CLINICAL TOXICOLOGY, 18( 9), pp. 1033- 1041 (1981)

P h e nc yc I id i ne (P C P) - S t ru c t u re versus Reactivity

ROBERT D. BUDD, BS

Los Angeles County Medical Examiner- Coroner Los Angeles, California 90033

A B S T R A C T

Seventeen different compounds having structural similari t ies to PCP (phencyclidine) were analyzed by radioimmunoassay (RIA) using '251-phencyclidine reagents (Roche). Affinities were com- pared with molecular structures and several observations were made. It was found that the three-ring structure (1-phenylcyclo- hexylpiperidine) i s essential for reactivity. Changes in and to the cyclohexyl ring and/or the piperidine ring reduced reactivity, while changes in the phenyl ring increased reactivity.

I N T R O D U C T I O N

PCP has been the most frequently abused drug (with the possible exception of alcohol and marijuana) by Los Angeles County proba- tioners for the last 2 years. Its popularity i s rapidly expanding to other parts of the country. Thus hospital and forensic laboratories a r e more and more frequently being asked to analyze for its presence.

To meet the needs of these laboratories for an analysis method, the Roche l2 'I-Phencyclidine Radioimmunoassay Procedure was de- veloped in 1979 [l]. It provides a rapid, sensitive method for the determination of PCP in biological fluids.

In using such a procedure for PCP analysis, it i s necessary to know its specificity-what other substances will cross-react. It i s important to know how reactive the PCP metabolites a r e and whether

1033 Copyright 0 1981 hy Marcel Dckkcr. Inc.

Clin

ical

Tox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Osa

ka U

nive

rsity

on

11/1

0/14

For

pers

onal

use

onl

y.

1034 BUDD

PCP-related substances such as PHP (which i s growing in popularity as the operators of PCP synthesis laboratories t ry to beat the regula- tions being placed on the chemicals necessary for PCP synthesis [2, 31 ) and PCC (an intermediate product in PCP synthesis [4] ) can be expected to cross-react. Some of these substances may cross-react to a sufficient degree that levels found i n biological fluids from users will produce a positive result when PCP itself i s absent.

Although a small amount of initial work has been done [ 13 , this ex- panded study has been done to determine the cross-reactivity of vari- ous PCP metabolites and related substances. A total of 17 different substances containing various components of the PCP structure were analyzed at several concentrations by the RIA system [ 11 and corre- lations between structure and affinity were determined. The effects on reactivity of various changes in the PCP-tricyclic structure a re reported. The data obtained allow predictions as to the reactivity of other PCP derivatives that someday may be produced for sale on the street.

M A T E R I A L S AND M E T H O D S

R e a g e n t s

Phencyclidine antibody reagent (Roche)

Ammonium sulfate solution Phencyclidine-related substances standards:

I- Phencyclidine derivative reagent (Roche) 1 2 5

0.5, 1.0, 3.0, and 5.0 pg/mL solutions in water of each phen- cyclidine-related substance (Table l), prepared fresh just prior to analysis.

A p p a r a t u s

Test tubes: Disposable polystyrene tubes, 12 X 75 mm (Lancer) Automatic pipetting station ( Micromedic Model 24004) Centrifuge: IEC Model K with 418 head Gamma scintillation counter with printer (Packard 5160) High-speed automatic pipette ( Micromedic Model 25004F) Sample and receiving racks (Micromedic)

A N A L Y S I S P R O C E D U R E

Each PCP standard was assayed using the Roche RIA procedure [ 11. A Micromedic automatic pipetting station was used to prepare all dilutions. Standards were analyzed 10 times at each of three concentrations to obtain an average count per minute.

Clin

ical

Tox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Osa

ka U

nive

rsity

on

11/1

0/14

For

pers

onal

use

onl

y.

PHENCYCLIDINE 1035

The analysis was done as follows: Twenty-one se t s of tubes were se t up for each concentration of each PCP standard to be analyzed. Add about 1 mL of solution to the appropriate tube for each standard concentration. Using an automatic pipetting station, add 0.1 mL of each solution to be analyzed from the above tubes, 0.2 mL of lZ5I-

phencyclidine antigen, and 0.2 mL of phencyclidine antibody to a se t of tubes. Repeat this procedure 10 times to generate 10 reaction mixtures for each concentration of each standard to be analyzed. Incubate the reaction mixture a t room temperature for 1 h. After incubation, add 0.5 mL of saturated ammonium sulfate solution to precipitate the protein complexes. Allow the reaction mixture to incubate at room temperature for 10 min. After incubation, centri- fuge the tubes at 2700 rpm for 10 min. Af te r centrifugation, use an automatic pipetting station to withdraw 0.5 mL of the supernatant fluid followed by 0.5 mL of distilled water to another tube for each tube of the reaction mixture. Count the tubes of supernatant fluid for 1 min in a gamma scintillation counter. Average the 10 results obtained for each standard concentration.

I N T E R P R E T A T I O N O F R E S U L T S

The PCP RIA reaction is a competitive reaction. The PCP i n the solution being analyzed competes with the limited amount of lZ5I- phencyclidine antigen for the limited amount of the PCP antibody. The resulting antigen-antibody and PCP-antibody complexes are pre- cipitated by the ammonium sulfate. The amount of unreacted lZ5I-

phencyclidine antigen which remains in solution is actually measured when an aliquot of the supernatant fluid is separated, diluted, and counted.

of the antigen will be used up and little will be left in the supernatant fluid, resulting in a low count. If a large concentration of PCP is analyzed, a larger amount of antigen wi l l be unreacted and left in the supernatant fluid, resulting in a higher count. Thus the larger the concentration of PCP in the solution being analyzed, the higher the count until a maximum is reached where further increases i n con- centration of PCP have little effect on the count produced.

is less reactive than PCP, a certain concentration of the substance will not compete as effectively with the antigen for the antibody as would the same concentration of PCP, resulting i n a lesser amount of unreacted antigen left in the supernatant fluid, yielding a lower count than would the same concentration of PCP. If the PCP-related sub- stance is more reactive than PCP, it will compete very effectively with the antigen for the antibody, more effectively than would the same concentration of PCP. This results in a greater amount of un- reacted antigen left in the supernatant fluid and yields a higher count.

For PCP, if none is present i n the solution being analyzed, most

For other PCP-related substances, if the substance being analyzed Clin

ical

Tox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Osa

ka U

nive

rsity

on

11/1

0/14

For

pers

onal

use

onl

y.

1036 BUDD

h 2 * u cd

.r( Y

$

h

k=

0 cd " k I

m 0 k 0

U .* .,-I Y

m

9

h

J E Y!

E

\

rl v

PI u

E3

N

rl

0) 0 E cd m Y

2

cd

8 I+

cd

2

I I

X

000 I

I X

PI u PI

m w In

m cn cn rl

Q (3 I

I X

ei (D

N I+ 0 N

Q 0 I

I X

PI X PI

Clin

ical

Tox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Osa

ka U

nive

rsity

on

11/1

0/14

For

pers

onal

use

onl

y.

PHENCYCLIDINE

m

m c-’

0 0 ID

m

5

t- m Q,

P E 2

a d r(

r(

I X

I X

I X

d 2

2 u d a,

h Q) Jz 0 a, 0 h C

d 5

u c PI PI

Q x

I X

I X

PI u t.l

m CU a 4

I X

Clin

ical

Tox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Osa

ka U

nive

rsity

on

11/1

0/14

For

pers

onal

use

onl

y.

Subs

tanc

e 1

2 3

CPM

(1

pg/m

L)

%I cr

oss-

reac

tivi

ty

1-P

iper

idin

e cy

clo-

H-

-CfN

NR

0

0

ca

NR

-N3

hexa

ne c

arbo

nitr

ile

Ket

amin

e

Phen

e tam

ine

@ca

2-J3

1-

Ben

zylp

yrro

lidin

e

NR

NR

NR

W C

U

U

1- (

1-C

yclo

hexe

ny1)

- pi

peri

dine

aRea

ctiv

ity

esti

mat

e by

pro

duct

man

ufac

ture

r [5

].

bNon

reac

tive

; re

acti

vity

les

s th

an 1

%.

Clin

ical

Tox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Osa

ka U

nive

rsity

on

11/1

0/14

For

pers

onal

use

onl

y.

PHENCYCLIDINE 1039

R E S U L T S AND D I S C U S S I O N

The structures and reactivities of the 17 PCP-related substances studied a r e illustrated in Table 1. Table 2 l ists the various structural factors affecting reactivity.

In order for a molecule to have affinity for the Roche RIA P C P anti- body, it must possess most of the aspects of the PCP tricyclic struc- ture ( 1-phenylcyclohexylpiperidine ) ( Table 1). Amphetamine-related drugs, benzodiazepines, caffeine, cocaine, diphenylhydantoin, ethinamate, glutethimide, meprobamate, methadone, methaqualone, methyprylon, nicotine, opiates, pentazocine, phenothiazines, propoxyphene, and tetra- hydrocannabinol, which all have vastly different s t ructures from PCP, were found to be totally unreactive a t concentrations up to 1 mg/rnL. The cross-reactivities of various PCP-related substances (Table 1) indicate that while the basic PCP structure is essential for high reac- tivity with the antibody, minor variations in structure effect affinity considerably.

using a protein conjugate of a PCP derivative. The binding site of the antibody consists of a hydrophobic pocket with some few charged s i tes and hydrogen-bond donor o r acceptor groups. The chemical properties and size of the binding s i te depends upon the functional groups of the hapten, i.e., there wil l be complimentary groups on the binding s i te for each functional group of the hapten. Therefore, the antibody binding site would be expected to recognize the PCP structure and the various groups contained therein: the cyclohexyl, phenyl, and piperidine rings. The presence of these groups is essential for strong reactivity.

Changes in the basic structure of PCP affect reactivity to varying extents. Specifically, changes a t any position of the PCP cyclohexane and piperidine rings reduce reactivity. The substitution of la rger groups will prevent binding due to s ter ic hindrance while the substitution of a smaller group will reduce the effective binding due to decreased binding area, decreased number of atoms available for binding, and increased ability of the group to more easily sl ip out of the binding site. The sub- stitution of a group of differing polarity (greater o r less) will a lso re- duce affinity because the antibody does not possess a complimentary group. The effects of various specific changes in the PCP structure will now be discussed in detail (Table 1).

of any one of the three rings (such as conversion of PCP to cyclohexyl- benzene, 1-piperidine cyclohexane carbonitrile, 1-benzylpyrrolidine, ketamine, o r 1- ( 1-cyclohexenyl kiperidine) eliminates reactivity. In a s imilar manner addition of one o r more ring structures would be ex- pected to greatly reduce o r totally eliminate reactivity.

The cyclohexane ring i s vital for reactivity. The addition of a group, such as a p-hydroxy group (conversion of PCP to i ts major metabolite), greatly reduces reactivity [ 51.

group with smaller groups, such as a pyrrolidine ring, an ethylamino

The PCP antibody i s a large protein molecule, generated i n goats,

The tricyclic structure of PCP is vital for reactivity. Elimination

The piperidine ring is also vital for reactivity. Replacement of the

Clin

ical

Tox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Osa

ka U

nive

rsity

on

11/1

0/14

For

pers

onal

use

onl

y.

1040

TABLE 2. Changes Affecting Affinity

BUDD

I. Changes increasing affinity A. Phenyl group

1. Replacement with a 2-thienyl group 11. Changes reducing affinity

A. Phenyl group 1. Elimination of the group 2. Replacement with a cyano group

B. Cyclohexyl group 1. Elimination of the group 2. Addition of a p-hydroxy group

C. Piperidine group 1. Elimination of the group 2. 3. 4.

5. 6.

Replacement with an amino group Replacement with an ethylamino group Replacement with a pyrrolidine ring Replacement with a morpholine ring Addition of a 4-hydroxy group

group, o r an amino group, greatly reduces reactivity, and the g rea t e r the s ize reduction, the g rea t e r the decrease in reactivity. Replacement of the group with a morpholine ring which is of s imilar s ize , but differ- ent in polarity and chemistry, substantially reduces reactivity. The ad- dition of groups to the ring such as a p-hydroxy group (conversion of P C P to i ts minor metabolite) greatly reduces reactivity [ 11.

The phenyl group is important for reactivity, but not as vital as the other two rings. Changes in i t had differing effects on reactivity. Re- placement of the group with a cyano group, which is much smaller , en- t irely eliminates reactivity; while replacement of the group with a 2- thienyl group, which is s imi l a r in s ize and shape but slightly more polar, increased reactivity. In fact, this is the only s t ructural change studied found to increase reactivity. The TCP molecule is apparently more closely structurally related to the P C P hapten than P C P itself.

The resul ts of this study lead to several interesting conclusions: Firs t , the metabolites are very slightly reactive and when present in a biological fluid being analyzed by RIA for PCP will yield a negligible affect on the final counts pe r minute measured. Second, the resul ts point to the fact that the P C P hapten-protein complex is a derivative

Clin

ical

Tox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Osa

ka U

nive

rsity

on

11/1

0/14

For

pers

onal

use

onl

y.

PHENCYCLIDINE 104 1

of P C P with the protein attached somewhere on the phenyl ring o r an- other ring form in that position of the P C P molecule. Third, P C P derivatives with changes to either the cyclohexyl r ing o r the piperi- dine ring will be, a t best , only weakly reactive with the Roche P C P antibody, while PCP derivatives with changes to the phenyl group may be quite reactive if a large group is maintained in that position. Thus d rug use r s who wish to avoid laboratory testing detection by Roche RIA will further gravitate toward derivatives such as PHP [2] r a the r than TCP o r P C P itself. Fourth, the resul ts of this study indicate that the Roche RIA method is very specific for P C P with interference f rom only very closely structurally related derivatives, but none f rom other drugs o r biological substances.

R E F E R E N C E S

Abuscreen, Radioimmunoassay for Phencyclidine (PCP) , Roche Diagnostics, Nutley, New J e r s e y 07110. R. D. Budd, PHP, a new drug of abuse, N. Engl. J. Med., 303, 588 (1980). G. R. Nakamura, E. C. Griesemer, L. E. Joiner , and T. T.

-

Noguchi, Determination of 1- ( 1-phenylcyclohexyl Ipyrrolidine (PHP) in postmortem specimens: A case report , Clin. Toxicol., 14, 383-388 (1979). - J. 'R. Ballinger and J. A. Marshman, GLC quantitation of 1- piperidinocyclohexanecarbonitrile ( PCC) in illicit phencyclidine (PCP) , J. Anal. Toxicol., 3, 158-160 (1979). Heveran, Personal Communication, Roche Diagnostics, 1980.

Clin

ical

Tox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Osa

ka U

nive

rsity

on

11/1

0/14

For

pers

onal

use

onl

y.