06/05/0089)

MINECO (SAF2015-68580-C2-1-R)

RETOS-COLABORACION (RTC-2014-1877-1)

cannabidiol, a broad-spectrum therapeutic … · 2018-11-28 · • isolated from cannabis plant in...

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