Introduction

Sativex® is an oromucosal medicine formulated from plant-based extracts prepared from genetically distinct varieties (chemovars) of Cannabis Sativa L. The two majortherapeutic components in Cannabis Sativa L extracts are the cannabinoids ∆9tetrahydrocannabinol (THC) and cannabidiol (CBD). Proprietary strains of Cannabis Sativa L havebeen cultivated by GW Pharma Ltd, that contain specified levels of these cannabinoids, one strain containing predominantly THC and one predominantly CBD. These chemovarsare then used as the Botanical Raw Material (BRM), from which partially purified whole plant extracts are produced (Botanical Drug Substances, BDSs). The THC-rich BDS istermed Tetranabinex®, and the CBD-rich BDS is termed Nabidiolex®. Sativex® contains Tetranabinex® and Nabidiolex® in an approximate 1:1 ratio, delivering a THC concentrationof 27mg/ml and a CBD concentration of 25mg/ml. The BDSs also contain smaller amounts of other compounds such as other minor cannabinoids e.g. cannabigerol, terpenoids,flavonoids and sterols.

Cytochromes P450 (CYP450) are a family of haem-containing enzymes responsible for the metabolism of many drugs in man. The major human drug metabolising cytochromeP450 enzymes comprise CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 isoforms. Of these, CYP3A4 is the major cytochrome P450 enzyme present in human liver andcatalyses the biotransformation of a wide variety of drugs. Many drugs can act as inhibitors of cytochrome P450 in vivo, thereby altering the disposition of other co-administereddrugs which are themselves metabolised by the same enzymes. The inhibition of important drug metabolising enzymes such as CYP450 has been implicated in clinically relevantdrug-drug interactions.

The cytochrome P450 (CYP) isozymes involved in metabolism of ∆9-THC are presented in Table 1.

A list of CYP450 Isoforms, selective substrates and inhibitors are presented in Table 2.

P450 Selective Reaction * Selective inhibitorIsoform substrate

CYP1A2 Phenacetin O-deethylation to acetaminophen Furafylline

CYP2C9 Tolbutamide hydroxylation to 4-methylhydroxy Sulphaphenazoletolbutamide

CYP2C19 S-mephenytoin hydroxylation to 4-hydroxy S- -mephenytoin

CYP2D6 Bufuralol hydroxylation to 1-hydroxy bufuralol Quinidine

CYP3A4 Testosterone hydroxylation to 6ß-hydroxy Ketoconazoletestosterone

Table 2 – List of CYP450 Isoforms, selective substrates and inhibitors

* S.E. Clarke., In vitro assessment of human cytochrome P450, Xenobiotica, 1998, vol.28, 12, 1167-1202.

THE EFFECTS OF CANNABIS EXTRACTS TETRANABINEX® & NABIDIOLEX®

ON HUMAN CYTOCHROME P450-MEDIATED METABOLISMColin G. Stott, Geoffrey W. Guy, Stephen Wright & Brian A. Whittle

GW Pharma Ltd, Porton Down Science Park, Salisbury, SP4 0JQ, UK

11-OH-THC 8ß-OH-THC 8αα-OH-THC 3’-OH-THC Minor Metabolites

CYP2C9

Human CYP2C19 CYP3A4CYP2C19

CYP3A4/5

CYP2C8*CYP3A4

-

CYP2C11

Rat CYP2C13*CYP2A2 CYP2C11

Male CYP2A2*

CYP2D6

Rat

FemaleCYP2C6

Mouse CYP2C29 CYP3A11 CYP2C29 -

Guinea Pig CYP2B CYP3A -

Objective:

The objective of this study was to determine the effect of Tetranabinex® (THC Botanical Drug Substance (BDS)) & Nabidiolex® (CBD Botanical Drug Substance (BDS)) and 1:1 %(v/v) mixture of Tetranabinex® & Nabidiolex® on the activity of human hepatic cytochromes P450 (CYP450) 1A2, 2C9, 2C19, 2D6 and 3A4.

Methods

The effects of the 3 test articles (TAs) on 5 cytochrome-P450 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP3A4 and CYP2D6) were investigated. Tetranabinex®, Nabidiolex®

and 1:1 mixture (10 concentrations) were incubated at 37°C with human liver microsomal protein, buffer, enzyme co-factor solution and CYP450-selective substrates. The extentof metabolism of each CYP450-selective substrate in the presence and absence of the TAs was compared. The concentration range of TAs under investigation was = 0, 0.01,0.03, 0.1, 0.3, 1, 3, 10, 30 & 100 µM.

CYP450 Isoforms – incubation with 14C-radiolabelled selective inhibitors

Cannabinoid extracts, THC, CBD and THC/CBD were incubated at final incubation concentrations of 0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30 and 100 µM with pooled human livermicrosomes (final incubation concentration of 1 mg microsomal protein/mL incubation mixture) for 40 min in the presence of the specified, 14C-radiolabelled, selective P450substrate (final incubation concentration of 20 µM).

Incubation mixtures contained THC, CBD or THC/CBD mixture (2.5µI of 2 – 20,000 µM solutions in DMSO), microsomal protein (defined volume µl), 14C-substrate (definedvolume (µl) of defined concentration in methanol (mM)) and phosphate buffer (421.2µl, 100 mM, pH 7.4) to give a pre-incubation volume of 450 µl. Following a pre-incubationperiod of 5 min, metabolic reactions were initiated by the addition of enzyme co-factor solution (50µI of ß-NADPH regenerating system) to give a final defined incubation volume(500µl).

Control incubations had DMSO in place of cannabinoid extracts (no test compound control, 0 µM THC, CBD, THC/CBD), phosphate buffer in place of microsomes (no proteincontrol) and NaHCO3 (2% (w/v)) in place of enzyme cofactor solution (no co-factor control).

Positive control incubations contained the specified P450 inhibtor (a chemical inhibitor selective for the relevant CYP450 isoform enzyme) in place of THC, CBD or THC/CBD atan appropriate final concentration (range 2 – 10 µM (these incubations were preincubated for a specified pre-incubation time prior to starting metabolic reactions, depending upon the inhibitor).

All pre-incubation and incubation stages were performed at ca 37°C in an oscillating water bath with vials open to the atmosphere.

Microsomal reactions were terminated after the desired time period by the addition of the appropriate agent (methanol, acetonitrile, perchloric acid, phosphoric acid, methanol/ water).

Samples were centrifuged at 10000 rpm using a Hermle Z230MR bench top centrifuge for 5 minutes at ambient temperature to pellet the precipitated microsomal protein. Analiquot of each sample supernatant (100 IL) was analysed by HPLC (Appendix 7) using on-line radiodetection (after storage at ca -20QC).

Endpoint:

IC50 was defined as the concentration of inhibitor that affords 50% inhibition of the control activity under the stated assay conditions. The results of the effects of the 3 TAs on

the 5 isoforms are presented in Table 3.

Results

The results of the assay are presented as IC50s in Table 3.

Inhibition by Tetranabinex®

Tetranabinex® is a relatively weak inhibitor of CYP3A4 and a weak inhibitor of CYP1A2, CYP2C9 and CYP2C19. THC is not an inhibitor of CYP2D6.

Figure 1: IC50 plot for THC against Figure 2: IC50 plot for THC against

14C-S-mephenytoin 4-hydroxylation 14C-testosterone 6H-hydroxylation(CYP2C19 activity) (CYP3A4 activity)

Discussion

Under these in vitro conditions, it appears that at high concentrations, CBD has greater inhibitory potential than THC, and that this results in the inhibitory potential which maybe associated with a 1:1 mixture of the TAs.

Other investigators (Jaeger, et al, 1996) have reported that CBD inhibits human hepatic microsomal CYP3A4 activity, observing a ca 25% reduction in cyclosporine A metabolismat a final incubation concentration of 30 µM.

It has long been recognised that substrate characteristics for CYP3A4 are known to vary widely and different inhibition potency is often observed with different substrates.Furthermore, if the inhibition observed results from some form of CBD metabolic activation (i.e. mechanism-based enzyme inhibition), the source and activity of the human livermicrosomes used for the assays will lead to varying degrees of inhibition being observed.

However, in human clinical studies, therapeutic doses of Sativex“, (a cannabis-based medicine containing Tetranabinex® and Nabidiolex® in a 1:1 ratio), produce human plasmalevels of THC and CBD of approximately 5-30ng/ml (ca. 15-100nM).

The most potent interaction observed in this study for an individual extract was an IC50 of 7 µM for Nabidiolex® against testosterone 6-hydroxylation, a marker reaction for CYP3A4

activity. This in vitro micromolar concentration is equivalent to 2201.3 ng/mL, a concentration some 440 fold greater than maximum observed plasma concentrations in humanclinical studies.

Conclusion

• Tetranabinex® does not inhibit CYP2D6, and is a relatively weak / weak inhibitor of CYP3A4, CYP1A2, CYP2C9 & CYP2C19.

• Nabidiolex® is a relatively weak / weak inhibitor of CYP1A2, CYP2C9 & CYP2D6.

• Although Nabidiolex® may inhibit CYP2C19 & CYP3A4 in vitro, inhibition only occurs at high doses: 7-9 µM (2,200 - 2,800 ng/ml)

• At levels of plasma exposure achieved with therapeutic doses of Sativex® (5 - 30 ng/ml), it is highly unlikely that Tetranabinex® or Nabidiolex® would contribute to CYP450-derived inhibitory drug-drug interactions in humans.

Based upon the IC50 values obtained in this study (without pre-incubation), it is unlikely that either Tetranabinex® and Nabidiolex® would contribute to cytochrome P450-derived

inhibitory drug-drug interactions in vivo considering the far lower levels of both components observed in plasma during clinical studies (some 440-fold lower than the most potentinteraction (IC50) observed in this study).

References

1. Hawksworth G and McArdle K (2004) Metabolism and pharmacokinetics of cannabinoids. The medicinal uses of cannabis and cannabinoids. Edited by G W Guy, B A Whittleand P J Robson, pp 205-228. Pharmaceutial press.

2. S.E. Clarke., In vitro assessment of human cytochrome P450, Xenobiotica, 1998, vol.28, 12, 1167-1202.

3. Jaeger, W., Benet, L. Z and Bornheim, L. M. Inhibition of cyclosporine and tetrahydrcannibol metabolism by cannabidol in mouse and human microsomes, Xenobiotica., 26,3, 275-284, 1996.

Test Article (TA) Solutions

Stock solutions of THC and CBD were prepared in dimethyl sulphoxide (DMSO) for use in this study. The solutions for the individual extract incubations were prepared to aninitial concentration of 40,000 µM (40 mM), relative to the amount of principal cannabinoid (i.e. THC or CBD).

A portion of each 40 mM extract solution (300 µl) was then diluted with an equal volume of DMSO to give Tetranabinex® and Nabidiolex® solutions of 20 mM of principalcannabinoid. Each 20 mM extract solution was then serially diluted with DMSO to give solutions of 6000, 2000, 600, 200, 60, 20, 6 and 2 µM.

An aliquot of the remaining THC 40 mM solution (300 µl) was mixed with an equal volume of the remaining 40 mM CBD solution, to give a 1:1 (% (v/v)) mixture of THC/CBD witha concentration of 20 mM for each principal cannabinoid. This mixture was then serially diluted with DMSO to give solutions of 6000, 2000, 600, 200, 60, 20, 6 and 2 µM.

IC50

CytochromeP-450 Tetranabinex® Nabidiolex® 1:1 Tetranabinex® :

Isoform Nabidiolex®

µM ng/ml µM ng/ml µM ng/ml

CYP1A2 40 12,579 14 4,403 12 3,774

CYP2C9 44 13,837 72 22,642 20 6,289

CYP2C19# 34 10,692 9 2,830 7 2,201

CYP2D6 125 39,309 84 26,416 38 11,950

CYP3A4 26 8,176 7 2,201 6 1,887

Table 3 – IC50 concentrations for Inhibition of Cytochrome P450 isoforms

#Investigations were performed to investigate the use of Tranylcypromine as a possible selective chemical inhibitor of CYP2C19 (data not shown). However, these were unsuccessful and illustrate the difficulty in obtaining selective inhibition of this enzyme.

Inhibition by Nabidiolex®

Nabidiolex® is a relatively weak inhibitor of CYP1A2, and is a weak inhibitor of CYP2C9 and CYP2D6. However, based on the above data the CBD present in Nabidiolex® maybe a relatively potent inhibitor of CYP2C19 and CYP3A4 activity (IC50 = <10 µM).

Figure 3: IC50 plot for CBD against Figure 4: IC50 plot for CBD against

14C-S-mephenytoin 4-hydroxylation 14C-testosterone 6H-hydroxylation(CYP2C19 activity) (CYP3A4 activity)

* slight activity

Table 1: Summary of major cytochrome P450 (CYP) isozymes involved in metabolism of D9-THC1