Metabolites of cannabidiol identified in human urine.

XENOBIOTICA,

1990, VOL. 20,

NO.

3, 303-320

Metabolites of cannabidiol identified in human urine

Xenobiotica Downloaded from informahealthcare.com by Universitaets- und Landesbibliothek Duesseldorf on 06/25/13 For personal use only.

D. J. HARVEYt and R. MECHOULAMS

t University Department of Pharmacology, South Parks Road, Oxford OX1 3QT, UK $ Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 91 120 Israel Received 30 March 1989; accepted 10 November 1989 1. Urine from a dystonic patient treated with cannabidiol (CBD) was examined by g.l.c.-mass spectrometry for CBD metabolites. Metabolites were identified as their trimethylsilyl (TMS), ['H,]TMS, and methyl ester/TMS derivatives and as the T M S derivatives of the product of lithium aluminium deuteride reduction. 2. Thirty-three metabolites were identified in addition to unmetabolized CBD, and a further four metabolites were partially characterized. 3. The major metabolic route was hydroxylation and oxidation at C-7 followed by further hydroxylation in the pentyl and propenyl groups to give 1"-, 2"-, 3"-, 4"- and 10-hydroxy derivatives of CBD-7-oic acid. Other metabolites, mainly acids, were formed by 8-oxidation and related biotransformations from the pentyl side-chain and these were also hydroxylated at C-6 or C-7. The major oxidized metabolite was CBD-7-oic acid containing a hydroxyethyl side-chain. 4. T w o 8,9-dihydroxy compounds, presumably derived from the corresponding epoxide were identified. 5. Also present were several cyclized cannabinoids including delta-6- and delta-ltetrahydrocannabinol and cannabinol. 6. This is the first metabolic study of CBD in humans; most observed metabolic routes were typical of those found for CBD and related cannabinoids in other species.

Introduction Cannabidiol (CBD, I) is one of over 60 cannabinoids found in the plant Cannabis satiwa L. (Turner et al. 1980)) the source of the drug marihuana. Although nonpsychoactive, the compound has been shown to possess anticonvulsant (Karler and Turkanis 1981) and neurological properties such as the ability to block the anxiety caused by the major psychoactive constituent of Cannabis, delta-9tetrahydrocannabinol (THC) (Zuardi et al. 1982). Metabolism of CBD has been studied in several animal species (reviewed by Harvey and Paton 1984; Samara et al. 1989 a, b) but so far, there has been no report of its metabolism in humans although CBD has been administered to human patients with epilepsy (Cuhna et al. 1980) and dystonia (Consroe and Snider 1986)with considerable success. We recently acquired a large urine sample from a patient treated with CBD for dystonia and analysed it for CBD and its metabolites. A preliminary report on this work has appeared (Harvey and Mechoulam 1987); the full results are reported in this paper.

Materials and methods Dosing of patient and acquisition of urine sample Urine (1.5 I) was collected over 24h from a dystonic patient treated chronically with CBD (600mg daily) at the Neurology Department, Hadassah-Hebrew University Hospital. Jerusalem (Consroe and Snider 1986). Metabolites were extracted from the urine with ethyl acetate (3 x 300ml), the solution was 0049-8254/90 $3.00

0 1990 Taylor & Francis Ltd.

304

D. J . Harvey and R. Mechoulam


dried dver MgSO, and evaporated to dryness under reduced pressure at S O T , leaving 68 mg of residue. Part of the extract was converted directly into derivatives for g.1.c.-mass spectrometry as described below. Another part of the ethyl acetate extract was hydrolysed with B-glucuronidase (Type VII from E. Coli, Sigma Chemical Co.) at pH 6.8 for 3 h at 37°C prior to conversion into derivatives. Preparation of derivatives TMS derivutives. Samples, of about 100pg, of both the unhydrolysed and hydrolysed ethyl acetate extract were heated with N,O-6is-(trimethylsilyI)-trifluoroacetamide(BSTFA, 1 0 ~ 1for ) lOmin at 60°C. ['H,]TMS derivatiwes. These were prepared as for the T M S derivatives with [2H,8]biStrimethylsilylacetamidereplacing the BSTFA. Methyl ester/ TMS derivatiwes. Methanol (10 pl) was added to the dried sample from the ethyl acetate extract followed by a fresh, ethereal solution (05 ml) of diazomethane (prepared from Diazald, Sigma Chemical Co.). The mixture was well stirred and allowed to stand at room temperature for 2 min. It was then centrifuged to remove the precipitate and the reagent and solvents were removed with a stream of nitrogen. The residue was converted into T M S derivatives as described above. Reduction with lithium aluminium deuteride. An aliquot of the dried ethyl acetate extract from both hydrolysed and unhydrolysed samples was dissolved in dry (sodium) ether (1.0ml) and heated at reflux temperature with an excess of lithium aluminium deuteride for 1 h. After destruction of the excess of reagent with ethyl acetate, the aluminium salts were dissolved in 0 5 M H,SO, (1 ml) and the products were extracted with ethyl acetate ( x 3), and washed with water and saturated aqueous NaCI. The solvent was removed with a stream of nitrogen and the residue was converted into T M S derivatives. Gas chromatography G.1.c. retention data and the relative concentration of the metabolites were measured with a HewlettPackard 5890A gas chromatograph fitted with a 50m x 0 3 mm OV-1 bonded-phase fused silica capillary column (film thickness 052 pm). Helium at 2 ml/min was used as the carrier gas with a split ratio of 10 :1. The injector and detector (FID) temperatures were both 300°C and the column oven was temperature programmed from 130°C to 350°C at 2"C/min. Data were recorded with a Servoscribe flat-bed recorder and with a Hewlett-Packard 3390A recording integrator. G.1.c.-mass spectrometry Two systems were used for g.1.c.-mass spectrometric analysis. The first was a VG 12B single focusing mass spectrometer interfaced via a glass jet separator to a Varian 2440 gas chromatograph fitted with a 2 m x 2 mm (int. diam.) glass column packed with 3% SE-30 on 10&120 mesh Gas Chrom Q (Applied Science Laboratories, State College, PA, USA). The carrier gas was helium (30 ml/min), and the column oven was temperature-programmed from 180°C to 300" at 2"lmin. The injector, separator and ion source temperatures were 300, 300, and 260°C respectively. Other operating conditions were: accelerating voltage, 2.4kV; trap current, 100pA; electron energy, 25 eV; scan speed, 3 sec/decade. Spectra were acquired with a VG 2050 data system and processed with a linked VG 11 :250 data system. The second g.1.c.-mass spectrometric system consisted of a VG 70/70F double focusing-mass spectrometer connected to a Varian 2440 gas chromatograph. The column was a 30 m x 0 2 mm OV-1 bonded-phase fused silica capillary (film thickness 033 pm) terminating 1 cm inside the ion source. Helium at 1 ml/min (measured in the absence of the vacuum) was used as the carrier gas. The injector was an SGE splitlsplitless system used in the split mode with a split ratio of 10 : 1. The column oven was temperature programmed from 220°C to 320" at 2"/min. Other operating conditions were: injector, transfer line and ion source temperatures, 300, 300, and 250°C respectively, electron energy, 70 eV, trap current, 1 mA; acceleratingvoltage, 4 kV. The instrument was scanned repetitively at 1 sec/decadeunder the control of the VG 11 :250 data system.

Results Identification of metabolites General. Metabolites were identified by g.1.c.-mass spectrometry. Many of the metabolites found in this sample have been reported previously in other species and so g.1.c.-mass spectrometric data could be compared directly with literature values. In all cases, functional groups were identified by the reactivity of the metabolites towards the various derivatizing reagents. Thus, comparison between the mass shifts produced by ['H,]TMS derivatives (McCloskey et al. 1968) and the masses observed from the T M S derivatives allowed the number of T M S and thus the number of hydroxy and acid groups to be determined. Carboxylic acids were identified by preparation of methyl ester/TMS derivatives and by their reduction to


Cannabiodol metabolites in human urine

305

alcohols with lithium aluminium deuteride. The purpose of the deuterium was to allow differentiation of the alcohols produced by reduction from those occurring naturally (Harvey and Paton 1980). Fragmentation of CBD and its metabolites produced several very abundant, diagnostic ions which readily allowed the structures of the compounds to be determined (Harvey 1987). Thus, the retro-Diels-Alder cleavage of the terpene ring (ion a, Scheme 1) resulted in loss of the five carbon atoms shown in Scheme 1, together with their substituents, and localized the metabolically added group either to the lost fragment or to ion a. The presencepf a substituent at C-7 invariably led to cleavage of the C-1-C-7 bond with loss of C-7 and its substituent (ion b), allowing ready identification. This fragmentation was, however, more prominent in the spectra of T M S derivatives of 7-carboxylic acids than in that of their corresponding methyl esters. In addition, the mass of the tropylium ion (c) and its shift in the spectra of the different derivatives, enabled the structure of the side-chain to be determined. Figure 1 shows a limited ion chromatogram (masses 300-700) of the metabolites present in the ethyl acetate extract of the unhydrolysed urine. The corresponding extract from hydrolysed urine was similar but without peaks corresponding to the intact glucuronides. Identified metabolites are listed in tables 1, 2 and 3 and mass spectral data for the various derivatives are listed in table 4. Thirty-three metabolites were identified in addition to unmetabolized CBD; several of these compounds have not been reported before as metabolites of this drug. Several other metabolites were detected but not fully identified. In an earlier report on the metabolism of CBD in this sample using packed column g.1.c.-mass spectrometry, fewer metabolites could be identified (Harvey and Machoulam 1987). Metabolites were identified as follows.

CBD and CBD-glucuronide. One of the major excreted compounds (peak 5, 12.1% of the total excreted cannabinoids as determined by g.1.c.) was identified as unmetabolized CBD by the identity of its mass spectrum (TMS derivative) with that of an authentic sample. This compound was also excreted as its 0-glucuronide (peak 50, 13.3%) which was identified by comparison of its mass spectrum (TMS and Me/TMS derivatives) with published data (Harvey et al. 1976, Lyle et al. 1977, Pallante et al. 1978). The presence of the aglycone ion at m / z 458, corresponding to the molecular ion of CBD T M S ether, indicated that the compound was an 0- and not a C-glucuronide (Lyle et al. 1977, Pallante et al. 1978). Other non-oxidized cannabinoids. The compounds producing peaks 9 (1.97%) and 10 (0.69%) were identified, by their mass spectra (TMS derivatives) and retention times, to be delta-6- and delta-1-THC respectively and were presumably formed by cyclisation of CBD. The aromatic compound, cannabinol (CBN) was also identified (peak 12, 0.6%). The compound producing peak 6 (1.2%) was not identified, but appeared to be a related compound in which one of the phenolic hydroxyl groups had been incorporated into a ring. Its molecular weight (TMS derivative) was the same as that of T H C (m/z 386,2%) and the base peak in its mass spectrum was at m/z 303. This spectrum was similar to that of the TMS derivative of cannabichromene (Harvey 1987), but the retention time was different. 7-Hydroxy-CBD, 11.This compound (peak 15), formed a tris-TMS derivative (molecular weight 546) and did not react with diazomethane or lithium aluminium hydride. The presence of the retro-Diels-Alder (RDA) ion (a) at m/z 478 ([M-68] '), the tropylium ion (c) at mlz337 and the [M-C-7]+ ion (b) at m/z443 (table 4)

D . J . Harvey and R. Mechoulam


306

CBD (I)

L-L 1:

I

1:

I

TMSO OTMS

QyJ-

a

A '

R

TMSO ; H 2 %

~TMS ion

Ion

R

=

5

H o r OTMS

Scheme 1. Structures of CBD and ions a-d.


307

39


I

3 3

-1 I

Scan Time

(rnins)

400 17.49

600

800

26.35

35.20

1000 44.06

J

Figure 1. Reconstructed ion chromatogram ( 4 2 3W700) of the compounds extracted from human urine. Separation was made with a 2 5 m x 0 2 m m OV-1 fused-silica capillary column operated as described in the Materials and methods section. Most peaks were produced by metabolites as identified in tables 1-3 and in the text. Peaks identified as not being metabolites included peak 1 (16:0 alcohol), peak 2 [18:2 carboxylic acid (linoleic acid)], peak 3 [18: 1 carboxylic acid (oleic acid)], peak 4 [18:0 carboxylic acid (stearic acid)], peak 11 (20: 0 alcohol), peak 18 (22 :O alcohol), peak 22 (22 :0 carboxylic acid), peak 47 (cholesterol) and peaks 44,46and 49 which were sikiconcontaining ‘bleed’ peaks from the g.1.c. column.

confirmed the structure as 7-hydroxy-CBD (11). The spectrum of this derivative was the same as that reported by Martin et al. (1976 a) for this compound. 6-Hydroxy-CBD, III. The mass spectrum of this metabolite (peak 13, TMS derivative) was characterized by very abundant RDA ion (a, m/z478), a tropylium ion (c) at m/z337, weak molecular and [M-CHJ’ ions, and the absence of a [M-TMSOCH,] + ion. The structural assignment was confirmed by published data (Martin et al. 1976a). 6,7-Dihydroxy-CBD, XIII. The mass spectrum of this compound (peak 29) was similar to that of the 6-hydroxy metabolite in that it had weak molecular and [M-CH3]+ ions, a very abundant RDA fragment ion (a) at m / z 566 and a tropylium ion (c) at mlz 337 showing no hydroxylation of the side-chain. The presence of the ion at m/z531 ([M-103]+) localized the second hydroxy group at C-7. This compound has previously been reported as a metabolite of CBD in the rat (Martin et al. 1976b). CBD-7-oic acid, IV.The mass spectrum (table 4)of the TMS derivative of this compound (peak 21) was similar to that of the 7-hydroxy-CBD except that the molecular and RDA ions (a) were shifted by 14 mass units to higher mass. The compound formed a methyl ester and was reduced by lithium aluminium deuteride to 7-hydroxy-CBD, thus, characterizing it as CBD-7-oic acid (IV), a previously identified metabolite (Martin et al. 1977).

D . J . Harvey and R . Mechoulam

308

Table 1 . Structures and quantities present in urine of the metabolites of CBD containing an intact side-chain.


R'

Compound 7-OH 6-OH 7-COOH l"-OH,7-COOH l"-OH,7-COOH 2"-OH,7-COOH 2"-OH,7-COOH 3"-OH,7-COOH 3"-OH,7-COOH 4"-OH,7-COOH 10-OH,7-COOH 6,7-di-OH 7-OH,Sf'-COOH 6-OH,Sf'-COOH

No.

I1 111

IV V VI VII VIII IX X XI XI1 XI11 XIV XV

R2 R3 R4 R5 R6 R7

Peak"

R'

15 13 21 24 26 32 33 37 38 39 34 29 43 41

CH,OH CH, COOH COOH COOH COOH COOH COOH COOH COOH COOH CHZOH CHZOH CH3

H OH H H H H H H H H H OH H OH

H H H H H H H H H H OH H H H

H H H H H OH OH H H H H H H H H H

H H H OH OH H H H H H H H

H H H H H H H OH OH H H H H H

R8

Quantityb

H CH3 H CH3 H CH, H CH3 H CH3 CH3 H CH3 H CH3 H CH, H OH CH3 CH, H CH3 H H COOH H COOH

064 0.07 1.34 262 1.02 5.83 5.19 327 1.61 11.80 5.77 010 007 Trace

'See figure 1. bAs a percentage of the total peak area of the identified metabolites as determined by g.1.c.

Table 2.

Structures and quantitiespresent in urine of the metabolitesof CBD containinga reduced side-chain.

R'

OH Compound

No.

Peak"

XVI XVII XVIII XIX

8 11 14 20 19 30 35 16 28 31 24 19 16a

XX XXI XXII XXIII XXIV XXV XXVI XXVII XXVIII

R'

R2

R3

Quantityb

H H H H H H H OH OH OH H H OH

COOH CH2COOH (CH,),COOH (CH,),COOH COOH (CH,),COOH (CH,),COOH COOH (CH,),COOH (CH2),COOH CH2CH,0H CH2CH20H CH,CH20H

007 022 0.3 1 069 010 4.01 099 007 2.08 244 13.40 014 014

"See figure 1. bAs a percentage of the total peak area of the identified metabolites as determined by g.1.c.


309

Table 3. Structuresof the 8,9-dihydro-8,9-dihydroxy-metabolitesof CBD.

R’


I

Compound

No.

Peak

R’

7-COOH,8,9-di-OH 7,8,9-tri-OH

XXIX

42 36

COOH CH,OH

xxx

Side-chain hydroxy derivatives of CBD- 7-oic acid. These metabolites formed methyl esters and produced tropylium ions (c)at m / z 425 indicating hydroxylation in the pentyl side-chain (Martin et al. 1976b, 1977). Prominent ions in the spectra representing loss of the carboxy ester group ( m / z531, b) localized this to C-7. The position of the hydroxy group on the side-chain was determined to be at C-1” (V, VI, minor constituent of peak 24, clearly identified by single-ion plots of its constituent ions, and peak 25), C-2” (VII, VIII, peaks 32 and 33), C-3” (IX, X, peaks 37 and 38) and C-4” (XI, peak 39) by the presence of the diagnostic fragment ions reported earlier (Binder et al. 1974, Harvey 1981, 1987). The mass spectra of these metabolites were identical to those of the same metabolites previously identified in mice (Martin et al. 1977). An interesting feature of the side-chain hydroxy metabolites present in this sample was that two peaks with almost identical mass spectra were produced by l’l-, 2”- and 3”-hydroxy-CBD-7-0ic acid. This behaviour has not been observed before with packed g.1.c. columns except for 1”-hydroxy metabolites, and was shown to be due to separation of the two chiral alcohols. It is significant that separation of these isomeric pairs becomes less distinct with increasing distance from the aromatic ring. 10-Hydroxy-CBD-7-oic acid (XII).The spectrum of this hydroxy acid (peak 34) had an ion formed by loss of the carboxy ester group ( m / z531, b) and a RDA ion ( a )at mlz 492 showing that the hydroxy group was located in the eliminated fragment. The presence of the tropylium ion (c)at m / z 337 showed no metabolism of the side-chain and confirmed the presence of the acid group at C-7. The relative retention time and mass spectrum of this metabolite, which has not been reported previously, was similar, after allowance for the carboxy ester group, to a synthetic sample of 10-hydroxy-CBD. This allylic alcohol has recently been identified as a metabolite of CBD in liver microsomal incubates from several species (unpublished). Acid metabolites produced by oxidative degradation of the side-chain. Four monocarboxylic acids (compounds XVI-XIX, peaks 8, 11, 14 and 20 respectively) were found. These had, respectively, one, two, three and four carbon atoms in the side-chain. Their mass spectra are listed in table 4 and were similar to those of the monocarboxylic acids reported previously (Martin et al. 1977). The masses of the RDA (a) and tropylium (c)ions clearly showed the presence of the acid group in the side-chain.

TMS TMS TMS

I1

I11

IV

V

VI

VII

VIII

IX

7-OH

6-OH

7-COOH

l”-OH,7-COOH

lf’-OH,7-COOH

2”-OH,7-COOH

2-OH,7-COOH

3”-OH,7-COOH Me/TMS

TMS

Me/TMS

TMS

Me/TMS

TMS

Me/TMS

TMS

Me/TMS

TMS

Me/TMS

Deriv.

No.

34.0

329

325

29.0

28.5

271

21.1

24.3

Ret. timeb Mt

[M-CH,]+

RDA‘ Tropd

GC/MS data for the metabolites of CBD found in human urine.

Compound

Table 4. Ions” [M-R]“


Other

I-

P

TMS

XI

TMS

XVII

XVIII

XIX

2"-COOH

3"-COOH

4"-COOH

xx

TMS

XVI

1"-COOH

Me/TMS

TMS

Me/TMS

TMS

Me/TMS

TMS

Me/TMS

TMS

Me/TMS

TMS

XI11

XI1

TMS

X

Me/TMS

Deriv.

No.

6,7-di-OH

Compound

Table 4 (continuad)

260

262

23.4

200

18.2

-

33.1

344

34.1

Ret . timeb Mt

619 (1) 489 (36) 503 (13) 445 (4) 517 (7) 459 (4) 531 (3) 473 (4) 577 (5) 519 (2)

(5)

633 (35) 633 (27) 575 (16) 633 (9) 575

[M-CH3]*

RDA'

Tropd

489 (78) 43 1 (59)

[M-R]"

Ions"


Other

xv

XXVI

XXVII

XXVIII

2”-OH,7-COOH, nor

2”,7-di-OH, tlor

2“,6-di-OH, nor

XXV

XXIV

TMS

XXIII

TMS

TMS

Me/TMS

TMS

TMS

Me/TMS

TMS

Me/TMS

TMS

Me/TMS

TMS

Me/TMS

TMS

XIV

XXII

TMS

XXI

Me/TMS

Deriv.

No.

6-OH,Sf’-COOH

Compound

Table 4 (continued)

-

346

28.5

35.2

28.1

285

24.5

363

33.4

31.1

timeb

Ret. M? [M-CH3]+

RDA’ Tropd

[M-R]+‘

Ions’


Other

0

EL

a

h)

w

1

TMS

TMS TMS TMS TMS

XXIX

xxx

XXXIII

XXXI

XXXII

8,9-di-OH,7-COOH

7,8,9-tri-OH, di-H

'

amla and (relative abundance). In minutes. Retro-Diels-Alder cleavage ion (a). "Tropylium ion (c). [M-7-carbon atom with its substituents]+ (ion b). [M-C,H9]+. Retro-Diels-Alder ion-C,H,. [TMSO=CH-C,H,]+.

MelTMS

Deriv.

Compound

No.

Table 4 (continued)

33.4

292

30.1

33.5

362

Ret. timeb

-

531 (15) 541 (29) 619 (8)

(37) 634 (4)

w9

\

\ -

723

[M-CH3]+

546 (16) 556

724 -

-

738

Mt

(39) 53 1 (32)

(29) 337 (22) 41 1 (100) 333

337

Trop"

(100) 425 (58)

439

-

(4)

(1001 531"

635"

Ions" [M-R]"

143 (100)

143

(32)

505

518"

Other

'

'

Retro-Diels-Alder ion-[C,H80]+. [M-C4H70TMS]+, Retro-Diels-Alder ion-C,H,OTMS. [M-TMSOH-CH,-CH = OTMS]'. [M-TMSO=CK,]+. "Lossof C-8 to C-10 with hydroxy substituents. [M-TMSOH-TMSO=CHJ+.

(79) 566 (100)

(93) 478 (20) 478 (95) 488

492

RDA'


iF b'

c.

P

3

2.

2

8 0

h

3

2

3


314

D. J . Harvey and R . Mechoulam

Mono-hydroxy derivatives of metabolites containing a carboxy-alkyl side-chain. Two groups of metabolites of this general structure were found; they had hydroxy groups at C-7 and C-6 respectively and side-chains of 1, 3, 4, and 5 carbon atoms long. Structures are shown in table 2 for the metabolites with shortened side chains and table 1 for the acids with a chain length of five carbon atoms. All structures are shown in Scheme 2. The masses and shifts on methylation of compounds in the first group showed that the carboxy group was in the side-chain, and the masses of the RDA ( a )and [M-C-7 substituent] + (b)ions confirmed hydroxylation at C-7, and the structures as 7-hydroxy-2",3",4",5"-tetrakis,nor-CBD-lrr-oic acid (XX, constituent of peak 19), 7-hydroxy-4",5"-bis,nor-CBD-3"-oic acid ( X X I , peak 30), 7-hydroxy5"-nor-CBD-4"-oic acid (XXII, peak 35) and 7-hydroxy-CBD-S"-oic acid (XIV, peak 43). Compound XX forming part of peak 19 was identified and distinguished from compound XXVII, also forming part of peak 19, by a slight difference in retention time seen in single ion plots of the ions shown in table 4.Also, compound XXVII did not form a methyl ester whereas compound XX did. The spectra of the other four metabdites [6-hydroxy-2",3",4",5"-tetrakis,norCBD- 1"-oic acid (XXI I I, peak 16), 6-hydroxy-4",5"-bis,nor-CBD-3"-oicacid (XXIV, peak 28), 6-hydroxy-5"-nor-CBD-4"-oicacid (XXV, peak 31) and 6hydroxy-CBD-S"-oic acid (XV, peak 41) did not possess an ion at [M-TMSOH]' (6) and were typical of those of 6-hydroxy-containing metabolites. Most of these compounds, particularly the hydroxy-4",5"-bis,nor-CBD-3"-oic acids have recently been found as the major urinary CBD metabolites in the dog (Samara et al. 1990 a) and rat (Samara et al. 1990b). Metabolites containing a shortened side-chain containing a hydroxy group The major metabolite producing the major constituent of peak 24 was identified as 2"-hydroxy-3",4",5"-tris,nor-CBD-7-oic acid (XXVI). It formed a tetrakis-TMS derivative and a monomethyl ether showing that it was a hydroxy-acid metabolite. However, the molecular weights of these derivatives indicated a mass loss of 42 units from the molecular weight of a hydroxy-acid metabolite of CBD itself. The masses of the RDA ( a ) ,tropylium (c) and [M-C-7]+ (b) ions clearly located the acid group at C-7 and indicated loss of three carbon atoms from the side-chain. In addition, reduction with lithium aluminium deuteride reduced only the acid group and the mass spectrum of the T M S derivative of the derived dihydroxy compound showed a prominent loss of the 7-CH2-OTMS group together with the two incorporated deuterium atoms. Additional ions involving loss of TMSOH from the RDA and [M-C-7]+ ions (see table 4) excluded the possibility of aromatic hydroxy substitution and located the second hydroxy group in the side-chain. A weak ion at mlz 103 confirmed its primary nature (substitution at C-5"). The structure of this metabolite was thus 2"-hydroxy-3",4",5"-tris,nor-CBD-7-oicacid (XXVI). Two other related metabolites with side-chains containing two carbon atoms were identified. The compound producing the second constituent of peak 19 was 2",7-dihydroxy-3",4",S"-tris-nor-CBD(XXVII) as it did not react with diazomethane and had a mass spectrum (TMS derivative) identical to that of the reduction product of the 7-oic acid metabolite identified above. The third metabolite of this type (peak 16a), present in trace concentration, had a mass spectrum typical of that of the 6-hydroxy derivative of 2-hydroxy-3",4",5"-tris-nor-CBD(XXVI 11, table 4). Dihydrodiol metabolites. The compounds producing peaks 42 and 36 (listed in table 3) had mass spectra consistent with their being the 8,9-dihydroxy derivatives of



315

Scheme 2. Metabolites identified from CBD in the human. Metabolic pathways shown by thick arrows have been proven to occur in cannabinoid metabolism. Most other pathways, shown by thin arrows, are the result of multiple hydroxylationsor oxidations where the order of metabolic transformation has not been established. Compounds enclosed in square brackets are intermediates, shown to occur for cannabinoids in other studies, but not observed in this study.

8,9-dihydro-CBD-7-oic acid (XXIX) and of 7-hydro~y-8~9-dihydro-CBD (XXX) respectively. Reduction of the acid (XXIX) with lithium aluminium deuteride converted it into the alcohol (XXX) demonstrating the common sites of metabolism. The presence of the tropylium ions (c) in the mass spectra (TMS derivatives) of both compounds at m / z 337 excluded the possibility of metabolism in the side-chain and the presence of the RDA ions (a) at mlz492 and 478 (TMS derivatives) for the acid and alcohol metabolites respectively located one of the metabolic sites as C-7. Loss of the other metabolically added groups during the formation of the tropylium ion thus located them at C-4,5,8,9 or 10. Both compounds had base peaks at mlz 143 (ion d), formed by loss of TMSOCH, from the smaller fragment of the RDA cleavage [charge localization occurs on this fragment when hydroxylation is present in the


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D. J. Harvey and R. Mechoulam

propenyl group, (unpublished)]. Ions were also present as the result of loss of the dihydroxypropyl group (C-8,9 and lo), behaviour similar to that of 8,9-dihydroxy8,9-dihydro-CBD. Full spectral details and fragmentation mechanisms for metabolites of this type will be published later. Metabolites not fully identified. The metabolite producing peak 27 was a monocarboxylic acid with the acid function at C-7. All of the diagnostic ions (compound XXXI, table 4) were of similar relative abundance to, but four mass units lower than, those of CBD-7-oic acid T M S ether indicating the presence of a metabolically altered side-chain. However, the structure of this altered side-chain was not determined. The most likely possibility would be that the side-chain contained two double bonds; dehydrogenation of aliphatic chains is well known for compounds such as valproic acid (Kochen and Scheffner 1980; Rettenmeier et al. 1986). A second component of peak 35 had a molecular weight (tetrakis-TMS derivative) of 634, did not react with diazomethane and was, thus, a dihydroxyCBD. The masses of the diagnostic ions (table 4) showed the presence of one of the additional hydroxy groups at C-7 and the other in the side-chain. No ions diagnostic of side-chain hydroxylation were present, indicating substitution at C-5“. The relative retention time of the metabolite indicated substitution at C-5”. The compound, thus, appeared to be Sr,7-dihydroxy-CBD (compound XXXII, table 4). The other incompletely identified metabolite (eluting just after the compound producing peak 29) had a molecular weight (tris-TMS derivative) of 546 and formed a monomethyl ester. The tropylium ion at m / z 41 1 indicated the presence of a 4”,5”bis,nor-3”-oicacid side-chain and the presence of the RDA ion (a)at mlz 478 showed that a further 14 mass units had been added to C-6 or C-7 (compound XXXIII, table 4). A compound containing a carbonyl function at either position would be the most likely structure but the compound was present in too low a concentration for other information to be obtained. Peaks identified as not being metabolites. Peaks in figure 1 identified as not being metabolites included peak 47 (cholesterol), 1 (16 :0 alcohol), 11 (20 :0 alcohol), 18 (22 : 0 alcohol), 2 (18 : 2 acid), 3 (18 :1 acid), 4 (18 :0 acid), 22 (22: 0 acid) and peaks 44,46 and 49 which were silicon-containing compounds from the g.1.c. column. The compound producing peak 17 had a molecular ion at mlz442 and a base peak at m / z 359. Although the spectrum resembled that of a cannabinoid, the compound was not identified.

Discussion Metabolism of CBD in this patient showed biotransformation routes reasonably typical for this drug, as outlined in Scheme 2. This is interesting as the compound is known to be a microsomal inhibitor (Paton and Pertwee 1972, Fernandes et al. 1973, Siemens et al. 1974). Although the mechanism of inhibition is not known, it has been shown that the resorcinol part of the molecule is probably responsible, and that binding is of the CO-type (Yamamoto et al. 1988). As CBD appears to inhibit certain cytochrome P-450 enzymes to a greater extent than others (Narimatsu et al. 1988) it is possible that the ratio of the metabofites of CBD reported in this paper from a patient treated chronically with the drug may not reflect the situation after a single dose. However, work in progress in this laboratory has shown that inhibition can be detected within 10min of drug administration and the similarity between the



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metabolites reported here and those observed in animals suggests that the present results are a reasonable reflection of the human situation and that any differences will be in the relative concentrations of the individual compounds rather than in different metabolic routes. One of the major metabolic routes seen in this experiment was hydroxylation at C-7 and oxidation to a carboxylic acid; this is the major metabolic pathway demonstrated by most cannabinoids (Harvey and Paton 1984). Further hydroxylation in the side-chain to give hydroxy-acid metabolites is also typical, but this sample was unusual in that more 2”-hydroxylation was present than is usually seen. Only in the mouse has a significant amount of hydroxylation at this position been reported to date and, in this case, no hydroxylation was seen at C-1” (Martin et al. 1977). The other noteworthy feature of the side-chain hydroxylation seen in these metabolites was the observation of both stereoisomers of the 1”-, 2”- and 3”hydroxy-CBD-7-oic acids (V-X). Previous studies on cannabinoid metabolism conducted in this laboratory have used packed g.1.c. columns with insufficient resolution to detect these isomers so it is not certain if this is unusual. The g.1.c. behaviour of the isomers was typical of compounds containing stereoisomeric hydroxy groups as demonstrated by the separation of reference samples of racemic 1”-, 2”-, 3”- and 4”-hydroxy-delta-l-THC (unpublished). In particular, g.1.c. separation decreased with increasing distance of the hydroxy-group from the aromatic ring so that separation of putative 4”-isomers could not be obtained. However, even though packed columns were used in previous studies, one sample of metabolites of delta-1-tetrahydrocannabinol was found to contain two isomers of the 1”-hydroxy metabolite although in unequal proportions (Harvey et al. 1980). Asymmetric hydroxylation at C-1” and C-3” was also found in the present CBD study although the absolute configuration of the alcohol groups was not determined. This behaviour appears typical; debrisoquin, for example, undergoes asymmetric benzylic hydroxylation to give a high proportion of the S-(+)-isomer (Meese et al. 1988). Another prominent metabolic route was j?-oxidation of the side-chain giving acids with reduced but odd numbers of carbon atoms. This probably also accounted for the low concentration of metabolites oxidized at C-5” as these had been metabolised further. The pathway for the formation of metabolites with even numbers of carbon atoms in the side-chain is less clear. Experiments on the metabolism of 5“-oxygenated derivatives of delta-6- and delta-1-THC have shown that, although j?-oxidation is a major metabolic route, and leads to compounds having three and one carbon atoms in the chain, no metabolites having even numbers of carbon atoms are formed (Harvey and Leuschner 1985). Recent experiments (Harvey 1989a) indicate that the precursor to these compounds is 4“-hydroxy-CBD. In an experiment in which metabolism of 4“-hydroxy-delta-1-THC was investigated in vivo in mice, 5”-bis-nor-4”-oic acids were identified as hepatic metabolites. A possible route could be a-oxidation. A further stage of b-oxidation would yield a compound containing a carboxymethyl side-chain. Also found in this experiment on 4”-hydroxy-delta-1-THC were metabolites containing carboxy-ethyl side-chains, the normal products of 8-oxidation indicating at least two biochemical pathways to these compounds. The major urinary metabolite of CBD (XXVI) in the human contained a hydroxy-ethyl side-chain and, therefore, does not appear to be the product of a 8-oxidation-type reaction, which would yield an acid. In another experiment


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(Harvey 1989b), the precursor to metabolites of this type in the T H C series has been shown to be a 3"-hydroxylated metabolite. It is most likely that the same situation pertains to CBD, particularly as 3"-hydroxy compounds are formed in abundance. However the biochemistry of this transformation has not been determined. Also of interest in the experiment with 3"-hydroxy-delta- 1-THC was the observation that 3"-hydroxy-metabolites constituted a third precursor for the products of 8oxidation. Two new metabolic pathways for CBD were found in this sample. Hydroxylation at C-10 to give 10-hydroxy-CBD-7-oic acid (XII) is only the second example of a metabolite hydroxylated at this position found for CBD. The alcohol itself has, however, recently been identified in microsomal incubations and identified by comparison with a synthetic sample (unpublished). The other new metabolic pathway involved dihydroxylation at C-8 and C-9, presumably via the intermediate epoxide and, again, the corresponding metabolite, not additionally oxidized at C-7, has been identified as a metabolite using liver microsomal preparations. Although CBD itself was found both in the free state and as its 0-glucuronide conjugate, none of the other metabolites were found as conjugates; these compounds are amenable to g.1.c.-mass spectrometric analysis as demonstrated previously (Harvey et al. 1976). Unmetabolized CBD and its glucuronide conjugate accounted for 22.4% of the excreted cannabinoids, a situation atypical for cannabinoids, where oxidative metabolism is normally involved in most of the excreted fraction. The high dose of drug given to the patient is probably a major contributory factor to this observation. The formation of cyclized cannabinoids and, in particular, the THCs is also of interest. The mechanism for their formation is unknown and formation in the urine itself cannot be ruled out. Indeed, the absence of any known metabolite of these compounds in the sample strongly indicates that this may be the case. If not, their concentration in vivo may be sufficient to produce psychoactivity with obvious adverse effects for the patient. However, a blood sample was not available to check circulating concentrations of the cannabinoids.

Acknowledgements This work was supported by the National Institute on Drug Abuse (NIDA) (Grant no. DA04005). We thank Dr R. L. Hawkes of NIDA for supplies of the reference cannabinoids. The mass spectrometric equipment was originally purchased with a grant from the Medical Research Council. Funds from the Wellcome Trust are also acknowledged. References BINDER,M., AGURELL, S., LEANDER, K., and LINDGREN, J.-E., 1974, Zur Identifikation potentieller Metabolite von Cannabis-Inhaltstoffen: Kernresonanz- und massenspektroskopische Untersuchungen an seitenketten-hydroxyliertenCannabinoiden. Helweticu Chimica Acta, 57, 1626-1641. P., and SNIDER, S. R., 1986, Therapeuticpotential of cannabinoids in neurological disorders. In CONSROE, Cannabinoids us Therupeutic Agents, edited by R. Mechoulam (Boca Raton, FL: CRC Press), pp. 21-49. CUNHA,J. M., CARLINI,E. A., PERIERA, E. E., RAMOS,0. L., PIMENTEL, C., GAGLIARDI, R., SANVITO, W. L., LANDER, N., and MECHOULAM, R., 1980, Chronic administration of cannabidiol to healthy volunteers and epileptic patients. Pharmacology, 21, 175-185. M., WARING, N., CHRIST, W., and HILL,R., 1973, Interactions of several cannabinoids with FERNANDES, the hepatic drug metabolising system. Biochemicul Phurmucology, 22, 2981-2987.



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Determination of aromatic hydrocarbons and their metabolites in human blood and urine.

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