88
Specialia
t i o n : K = - 0 , 00037 C-0,0174 o h s o n t r e p r 6 s e n t 6 s : l ' i n d e x p h a g o c y t a i r e : K, la dose de c a r b a r y l a d m i n i s t r 6 e : C, la p e n t e de la d r o i t e : - 0 , 0 0 0 3 7 4- 0,00006, l ' i n d e x p h a g o c y t a i r e lorsque l ' a d m i n i s t r a t i o n de c a r b a r y l est n u l l e : - 0 , 0 1 7 4 • 0,0003. L ' 6 t u d e s t a t i s t i q u e de la p e n t e de c e t t e d r o i t e (test de t) m o n t r e q u e la v a r i a t i o n de l ' i n d e x p h a g o c y t a i r e est f o n c t i o n de la dose de c a r b a r y l a d m i n i s t r ~ e (t = 6; p < 0,001). L a c o m p a r a i s o n des v a r i a n c e s e n t r e les doses de c a r b a r y l e t de la v a r i a n c e t o t a l e des d o n n 6 e s m o n t r e q u e la d r o i t e de r6gression est bien lin6aire (F : 0 , 6 5 < F ~ 5 ~ 5%). Ainsi, d a n s n o s c o n d i t i o n s e x p ~ r i m e n tales, il y a u n e r e l a t i o n lin6aire e n t r e la dose de c a r b a r y l a d m i n i s t r 6 e et l ' i n h i b i t i o n de la p h a g o c y t o s e observ~e 1 h apr~s t r a i t e m e n t . Le c a r b a r y l s e m b l e d o n c a v o i r u n i m p a c t sur les cellules du S R E e t n o t a m m e n t sur les cellules de Kiipffer d u toie r e s p o n s a b l e s ~t elles seules de 8 0 - 9 0 % de l ' 6 p u r a t i o n s a n g u i n e d u c a r b o n e colloidal s. C e t t e dimin u t i o n progressive de la p h a g o c y t o s e en f o n c t i o n de la dose a d m i n i s t r 6 e laisse s u p p o s e r que le c a r b a r y l , s u b s t a n c e a n t i c h o l i n e s t ~ r a s i q u e , p o u r r a i t ~tre r e s p o n s a b l e de l ' i n h i b i t i o n de s6rine est6rases p o u v a n t j o u e r u n r61e d a n s les processus m e m b r a n a i r e s a c c o m p a g n a n t la p h a g o c y t o s e 4. Ce r 6 s u l t a t p e u t ~tre r a p p r o c h 6 de l ' i n h i b i t i o n de la p h a g o c y t o s e des globules rouges p a r des leucocytes e n pr6sence d ' i n h i b i t e u r s de la s6rine est6rase 3 off l ' i n t e n s i t 6 de l ' i n h i -
EXPERIENTIA
34/1
b i t i o n est 6 g a l e m e n t f o n c t i o n de la dose. L a d i m i n u t i o n p r o g r e s s i v e de la vitesse de p h a g o c y t o s e d u c a r b o n e colloidal p o u r r a i t ~tre 6 g a l e m e n t le r 6 s u l t a t d ' u n e comp6t i t i o n c r o i s s a n t e e n t r e les p a r t i c u l e s de c a r b a r y l q u e n o u s a v o n s a d m i n i s t r 6 e s e n fine s u s p e n s i o n e t le c a r b o n e colloidal. U n tel m 6 c a n i s m e a ~t6 m o n t r 6 lorsque le c a r b o n e colloidal et u n e a u t r e s u b s t a n c e p a r t i c u l a i r e se t r o u v e n t en c o m p 6 t i t i o n p o u r les sites m e m b r a n a i r e s des m a c r o p h a g e s 9. Des 6 t u d e s plus a p p r o f o n d i e s s e r a i e n t n6cessaires p o u r ~lucider la cause de c e t t e i n h i b i t i o n et les m 6 c a n i s m e s m e m b r a n a i r e s r ~ g i s s a n t la p h a g o c y t o s e . C o n n a i s s a n t l ' i m p o r t a n c e de la f o n c t i o n p h a g o c y t a i r e des m a c r o p h a g e s d u S R E darts la d6fense de l ' o r g a n i s m e , il s e r a i t e n effet imp o r t a n t de pr6ciser ces fairs s u r t o u t l o r s q u ' o n sait q u e le c a r b a r y l , p r 6 s e n t d a n s l ' e n v i r o n n e m e n t , p e u t ~tre absorb6 en t a n t q u e r6sidu d a n s les a l i m e n t s ou ~tre e n cont a c t c u t a n 6 accidente110.
8
B.N. Halpern, G. Biozzi, G. Pequignot, B. Delaloye. C. Stiffel et D. Mouton, Path. Biol. 7, 1637 (1959). 9 S.J. Normann, Lab. Invest. 31, 161 (1974). 10 R . J . Feldmann et H. I. Maibach, Toxic. appl. Pharmac. 28, 126 (1974).
D i f f e r e n t e f f e c t s of s y n t h e t i c A 9 - t e t r a h y d r o c a n n a b i n o l o n s t e r o i d m e t a b o l i s m in m a l e r a t s 1
and cannabis extract
M. P. M a s k a r i n e c , G. Shipley, M. N o v o t n y S , D. J. B r o w n a n d R. B. F o r n e y
Department o/ Chemistry, Indiana University, Bloomington (Indiana 47401), and Department o/ Toxicology, Indiana University School o/Medicine, Indianapolis (Indiana 46202, USA), 7 April 7977 Summary. R e p e a t e d oral a d m i n i s t r a t i o n of c a n n a b i s e x t r a c t as well as s y n t h e t i c A D - t e t r a h y d r o c a n n a b i n o l to m a l e r a t s p r o d u c e d Significant c h a n g e s in e x c r e t i o n of a n d r o g e n i c s t e r o i d s a n d t h e i r m e t a b o l i t e s as d e t e c t a b l e in b l o o d a n d urine. C a n n a b i s e x t r a c t s were f o u n d t o be s i g n i f i c a n t l y m o r e a c t i v e t h a n t h e m i x t u r e s c o n t a i n i n g s a m e a m o u n t s of s y n t h e t i c cannabinoids. T h e r e have b e e n c o n f l i c t i n g r e p o r t s 3-5 o n c a n n a b i s aff e c t i n g t h e n o r m a l r e p r o d u c t i v e a b i l i t y of h u m a n males. S t u d i e s w i t h r o d e n t s , u s i n g g r e a t e r a m o u n t s of c a n n a b i s resin or s y n t h e t i c A g - t e t r a h y d r o c a n n a b i n o l (THC), h a v e p r e s e n t e d a m o r e conclusive e v i d e n c e for r e d u c e d copul a t o r y b e h a v i o r e, 7 g r o w t h i n h i b i t i o n s, 9 a n d a r e t a r d e d d e v e l o p m e n t of testes, s e m i n a l vesicles, a n d p r o s t a t e s, 10,11 S i g n i f i c a n t a c c u m u l a t i o n of t h e r a d i o a c t i v e T H C in r a t t e s t e s h a s also b e e n n o t e d 12, Since t h e ' d e m a s c u l i n i z i n g f a c t o r ' of c a n n a b i s does n o t a p p e a r t o h a v e d i r e c t l y a n estrogenic a c t i v i t y n , a c o m p l e x m e c h a n i s m of a l t e r e d s t e r o i d a l p a t h W a y s a p p e a r s t o b e i n v o l v e d . D e t a i l e d biochemical studies can potentially elucidate the consequences of a d m i n i s t r a t i o n of t h e drug. D u r i n g t h e course of s u c h s t u d i e s 13, we h a v e o b s e r v e d t h a t t h e a l t e r a t i o n in t h e exc r e t i o n of s t e r o i d s is s i g n i f i c a n t l y d i f f e r e n t for c a n n a b i s e x t r a c t s as c o m p a r e d t o s y n t h e t i c THC. S u c h differences were o b s e r v e d w i t h b o t h u r i n a r y a n d p l a s m a steroids. Materials and methods. Male W i s t a r r a t s (100-200 d a y s old) w e i g h i n g 300-450 g were o r a l l y a d m i n i s t e r e d for 7 d a y s e i t h e r t h e e x t r a c t of M e x i c a n c a n n a b i s or s y n t h e t i c T H C (doses c o r r e s p o n d i n g u p to 50 m g / k g of THC) u s i n g prop y l e n e glycol as t h e vehicle, w i t h c o n t r o l s g i v e n vehicle only. All d r u g s were o b t a i n e d f r o m t h e N a t i o n a l I n s t i t u t e on D r u g Abuse, Rockville, M a r y l a n d . T h e a n i m a l s were first allowed to a c c l i m a t e t h e m s e l v e s in t h e m e t a b o l i s m cages for 3 days. As a rule, 4 a n i m a l s were used in e a c h
e x p e r i m e n t , b u t t h e d e s c r i b e d e x p e r i m e n t s were r e p e a t e d a t l e a s t 3 t i m e s w i t h q u i t e r e p r o d u c i b l e results. U r i n e s a m p l e s were collected (total of 11 d a i l y collections) o v e r d r y ice a n d k e p t frozen u n t i l a n a l y z e d . V e n o u s b l o o d 1 2 3 4 5 6 7 8 9 10 11 12 13
Acknowledgment. This work was supported by Grant No. R01-DA-00507 from the National Institute on Drug Abuse, US Public Health Service. Correspondence should be addressed to: M. N., Department of Chemistry, Indiana University, Bloomington, Indiana 47401. J. Harmon and M. A. Aliapoulios, New Engl. J. Med. 287, 936 (1972). R.C. Kolodny, W. H. Masters, R. M. Kolodner and G. Toro, New Engl. J. Med. 290, 872 (1974). J . H . Mendelson, J. Kuenhle, J. Ellingboe and T. F. Babor, New Engl. J. Med. 291, 1051 (1974). A. Merari, A. Barak and M. Plaves, Psychopharmacologia 28, 243 (1973). M.E. Corcoran, A. Amit, C. W. Malsbury and S. Daykin, Res. Commun. chem. Path. Pharmac.7, 779 (1974). G . R . Thompson, M. M. Mason, H. Rosenkrantz and M. C. Braude, Tdxic. appl. Pharmac. 25, 373 (1973). N. Kokka and J. F. Garcia, Life Sei. 15, 329 (1974). V.P. Dixit, V. W. Sharma and W. K. Lohiya, Eur. J. Pharmac. 26, 111 (1974). A.B. Okey and G. S. Truant, Life Sci. 77, 1113 (1975). B.T. Ho, G. E. Fritehie, P. M. Kralik, L. F. Englert and W. M: MeIsaak, J. Pharm. Pharmae. 22, 538 (1970). M. P. Maskarinee, G. Shipley, M. Novotny, D. j. Brown and R. B. Forney, in press.
Specialia
15. 1. 1978
(typically, 5 ml) was collected f r o m t h e tail, using E D T A as a n a n t i c o a g u l a n t . A n i m a l s were b l e d a t t h e e n d of t h e 7th d a y (bleeding c o n s i s t e n t l y d o n e a t t h e e n d of d a y t i m e period, a t 20.00, t o m i n i m i z e d a i l y f l u c t u a t i o n in h o r m o n e levels), a n d urine was n o t collected d u r i n g a 24-h-period following blood collection. U r i n a r y steroids were isolated b y a c o m b i n a t i o n of t h e p r o c e d u r e s of H o m i n g a n d H o r n i n g 14 a n d Luyten15 a n d t h e profiles of t h e i r volatile d e r i v a t i v e s r e c o r d e d b y h i g h r e s o l u t i o n gas c h r o m a t o g r a p h y using a 20 m • 0.28 ram, ID, glass capillary c o l u m n c o a t e d w i t h G E SE-30 silicone g u m s t a t i o n a r y phase. P l a s m a steroids were isolated a n d analyzed by the gas-chromatographic method developed in our l a b o r a t o r y ~6. T h e i d e n t i t y of s t u d i e d c o m p o u n d s was verified b y m e a n s of a c o m b i n e d gas c h r o m a t o g r a p h / m a s s s p e c t r o m e t e r ( H e w l e t t - P a c k a r d Model 5980 A) a n d t h r o u g h c h r o m a t o g r a p h i c r e t e n t i o n of a u t h e n t i c samples. T e s t o s t e r o n e was d e t e r m i n e d b y m e a n s of electron c a p t u r e d e t e c t o r as its m e t h o x i m e - h e p t a f l u o r o b u t y r y l d e r i v a t i v e . Results and discussion. The n o r m a l c o n c e n t r a t i o n s of t e s t o s t e r o n e in r a t p l a s m a were f o u n d to be 452 • ng/ 100 m l (an average of 11 d e t e r m i n a t i o n s ) . The values are s o m e w h a t h i g h e r t h a n t h o s e o b t a i n e d for m a l e r a t s b y m e a n s of a r a d i o i m m u n o a s s a y p r o c e d u r e a n d r e p o r t e d b y G u p t a et al.~L W h e r e a s t e s t o s t e r o n e was u n a l t e r e d b y T H C a d m i n i s t r a t i o n e x c e p t for t h e r e l a t i v e l y h i g h dose
Testosterone levels in marijuana treated rats Dosage regimen
Plasma testosterone level ~ 1 SD
Control (11 animals) 1 mg/kg THC 5 mg/kg THC 10 mg/kg THC 25 nlg]kg THC 50 mg/kg THC 5 mg/kg THC in cannabis extract 10 mg/kg THC in cannabis extract 10 mg/kg THC+7.8 mg/kg CBD+ 3.4 mg]kg CBN
452 ng/mI J= 37.7 472 ng/ml -~ 35.0 443 ng/ml • 39.8 467 ng/ml i 45.0 427 ng/ml • 36.3 361 ng/ml • 23.6 345 ng/ml -t= 29.8 160 ng/ml i 18.5 453 ng/mI -~ 32.7
3O
89
of 50 mg/kg, t h e r a t s given a l r e a d y 5 m g / k g of t h e cann a b i s e x t r a c t s h o w e d s i g n i f i c a n t (99.9% confidence using t - d i s t r i b u t i o n ) decrease of t e s t o s t e r o n e c o n c e n t r a t i o n . A m i x t u r e of t h e m a i n s y n t h e t i c c a n n a b i n o i d s (THC, 10.0 m g / k g ; c a n n a b i n o l (CBN), 3.4 m g / k g ; a n d c a n n a b i d i o l (CBD), 7.8 m g / k g in a single d o s e ) b l e n d e d t o s i m u l a t e t h e c a n n a b i n o i d c o n t e n t of tile used c a n n a b i s e x t r a c t failed t o p r o d u c e a n y s u b s t a n t i a l d e c r e a s e of t e s t o s t e r o n e . One of t h e m a j o r u r i n a r y s t e r o i d s t h a t was i d e n t i f i e d t h r o u g h its m a s s s p e c t r u m a n d c h r o m a t o g r a p h i c r e t e n t i o n as Al~-androstene-3/~-ol, s h o w e d m a r k e d d o s e - r e s p o n s e rel a t e d e l e v a t i o n in r a t s given THC. H o w e v e r , t h i s e l e v a t i o n is e v e n m o r e p r o n o u n c e d a f t e r t h e d a i l y a d m i n i s t r a t i o n of c a n n a b i s e x t r a c t (figure). I t should be n o t e d t h a t t e s t o s t e r o n e can be c o n s i d e r e d a likely p r e c u r s o r of t h i s steroid. I n a similar fashion, a 3fold increase of a n d r o s t a n e d i o l c o n c e n t r a t i o n o v e r t h e c o n t r o l was f o u n d following t h e a d m i n i s t r a t i o n of c a n n a b i s as c o n t r a s t e d b y a p p r o x i m a t e l y 1.5 t i m e s increase due to s y n t h e t i c THC. A c c o r d i n g t o M a t s u i et al. is, 5~-androstene-3fl, 17/~-diol is t h e m a j o r m e t a b o l i t e of t e s t o s t e r o n e sulfate f o u n d in male r a t bile. Again, none of t h e o b s e r v e d differences could be a t t r i b u t e d to t h e o t h e r m a j o r c a n n a b i n o i d s . E l e v a t i o n of u r i n a r y a n d r o s t e r o n e (from a p p r o x i m a t e l y 10 for c o n t r o l s to 80 txg/ 24 h) for T H C - t r e a t e d a n d c a n n a b i s - t r e a t e d r a t s was n o t s i g n i f i c a n t l y different. Also, a t r a c e a m o u n t of etiochola n o l o n e t h a t is a d j a c e n t to t h e a n d r o s t e r o n e c h r o m a t o g r a p h i c p e a k d r o p p e d b e l o w its d e t e c t i o n l i m i t a f t e r t h e t r e a t m e n t of r a t s w i t h b o t h T H C a n d c a n n a b i s e x t r a c t . A l t h o u g h t h e a n a l y s e s of p l a s m a c o n j u g a t e d steroids following a c o m p l i c a t e d e x t r a c t i o n a n d f r a c t i o n a t i o n scheme16 h a v e to be c o n s i d e r e d less a c c u r a t e t h a n t h o s e of u r i n a r y steroids, m a r k e d l y i n c r e a s e d A16-androstenol m o n o s u l f a t e and a n d r o s t a n e d i o i disulfate were c o n s i s t e n t l y o b s e r v e d ; again, t h i s effect was m o r e p r o n o u n c e d in cann a b i s - t r e a t e d as c o m p a r e d to T H C - t r e a t e d rats. A l t h o u g h t h e m e c h a n i s m of d r u g i n t e r f e r e n c e w i t h t h e n o r m a l g o n a d a l a n d a d r e n a l m e t a b o l i s m has y e t to be inv e s t i g a t e d in detail, t h e p r e s e n t e d results s t r o n g l y indicate t h a t t h e actions of s y n t h e t i c T H C a n d c a n n a b i s resin are n o t s y n o n y m o u s . W h e r e a s t h i s m a y n o t be h a r d t o u n d e r s t a n d in view of t h e m a n y h i t h e r t o u n i d e n t i f i e d c a n n a b i s c o n s t i t u e n t s , it should be c a u t i o n e d t h a t m a j o r i t y of r e l a t e d endocrinological s t u d i e s are usually p e r f o r m e d w i t h s y n t h e t i c T H C p r e p a r a t i o n s . Since t h e r e m a i n i n g m a j o r c a n n a b i n o i d s of t h e u s e d e x t r a c t were n o t f o u n d responsible for t h e e n h a n c e d effects, o t h e r c a n n a b i s cons t i t u e n t s m u s t be considered. Our p r e l i m i n a r y s t u d i e s h a v e also ruled o u t olivetol t h a t was p r e v i o u s l y f o u n d a c t i v e as an i n h i b i t o r of p r o s t a g l a n d i n s y n t h e t a s e 1~.
20
"q
o
10
Treatment period L
i
i
i
,
i
i
1 2 3 4 5 6 7 8 ; Time
10 11Days
Excretion of zJl~-androstenol due to the treatment of rats with the drug. [] control; 9 10 ing/kg THC; 9 5 mg/kg THC in the cannabis extract; 9 10 mg/kg THC in the cannabis extract.
14 E.C. Horning and M. G. Horning, Meth. reed. Res. 12, section 5 (1970). 15 J. A. Luyten, Doctoral Thesis, Technological University of Eindhoven, The Netherlands 1973. 16 M. Novotny, M. P. Maskarinec, A. T. G. Steverink and R. Farlow, Analyt. Chem. 48, 468 (1976). 17 D. Gupta, J. Zarzyeki and K. Rager, Steroids 25, 33 (1975). 18 M. Matsui, Y. Kinuyama and M. Hakozaki, Steroids 25, 637 (1975). 19 S. Burstein, E. Levin and C. Varanelli, Bioehem. Pharmac. 22, 2905 (1973).
Specialia
t i o n : K = - 0 , 00037 C-0,0174 o h s o n t r e p r 6 s e n t 6 s : l ' i n d e x p h a g o c y t a i r e : K, la dose de c a r b a r y l a d m i n i s t r 6 e : C, la p e n t e de la d r o i t e : - 0 , 0 0 0 3 7 4- 0,00006, l ' i n d e x p h a g o c y t a i r e lorsque l ' a d m i n i s t r a t i o n de c a r b a r y l est n u l l e : - 0 , 0 1 7 4 • 0,0003. L ' 6 t u d e s t a t i s t i q u e de la p e n t e de c e t t e d r o i t e (test de t) m o n t r e q u e la v a r i a t i o n de l ' i n d e x p h a g o c y t a i r e est f o n c t i o n de la dose de c a r b a r y l a d m i n i s t r ~ e (t = 6; p < 0,001). L a c o m p a r a i s o n des v a r i a n c e s e n t r e les doses de c a r b a r y l e t de la v a r i a n c e t o t a l e des d o n n 6 e s m o n t r e q u e la d r o i t e de r6gression est bien lin6aire (F : 0 , 6 5 < F ~ 5 ~ 5%). Ainsi, d a n s n o s c o n d i t i o n s e x p ~ r i m e n tales, il y a u n e r e l a t i o n lin6aire e n t r e la dose de c a r b a r y l a d m i n i s t r 6 e et l ' i n h i b i t i o n de la p h a g o c y t o s e observ~e 1 h apr~s t r a i t e m e n t . Le c a r b a r y l s e m b l e d o n c a v o i r u n i m p a c t sur les cellules du S R E e t n o t a m m e n t sur les cellules de Kiipffer d u toie r e s p o n s a b l e s ~t elles seules de 8 0 - 9 0 % de l ' 6 p u r a t i o n s a n g u i n e d u c a r b o n e colloidal s. C e t t e dimin u t i o n progressive de la p h a g o c y t o s e en f o n c t i o n de la dose a d m i n i s t r 6 e laisse s u p p o s e r que le c a r b a r y l , s u b s t a n c e a n t i c h o l i n e s t ~ r a s i q u e , p o u r r a i t ~tre r e s p o n s a b l e de l ' i n h i b i t i o n de s6rine est6rases p o u v a n t j o u e r u n r61e d a n s les processus m e m b r a n a i r e s a c c o m p a g n a n t la p h a g o c y t o s e 4. Ce r 6 s u l t a t p e u t ~tre r a p p r o c h 6 de l ' i n h i b i t i o n de la p h a g o c y t o s e des globules rouges p a r des leucocytes e n pr6sence d ' i n h i b i t e u r s de la s6rine est6rase 3 off l ' i n t e n s i t 6 de l ' i n h i -
EXPERIENTIA
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b i t i o n est 6 g a l e m e n t f o n c t i o n de la dose. L a d i m i n u t i o n p r o g r e s s i v e de la vitesse de p h a g o c y t o s e d u c a r b o n e colloidal p o u r r a i t ~tre 6 g a l e m e n t le r 6 s u l t a t d ' u n e comp6t i t i o n c r o i s s a n t e e n t r e les p a r t i c u l e s de c a r b a r y l q u e n o u s a v o n s a d m i n i s t r 6 e s e n fine s u s p e n s i o n e t le c a r b o n e colloidal. U n tel m 6 c a n i s m e a ~t6 m o n t r 6 lorsque le c a r b o n e colloidal et u n e a u t r e s u b s t a n c e p a r t i c u l a i r e se t r o u v e n t en c o m p 6 t i t i o n p o u r les sites m e m b r a n a i r e s des m a c r o p h a g e s 9. Des 6 t u d e s plus a p p r o f o n d i e s s e r a i e n t n6cessaires p o u r ~lucider la cause de c e t t e i n h i b i t i o n et les m 6 c a n i s m e s m e m b r a n a i r e s r ~ g i s s a n t la p h a g o c y t o s e . C o n n a i s s a n t l ' i m p o r t a n c e de la f o n c t i o n p h a g o c y t a i r e des m a c r o p h a g e s d u S R E darts la d6fense de l ' o r g a n i s m e , il s e r a i t e n effet imp o r t a n t de pr6ciser ces fairs s u r t o u t l o r s q u ' o n sait q u e le c a r b a r y l , p r 6 s e n t d a n s l ' e n v i r o n n e m e n t , p e u t ~tre absorb6 en t a n t q u e r6sidu d a n s les a l i m e n t s ou ~tre e n cont a c t c u t a n 6 accidente110.
8
B.N. Halpern, G. Biozzi, G. Pequignot, B. Delaloye. C. Stiffel et D. Mouton, Path. Biol. 7, 1637 (1959). 9 S.J. Normann, Lab. Invest. 31, 161 (1974). 10 R . J . Feldmann et H. I. Maibach, Toxic. appl. Pharmac. 28, 126 (1974).
D i f f e r e n t e f f e c t s of s y n t h e t i c A 9 - t e t r a h y d r o c a n n a b i n o l o n s t e r o i d m e t a b o l i s m in m a l e r a t s 1
and cannabis extract
M. P. M a s k a r i n e c , G. Shipley, M. N o v o t n y S , D. J. B r o w n a n d R. B. F o r n e y
Department o/ Chemistry, Indiana University, Bloomington (Indiana 47401), and Department o/ Toxicology, Indiana University School o/Medicine, Indianapolis (Indiana 46202, USA), 7 April 7977 Summary. R e p e a t e d oral a d m i n i s t r a t i o n of c a n n a b i s e x t r a c t as well as s y n t h e t i c A D - t e t r a h y d r o c a n n a b i n o l to m a l e r a t s p r o d u c e d Significant c h a n g e s in e x c r e t i o n of a n d r o g e n i c s t e r o i d s a n d t h e i r m e t a b o l i t e s as d e t e c t a b l e in b l o o d a n d urine. C a n n a b i s e x t r a c t s were f o u n d t o be s i g n i f i c a n t l y m o r e a c t i v e t h a n t h e m i x t u r e s c o n t a i n i n g s a m e a m o u n t s of s y n t h e t i c cannabinoids. T h e r e have b e e n c o n f l i c t i n g r e p o r t s 3-5 o n c a n n a b i s aff e c t i n g t h e n o r m a l r e p r o d u c t i v e a b i l i t y of h u m a n males. S t u d i e s w i t h r o d e n t s , u s i n g g r e a t e r a m o u n t s of c a n n a b i s resin or s y n t h e t i c A g - t e t r a h y d r o c a n n a b i n o l (THC), h a v e p r e s e n t e d a m o r e conclusive e v i d e n c e for r e d u c e d copul a t o r y b e h a v i o r e, 7 g r o w t h i n h i b i t i o n s, 9 a n d a r e t a r d e d d e v e l o p m e n t of testes, s e m i n a l vesicles, a n d p r o s t a t e s, 10,11 S i g n i f i c a n t a c c u m u l a t i o n of t h e r a d i o a c t i v e T H C in r a t t e s t e s h a s also b e e n n o t e d 12, Since t h e ' d e m a s c u l i n i z i n g f a c t o r ' of c a n n a b i s does n o t a p p e a r t o h a v e d i r e c t l y a n estrogenic a c t i v i t y n , a c o m p l e x m e c h a n i s m of a l t e r e d s t e r o i d a l p a t h W a y s a p p e a r s t o b e i n v o l v e d . D e t a i l e d biochemical studies can potentially elucidate the consequences of a d m i n i s t r a t i o n of t h e drug. D u r i n g t h e course of s u c h s t u d i e s 13, we h a v e o b s e r v e d t h a t t h e a l t e r a t i o n in t h e exc r e t i o n of s t e r o i d s is s i g n i f i c a n t l y d i f f e r e n t for c a n n a b i s e x t r a c t s as c o m p a r e d t o s y n t h e t i c THC. S u c h differences were o b s e r v e d w i t h b o t h u r i n a r y a n d p l a s m a steroids. Materials and methods. Male W i s t a r r a t s (100-200 d a y s old) w e i g h i n g 300-450 g were o r a l l y a d m i n i s t e r e d for 7 d a y s e i t h e r t h e e x t r a c t of M e x i c a n c a n n a b i s or s y n t h e t i c T H C (doses c o r r e s p o n d i n g u p to 50 m g / k g of THC) u s i n g prop y l e n e glycol as t h e vehicle, w i t h c o n t r o l s g i v e n vehicle only. All d r u g s were o b t a i n e d f r o m t h e N a t i o n a l I n s t i t u t e on D r u g Abuse, Rockville, M a r y l a n d . T h e a n i m a l s were first allowed to a c c l i m a t e t h e m s e l v e s in t h e m e t a b o l i s m cages for 3 days. As a rule, 4 a n i m a l s were used in e a c h
e x p e r i m e n t , b u t t h e d e s c r i b e d e x p e r i m e n t s were r e p e a t e d a t l e a s t 3 t i m e s w i t h q u i t e r e p r o d u c i b l e results. U r i n e s a m p l e s were collected (total of 11 d a i l y collections) o v e r d r y ice a n d k e p t frozen u n t i l a n a l y z e d . V e n o u s b l o o d 1 2 3 4 5 6 7 8 9 10 11 12 13
Acknowledgment. This work was supported by Grant No. R01-DA-00507 from the National Institute on Drug Abuse, US Public Health Service. Correspondence should be addressed to: M. N., Department of Chemistry, Indiana University, Bloomington, Indiana 47401. J. Harmon and M. A. Aliapoulios, New Engl. J. Med. 287, 936 (1972). R.C. Kolodny, W. H. Masters, R. M. Kolodner and G. Toro, New Engl. J. Med. 290, 872 (1974). J . H . Mendelson, J. Kuenhle, J. Ellingboe and T. F. Babor, New Engl. J. Med. 291, 1051 (1974). A. Merari, A. Barak and M. Plaves, Psychopharmacologia 28, 243 (1973). M.E. Corcoran, A. Amit, C. W. Malsbury and S. Daykin, Res. Commun. chem. Path. Pharmac.7, 779 (1974). G . R . Thompson, M. M. Mason, H. Rosenkrantz and M. C. Braude, Tdxic. appl. Pharmac. 25, 373 (1973). N. Kokka and J. F. Garcia, Life Sei. 15, 329 (1974). V.P. Dixit, V. W. Sharma and W. K. Lohiya, Eur. J. Pharmac. 26, 111 (1974). A.B. Okey and G. S. Truant, Life Sci. 77, 1113 (1975). B.T. Ho, G. E. Fritehie, P. M. Kralik, L. F. Englert and W. M: MeIsaak, J. Pharm. Pharmae. 22, 538 (1970). M. P. Maskarinee, G. Shipley, M. Novotny, D. j. Brown and R. B. Forney, in press.
Specialia
15. 1. 1978
(typically, 5 ml) was collected f r o m t h e tail, using E D T A as a n a n t i c o a g u l a n t . A n i m a l s were b l e d a t t h e e n d of t h e 7th d a y (bleeding c o n s i s t e n t l y d o n e a t t h e e n d of d a y t i m e period, a t 20.00, t o m i n i m i z e d a i l y f l u c t u a t i o n in h o r m o n e levels), a n d urine was n o t collected d u r i n g a 24-h-period following blood collection. U r i n a r y steroids were isolated b y a c o m b i n a t i o n of t h e p r o c e d u r e s of H o m i n g a n d H o r n i n g 14 a n d Luyten15 a n d t h e profiles of t h e i r volatile d e r i v a t i v e s r e c o r d e d b y h i g h r e s o l u t i o n gas c h r o m a t o g r a p h y using a 20 m • 0.28 ram, ID, glass capillary c o l u m n c o a t e d w i t h G E SE-30 silicone g u m s t a t i o n a r y phase. P l a s m a steroids were isolated a n d analyzed by the gas-chromatographic method developed in our l a b o r a t o r y ~6. T h e i d e n t i t y of s t u d i e d c o m p o u n d s was verified b y m e a n s of a c o m b i n e d gas c h r o m a t o g r a p h / m a s s s p e c t r o m e t e r ( H e w l e t t - P a c k a r d Model 5980 A) a n d t h r o u g h c h r o m a t o g r a p h i c r e t e n t i o n of a u t h e n t i c samples. T e s t o s t e r o n e was d e t e r m i n e d b y m e a n s of electron c a p t u r e d e t e c t o r as its m e t h o x i m e - h e p t a f l u o r o b u t y r y l d e r i v a t i v e . Results and discussion. The n o r m a l c o n c e n t r a t i o n s of t e s t o s t e r o n e in r a t p l a s m a were f o u n d to be 452 • ng/ 100 m l (an average of 11 d e t e r m i n a t i o n s ) . The values are s o m e w h a t h i g h e r t h a n t h o s e o b t a i n e d for m a l e r a t s b y m e a n s of a r a d i o i m m u n o a s s a y p r o c e d u r e a n d r e p o r t e d b y G u p t a et al.~L W h e r e a s t e s t o s t e r o n e was u n a l t e r e d b y T H C a d m i n i s t r a t i o n e x c e p t for t h e r e l a t i v e l y h i g h dose
Testosterone levels in marijuana treated rats Dosage regimen
Plasma testosterone level ~ 1 SD
Control (11 animals) 1 mg/kg THC 5 mg/kg THC 10 mg/kg THC 25 nlg]kg THC 50 mg/kg THC 5 mg/kg THC in cannabis extract 10 mg/kg THC in cannabis extract 10 mg/kg THC+7.8 mg/kg CBD+ 3.4 mg]kg CBN
452 ng/mI J= 37.7 472 ng/ml -~ 35.0 443 ng/ml • 39.8 467 ng/ml i 45.0 427 ng/ml • 36.3 361 ng/ml • 23.6 345 ng/ml -t= 29.8 160 ng/ml i 18.5 453 ng/mI -~ 32.7
3O
89
of 50 mg/kg, t h e r a t s given a l r e a d y 5 m g / k g of t h e cann a b i s e x t r a c t s h o w e d s i g n i f i c a n t (99.9% confidence using t - d i s t r i b u t i o n ) decrease of t e s t o s t e r o n e c o n c e n t r a t i o n . A m i x t u r e of t h e m a i n s y n t h e t i c c a n n a b i n o i d s (THC, 10.0 m g / k g ; c a n n a b i n o l (CBN), 3.4 m g / k g ; a n d c a n n a b i d i o l (CBD), 7.8 m g / k g in a single d o s e ) b l e n d e d t o s i m u l a t e t h e c a n n a b i n o i d c o n t e n t of tile used c a n n a b i s e x t r a c t failed t o p r o d u c e a n y s u b s t a n t i a l d e c r e a s e of t e s t o s t e r o n e . One of t h e m a j o r u r i n a r y s t e r o i d s t h a t was i d e n t i f i e d t h r o u g h its m a s s s p e c t r u m a n d c h r o m a t o g r a p h i c r e t e n t i o n as Al~-androstene-3/~-ol, s h o w e d m a r k e d d o s e - r e s p o n s e rel a t e d e l e v a t i o n in r a t s given THC. H o w e v e r , t h i s e l e v a t i o n is e v e n m o r e p r o n o u n c e d a f t e r t h e d a i l y a d m i n i s t r a t i o n of c a n n a b i s e x t r a c t (figure). I t should be n o t e d t h a t t e s t o s t e r o n e can be c o n s i d e r e d a likely p r e c u r s o r of t h i s steroid. I n a similar fashion, a 3fold increase of a n d r o s t a n e d i o l c o n c e n t r a t i o n o v e r t h e c o n t r o l was f o u n d following t h e a d m i n i s t r a t i o n of c a n n a b i s as c o n t r a s t e d b y a p p r o x i m a t e l y 1.5 t i m e s increase due to s y n t h e t i c THC. A c c o r d i n g t o M a t s u i et al. is, 5~-androstene-3fl, 17/~-diol is t h e m a j o r m e t a b o l i t e of t e s t o s t e r o n e sulfate f o u n d in male r a t bile. Again, none of t h e o b s e r v e d differences could be a t t r i b u t e d to t h e o t h e r m a j o r c a n n a b i n o i d s . E l e v a t i o n of u r i n a r y a n d r o s t e r o n e (from a p p r o x i m a t e l y 10 for c o n t r o l s to 80 txg/ 24 h) for T H C - t r e a t e d a n d c a n n a b i s - t r e a t e d r a t s was n o t s i g n i f i c a n t l y different. Also, a t r a c e a m o u n t of etiochola n o l o n e t h a t is a d j a c e n t to t h e a n d r o s t e r o n e c h r o m a t o g r a p h i c p e a k d r o p p e d b e l o w its d e t e c t i o n l i m i t a f t e r t h e t r e a t m e n t of r a t s w i t h b o t h T H C a n d c a n n a b i s e x t r a c t . A l t h o u g h t h e a n a l y s e s of p l a s m a c o n j u g a t e d steroids following a c o m p l i c a t e d e x t r a c t i o n a n d f r a c t i o n a t i o n scheme16 h a v e to be c o n s i d e r e d less a c c u r a t e t h a n t h o s e of u r i n a r y steroids, m a r k e d l y i n c r e a s e d A16-androstenol m o n o s u l f a t e and a n d r o s t a n e d i o i disulfate were c o n s i s t e n t l y o b s e r v e d ; again, t h i s effect was m o r e p r o n o u n c e d in cann a b i s - t r e a t e d as c o m p a r e d to T H C - t r e a t e d rats. A l t h o u g h t h e m e c h a n i s m of d r u g i n t e r f e r e n c e w i t h t h e n o r m a l g o n a d a l a n d a d r e n a l m e t a b o l i s m has y e t to be inv e s t i g a t e d in detail, t h e p r e s e n t e d results s t r o n g l y indicate t h a t t h e actions of s y n t h e t i c T H C a n d c a n n a b i s resin are n o t s y n o n y m o u s . W h e r e a s t h i s m a y n o t be h a r d t o u n d e r s t a n d in view of t h e m a n y h i t h e r t o u n i d e n t i f i e d c a n n a b i s c o n s t i t u e n t s , it should be c a u t i o n e d t h a t m a j o r i t y of r e l a t e d endocrinological s t u d i e s are usually p e r f o r m e d w i t h s y n t h e t i c T H C p r e p a r a t i o n s . Since t h e r e m a i n i n g m a j o r c a n n a b i n o i d s of t h e u s e d e x t r a c t were n o t f o u n d responsible for t h e e n h a n c e d effects, o t h e r c a n n a b i s cons t i t u e n t s m u s t be considered. Our p r e l i m i n a r y s t u d i e s h a v e also ruled o u t olivetol t h a t was p r e v i o u s l y f o u n d a c t i v e as an i n h i b i t o r of p r o s t a g l a n d i n s y n t h e t a s e 1~.
20
"q
o
10
Treatment period L
i
i
i
,
i
i
1 2 3 4 5 6 7 8 ; Time
10 11Days
Excretion of zJl~-androstenol due to the treatment of rats with the drug. [] control; 9 10 ing/kg THC; 9 5 mg/kg THC in the cannabis extract; 9 10 mg/kg THC in the cannabis extract.
14 E.C. Horning and M. G. Horning, Meth. reed. Res. 12, section 5 (1970). 15 J. A. Luyten, Doctoral Thesis, Technological University of Eindhoven, The Netherlands 1973. 16 M. Novotny, M. P. Maskarinec, A. T. G. Steverink and R. Farlow, Analyt. Chem. 48, 468 (1976). 17 D. Gupta, J. Zarzyeki and K. Rager, Steroids 25, 33 (1975). 18 M. Matsui, Y. Kinuyama and M. Hakozaki, Steroids 25, 637 (1975). 19 S. Burstein, E. Levin and C. Varanelli, Bioehem. Pharmac. 22, 2905 (1973).
