Cannabis and Epilepsy from Recreational Abuse to Therapeutic Use B

School of Chemistry, Food Biosciences & Pharmacy

Cannabis and epilepsy from recreational abuse to therapeutic use B. Whalley

• Chronic, progressive neurological disorder characterised by spontaneous, recurrent seizures.

• ~10% of people will have a seizure in their lifetime of which ~30% will subsequently develop epilepsy.

• Lifetime prevalence ~1%.

• Third most prevalent neurological disorder after migraine and Parkinson’s Disease (both ~0.7- 1.2%).

• Affects 50 million people worldwide and accounts for 1% of the global burden of disease.

2 Banerjee & Hauser, 2006 Aetiology of epilepsy

• ~60% of cases are idiopathic (WHO, 2012)

• Remainder are cryptogenic or secondary to insults such as hypoxia (or other trauma) at birth, head trauma, drug use, stroke and CNS infection or tumour.

• Age is an independent risk factor and febrile seizure specific to childhood

• A small minority are due to specifically identifiable molecular/genetic causes.

Rate ratio <1 = protective

Banerjee & Hauser, 2006

3 Disease burden and co-morbidities

• Example disease burden: $15.5 billion per year in US (CDC, 2008)

• Premature mortality is 2-3 times higher in epilepsy patients (maximum reported: 8.8). – Significant causes: SUDEP, status epilepticus, accidents as a consequence of seizure, aspiration pneumonia after seizure, drug toxicity and idiosyncratic ADRs and suicides (Lhatoo et al, 2006) • Co-morbidities include: – Cognitive decline (drug and disease-related) – Anxiety – Depression – Agitation, anger and emotional outbursts – Suicide (5-15x more likely) – ADHD – Reproductive problems (make and female) – Insomnia – Migraine

• Co-morbidities more frequent and severe in refractory patients 4 Clinical need for new AEDs

• The introduction of new AEDs since 1990 onwards has had no effect upon the number of pharmacologically intractable/refractory epilepsy patients. – Of 525 people with newly diagnosed epilepsy with 2–16 years of follow up, 37% still exhibited seizures at the final clinic visit whilst the remainder were seizure free for Ʀ1 year. – Seizure-free rate did not differ significantly between those treated with a single established drug (67%) and those treated with a single new drug (69%). Thus, new AEDs have not reduced pharmacoresistance (Kwan & Brodie, 2000). • New AEDs achieve some benefit via improved side-effect profiles. • New, better tolerated and more effective AEDs are clearly required to benefit the 15- 20M people experiencing pharmacologically refractory seizures.

http://www.ncbi.nlm.nih.gov 5 Rational drug design

• How have successful drugs been discovered?

– Serendipity e.g. valproate, levetiracetam – Secondary use of existing drugs e.g. phenobarbital – Screening related compounds e.g. phenytoin, ethosuximide • ‘me too’ drugs – Modification of existing drugs e.g. oxcarbazepine, pregabalin – “Rational”/target oriented design e.g. vigabatrin, tiagabine

• The least successful have come from rational/target-based development.

• Related and modified compounds are typically only effective in epilepsies that already respond to existing treatments.

6 Historical use of cannabis in epilepsy

• 1100AD: al-Mayusi makes first written record of its use for this purpose

• C15th: Ibn al-Badri notes “the epileptic son of the caliph's chamberlain” was treated with Cannabis and “it cured him completely, but he became an addict who could not for a moment be without the drug”

• C19th: O’Shaughnessy, McMeens, Moreau and Reynolds independently tested the efficacy of a crude extract against seizures.

• J.R. Reynolds, Queen Victoria’s personal physician said Cannabis is “the most useful agent with which I am acquainted” in the treatment of “attacks or violent convulsions,” which“may recur two or three times in the hour,” claiming that such attacks “may be stopped with a full dose of hemp” 7 Evidence from preclinical models

Publis hed resu lts of cannabinoid effects upon w hole animal models of seiz ure and epileps y

Grosseffect ( pr oco nvul s an t = Si ngl e comp oun d o r co- C ann abi noi d dos e s RED, Speci es S t r a in A ge M odel ty pe D ise as e mode l Method ad ministration tested D os e t imi ng R o u te E ffe ct an ti con vul s ant = N ot es Ref er e nce GREEN,no effect = BLAC K)

Phy to can nab inoi ds

' 9-T HC 1 0mg/kg 5- 13 5 mi ns bef or e t es t s t i m ul us 1 0 mg/ k g 15- 45 m i ns bef or e t es t s t i m ul us re duce d n um be r of a ni m als s how i ng s i gns of sei z ur e s 2 .5- 10 m g / k g r e du c e d num b e r o f a n im a l s s h ow i ng s i g n s of Mouse C57B L/6 A dul t A cute G ene ra li sed sei zur e Audiogenic priming 1.25-10 mg/kg 15 m i ns bef or e t es t st i m ul us i. p. M od e l us e s a pr i m in g s t i m ul us a t P+ 1 9 Bog gan et al . , 1973 se izu r es followed by atest stimulus at P+28 1 0 mg/ k g 15- 45 m i ns bef or e pr i m in g st i m ul us s how ed THC 1 0mg/kg 15 - 90 m in s be f or e pr im i ng s t im u lu s r e duce d si gns of s ei z ur es 1 0- 50 mg/k g 15 - 90 m in s a f t e r pr im i ng s t im u lu s N o ef f ect Mouse QS A dul t A cute G ene ra li sed sei zur e PTZ 1-80mg/kg 30 m i ns bef or e t es t i ng N o ef f ect PT Z thr es h ol d m ode l 30 m i ns bef or e t es t i ng p. o. > 160 m g/ kg p r ot ect ed agai ns t hi ndl i m b e xt ens i on 2 5- 200mg /kg 30 m i ns bef or e t es t i ng 2 0- 75 m g / kg s i gni f i cant ly i ncr eas e d hin dl imb e xt ens i on St an dar d MES mo del 2 0mg/kg 30 m i ns bef or e t es t i ng i.v. Significantlydecreased hindlimb extension 5 0 m g / k g T H C, 50m g / kg TH C p. o. w as pr ocon vul s ant alo ne TH C pl us C BD pl us CBN 5 0mg/kg CBD , 50 30 m i ns bef or e t es t i ng p. o. Significantlydecreased hindlimb extension a n d 5 0 m g / k g CB D nor CBN p .o . a r e m g / kg C BN an ticon vulsant at these d oses Che sher et al . , 1974 Mouse QS A dul t A cute G ene ra li sed sei zur e MES 50mg/kg THC (with a Aproconvulsant THC dose (50 mg/kgp.o.) r ed uces AED ED 50. S uppo r ts ear l i er pr o pos al TH C pl us P HN r an ge on phe nyt oi n 30 m i ns bef or e t es t i ng p. o. S ign if i cant l y r e duce d p hen yt oi n E D50 t hat canna bi s an d p hen yt oi n i nt e ract d oses) synergistically (Loewe et al.,1947) 5 0 m g / k g T H C, 50 A ED ED50 f ur t he r r edu ced f ro m t hat mg/kgCBD (with a T H C pl us P HN p lu s CB D range ofphenytoin 30 m i ns bef or e t es t i ng p. o. S ign if i cant l y r e duce d p hen yt oi n E D50 ach ie ved by coad m ini s t r at io n of TH C wi t h ph eny to in d oses) Mouse CF-1 A dul t A cute G ene ra li sed sei zur e MES THC 1 -1 00mg/kg 12 0 mi ns bef or e t es t i ng i. p. S ign if i cant pr ot ect i on agai ns t s ei z ur es Bl ock ed by SR141 716A Wal lace e t al ., 2001 2 5- 50 mg/k g TH C pl us T H C: 1 20 m i ns ; P BL : 60 m i ns b e f or e THC:p.o., Po t ent i at io n g re at er t ha n th at caus e d by CBD TH C pl us P BL S ignificant potentiation of PBL effect 9 .3-40 mg/kg PB L testing PBL: i . p. r ep or t ed in s am e s t udy Mouse QS A dul t A cute G ene ra li sed sei zur e MES 2 5 m g / k g T H C, 25 Che sher et al . , 1975 mg /kg C BD, 9.3 -4 0 THC &CBD: 120 mins;PBL: 60 mins THC & Po t ent i at io n co m par abl e t o th at cau s ed by TH C pl us C BD pl us PBL mg /kg b ef or e t est in g CBD:p.o., S ignificant potentiation of PBL effect c o- a d m in is t r a t i on of T H C 50 m g / k g a lo ne PBL: i . p. phenobarbitone

MES Significantlydecreased hindlimb extension N o in di ca ti on o f t ol er ance af t er 3-4 da ys repeated administration Mouse Not stated A dul t A cute G ene ra li sed sei zur e 6Hz el ectrosho ck 1 00 mg/k g o .d . f or 3- 4 days be fo r e tes t i ng i. p. S ign ifi cant anti convu lsan t effect Kar l er e t al ., 1980 • Only whole animal models shown since 60H z el ectrosh ock T hr es ho ld t o s ei z ur e r educe d N one MES 15 mi ns to 24 ho ursb efore testing S ign if i cant in cre as e in l at ency t o hi ndl i m b e xt ens i on D ose t imi ng var i at io nrev eal ed pea k e ffe cts at 30 mi ns Mouse Not stated A dul t A cute G ene ra li sed sei zur e PTZ up to 80 mg/kg i. p. N o ef f ect Sof i a e t al ., 1974 Nicotine 30 m i ns bef or e t es t i ng N o ef f ect N one St r ychni ne N o ef f ect Los s of pr ocon vul s ant e f fe ct up on la te ncy Gerbil Mer io nes A dul t C hr oni c G ene ra li sed sei zur e Spo nt aneo us l y e pi le pt ic 2 0 & 50 m g / k g Si ng le do s e i. p. 5 0 mg/ k g si gni f ica nt ly de re as ed l at ency t o s ei z ur e Loskot a et al. , 19 75 ung ui cula t us wi th ch ronictreatmen t sugg ests o .d . f or 6 days be fo r e tes t i ng N o ef f ect ad apt at io n/t ol eran ce

0.25 mg/kg A t on s et o f ki ndl i ng S ign if i cant re duct i on of epi l ept i f or m af t er - di s char ge s Am yg dal a ki nd li ng Cat Not stated A dul t C hr oni c G ene ra li sed sei zur e (electrical) THC A t s t age 3 (h ead no ddi ng) i. p. N one Wada et al. , 19 75 0 .25 - 4 mg/kg A t st age 5 (cl on ic j um pi ng) N o ef f ect A t ki nd li ng end poi nt

Rat Spr agu e- Da wl ey A dul t C hr oni c Te m por al l ob e Pi lo car pi ne- i ndu ced SRS 0 .5-30mg /kg Si ng le do se i mmed ia te ly be fo r e i. p. Spontaneousseizures completely prevented E f f e c t blo c k e d by A M2 51 Wal lace e t al ., 2003 e pilep sy EE G and beh avi our al mo ni t or in g A ntico nvulsan t effectsd escri bed as Cor cor an e t al ., 1973 & Rat Not stated A dul t A cute G ene ra li sed sei zur e PTZ 1 5- 200 mg /kg 45 m i ns bef or e t es t i ng i. p. S ign ifi cant anti convu lsan t effect 'unreliable' Fr ie de t al . , 1973

Pho t og e n ic N o ef f ect Sp eci es e xhi bi t s ph ot om yo clo nus and i s Babo on Pap io pa pi o A dul t C hr oni c G ene ra li sed sei zur e 0 .25 -1 mg/kg ~6 0 mi nut e s be f or e te s ti ng i. p. Wada et al. , 19 75 Am yg dal a ki nd li ng S ign if i cant re duct i on i n se izu r es a nd epi l ept i f or m af t er - s us ce pt ib le t o am ygd ala ki nd li ng (electrical) discharges S ign ificant anticonvu lsan t effect aftertreatment at 0.5 but Pho t og e n ic n ot 2 ho ur s Gallus PTZ (35 mg/kgin epileptic Su bpo pul at io n of ch icke ns su s cept i bl e to Chi cken dome sticus A dul t A cute G ene ra li sed sei zur e f ow l) 0 .25 -1 mg/kg 0. 5 or 2 ho ur s b ef or e t est in g i.v. ph ot i c s t im u la ti on . S us c e pt ib le = 'e pi le pt ic ' J ohnso n et al . , 1975 N o ef f ect PTZ ( 80 mg / kg in no n- epilepticfowl)

CBD 5 0 m g / k g CB D pl us 9 .3- C BD: 2 h our s , PB L: 1 hour bef o re CBD:p.o., Mouse QS A dul t A cute G ene ra li sed sei zur e MES C BD pl us PBL 40mg/kg S ignificant potentiation of PBL effect N one Che sher et al . , 1975 phenobarbitone testing PBL: i . p. Mouse CF-1 A dul t A cute G ene ra li sed sei zur e MES C BD 1 -1 00mg/kg 12 0 mi ns bef or e t es t i ng i. p. S ign if i cant pr ot ect i on agai ns t s ei z ur es N ot b lo cked by SR1 41716A Wal lace e t al ., 2001 seizures and epilepsy can only be poorly MES Significantlydecreased hindlimb extension N o in di ca ti on o f t ol er ance af t er 3-4 da ys Mouse Not stated A dul t A cute G ene ra li sed sei zur e 6Hz el ectrosho ck 1 00 mg/k g o .d . f or 3- 4 days be fo r e tes t i ng i. p. S ign ifi cant anti convu lsan t effect repeated administration Kar l er e t al ., 1980

60H z el ectrosh ock S ign ifi cant anti convu lsan t effect I ncr eas e d th re s hol d t ose izu r e

Rat Not stated A dul t A cute G ene ra li sed sei zur e MES 1 .5-12 mg/kg 1 hourbefore testing i. p. S ign ifi cant anti convu lsan t effect N ot abl y lo w do s e re qui r ed Izq ui er do et al. , 197 3

PTZ Mouse Not stated A dul t A cute G ene ra li sed sei zur e 5 0- 200 mg /kg 30 m i ns bef or e t es t i ng p. o. N o ef f ect N one Che sher et al . , 1974 MES

Mouse I CR A dul t A cute G ene ra li sed sei zur e MES 1 20 mg/k g ( ED 50) 0. 5- 6 hou rs be fo r e te s ti ng i. p. S ign ifi cant anti convu lsan t effect N one Kar l er e t al ., 1978

Rat Spr agu e- Da wl ey A dul t C hr oni c Generalised seizure Limbic kindling (electrical) 0 .3-3 mg/kg 15 -3 00 mi nu te s i. p. S ign if i cant in cre as e in t hr es h ol d to af t er - di s char ge N one Tur kan is et a l. , 1979

Partialseizure with Corticalimplantation of Rat Not stated A dul t C hr oni c s e conda ry c oba lt 6 0mg/kg Twice daily after implantation i. p. N o ef f ect N one Col asan ti et al . , 1982 ge neral i sat io n

MES C BD S ign ifi cant anti convu lsan t effect

3-mercaptop ropri oni c a cid N o ef f ect Picrotoxin S ign ifi cant anti convu lsan t effect Mouse Not stated A dul t A cute G ene ra li sed sei zur e 5 -4 00 mg/k g 60 m i nut es be fo r e t e s ti ng i. p. A ppar e nt ly hi ghl y ef f ect iv e in m ode ls r el ia nt Con sro e et al . , 1982 Isonicotinic acid S ign ifi cant anti convu lsan t effect up on GABAergi csystems. Bicuculline S ign ifi cant anti convu lsan t effect Hydrazine S ign ifi cant anti convu lsan t effect PTZ S ign ifi cant anti convu lsan t effect St r ychni ne N o ef f ect

Rat Wistar-K yoto A dul t A cute G ene ra li sed sei zur e PTZ S ign ificant anticonvu lsan teffects at 1 &100 m g/kg N one J ones et al. , 20 10

Te m por al l ob e Rat Wistar-K yoto A dul t A cute Pi lo car pi ne ( acut e) N one J ones et al. , 20 12 se izure 1 , 10 & 100 mg/kg 1 hourbefore testing i. p.

S ign ificant anticonvu lsan t effects at ш1 mg/kg Partialseizure with Rat Wistar-K yoto A dul t A cute s e conda ry Penicillin N one J ones et al. , 20 12 modelled in vitro ge neral i sat io n Other phytocannabinoids Mouse A dul t A cute G ene ra li sed sei zur e MES C BN 2 30 mg/k g ( ED 50) 30 m i ns bef or e t es t i ng i. p. S ign ifi cant anti convu lsan t effect N one Kar l er e t al ., 1978

Rat Wistar-K yoto A dul t A cute G ene ra li sed sei zur e PTZ TH CV 2 .5 mg/kg 1 hourbefore testing i. p. S ign ifi cant anti convu lsan t effect A ntico nvulsan teffect lost Hi l l et al . , 2010

Mouse DBA/2 A dul t A cute G ene ra li sed sei zur e Au di ogen ic 1 hourbefore testing i. p. N one

Mouse I CR A dul t A cute G ene ra li sed sei zur e MES 1 hourbefore testing i. p. N one

C BDV 5 0- 200 mg /kg 1 hourbefore testing i. p. S ign ifi cant anti convu lsan t effect N one Hi l l et al . , 2012 ( s ubm i t te d) A cute G ene ra li sed sei zur e PTZ

Rat Wistar-K yoto A dul t 3. 5 hou r s be fo r e te s ti ng p. o. N one

Te m por al l ob e A cute Pi lo car pi ne 1 hourbefore testing i. p. N one se izure

Exog eno us s ynth et ic can nab inoi ds , end ocann abin oid s, der ivat ives an d pr ecur s or s

CB1 Agonists

WI N 55, 212 pl us 15mg /kg WI N 55, 212 sign if i cant l y affe cts 15 m i ns bef or e t es t i ng N o ef f ect m ot o r f unct i on w hen coad m in ist er e d (n o s cl ona z epam ef f ect not ed w i t hou t WI N55 ,21 2) 15mg /kg WI N 55, 212 el evat ed e th osuxi mi de br ai n l evel s; 15mg /kg WI N 55, 212 WI N 55, 212 pl us 45 m i ns bef or e t es t i ng 1 5m g/ kg WIN 55, 212 en hance d ant i convu lsan t ef f ect of si gni fican tly affects mo tor fun ction whe nco - e th os uxi m i de e t hos u ximi de administered (noeffect notedwithout A dul t A cute G ene ra li sed sei zur e PTZ 5-15mg/kg WI N 55, 212) Luszczki , 2011 a 15mg /kg WI N 55, 212 el evat ed va lpr o at e

1 5m g/ kg WIN 55, 212 en hance d ant i convu lsan t ef f ect of l eve ls in br ai n; 15mg/kg WI N55 ,212 e WI N 55, 212 pl us v alp ro at e 30 m i ns bef or e t es t i ng v alp roat e si gni fican tly affects mo tor fun ction whe nco - administered (noeffect notedwithout WI N 55, 212)

Mouse Swiss WI N 55, 212 pl us i. p. 1 5m g/ kg WIN 55, 212 en hance d ant i convu lsan t ef f ect of 15mg /kg WI N 55, 212 sign if i cant l y affe cts i Number of discrete No 60 m i ns bef or e t es t i ng m ot o r f unct i on w hen coad m in ist er e d (n o p heno bar bi t al phenobarbitone ef f ect not ed w i t hou t WI N55 ,21 2) 1 0m g/ kg WIN 55, 212 en hance d ant i convu lsan t ef f ect of 10m g / kg an d hi gher imp air e d mo to r f un cti on WI N 55, 212 pl us v alp ro at e 30 m i ns bef or e t es t i ng v alp roat e an dme mory.

WI N 55, 212 pl us 30 m i ns bef or e t es t i ng 1 0m g/ kg WIN 55, 212 en hance d ant i convu lsan t ef f ect of 10m g / kg an d hi gher imp air e d mo to r car ba m az epi ne 1 0m g/ kg and AE D dose- carbamazepine function. WI N 55, 212 pl us re sponse 1 0m g/ kg WIN 55, 212 en hance d ant i convu lsan t ef f ect of 10m g / kg an d hi gher imp air e d mo to r f un cti on c A dul t A cute G ene ra li sed sei zur e MES 60 m i ns bef or e t es t i ng Luszczki , 2011 b p heno bar bi t on e phenobarbitone an dme mory. Compound Dose Anticonvulsant Proconvulsant WI N 55, 212 pl us 1 0m g/ kg WIN 55, 212 en hance d ant i convu lsan t ef f ect of 10m g / kg an d hi gher imp air e d mo to r f un cti on 12 0 mi ns bef or e t es t i ng p heny to in phenytoin an dme mory. 10m g / kg an d hi gher imp air e d mo to r WI N 55, 212 2 .5-15mg /kg 20 m i ns bef or e t es t i ng 15mg/kg elevated threshold toseizure function. A l l dos e s r ed uced s ev er it y vs v ehi cl e co nt r ol s bu t hi ghe r WI N ef f ect on kai ni c aci d in duce d se izu r es i s Rat Wistar J uv eni l e ( P2 0) A cut e Generalised seizure Kainicacid WI N 55, 212 0 .5- 5mg/k g 90 m i ns bef or e t es t i ng i. p. Rud enko , 2012 W IN d oses exe rte da smal le r ef f ect on seve rit y. i nve rse ly r el at ed. e PMS F ( FA AH i nhi bt o r) us ed t o pr eve nt A CEA A CEA 1.25-15mg/kg i. p. I ncr e as ed t hr es ho ld t o s ei z ur e metab olism Mouse Swiss A dul t A cute G ene ra li sed sei zur e MES 10 mins priorto testing Luszczki , 2006 A CEA pl us 30mg/kg A CEA i ncrease ava il abl e val proat e conditions/models/designs effect 1 .25 -2.5mg/k g i. p. A CEA e nhan ced t he ant i conv ul s ant e ffe ct of val pr oat e c on c e nt r a t i on; A CE A h a d not e f f e c t on m o to r va lproate f un cti on

A ni m al s d id no t exh ib it to ni c se izu re s bu t pr o gr es s ed PE A di d n ot af f ect any ot he r sei z ur e Rat Long -E vans A dul t A cute G ene ra li sed sei zur e PTZ 4 0mg/kg 2 ho ur s pr i or to t es t i ng directly toclonic seizures featu res.

i. p. PE A onl y g ive n pr io r t o te s t s es s i on , not pr i or She er in , 2004

p Rat Long -E vans A dul t C hr oni c G ene ra li s ed s ei z ur e Am yg dal a ki nd li ng 1 , 10 & 100mg/kg 2 ho ur s pr i or to t es t i ng 1mg/kg PEA delayed onsetof seizures to kindlingstimuli. Effect on latency lostat 10 & 100mg /kg PTZ N- S ign if i cant l y r e duce d ton ic con vul s io ns 3-mercaptop ropi on ic acid palmitoylethanolamide S ign if i cant l y r e duce d ton ic con vul s io ns Bicuculline 2 5mg/kg 2 ho ur s pr i or to t es t i ng S ign if i cant l y r e duce d ton ic con vul s io ns I nef f ect i ve agai ns t clon ic con vul s io ns St r ychni ne S ign if i cant l y r e duce d ton ic con vul s io ns Mouse OF1 A dul t A cute G ene ra li sed sei zur e Picrotoxin i. p. S ign if i cant l y r e duce d ton ic con vul s io ns Lamber t , 20 01 NMDA S ign if i cant l y r e duce d ton ic con vul s io ns S Peak effectseen at2 hours after MES 5 0 & 10 0mg/kg 0.5 to4 hoursprior totesting 5 0 & 1 00m g/ kg s ig ni fi cant l y re duce d se izu r es i nci de nce a d m in is t r a t io n MES A nand am i de 5 0mg/kg 5 0m g/ kg s ign if i cant l y re duce d se izu r es i nci de nce WI N 55, 212 0 .5-4mg/k g S ign if i cant pr ot ect i on agai ns t s ei z ur es 0 -4 m g / k g o f e a c h i n 3 :1 r at io ( di azep am: WIN 55, 212) pr od uced syne r gist i c e ffe ct WI N 55, 212 pl us d iaze pam mixedratios u pon p ro t ect io n f r om s ei z ur e A M404 0.125-4mg/kg No significanteffect Mouse NMRI A dul t A cute G ene ra li sed sei zur e MES 0 -4 m g / k g o f e a c h i n 30 m i ns bef or e t es t i ng i. p. Na der i , 2008 A M404 pl us d ia z epam mixedratios No significanteffect URB597 0 .05 -1mg /kg S ign if i cant pr ot ect i on agai ns t s ei z ur es 0 -2 m g / k g o f e a c h i n URB597plus diazepam mixedratios U RB 597 ant agon ise d ef fe ct s of di az epa m URB597is a FAAH inhibitor WI N 55, 212 1 -1 00ug S ig n if i c a nt in c re a s e in l a t e nc y t o s e i z ur e WI N 55, 212 pl us 1 -1 00ug and 5- 50u g N o s i gni f ican t ef f ect upon i s ogu vaci ne- i ndu ced i ncr eas ed I sogu vacine is aGABAAR agonist isoguvacine respectively latencyto seizure Rat Wistar A dul t A cute G ene ra li sed sei zur e PTZ URB597 1 0- 100ug 5 mi ns b ef or e t est in g i.c.v. No significanteffect Na der i , 2011

URB597plus isoguvacine 1 0- 100ug pl us 5- 50ug N o s i gni f ican t ef f ect upon i s ogu vaci ne- i ndu ced i ncr eas ed I sogu vacine is aGABAAR agonist respectively latencyto seizure URB602 1 0- 500ug S ign ifi cant in cre ase in l at ency t o sei zur e at al l do ses U RB60 2 i s a M AG L i nh ib it or

A CEA pl us car bam az e pi ne No significanteffect

A CEA pl us l am ot r igi ne No significanteffect A CEA pl us oxca rb az epi ne No significanteffect Mouse Swiss A dul t A cute G ene ra li sed sei zur e MES 2.5mg/kg 10 mins priorto testing i. p. Luszczki , 2010 A CEA ph eno bar bi t one A ct io n of ph eno bar bi t one s i gni f i cant ly i ncr eas e d A CEA di d not af f ect ph eno bar bi t one availability A CEA pl us ph enyt oi n No significanteffect A CEA pl us t opi r am at e No significanteffect Significantlydecreased spike wave discharge incidence but Rat WAG/Rij A dul t C hr oni c A bsen ce Genetic WI N 55, 212 3-12mg/kg 2 ho ur s pr i or to t es t i ng s.c. c o nc om i t a nt in c re a s e in du r a t io n vanR ij n, 2 010 0.25-200 45 mins afterestab lishment of F re que ncy and am pl i t ude of epi l ept i fo r m act i vi ty Partial with A CEA 0.25ug e pi le pt if or m a c t iv it y . i.c.v. significantlyreduced

Rat Wistar A dul t A cute s e conda ry Pe n ic i l li n ( i. c . v .) Me mant in e 30 mi ns and A CEA 45 Caki l , 2011 ge neral i sat io n 0 .25 ug a nd 1- 20mg/kg A CEA pl us m em an ti ne respectively mins afterestab lishme nt of i.c.v. S ome potentiation of anticonvu lsa nt effect of me mantine Mem a nt in e is an NMD AR ant ag oni s t e pi le pt if or m a c t iv it y . Maxi m al de nt at e gyr us Mo nitored forup to 120 minsafter Rat Wistar A dul t A cute Partial seizure WI N 55, 212 1-21mg/kg i. p. S ign if i cant decr ea s e i n dur at i on an d i ncr ea s e i n l ate ncy Blocked by co-administration ofAM251 Ri zzo, 200 9 a c t iv a t io n ad ministration Rat Not stated A dul t A cute Partial with Pe n ic i l li n ( i. c . v .) A CEA 2 .5-15u g Mo nitored forup to 120 minsafter i.c.v. S ign if i cant decr ea s e i n f re que ncy onl y at hi gh es t do s e Blocked by co-administration ofAM251 Kozan , 2009 THC 6 31 61% 29% 10% A CEA 0 .1-8mg/k g U p to 60 mi ns b ef or e t est in g i. p. S ign if i cant in cre as e in t hr es h ol d to s ei z ur e Mouse NMRI A dul t A cute G ene ra li sed sei zur e PTZ Bah re mand, 2008 0 .1-8mg/k g an d 1- A CEA pl us nal t r exon e 5 00pg / k g r e s p e c t iv e ly U p to 60 mi ns b ef or e t est in g i. p. S ign ificant potentiation of ACEA anticonvu lsan teffect

Mouse NMRI A dul t A cute G ene ra li sed sei zur e PTZ A CEA 0 .1-4mg/k g 60 m i ns bef or e t es t i ng i. p. S ig n if i c a nt in c re a s e in s e i z ur e t hr e s h ol d a t 2 & 4 m g / k g A CE A E f f e c t blo c k e d by A M2 51 Bah re mand et al. , 200 9

Ef f ect enha nced by ul t r a- lo w do s e A M251 Mouse NMRI A dul t A cute G ene ra li sed sei zur e PTZ A CEA 0 .1-8mg/k g 15 m i ns bef or e t es t i ng i. p. S ignificant increase in threshold to seizure at 2-8mg/kg ( 100f g/kg -1 00ng/k g; f g = f emt ogram) Gh oli zad eh, 2 007 Mouse NMRI A dul t A cute G ene ra li sed sei zur e PTZ A CPA 0 .5-2mg/k g 60 m i ns bef or e t es t i ng i. p. S ignificant increase in threshold to seizure at 1.5-2mg/kg Bl ock ed by AM251 Shaf aroo di e t al ., 2 004 Te m por al l ob e Si ng le do se i mmed ia te ly be fo r e mg/kg Rat Spr agu e- Da wl ey A dul t C hr oni c e pilep sy Pi lo car pi ne- i ndu ced SRS WI N 55, 212 5 mg/kg EE G and beh avi our al mo ni t or in g i. p. Spontaneousseizures completely prevented E f f e c t blo c k e d by A M2 51 Wal lace e t al ., 2003 Mouse CF-1 A dul t A cute G ene ra li sed sei zur e MES WI N 55, 212 1 -1 00mg/kg 12 0 mi ns bef or e t es t i ng i. p. S ign if i cant pr ot ect i on agai ns t s ei z ur es Bl ock ed by SR141 716A Wal lace e t al ., 2001 WI N 55, 212 2 .5 & 4mg/kg 30 or 60 mins be fo r e tes t i ng i. p. S ei z ur e dur at i on in cr eas ed by 4m g/ k g at 60 mi ns onl y Pe r fo r me d on f ul ly ki ndl ed an imal s 30 m i ns bef or e ki ndl i ng s ti m ul us i. p. Kindlingsignificantly retarded Pe r fo r me d dur i ng ki ndl in g (e pi le pt ogen es i s ) Mouse NMRI A dul t C hr oni c G ene ra li s ed s ei z ur e Am yg dal a ki nd li ng 30 or 60 mins be fo r e tes t i ng i. p. 1 m g / k g a t 3 0 m i ns in c r e a s e d a f t e r di s c ha r g e t hr e s hol d Pe r fo r me d on f ul ly ki ndl ed an imal s Wend t et al ., 2 011 URB597 1 & 3m g / k g 30 m i ns bef or e ki ndl i ng s ti m ul us i. p. No significanteffect Pe r fo r me d dur i ng ki ndl in g (e pi le pt ogen es i s ) Synthe tic CB1 antago nists ( see also THCV) Ra ts no t s us cep t ib le t o audi og eni c se izu re Rat Wistar A dul t A cute G ene ra li sed sei zur e Au di ogen ic SR 141716 A 3 0mg/kg o.d.for 5 days p. o. S ei zur e sever i t y an d dur at i on in cr eased i n ra ts suscep t ib le t o sho w ed no sei zur e r espon se to SR 141716 A. vanR ij n, 2 011a se izu r e. H igh r i m onab ant dos e. A M251 0 .25 -5mg /kg No significanteffect Mouse NMRI A dul t A cute G ene ra li sed sei zur e MES 0 -4 m g / k g o f e a c h i n 30 m i ns bef or e t es t i ng Na der i , 2008 A M251 pl us d ia z epam No significanteffect mixedratios Rat WAG/Rij A dul t C hr oni c A bsen ce Genetic A M251 6-12mg/kg 2 ho ur s pr i or to t es t i ng s.c. No significanteffect vanR ij n, 2 010

A M251 7.5ug 45 mins afterestab lishment of i.c.v. F re que ncy and am pl i t ude of epi l ept i fo r m act i vi ty Caki l , 2011 Partial with e pi le pt if or m a c t iv it y . significantlyincreased Rat Wistar A dul t A cute s e conda ry Pe n ic i l li n ( i. c . v .) ge neral i sat io n Me mant in e 30 mi ns and A M251 45 A M251 pl us me m ant i ne 7 .5u g an d 1- 20mg/kg mins afterestab lishme nt of i.c.v. N o ef f ect on ant i convu lsan t ef f ect of m e m ant i ne Mem a nt in e is an NMD AR ant ag oni s t respectively e pi le pt if or m a c t iv it y . Maxi m al de nt at e gyr us Mo nitored forup to 120 minsafter Rat Wistar A dul t A cute Partial seizure a c t iv a t io n A M251 1 mg/kg ad ministration i. p. No significanteffect Ri zzo, 200 9 Partial with Mo nitored forup to 120 minsafter Rat Not stated A dul t A cute s e conda ry Pe n ic i l li n ( i. c . v .) A M251 0.125-1ug ad ministration i.c.v. S ign if i cant pr ocon vul s ant ef f ect at 0. 25 an d 0.5 ug onl y Kozan , 2009 ge neral i sat io n Mouse NMRI A dul t A cute G ene ra li sed sei zur e PTZ A M251 0 .01 -1mg /kg 15 m i ns bef or e t es t i ng i. p. No significanteffect Bah re mand et al. , 200 9

Rat Spr agu e- Da wl ey A dul t C hr oni c Ep il ep to gen esi s Kai ni c a cid SR 141716 A 1 0mg/kg Once at firstsign ofacute, kainic i. p. N o ef f ect Du dek, 2 010 aci d- i nd uced s ei z ur e J uv eni le h ead

t r aum a f ol lo w ed by S R1417 16A t re at m ent r educe d s ei z ur e su s cept i bi l it y to Rat Wistar A dul t / I mma tu r e C hr oni c acu te p ro conv ul s ant Kai ni c a cid SR 141716 A 1 & 10 mg/kg I m m ed iat el y f ol lo wi ng he ad tram a i. p. control levels Echeg oyen , 2009 1-400 ch all en ge 6wee ks l at er Mouse NMRI A dul t A cute G ene ra li sed sei zur e PTZ A M251 1 fg /kg- 1mg/kg 45 m i ns bef or e t es t i ng i. p. No significanteffect f g = fem t og ra m ( ul t r a-l ow dos e ) Gh oli zad eh, 2 007 S ign ificant proconvulsant effect at 0.75-3mg/kg (peak effect Mouse NMRI A dul t A cute G ene ra li sed sei zur e PTZ A M251 0 .5-3mg/k g 60 m i ns bef or e t es t i ng i. p. at1mg/kg) Shaf aroo di e t al ., 2 004

Not able eff ect s in no n-seizu re mo dels

None (healthy N ot e ver y hi gh SR1417 16A do se; ani mal s CBD 2 21 81% 19% 0% Rat Wistar A dul t N /A EEGrecording SR 141716 A 3 0mg/kg o .d . f or at le ast 20 day s p. o. 2 0% of ani m al s ( 4/ 20) exhi bi t ed s po nt ane ous s ei z ur es . were female and no note ofstage of vanR ij n, 2 011a an imal s) oe astrous cycle in original report. THC 1 0mg/kg I ncr e as ed in cid ence o f sei z ur es on han dl in g Mouse R6/1 A dul t C hr oni c H unt i ngt on 's di s eas e Beh avi our al o bs er va ti on URB597 0.3mg/kg o .d . f or 8 w eeks i. p. N o ef f ect on s ei z ur e in cide nce Do wi e, 2 010 mg/kg H U210 0.01mg/kg I ncr e as ed in cid ence o f sei z ur es on han dl in g

Other plant 27 N/A 100% 0% 0% cannabinoids

2% CB1 receptor 2 55 N/A 73% 18% (7% mixed agonists effect) • Results strongly support an overall anticonvulsant effect of plant cannabinoids and synthetic CB1R agonists. 8 Evidence from anecdotal use

• No modern, valid human clinical trials have been conducted

• Six modern case studies report clinically assessed anticonvulsant effects of cannabis

• Five large surveys concluded that some individuals using ‘medical marijuana’ do so to control symptoms of epilepsy

• Personal correspondence with ~50 UK PWE using cannabis for control of seizures.

• One small scale clinical trial (1981) demonstrated that CBD was anticonvulsant in 7/8 patients treated (no change in placebo group).

9 Cannabidivarin (CBDV) pharmacology

• Cannabidivarin (CBDV; also ‘cannabidivarol’) is a propyl analogue of cannabidiol (CBD).

• First isolated from hashish in 1969 (Vollner et al., 1969) although there is little extant evidence about pharmacological properties or therapeutic uses.

• Existing evidence of pharmacological effects:

– Stimulates recruitment of bone marrow mesenchymal stromal cells via a CB2 receptor-dependent mechanism (direct effect on CB2 not shown; Scutt & Williamson, 2007)

– Differential effects at transient receptor potential (TRP) channels in vitro:

• Acts as an hTRPA1, hTRPV1 and hTRPV2 agonist (EC50: 0.42, 3.6 and 7.3PM respectively) in transfected HEK-293 cells (De Petrocellis et al, 2011a, De Petrocellis et al, 2011b)

• Acts a TRPM8 antagonist (IC50: 0.90 PM) in transfected HEK-293 cells (De Petrocellis et al, 2011a).

• Relevance of TRP target in epilepsy unknown

• Inhibits diacylglycerol lipase a (DAGLa; IC50: 16.6PM) in vitro, the primary synthetic enzyme of the endocannabinoid, 2-arachidonoylglycerol (De Petrocellis et al, 2011a) but relevance to epilepsy unknown.

• Relevance of affinity for these targets for epilepsy remains unclear. 10 In vitro efficacy

11 Efficacy vs maximal electroshock seizures and audiogenic seizures • First line in vivo, mouse models for AED screening • Both reveal whether or not broad anticonvulsant effects are present.

MES

Audiogenic in DBA/2

12 Efficacy against acute PTZ and pilocarpine induced seizures • PTZ (panels A-D): model of generalised seizure also indicative of efficacy against absence seizures • Acute pilocarpine (panels E-H): model of temporal lobe seizures and status epilepticus

• Significant anticonvulsant effects against acute PTZ- induced generalised seizures

• No significant effect against acute, pilocarpine-induced TLS/status epilepticus (in this study).

13 Efficacy and tolerability retained with other AEDs (acute PTZ)

• Safe when co- administered?

• Study drug effect retained?

• Synergism of effects?

14 Efficacy and tolerability retained with other AEDs (acute pilocarpine)

• Safe when co- administered?

• Study drug effect present?

• Synergism of effects?

• Notably, in this more highly powered study (more animals received study drug), CBDV was anticonvulsant against acute, pilocarpine-induced TLS/status epilepticus

15 Tolerability and oral efficacy vs PTZ

16 Acknowledgements

• Collaborators: Dr C. Williams, Dr G. Stephens, Dr G. Woodhall (Aston, UK), Prof. L. Sander (NEC & UCL, UK), Prof. E. Williamson & Prof. R. Hampson (Wake, USA). • Researchers (past and present): Dr A. Hill, Mr N. Jones, Dr S. Weston, Dr J. Farrimond, Dr I. Smith, Dr X. Wang, Dr Y. Yamasaki, Mr T. Hill, Mrs C. Hill, Mr N. Amada, Miss I. Peres, Mrs R. Hadid, Mrs A. Al-Husaini, Mr S. Akiyama, Dr A. Futamura, Miss M. Mercier, Mr S. Marshall.

Sponsors: GW Pharmaceuticals and Otsuka Pharmaceuticals for continued financial and material support. 17 References

• Banerjee & Hauser (2006) in Epilepsy: a comprehensive textbook Lippincott & Williams (Eds: Engel & Pedley) Chapter 5, pp15 • Center for Disease Control (2008) Morbidity and Mortality Weekly Report 57; SS-6 • De Petrocellis, Ligresti, Moriello, Allara, Bisogno, Petrosino, Stott & Di Marzo (2011) Effects of cannabinoids and cannabinoid-enriched cannabis extracts on TRP channels and endocannabinoidmetabolic enzymes. Br J Pharmacol 163:1479-1494. • De Petrocellis, Orlando, Moriello, Aviello, Stott, Izzo & Di Marzo (2011) Cannabinoid actions at TRPV channels: effects on TRPV3 and TRPV4 and their potential relevance to gastrointestinal inflammation. Acta Physiol (Oxf). • EMEA (2000) Guidance on Clinical Investigation of Medicinal Products in the Treatment of Epileptic Disorders http://www.emea.eu.int/pdfs/human/ewp/056698en.pdf • Engel (2006) Report of the ILAE Classification Core Group. Epilepsia. 2006;47:1558-1568 • Farrimond, Mercier, Whalley and Williams (2011) Cannabis sativaand the endogenous cannabinoid system Phytotherapy Research 25(2):170-88. • Gastaut (1970) Clinical and electroencephalographicalclassification of epileptic seizures Epilepsia11; 102 • Hill, Jones, Williams, Stephens & Whalley (2010) Development of multi-electrode array screening for anticonvulsants in acute rat brain slices. J Neurosci Methods 185:246-256. • Hill, Williams, Whalley and Stephens (2012) Phytocannabinoids as novel therapeutic agents in CNS disorders. Pharmacology & Therapeutics 133(1):79-97. • Hill, Mercier, Hill, Glyn, Jones, Yamasaki, Futamura, Duncan, Stott, Stephens, Williams and Whalley* (2012; ePub ahead of print) Cannabidivarinis anticonvulsant in mouse and rat in vitro and in seizure models British Journal of Pharmacology • Kwan & Brodie (2000) Early identification of refractory epilepsy. N Engl J Med 342(5):314–9. • Lhatoo, Sander & Tomson (2006) in Epilepsy: a comprehensive textbook Lippincott & Williams (Eds: Engel & Pedley) Chapter 10, pp1 • Vollner, Bieniek, & Korte F (1969) Hashish. XX. Cannabidivarin, a new hashish constituent. Tetrahedron Lett 145-147. • WHO (2012) http://www.who.int/mediacentre/factsheets/fs999/en/index.html (last accessed 06/01/2012) • Scutt & Williamson (2007) Cannabinoids stimulate fibroblastic colony formation by bone marrow cells indirectly via CB2 receptors. Calcif Tissue Int 80:50-59.