Unheated Cannabis sativa extracts and its major compounds THC-acid have potential anti-inflammatory properties not mediated

by CB1 and CB2 receptor coupled pathways

Henrie Korthout1, Marcel de Wit2, Kitty Verhoeckx3, Mei Wang4 and Renger Witkamp3
1

Fytagoras B.V., Zernikedreef 9, 2333 CK Leiden, the Netherlands; 2Maripharm B.V. Nieuw Mathenesserstraat 33, 3029 AV Rotterdam, the Netherlands; 3TNO Pharma, Utrechtseweg 48, P.O.box 360, 3700 AJ Zeist, the Netherlands; 4SU BioMedicine, Zernikedreef 9, 2333 CK, Leiden, the Netherlands

There is a great interest in the pharmacological properties of cannabinoid like compounds that are not linked to the adverse effects of 9-tetrahydrocannabinol (THC), e.g. psychoactive properties. Here we present the anti-inflammatory properties of unheated Cannabis sativa extracts and its main non-psychoactivie constituent 9-tetrahydrocannabinoidic acid (THC-acid) in in vitro and in vivo studies Receptor binding activity
1,0 0 E 09

PC-PLC activity
18 16 14 PLC activity in mU/mL

TNF- release U937 macrophages

160 140 TNF-alpha in ng/mg protein 120 100 80 60 40 20

in vivo inflammatory response

3

CB1

CB2

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Histological score

12 10 8 6 4 2

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1 p=0.004 0 placebo THC -acid

p=0.006

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cold extract

extract cold extract heated

Extract cold

THC

Extract heated

extract cold extract heated

THC-acid THC

PMA

THC-acid

THC-acid

controls

Dissociation constants of pure THC, THC-acid and cold and heated cannabis extracts (normalized for the concentration of THC-acid and THC respectively) for CB1 and CB2 receptors. The heated extracts were heated at 200C for 7 minutes.

Phosphatidylcholine specific phospholipase C activity of U937 macrophages exposed to LPS alone (negative control) or in combination with THC, THC acid (both 30 g/mL), unheated cannabis extract (THC-acid = 30 g/mL) or D609 (200 M; positive control).

Release of TNF-alpha from U937 macrophages incubated for 4 hours with LPS (black column) or LPS in combination with THC, THC acid (both 30 g/mL) or heated or unheated (cold) cannabis extract (final THC or THC-acid concentrations 30 g/mL)

These results suggest that THC-acid and unheated extracts have anti-inflammatory properties and are acting via different metabolic pathways than THC and heated cannabis extracts. The anti inflammatory effect of THC-acid and unheated extracts was not only observed in vitro, but also in vivo. In a pilot EAE animal study the effetcs of THC-acid and unheated cannabis extracts on the clinical and histological signs of EAE collectively suggest that these may have therapeutic potential for Multiple Sclerosis, Arthritis etc.

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Extract cold

THC-acid THC

THC

LPS

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Histological score of the effect of THC-acid and unheated cannabis extract on the inflammatory response in the brain stem in Experimental Autoimmune Encephalomyelitis (EAE) induced mice (female SJL). : individual score; mean score (n=9). The severity of the inflammatory reaction was indicated using the following individual score; 0: no infiltrates, 1: sporadic, mild perivascular infiltration, 2: multifocal, mild perivascular infiltration, 3: multifocal, severe perivascular infiltration, 4: multifocal, severe perivascular infiltration accompanied by spreading into parachyme.