Radioimmunoassay of Cannabinoid Compounds - American

9 Radioimmunoassay of Cannabinoid Compounds C. E. COOK

Downloaded by PURDUE UNIV on September 26, 2017 | http://pubs.acs.org Publication Date: April 10, 1979 | doi: 10.1021/bk-1979-0098.ch009

Chemistry and Life Sciences Division, Research Triangle Institute, Research Triangle Park, NC 27709

Radioimmunoassay (or RIA) was first introduced as an analytical technique i n 19 59. Since then it has played an increasingly important role i n the quantitative analysis of hormones and drugs. Much of the recent rapid progress i n endocrinology may i n fact be attributed to the a v a i l a b i l i t y of this sensitive and accurate method for quantitation of steroidal and protein/peptide hormones. Since radioimmunoassay has permitted the quantitation of substances present i n concentrations as low as a few pg/ml of a b i o l o g i c a l f l u i d , and since the inherent s e l e c t i v i t y of antibodies often make sample preparation requirements minimal and assay methodology simple, RIA has naturally been considered as a means for measuring blood levels of cannabinoid compounds. A number of investigators have been interested i n the development of RIA procedures for the cannabinoids. Before beginning a discussion of the RIA of cannabinoid compounds, l e t us b r i e f l y review some general principles of radioimmunoassay. Figure 1 i l l u s t r a t e s the basic premise of RIA. A radiolabeled substance, the antigen or radioligand, binds to an antibody. Addition of unlabeled antigen results i n a competition with the radioligand for binding s i t e s , thus reducing the fraction of radioactivity which i s bound to the antibody. By measuring either the free or bound rad i o a c t i v i t y and establishing a standard curve, one can then determine the amount of antigen present i n an unknown sample. The first step i n the development of an RIA pro0-8412-0488-8/79/47-098-137$05.00/0 © 1979 American Chemical Society

Vinson; Cannabinoid Analysis in Physiological Fluids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

138

CANNABINOID ANALYSIS IN PHYSIOLOGICAL FLUIDS

cedure t h e r e f o r e i n v o l v e s g e t t i n g a n t i b o d i e s w h i c h w i l l b i n d t h e compound i n q u e s t i o n . F u r t h e r m o r e , a n t i b o d i e s w h i c h s e l e c t i v e l y b i n d t h e d r u g t o be a n a l y zed a r e o f t e n most d e s i r a b l e s i n c e t h e y p e r m i t a n a l y s i s w i t h l e s s f o r e x t e n s i v e sample p r e p a r a t i o n . A second s t e p i n RIA development i s c h o i c e o f the a p p r o p r i a t e r a d i o l i g a n d . Much o f t h i s p a p e r w i l l be d e v o t e d t o t h e s e two a s p e c t s o f RIA. LABELED ANTIGEN

+

SPECIFIC

LABELED

ANTIGEN-

ANTIBODY

COMPLEX

%

ANTIBODY

1

4Downloaded by PURDUE UNIV on September 26, 2017 | http://pubs.acs.org Publication Date: April 10, 1979 | doi: 10.1021/bk-1979-0098.ch009

UNLABELED ANTIGEN

jr U N L A B E L E D ANTIGENANTIBODY COMPLEX

10

10

2

10

3

10

4

A D D E D MASS (UNLABELED) Raven Press

Figure 1.

Basic premise of RIA (13)


9.

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Radioimmunoassay

IMMUNOGEN SYNTHESIS - GENERAL PRINCIPLES F i r s t l e t us l o o k a t a n t i b o d y f o r m a t i o n as i l l u s t r a t e d i n F i g u r e 2. S m a l l m o l e c u l e s i n g e n e r a l do n o t e l i c i t an immune r e s p o n s e when i n j e c t e d i n t o a n i m a l s . T h i s s i t u a t i o n , w h i c h i s f o r t u n a t e from t h e s t a n d p o i n t o f t h e r a p e u t i c use o f d r u g s , p r e s e n t s a s u b s t a n t i a l p r o b l e m f o r t h e a n a l y s t who wants t o assay them. Howe v e r , i n t h e e a r l y 1900 s L a n d s t e i n e r (1) showed t h a t i f s m a l l m o l e c u l e s w h i c h i n t h e m s e l v e s were n o t immunog e n i c were c o v a l e n t l y bonded t o a l a r g e m o l e c u l e such as a p r o t e i n , t h e r e s u l t i n g s u b s t a n c e ( t h e c o n j u g a t e ) was immunogenic. A n t i b o d i e s formed by i n j e c t i o n o f a n i m a l s w i t h t h i s h a p t e n - p r o t e i n c o n j u g a t e were found to s e l e c t i v e l y b i n d t h e o r i g i n a l s m a l l m o l e c u l e w i t h very high a f f i n i t y .


1



140

CANNABINOID ANALYSIS IN

PHYSIOLOGICAL FLUIDS

The f i t between a n t i b o d y and a n t i g e n i s o f t e n q u i t e good, p a r t i c u l a r l y i n a r e a s w h i c h are removed from the s i t e o f a t t a c h m e n t t o the p r o t e i n . However, i n t r o d u c t i o n o f the c o v a l e n t l i n k i n e v i t a b l y r e s u l t s i n a change i n the s t r u c t u r a l c h a r a c t e r i s t i c s o f the molec u l e , and the r e s u l t i n g a n t i b o d y o f t e n r e f l e c t s t h i s change as i s i l l u s t r a t e d by the f u z z y a r e a i n the f i g ure. Indeed the b i n d i n g s i t e can emcompass n o t o n l y the o r i g i n a l s m a l l m o l e c u l e b u t the l i n k a g e i t s e l f and even p o r t i o n s o f t h e a t t a c h e d p r o t e i n . I n c e r t a i n i n s t a n c e s i t has been shown t h a t t h e d r u g p l u s l i n k i s bound w i t h much g r e a t e r a f f i n i t y t h a n t h e d r u g a l o n e , a t l e a s t by a c e r t a i n p o p u l a t i o n o f a n t i b o d i e s p r e s e n t i n the antiserum. Thus, no l i n k i n g group can be cons i d e r e d i n e r t , and t h e r e i s no i d e a l l o c a t i o n f o r l i n k age o f a d r u g t o the p r o t e i n . However, c a r e f u l s e l e c t i o n o f the l i n k a g e group and the s i t e o f a t t a c h m e n t may m i n i m i z e t h e problems i n v o l v e d . I n g e n e r a l , i n d e s i g n i n g a mode o f l i n k a g e o f t h e d r u g t o the p r o t e i n , one t r i e s t o a v o i d c o v e r i n g up s i t e s on the d r u g w h i c h a r e m e t a b o l i c a l l y r e a c t i v e — t h u s l e a v i n g them f r e e t o i n f l u e n c e the f o r m a t i o n o f s e l e c t i v e a n t i b o d i e s . A l s o one t r i e s t o a v o i d c o v e r i n g up s t r u c t u r a l f e a t u r e s w h i c h may p e r m i t d e v e l o p ment o f s t r o n g a f f i n i t y b i n d i n g s i t e s . The f i r s t p o i n t , to leave m e t a b o l i c a l l y a c t i v e s i t e s f r e e , i s p a r t i c u l a r l y i m p o r t a n t s i n c e i n g e n e r a l t h e d r u g and i t s m e t a b o l i t e s w i l l have many s i m i l a r s t r u c t u r a l f e a t u r e s . T h e r e f o r e , the m e t a b o l i t e s w i l l , under u s u a l c i r c u m s t a n c e s , be the p r i n c i p a l i n t e r f e r i n g c o n s t i t u e n t s i n an RIA a n a l y s i s . I n a d d i t i o n , i n d e s i g n i n g t h e s y n t h e s i s o f the immunogen, one must c o n s i d e r the c h e m i s t r y i n v o l v e d and t h e ease o r d i f f i c u l t y i n r e l a t i o n t o the e x p e c t e d b e n e f i t . Depending upon t h e i r needs t h e n , i t can be e x p e c t e d t h a t d i f f e r e n t i n v e s t i g a t o r s w i l l s y n t h e s i z e immunogens i n d i f f e r e n t ways. IMMUNOGEN SYNTHESIS

8

9

- A - a n d A -THC

As d e s c r i b e d i n p r e v i o u s p a p e r s by W a l l ( 2 ) , most o f t h e m e t a b o l i c a l t e r a t i o n s o f A -THC o c c u r i n t h e c y c l o h e x a n e m o i e t y w i t h h y d r o x y l a t i o n o f the A -compound o c c u r r i n g a t the 11- and 8 - p o s i t i o n s a l o n g w i t h c o n v e r s i o n o f the 11-carbon t o a c a r b o x y l group. More r e c e n t l y i t has been found t h a t h y d r o x y l a t i o n can a l s o o c c u r i n t h e amyl s i d e c h a i n o f t h e p h e n o l i c r i n g . These l a t t e r m e t a b o l i t e s e x h i b i t b i o l o g i c a l a c t i v i t y , b u t r e s u l t s from Dr. W a l l ' s group i n our l a b o r a t o r y i n d i c a t e t h a t i n humans, a t l e a s t , t h e y a r e q u a n t i t a t i v e l y minor m e t a b o l i t e s . 9

9



9.

141

Radioimmunoassay

COOK

a)

CO-NH -BSA (Teale, et al.,

b)

C O - C H C H C O - N H - B S A (Teale,eta/., 1975)

c)

C O - C H C H C O - N H - P G G (MSA, SGG, or PLL) (Tsui, et al.,

d)

C H C O - N H - H S A (or SGG) (Tsui, et al., 1974)

e) f)

2

2

1974)

2

2

1974)

2

Ν=Ν-((^)ν N

=

f

CO-NH-PGG (HSAor SGG) (Tsui, et al.,

1974)

* \ Ç J / " C O - N H - K L H (Gross,eta/., 1974)

(+ 4—isomer) g

Figure 3.

)

10—I—9—NH—CO—NH—HSA (or PGG) (Tsui,efa/., 1974)

Some positions through which A -THC has been linked to protein (3) 9

A number o f p r e v i o u s l y r e p o r t e d immunogens a r e summarized i n F i g u r e 3. T e a l e , Marks and t h e i r c o w o r k e r s have p r e p a r e d immunogens by f o r m i n g a h e m i e s t e r l i n k a g e w i t h the p h e n o l i c h y d r o x y l group and c o u p l i n g t h i s compound t o b o v i n e serum a l b u m i n (4, 5 ) . T s u i and c o - w o r k e r s (6) a l s o formed a h e m i s u c c i n a t e and a c a r b o x y m e t h y l e t h e r from the p h e n o l i c h y d r o x y l . These p r o d u c t s were c o u p l e d t o a v a r i e t y o f p r o t e i n s . The p h e n o l i c group a p p a r e n t l y undergoes no m e t a b o l i c a l t e r n a t i o n s b u t would be e x p e c t e d t o b i n d w e l l t o an a n t i body, and s p a t i a l l y t h e a t t a c h m e n t t o p r o t e i n i s r e l a t i v e l y c l o s e t o the m e t a b o l i c a l l y i m p o r t a n t c y c l o h e x e n e r i n g . To a v o i d t h i s p r o b l e m T s u i e t a l . a l s o p r e p a r e d a 2-azophenylcarboxy d e r i v a t i v e . TKis s u b s t i t u t i o n had been p r e v i o u s l y employed by S o a r e s , Gross and cow o r k e r s ( 7 ) . F i n a l l y , T s u i e t a l . (6) a l s o p r e p a r e d a 1 0 - i o d o - 9 - u r e i d o l i n k e d THC. Our a t t e n t i o n was drawn t o the amyl s i d e c h a i n o f the a r o m a t i c r i n g as a p o t e n t i a l p o s i t i o n f o r a t t a c h ment t o the p r o t e i n . Such a l i n k a g e would f u l f i l l the



142


Figure 4. Synthetic route to immunogen based on 5'-carboxy-k -THC: (a) dicyclohexyl carbodiimide/CH Cl ; (b) bovine serum albumin/dioxane-water. 8

2

2


9. COOK

143

Radioimmunoassay

r e q u i r e m e n t o f d i s t a n c e from t h e m e t a b o l i c a l l y r e a c t i v e c y c l o h e x a n e r i n g , a l t h o u g h n o t , o f c o u r s e , from s i t e s o f h y d r o x y l a t i o n on t h e amyl s i d e c h a i n . F o r s y n t h e t i c reasons our i n i t i a l attempts d e a l t w i t h the Δ -THC a n a l o g as a s u b s t r a t e . S y n t h e s i s o f t h e A -THC a n t i g e n i s shown i n F i g u r e 4. 5 -Carboxy-A -THC l a b e l e d w i t h t r a c e r amounts o f carbon-14 i n t h e c a r b o x y l group (8) was c o n v e r t e d t o a r e a c t i v e e s t e r by t r e a t m e n t w i t h N - h y d r o x y - s u c c i n i m i d e and d i c y c l o h e x y l c a r b o d i i m i d e . T h i s a c t i v e e s t e r was r e a d i l y c o u p l e d w i t h b o v i n e serum a l b u m i n i n a m i x t u r e of d i o x a n e and w a t e r t o y i e l d t h e d e s i r e d a n t i g e n . R a d i o a c t i v i t y measurements i n d i c a t e d t h e i n c o r p o r a t i o n of about 33 r e s i d u e s o f A -THC/molecule o f b o v i n e serum a l b u m i n . We have found t h i s mode o f c o u p l i n g t o be a v e r y u s e f u l o n e , as i t i s r e l a t i v e l y easy t o c o n s i s t e n t l y c o n t r o l t h e number o f m o l e c u l e s o f d r u g m o i e t y incorporated i n t o the p r o t e i n . 8

8

,

8


8

ANTISERUM CHARACTERIZATION R a b b i t s were immunized w i t h t h e c o n j u g a t e ( d i s s o l v e d i n s t e r i l e sodium c h l o r i d e s o l u t i o n and homo g e n i z e d w i t h an e q u a l volume o f F r e u n d s complete a d j u v a n t ) . I m m u n i z a t i o n w i t h 200 yg o f a n t i g e n was c a r r i e d o u t by t h e i n t r a d e r m a l t e c h n i q u e o f V a i t u k a i t i s e t a l . ( 9 ) , f o l l o w e d a f t e r two weeks by a n o t h e r intraSTermal i m m u n i z a t i o n and then a t f o u r week i n t e r v a l s by subcutaneous b o o s t e r i n j e c t i o n s . The a n i m a l s were b l e d 7 1/2 weeks a f t e r t h e i n i t i a l dose and e v e r y 4 weeks t h e r e a f t e r . A f t e r t h e t h i r d b l e e d i n g , t h e i m m u n i z a t i o n program was d i s c o n t i n u e d f o r a t h r e e month p e r i o d . A n t i g e n b o o s t e r i n j e c t i o n s were t h e n resumed and t h e f o u r t h b l e e d i n g was t a k e n 10 days a f t e r immuni z a t i o n . Reasonable t i t e r s ( t h a t i s , 50% b i n d i n g o f about 125 pg o f t r i t i u m l a b e l e d A -THC a t a f i n a l d i l u t i o n o f 1200-2700) were a c h i e v e d i n two o u t o f f o u r r a b b i t s a t t h e f i r s t b l e e d i n g . These t i t e r s compare q u i t e f a v o r a b l y w i t h t h o s e which have been r e p o r t e d by o t h e r s and demonstrate t h a t w i t h t h i s immunogen t h e r a b b i t i s a r e a s o n a b l e a n i m a l t o use f o r a n t i s e r u m p r o duction. The assay p r o t o c o l i s shown i n F i g u r e 5. U s i n g t h i s p r o t o c o l , we measured t h e a v i d i t y o f t h e a n t i s e r a f o r v a r i o u s m e t a b o l i t e s and a n a l o g s o f A -THC by de t e r m i n i n g t h e r e l a t i v e amount o f compound r e q u i r e d f o r 50% d i s p l a c e m e n t o f i n i t i a l l y bound r a d i o l i g a n g . A l though on t h e o r e t i c a l grounds t h i s p r o c e d u r e i s n o t s t r i c t l y v a l i d , we have shown t h a t i n most i n s t a n c e s i t provides a u s e f u l guide t o antibody s e l e c t i v i t y (10) . f

8

9



0.1 M phosphate buffer, pH 6.8 Antiserum 3

H - A ® f H C -10,000 c p m / Ι Ο μ β Unlabeled Drug


1

Total Volume - 0.52 ml Vortex 10 sec Incubate 24 hrs (4°C)

2.5% Charcoal Suspension (1.0 ml) Vortex 10 sec Incubate 15 minutes

Centrifuge 1200 g — 15 minutes Decant

Scintillation Fluid (10 ml)

1

Count 1 Minute (Bound Fraction) Figure 5.

General procedure for RIA of A -THC 9


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STRUCTURE BOUND

(HHC)

A

Β

C

100% (100)

100% (100)

63 (100)

48

94

144

102

49 (47)

13

2 (3)

0.6

100%

271

CH-OH

244 (100) (as Δ ) 8

COOH

(

Radioimmunoassay of Cannabinoid Compounds - American

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