Biochimica et Biophysica Acta, 436 (1976) 889--894

889

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA Report BBA 71258

THE EFFECT OF HASHISH COMPOUNDS TRANSITION

ON PHOSPHOLIPID PHASE

D. BACH, A. RAZ and R. GOLDMAN

Laboratory o f Membranes and Bioregulation, Weizmann Institute o f Science, Rehovot (Israel) (Received April 2nd, 1976)

Summary The interaction of hashish compounds, A ~-tetrahydrocannabinol and cannabidiol, with dipalmitoyl phosphatidylcholine was investigated using differential scanning calorimetry. Both drugs affect the transition of dipalmitoyl phosphatidylcholine from the gel to liquid crystalline state, decreasing both the melting temperature and the enthalpy of melting. At a drug to dipalmitoyl phosphatidylcholine ratio of approx. 1:5, two peaks appear in the transition profile, suggesting a phase separation in the drug dipalmitoyl phosphatidylcholine mixture.

In addition to the psychomimetic activity exhibited by hashish extracts, hashish compounds (cannabinoids) have been shown to affect a variety of parameters of biological and artificial phospholipid membranes. The psychomimetic effect is mainly due to the activity of A ~-tetrahydrocannabinol [ 1 ], while in interaction with membraneous systems leading to changes in membrane permeability and inhibition of membrane functions, both A 1-tetrahydrocannabinol and cannabidiol have a similar potency. Cannabidiol is a nonpsychoactive hashish component comprising 40% of the cannabis extracts [2]. It is the precursor in the synthesis and probably also in the biogenesis of A ~-tetrahydrocannabinol [1 ]. Cannabinoids are lipid-soluble neutral compounds with a very high membrane/aqueous solution partition coefficient [3]. As such they can insert into lipid bilayer regions of synthetic and biological membranes and probably cause perturbation of ordered phospholipid regions. Both hashish compounds, A1-tetrahydrocannabinol and cannabidiol show the well-documented biphasic interaction pattern of lipid-soluble compounds with membranes, i.e. at low concentrations they act as membrane stabilizers and at high concentrations as membrane labilizers [4,5]. Membrane stabilization against hypoosmotic lysis could stem from a fluidizing effect on membrane phospholipids enabling the membrane to accommo-

890 date a larger volume within its boundaries. At high drtua (.~,~, u~ltr~,t~(,~,+ ~+~,: compounds probably have a disordering effect on membrane lit>ids in a way leading to damage to the membrane permeability barrier. Indeed, permeability changes observed in lipid bilayer ,nemb~anes It;I. damage to organelles (mitochondria [7], lysosomes [5]) and c:ytot(,xJ~+ity towards maerophages [8] may all reflect initial drug-induced re,organization of lipids leading to impairment of the membrane-permeability I~arrie~ Drug-induced disorganization of the lipid environment.... i membrane enzymes could possibly explain also the inhibition of (Na + ~ K' }ATl'asv activity by hashish compounds (ref. 9 and Hersuhkovitch, M., Raz, A+ at~+l Goldman, R., unpublished). To probe the changes in the physical state of phospholipuis thdt 111ighl~ be induced by hashish compounds we have studied the influence' ~f .5~ tetrahydrocannabinol and cannabidiol on the electrical resistatwc of plaaar lipid bilayer membranes [6]. We now present evidence that b,)th A '.-tolru hydroeannabinol and cannabidiol can affect the transition of l~hospholip£ds from crystalline gel to liquid crystalline state as revealed by differential scanning calorimetry measurements. L-fl,7-Dipalmitoyl-a-lecithin puriss grade, was purchased from Fluka (Buehs, Switzerland). Dipalmitoyl phosphatidylcholine was freshly dissoh, ed in distilled chloroform before each experiment. A hTetrahydrocannabinol and cannabidiol were obtained from Makor Chemicals, Jerusalem, Israel. Stock solutions of the drugs (5 mg/ml in spectroscopic grade ethanol) were kept at --18°C. Mixtures of dipalmitoyl phosphatidyleholine and cannaim~oids at the desired molar ratios were prepared. The solvents were evaporated by a stream of N2 and high vacuum (2 h) and the material was dispersed in 0.2 ml of 1 • 10 -2 M NaC1 in 5 . 1 0 - + M Tris. HCl buffer pH 7.4, and incubated with shaking for 2 h at 48°C. The samples were concentrated about ten fold by evaporation of water with a stream of N2, and transferred to aluminum pans which were subsequently sealed. The same a m o u n t of ethanol as that present initially at the drug: dipalmitoyl phosphatidyleholine ratios was added to dipalmitoyl phosphatidylcholine as a control. The calorimetric measurements were performed in the Perkm Elmer DSC 1B apparatus. The apparatus was calibrated with stearic acid and radium, operated at a scanning rate of 8°C/min and a sensitivity scale of 4 mcal/s for full scale response. The experiments were performed at least in triplicates. The heat of transition (AH) was calculated from the areas under the curves of the thermograms and from the phospholipid content as assessed by phosphate determination [10] after digestion of the pan with perchloric acid. The respective structures of A t-tetrahydrocannabinol and cannabidiol are given in Fig.1. Fig.2 represents differential scanning calorimetry thermograms of dipalmitoyl phosphatidylcholine and of dipalmitoyl phosphatidyb choline/cannabinoid mixtures. At low ratios of the cannabinoids to dipalmitoyl phosphatidylcholine (1:20) the pretransition peak of dipalmitoyl phosphatidylcholine disappears with a concomitant small decrease of the melting temperature. At higher ratios of cannabinoids to dipalmitoyl phosphatidylcholine the

891

OH

,

~r

3, C5HI7

z~? U ' t r a r y d r oconn@b,nc~l

Cannabld~ol

Fig.1. The structure of AX-tetrahydrocannabinol and cannabidiol. I - - T = - - -

a

O

d-THC

CBD

! I

I

I

I

I

20 50 40 50

I

I

I

L

20 30 40 50

TEMPERATURE

(°c)

Fig.2. T h e e f f e c t o f ~ l . t e t r a h y d r o c a n n a b i n o l ( Az - T H C ) a n d c a n n a b i d i o l ( C B D ) o n t h e d i f f e r e n t i a l scanning calorimetry thermograms of dipalmitoyl phosphatidylcholine (DPL).

Al THC (a) DPL alone (b) l f l:22(mola]cratio) (e) AI.THC/DPL i:Ii (d) 1:5 (e) 1:2.4

[DPL] 2.4 mg 2.1mg 3.8 mg 2.4 mg 2.2 mg

CBD (a) DPL alone (b) I 1 (c) CBDIDPL (d) (e)

l:20 (molar ratio) I:I0 1:5 1:2

2.3 mg 2.2mg 2.5 mg 2.0 mg 2.2 mg

892 transition curves b r o a d e n and the melting tempt, raturt~ lutii~ .... , . . . . ,,:, ;~ a m o l a r ratio o f 1:5 an additional peak appears iH the t r a n m t i , , , [)fti|'ih? 1[,,' s e c o n d p e a k could n o t stem f r o m melting o f pure cannabidl,;! .,mu(, th( ~ melting p o i n t o f the latter was f o u n d to be 68°C. The generat~(,~ ,)l' ih~ . ,~,, o n d p e a k might indicate a phase s e p a r a t i o n occurring at high ~ almmHi~(a~l i~ d i p a l m i t o y l p h o s p h a t i d y l c h o l i n e ratios. This p h e n o m e n o n c~)Hh l t)()ssiJ,I 2 i~, e x p l a i n e d o n the basis of the limited solubility of cannabinui,/s l~ (HpaHH,l~5'i p h o s p h a t i d y l c h o l i n e . A f u r t h e r increase in the ratio o f cam~alm,,)ids t,, d i p a l m i t o y l p h o s p h a t i d y l e h o l i n e does n o t lead to additional chaHges m t i,,. melting t e m p e r a t u r e s and the t h e r m o t r o p i c prufile. Fig.3 shrews tht. ,icl),~H d e n e e o f the m i d p o i n t melting t e m p e r a t u r e s on the ratio of the drugs to d i p a l m i t o y l p h o s p h a t i d y l e h o l i n e . T h e m a x i m u m shift m Lt~, ml, lt),nHi iem p e r a t u r e a m o u n t s to a b o u t 15°C.

50

-THC i

AI

40,

$

'

•

i /

30 Ud C1:2 :D

20

...... - - ~ + ...... ~- .....~. . . .

{ i

+--

t

CBD

a::: b,.J 12-

50F~o~_~o

IM k-

40~-~.

•

0,1

0.2 0.5 0,4 Drug ( tool DPL xm--~ )

I

• .

0.5

-i

11

0.6

Fig.3, The dependence of the midpoint temperature (Tin) of the phase trallsition ot dipaimjto5 1 p h o s p h a t i d y l e h o l i n e ( D P L ) o n t h e m o l a r r a t i o o f c a n n a b i n o i d s t o p h o s p h o l i p i d . ~. . . . . . , T m ,,~ ~ l l r s t p e a k (I): ~ - - - - - - ~ , T m o f t h e s e c o n d p e a k ( l I ) . A I - T H C , A l - t e t r a h y d r o c a n n a b i n o l ; C B D , c a n n a ~ bidiol.

Table I presents the heats o f transition of p u r e and o f d i p a h m t o y l phosp h a t i d y l c h o l i n e interacting with A l - t e t r a h y d r o e a n n a b i n o l or cannabidiol. T h e heat o f transition (AH) o f d i p a l m i t o y l p h o s p h a t i d y l c h o l i n e decreases u p o n i n t e r a c t i o n with t h e c a n n a b i n o i d s f r o m a value o f 8.6 k c a l / m o l for pure d i p a l m i t o y l p h o s p h a t i d y l c h o l i n e t o a b o u t 5.4 kcal/mol. Several r e p o r t s have a p p e a r e d dealing with t h e e f f e c t o f various drugs o n b o t h t h e t r a n s i t i o n t e m p e r a t u r e and e n t h a l p y o f the transition of phospholipids. Cater et al. [11] have e x a m i n e d t h e e f f e c t o f m o r p h i n e and tricyclic a n t i d e p r e s s a n t derivatives on t h e melting b e h a v i o u r o f s y n t h e t i c phosphatidylcholines. Certain drugs o f these series e x h i b i t a shift in transition t e m p e r a t u r e a c c o m p a n i e d b y the a p p e a r a n c e o f a d d i t i o n a l transition peaks. No appreciable change in t h e heat o f t r a n s i t i o n c o u l d h o w e v e r be d e t e c t e d . On the o t h e r

893

TABLE l H E A T S OF D I P A L M I T O Y L P H O S P H A T I D Y L C H O L I N E ( D P L ) P H A S E T R A N S I T I O N S A T D I F F E R E N T M O L A R R A T I O S O F P H O S P H L I P I D T O A t - T H C OR CBD AH (kcal/mol DPL) DPL only DPL/A~-THC DPL/A~-THC D P L / A 1-THC D P L / A I-TItC

22:1 11:1 5:1 * 2.4:1

8.6 7.5 6.4 5.2 5.4

AH (keal/mol DPL) DPL/CBD DPL/CBD DPL/CBD DPL/CBD

20:1 10:1 5:1 2:1

7.4 5.4 6.0 5.7

~Wben t w o peak~ a~pea~ed in t h e t r a n s i t i o n p r o f i l e , t h e t o t a l a r e a w a s t a k e n f o r t h e c a l c u l a t i o n .

hand, the interaction of chlorothricin with dipalmitoyl phosphatidylcholine caused a decrease in the heat of transition of dipalmitoyl phosphatidylcholine with almost no effect on the melting temperature [12]. The interaction of a wide range of drugs with dipalmitoyl phosphatidylcholine results in small shifts in the melting temperature [13,14] and in no change in the enthalpy of transition. Lawrence and Gill [15] studied the effects of Al-tetrahydrocannabinol and other cannibinoids on spin-labelled phosphatidylcholine/cholesterol liposomes. They found that A l-tetrahydrocannabinol fluidized the lipid bilayer whereas cannabidiol decreased the fluidity of the bilayer, and have suggested that the psychoactive cannabinoids may be classified as "partial anaesthetics" producing a perturbation of the membrane structure. They suggest that due to their limited solubility in the lipid phase of cell membranes the cannibinoids are unable to produce the degree of membrane disorder corresponding to clinical anaesthesia. The studies described in this communication show a remarkable fluidizing effect of both drugs on dipalmitoyl phosphatidylcholine liposomes. Cannabinoids could possibly trigger a localized phase transition of phospholipids at a constant temperature. This might be of relevance for the activity of functional membrane entities such as enzymes and carriers, many of which have been shown to be associated with phospholipids. No significant difference in the perturbation exerted on a phosphatidylcholine bilayer by A l-tetrahydrocannabinol and cannabidiol was observed. Moreover, our previous studies both on black lipid membranes [6] and on biological membranes (refs. 5 and 8, and Herschkovitch, M., Raz, A. and Goldman, R., unpublished} suggest that both A 1-tetrahydrocannabinol and cannabidiol are similarly potent in damaging the permeability of phospholipid and biological membranes. A LTetrahydrocannabinol and cannabidiol exhibit also similar cytotoxicity towards macrophages in culture [8]. We should like to thank the Plastics Department of the Weizmann Institute of Science for enabling us to use the differential scanning calorimetry apparatus. References 1 M e c h o u l a m , R. ( 1 9 7 0 ) Science 168, 1 1 5 9 - - 1 1 6 6 . 2 Grli6, L. ( 1 9 6 2 } Bull. N a r c o t . 14, 3 7 - - 4 6 .

894

3 4 5 6 7 8 9 10 11 12 13 14 15

S e e m a n , P., C h a u - W o n g , M. a n d M o y y e n , S. ( 1 9 7 2 ) Can. J. Physiol. P h a r m a c o l . 50, ! ] 93 1200. Raz, A., S e h u r r , A. a n d Livne, A. ( 1 9 7 2 ) Biochim. Biophys. A c t a 274, 269 271. R a z , A., S e h u r r , A., L i v n e , A. a n d G o l d m a n , R. ( 1 9 7 3 ) B i o e h e m . P h a r m a e o l . 22, 3l 2 9 - - 3 1 3 1 . Bach, D., Raz, A. and G o l d m a n , R. ( 1 9 7 6 ) B i o e h e m . P h a r m a c o l . , in press. Bino, T., Chari-Bitron, A. and S h a h a r , A. ( 1 9 7 2 ) Biochim. Biophys. Aeta 288, 195 202. Raz, A. a n d G o l d m a n , R. ( 1 9 7 6 ) Lab. Invest. 34, 69. G i b e r m a n n , E., C h a r i - B i t r o n , A., Milo, S. a n d Gothilf, S. ( 1 9 7 4 ) E x p e r i e n t i a 30, 68 69. King, E.J. a n d W o o t t o n , I.D.P. ( 1 9 5 6 ) Mieroanalysis in Medicinal B i o c h e m i s t r y , G r u n e and S t r a t t o n , New York. Cater, B.R., C h a p m a n , D., H a w e s , S.M. and Saville, J. ( 1 9 7 4 ) Biochim. Biophys. A c t a 363, 54 69. Paehe, W. a n d C h a p m a n , D. ( 1 9 7 2 ) Biochim. Biophys. Acta 255, 3 4 8 - - 3 5 7 . J a i n , M.K., Wu, N.Y.-M. a n d Wray, L.V. ( 1 9 7 5 ) N a t u r e 255, 4 9 4 - - 4 9 5 . P a p a h a d j o p o u l o s , D., J a e o b s o n , K,, Poste, G. a n d S h e p h e r d , G. ( 1 9 7 5 ) Bioehirn. Biophys. A c t a 394~ 504--519. L a w r e n c e , D.K. a n d Gill, E.W. ( 1 9 7 5 ) Mol. P h a r m a c o l . 11, 5 9 5 - - 6 0 2 .