The alpha carbon oxidation of some phencyclidine analogues by rat tissue and its pharmacological implications.

XENOBIOTICA,

1990, VOL. 20,NO. 6, 591-600

The alpha carbon oxidation of some phencyclidine analogues by rat tissue and its pharmacological implications M. STEFEKt, R. W. RANSOMS, E. W. DiSTEFANOt and A. K. CHOtO

Xenobiotica Downloaded from informahealthcare.com by Nyu Medical Center on 11/11/14 For personal use only.

t Department of Pharmacology and the Jonsson Comprehensive Cancer Center, UCLA School of Medicine, Center for the Health Sciences, Los Angeles, California 90024-1735, USA $ Merck, Sharp and Dohme Research Laboratories W26A-3025 West Point, PA 19486, USA Received 4 August 1989; accepted 10 February 1990

1. The metabolism of phencyclidine (PCP) and three congeners, differing in the structure of the m i n e moiety, by liver microsomesfrom phenobarbital-pretreated rats, was determined. 2. The metabolites generated by sequential oxidation of the two carbons alpha to the nitrogen were measured for PCP and its diethyl analogue (PCDE). 3. Alpha hydroxylation was a dominant metabolic pathway for PCDE, but less so for PCP. 4. Evaluation of affinities for the N-methy1-D-aspartate (NMDA) and sigma receptors in Vitro showed that the product of alpha-hydroxylation of PCDE, phenylcyclohexylethylamine (PCE), was very potent. 5. Therefore, the in vivo actions of PCDE could include a significant contribution by PCE. 6. All congeners formed phenylcyclohexylamine(PCA), the product of a second alphahydroxylation, with PCDE and the pyrrolidine analogue generating the largest proportion.

Introduction Phencyclidine (PCP) is a hallucinogenic drug of abuse that is available primarily through synthesis in illicit laboratories. The simplicity and ready application of its synthesis to closely related compounds, together with restrictions that have been placed on some of its starting materials, have resulted in the manufacture and sale of closely related congeners. For example, N-ethyl-phenylcyclohexylamine (PCE) (Smialek et ul. 1979), and phenylcyclohexylpyrrolidine (PCPY) (Budd 1980) have been identified in samples of both fluids in the course of forensic analysis. Although PCPY is similar to PCP in potency when compared in drug discrimination(Shannon 1981) and rotarod procedures (Vaupel et ul. 1984), PCE is more potent in these procedures. As this group of chemicals continues to be abused, an understanding of the changes in pharmacology associated with structural modifications is important. Such psychopharmacological agents must be studied in wiwo, but to interpret the results from such experiments, information on the metabolic disposition and affinities for known receptors is essential. Recent studies (e.g. Gole et al. 1988) have identified the NMDA and sigma receptor systems as sites of PCP action. Since the structures of two receptor systems differ, it is likely that the binding affinities of §Author to whom correspondence should be addressed. 0049-8254/90 (3.00

0 1990 Taylor & Francis Ltd.

M. Stefek et al.

592

Substrate

Phencyclidine (PCP)

Amine

-3

Phenylcyclohexylpyrrolidine (PCPY)


Amine

75

Phenylcyclohexylhexamethyleneimine (PCHM)

Phenylcyclohexyldiethylamine (PCDE)

Phenylcyclohexylethylamine (PW Phenylcyclohexylamine (PCA)

.3 /.-

-N

b

-N 4 \ H

-Nn,

Figure 1. Structures of the phencyclidine analogues studied.

analogues would also differ, and differences in overall pharmacology would be expected. Structural changes also alter pharmacokinetic properties of a drug which would also alter the in o;wo pharmacology. The report describes studies with rat tissues comparing the metabolism and pharmacology of several PCP analogues (figure 1) including PCPY and PCE. These compounds are extensively metabolized at the m i n e portion, and the carboxylic acid generated by oxidation of the alpha-carbon in the heterocyclic compounds is a major terminal metabolite in mammals (Baker et al. 1981, Hallstrom et al. 1983, Cohen et al. 1982). Alpha-carbon oxidation also leads to a pharmacologically active metabolite, phenylcyclohexylamine (PCA), and previous studies (Cho et al. 1983) suggested that it could accumulate in the brain after administration of PCP. PCE is a metabolite of phenylcyclohexyldiethylamine (PCDE) and an intermediate in its conversion to PCA.

Methods Chemicals PCPY, phenylcyclohexylhexaethyleneimine (PCHM), and PCA were synthesized in this laboratory (Brady et al. 1987). PCP, PCE and PCDE were obtained from the National Institute of Drug Abuse (Rockville, MD). Benzphetarnine HCI was donated by Upjohn Co. (Kalamazoo, MI). Hexobarbital, NADP, glucose 6-phosphate, glucose 6-phosphate dehydrogenase, and sodium phenobarbital were purchased from Sigma Chemical Co. (St. Louis, MO). Other chemicals and reagents were obtained from common commercial sources. Radiolabelled (+)-5-methyl-lO,l 1-dihydro-5Hdibenzo[a,d]cycloheptene-5,10-imine (MK801) and 1,3-di(2-tolyl)guanidine (DTG) were obtained from New England Nuclear Corp. (Boston, MA, USA).

a-Carbon oxidation of phencyclidines

593


Liver preparations Livers were obtained from male Sprague-Dawley rats (220-270 g) that had been fasted overnight. T o induce enzyme activity the rats were pretreated for 3 days with sodium phenobarbital (3 x 80mg/kg i.p.). The livers were homogenized in 3 vol. of 1.15% KCI and centrifuged at 9000g for 20 min. The supernatant was then centrifuged at 1000OOOg for 60min and the microsomal pellet collected and washed by resuspension in 3 vol. of 1.15% KCI and recentrifugation. The washed pellet was stored under 1 ml of 1.15% KCI at -80°C for up to 2 weeks. For incubation the thawed pellet was resuspended in 1.15% KCI and diluted to a concentration equivalent to 033 g of liver, wet weight, per ml. Incubations Incubations were conducted aerobically at 37°C in 25 ml Erlenmeyer flasks for the indicated times. Unless otherwise stated, microsomal suspension equivalent to 0 3 3 g of wet liver was incubated in a total volume of 5 ml of 0 15 M potassium phosphate buffer (pH 7 5 ) in the presence of 2-6pmol of NADP, 30pmol of glucose 6-phosphate, 1 2 p o l of MgCI,, and 4 units of glucose 6-phosphate dehydrogenase and substrate as indicated. Microsomal cytochrome P-450 concentrations of approx. 2 4 p ~ substrate , concentration in the region of 5-100p~,and a reaction time of 2min were found to be optimal for the determination of the kinetic constants. The material balance and kinetic studies were performed at cytochrome P-450 concentrations of 5 . 5 f 0 9 p ~ .Incubations were started by the addition of the microsomal suspension. The reaction was stopped by transferring the incubation mixture to an extraction tube containing 10mI.of ice-cold dichloromethane (CH,CI,), 1.0ml of 1.5 M Na,CO, solution (pH 9 5 ) and 40 nmol of benzphetamine as an internal standard. For trapping of the aminoaldehyde metabolite of phencyclidine the incubation mixture was instead transferred to a tube containing methoxyamine hydrochloride ( 5 mg), mixed, and immediately heated in a boiling water bath for 5 min. The basified incubation mixture and dichloromethane were shaken for 15rnin and centrifuged at 200g for 10min. The organic layer was filtered through silanized glass wool, evaporated under nitrogen to a small volume and subjected to g.1.c. analysis for the unchanged substrate, PCA, and in the case of the PCDE substrate, for PCE as a metabolite as well. For analysis of the aminoaldehyde metabolite of phencyclidine as its 0methyloxime, the boiled incubation mixture containing methoxyamine was allowed to cool to room temperature (30 min), and then basified and extracted with dichloromethane (CH,Cl,) as described above. The concentrated organic extract was derivatized with l00pl of bis(trimethylsily1)trifluoroacetamide (BSTFA) and analysed by g.1.c. Gas chromatography A Hewlett-Packard 5830A gas chromatograph fitted with a 1.8 x 2 mm (int. diam.) glass column packed with 3% OV-101 on 80/100 Supelcoport was used with the following conditions: carrier gas flow (He), 25 ml per min; injection port temp., 210°C; flame-ionization detector temp., 220°C; oven temp. programmed 170-230°C at 8"C/min. The retention times (min) of the measured compounds were: PCP (4-33), PCCHM (532), PCDE (3-09), PCE (1-97), phenylcyclohexylaminovaleraldehyde methoxime (7-47), PCA (1.59). A glass column (1%mx2mm, int. diam.) packed with 5% QF-1 on 100/120 Supelcoport, under isothermal conditions at 165°C was used for analysis of PCPY (retention time 2.39 min). The area ratio of the compounds analysed to the internal standard (40 nmol of benzphetamine) was calculatedand compared to a standard curve, which was carried through each experiment. The slopes of the standard curves from at least four assays had a standard deviation of less than 10%. The recoveries for the measured compounds ranged from 85% for PCCHM to 100% for PCP. Recording of different spectra Absorption spectral changes produced on addition of the substrates to microsomal suspensions were measured according to Schenkman et al. (1967) using a UVIKON 810 double-beam spectrophotometer. A volume of 5 ml of diluted microsomal suspension (approx. 2 p P-450) ~ in 0 1 M postassium phosphate buffer (pH 7-5) was divided equally into two cuvettes, and a baseline was recorded. Then aliquots (25100pl) of a substrate solution were added to the sample cuvette, and a corresponding volume of distilled water was added to the reference cuvette. The absorbance from 470 nm to 360 nm was scanned at ambient temperature. Pharmacological procedures The effect of the compounds on 'H-MK-801 binding to rat cortex homogenates was determined by the procedure of Wong et al. (1988) except that the incubation buffer was lOmM Tris HCI, pH75. All experiments were conducted using 2 nM ,H-MK-801 in a final volume of 1 ml. One hundred micromol MK801 was used to define nonspecific binding. Incubations were terminated by filtration (Whatman GF/B) after 1 h at 23°C. ,H-ditolyl guanidine (DTG) binding to rat pons-medulla membranes also followed the procedure of Wong et al. (1988). Membranes were incubated with 5 nM jH-ditolyl guanidine (DTG) for 90 rnin at 23°C in 1OmM Tris HCl prior to filtration. Ten micromolar haloperidol was used to determine nonspecific binding.

M . Stefek et al.

5 94

OTHER METABOLITES

H

0

I

I1

1x1


OTHER METABOLITES

%

k2

H

I Figure 2.

XI

1x1

Kinetic model used to analyse phencyclidine (PCP) and diethylphenylcyclohexylamine (PCDE) metabolism.

The concentrations of the substrates and two of their metabolites (11,111) were determined over a 60min period and the data points fitted to the model shown above by%nonlinearregression procedures as described in Methods. The values for the apparent rate constants are shown in table 3.

Data analysis The time-concentration data for the conversion of PCP and PCDE to PCA were analysed for the individual rate constants by nonlinear regression procedures using the BMDP software AR program (Dixon 1988). The data were fitted to the model shown in figure 2, for which the concentrations of substrate, intermediate and PCA are given by the equations: (CI, figure 2 ) = ~ e - ~ l ~ l (CI I, figure 2) =Ak,

(A)

-k,,){e -'z5' -e -

(CIII, figure 2) =Aklkz{e-k14'/(k25-k14) +e-k251/(k14-kz5)(k3 -k,J

1

+e-'"/(kl4-

(B) k3)(k,, -k 3 ) } (C)

k,,=k,+k, kz5 =kz

+k5

Estimates of k,, the rate constant for PCA disappearance, k,, PCE and aldehyde conversion to PCA, and k,, alternate pathways, were also obtained independently. The values of k, and k, obtained by this method were used to estimate the other rate constants. Although the estimated value of k, for PCP was very close to the value obtained directly, this approach was not as reliable for PCDE. Competition by parent drug for cytochrome P-450binding sites could be responsible for this discrepancy.

Results Metabolic studies The metabolism of the compounds was examined at two levels. Initially, their overall metabolism was determined by monitoring substrate disappearance at different concentrations, In addition, the interaction of the substrates with cytochrome P-450 was measured by spectral changes with binding to the oxidized cytochrome. In the second level the formation of phenylcyclohexylamine was



595

examined with PCP and PCDE. These compounds differ by the cyclic nature of the amine moiety and a comparison would assess the role of ring closure on metabolic disposition. The overall metabolism of the analogues was rapid, and except for PCP, essentially all substrate was consumed within the first 6-15min. The enzymic parameters for disappearance of PCP and its structural congeners by liver microsomes were determined and compared with those for interaction with cytochrome P-450, measured by analysis of spectral changes from binding to oxidized cytochrome. At low concentrations (5-65 p ~ the ) Eadie Hofstee plots for both sets of data were linear (5’ >097) for each substrate and values of these apparent parameters are summarized in table 1. Except for PCP itself the maximal rates were very similar. The compounds generated typical type I difference spectra (Schenkman et al. 1967)with absorbance maxima at 388 nm and absorbance minima at 420 nm (figure 3) over this concentration range. The parameter values for these spectral shifts are also summarized in table 1. The values for K,and K, correlated at the 1’ 5093 level. The intermediate metabolites, aldehyde and PCE, also gave type I spectral changes. The K,values obtained for aldehyde and PCE were 16.9 and 37-4pM, resepctively (an average from two experiments). The interaction of PCA with microsomal cytochrome P-450 was dual in nature. When added to a suspension of microsomes this primary amine gave rise to an asymmetrical spectral change (figure 4) whose character did not change when concentration varied from 5 to 500 p ~However, . addition of hexobarbital(05 mM) to both cuvettes resulted in a typical type I1 (Schenkman et al. 1967) spectrum with an absorbance maximum at 425 nm and an absorbance minimum at 394 nm (figure 4, curve b). When curve b was subtracted from the overall spectral change a type I spectral change was obtained (curve c). Thus, the observed spectral asymmetry appeared to be due to a combination of type I and type I1 spectral changes. When type I binding sites were blocked with 05 mM hexobarbital, a spectral dissociation constant of 1 8 0 for ~ the ~ type I1 spectral component of PCA was obtained (an average from two experiments). Table 1 . Parameters for overall metabolism and spectral binding of some phencyclidine analogues. A: Apparent Michaelis-Menten constants Metabolism substrate (amine) PCDE PCPY PCP PCHM Spectral bmding substrate (amine) PCDE PCPY PCP PCHM

.,v

Km(PM)

(nmol/min per nmol P-450)

3 8 7 f 6 3 (n=3) 37.2 f5.1 (n= 3) 486 & 4.9 (n= 3) 2Q6f3.8 (n=3)

8 9 f1.8 (n = 3) 8.8 f1.2 (n = 3) 68k1.4 (n-3) 8.3f0.8 (n=3)

K. (PM)

A,, (a.u./nmol P-450)

126+1-4 (n=3) 124+1-9 (n=3) 13.7k1.7 (n=6) 70f 1.8 (n=3)

19-4k4.0 (n=3) 17.7f3.2 (n=3) 9-1k21 ( n = 6 ) 21.5f2.1 (n=3)

Results are mean values fSD, with the number of experiments in parantheses. The substrates are identified by the amine substituent(see figure 1). The rates of overall metabolism were determinedin two minute incubations of the substrate at concentration ranges of 5 to 1 0 0 as~described ~ in Methods. The spectral binding data were obtained as described in Methods. The constants were calculated by linear regression of the reciprocals of substrate concentration and concentration as absorbance.

M. Stefek et al.


596

-1oo-I -

' . ' . ' . ' . ' . ' - ' 350 370 390 410 430 450 470 490 Wavelength (nm)

'

Figure 3. Spectral binding curve of phencyclidine (PCP). , incubated with The U.V. difference spectrum of PCP, at a concentration of O l r n ~ when rnicrosornes.

b a C

-20-40

360

380

400

420

440

Wavelength (nm) Figure 4.

Spectral binding curve of phenylcyclohexylamine (PCA).

(a)PCA at 1 rnM, (6) PCA) (1 rnM)+hexobarbital (O.SmM), (c) calculated curve, obtained by

subtracting (6) from (a).

Of the approximately 20&350 nmol of the substrate consumed during the first 2 min, 10-17% appeared as PCA (table 2) and after 15 minutes it accounted for 1975% of the consumed substrates (table 2). PCA is generated from the substrates by a sequential oxidation of two C-N bonds and is relatively stable in this preparation as shown by its small rate constant for metabolism (table 3). In order to analyse the rates of PCA formation, levels of intermediate were determined for PCP and PCDE. For PCP the aldehyde generated by alpha-carbon oxidation was measured as the oxime (Hallstrom et al. 1983). The intermediate in the conversion of PCDE to PCA is PCE. The temporal relationships between substrate, intermediate and PCA generated from PCP and PCDE are shown in figure 5.


597

Table 2. Overall metabolism of phenycyclidine (PCP) congeners and formation of phenylcyclohexylamine (PCA). PCA formed in (percentage of metabolized substrate) Substrate (amine structure) PCDE PCPY PCP PCHM

k (disapp)

2min

15 min

16.0k1.3 (3) 13.7 14.0k3.8 (9)

5 7 5 * 2 5 (3) 56.9 22.7 f 2.3 (9)

9.6

19

(min- ')'

0.55+0.01 0.30 0.13f002 0.53

The initial substrate concentration was 1 0 0 ~ Substrates ~. are identified by the amine substituent Xenobiotica Downloaded from informahealthcare.com by Nyu Medical Center on 11/11/14 For personal use only.

(see figure 1).

T h e apparent first-order decay constants. Results are mean values & SD, with the number of experiments in parentheses. Where no S D is given the figure is the mean value from two experiments. a

100

. I .

0

5

10

15

0

5

10

15

Time (min) Figure 5. Conversion of phencyclidine (PCP) and diethylphenylcyclohexylamine (PCDE) to phenylcyclohexylamine (PCA). (a)PCP at a concentration of 0.1 mM was incubated over 15 minutes as described in Methods and levels of PCP ( O ) ,aldehyde (0)and PCA ( A ) determined. The concentrations of the compounds are plotted against the time of incubation. (b) PCDE at a concentration of 0.1m~ was incubated over 15 minutes as described in Methods and levels of PCDE ( O ) , PCE (0)and PCA ( A ) determined. The concentrations of the compounds are plotted against the time of incubation. The solid lines represent the predicted curves generated from the constants of table 3.

M. Stefek et al.

598

Table 3. Rate constants for sequential alpha-carbon hydroxylation of phencyclidine (PCP) and diethylphenylcyclohexylamine(PCDE). Substrate

n

k,

k2

k3

k4

k,

PCP PCDE ALD PCE PCA

9 3 2

0.090+0013 0471 f0107

-

0-251*O-059 0095f0022 0.273 0283

-

-

0011 0011 0011 f0002

0071 f0021 0052f O W 7 -

0244 0001 0244 0001 -

2 3


Values for the rate constants (min-') of figure 2 determined from the substrates indicated (fasymptotic standard deviation). The initial concentrations of PCP and PCDE were 100p~, those of PCA, aldehyde, and PCE were 50 p ~ The . substrates are identified by the amhe substituent (see figure 1). Table 4. Pharmacological properties of phencyclidine (PCP) analogues: IC,, values for affinities for NMDA and sigma receptors. Compound (amine) PCP PCHM PCPY PCDE PCE PCA

MK 801

Ditolyl guanidine

0.110f002 0.060f0012 029f0.05 1.90f0.64 0.184f0.022 1-00& 021

1-69m 3 0 062k008 204+0-62 870k 1.60 539+ 1.43 27-51& 523

IC,, values for inhibition of tritiated ligand binding. 'H-MK801 was used to measure NMDA receptor affinity and 'H-ditolyl guanidine was used to measure sigma receptor affinity of the compounds. The values are expressed as the meansf SEM for at least three separate determinations. The substrates are identified by the amine substituent (see figure 1).

The temporal relationships between the alpha-carbon oxidation products of PCP and its ring-opened diethylamine analogue (PCDE) were estimated from changes in substrate and product levels with time over a 15 min incubation. The rate constants for conversion of the intermediates to the primary amine were obtained separately by incubation for the appropriate intermediate. These rate constants and the data obtained were fitted by nonlinear regression procedures to the model shown in figure 2 and the estimated values are shown in table 3. The values of the ratio k,/R, for the two substrates show that a higher proportion of N-dealkylation occurs with the diethylamine derivative. The oxidation of the alpha-carbon of the amine represents about 55% and 90% of the overall metabolism of PCP and phenlcyclohexydiethylamine, respectively. Thus ring closure can result in substantial quantitative changes in the site of oxidation by cytochrome P-450. Pharmacological studies The interaction of the compounds with two binding sites associated with the actions of PCP was also determined. One site is that associated with the NMDA receptor system and is characterized by the ability to inhibit the binding of 3H-MK801 (Wong et al. 1988)and the second is the sigma receptor, characterized by competition with 3H-DTG binding (Weber et al. 1986). The results, shown in table 4, reveal changes in both affinities for the two binding sites and changes in the selectivity to the binding sites. Although PCE and PCA exhibited high selectivity for the NMDA receptor system, as evidenced by the large differences in their affinities for the two receptors, the actual affinity of N-ethyl phenylcyclohexylaminefor each receptor was much greater.


599


Discussion The correlation between the apparent affinity constants of phencyclidines for cytochrome P-450, measured by spectral changes and by overall metabolism, is consistent with the notion that this monooxygenase is responsible for the majority of the microsome-mediated oxidation of these drugs. Differences in maximal binding and maximal metabolic activity would be expected since the measurements reflect processes which are dependent on different substrate properties. Phenylcyclohexylamine interaction with cytochrome P-450 demonstrated an unusual duality. In the absence of other type I substrates the lipophilic character of this compound caused a type I interaction. However, when another type I substrate such as hexobarbital was added, either the orientation on a particular isozyme changed or binding to another isozyme occurred resulting in a type I1 spectral change characteristic of many amines. The metabolic formation of this m i n e was of pharmacological interest because of its activity and its commonality as a secondary metabolite of phenycyclidine analogues. Although 5-( 1-phenylcyclohexylamino)valeric acid represents a major elimination pathway (Hallstrom et al. 1983, Cohen et al. 1982, Baker et al. 1981) the persistence of an alternative metabolite, PCA, of the same pathway in the brain (Cho et al. 1983) indicates that the alpha-carbon oxidation is pharmacologically relevant. In addition to its activity at the NMDA receptor, PCA antagonizes electroshock seizures in rats by a mechanism that appears to be different from NMDA blockade (Rogawski et al. 1989). Thus, the formation of this metabolite in varying amounts could contribute to the complexity of phencyclidine analogues. The studies described here showed a three-fold difference in PCA levels among the compounds examined. Metabolism of PCA, even by microsomes from phenobarbital-induced animals, is slow, which accounts for its persistence in the brain after PCP administration (Cho et al. 1983). In spite of its low in witro activity, PCA has about one-half the potency of PCP in viwo, as measured by a discriminitive stimulus assay in rats (Shannon 1981). This discrepancy could be due to its greater bioavailability . The rate data of table 3 show a large difference in alpha-carbon hydroxylation between PCDE and PCP. The diethyl compound is rapidly converted to PCE and this pathway is a major pathway for its metabolism. In contrast, the overall metabolism of PCP is lower, and the rate of alpha-hydroxylation is less than one fifth of that for PCDE. Although both compounds have similar alternative sites for oxidation, the other metabolic pathways are much more important for PCP. Thus, one difference in metabolism between the cyclic m i n e and its open-chain analogue appears to be oxidation at other sites in the molecule. The high rate of alpha-carbon oxidation for PCDE may reflect better access to the haem of cytochrome P-450. A second difference is the formation of an inactive, valeric acid metabolite by the ringclosed compound. The diethylamine compound, on the other hand, is converted to two active compounds, the monoethylamine derivative and phenylcyclohexylamine. The monoethyl metabolite, called rocket fuel, has been found in illicit preparations (Smialek 1979) and is about 5 times more potent than PCP in wiwo (Shannon 1981) and is comparable in witro (table 4). PCDE is similar to PCP in wiwo, but in the in witro evaluations used here the compound is substantially weaker. The greater in viwo activity of PCDE can be attributed to the formation of the active monoethyl metabolite. The verification of this possibility will require pharmacokinetic studies.

600


Acknowledgement This work was supported by USPHS grant DA 0241 1.


References BAKER, J . F., WOHLFORD, J. G., BRADBURY, B. J., and WITH,P. W., 1981, Mammalian metabolism of phencyclidine. Journal of Medicinal Chemistry, 24, 666469. BRADY, J. F., DOKKO, J., DISTEFANO, E. W., and CHO,A. K., 1987, Mechanism-based inhibition of cytochrome P450 (P450) by heterocyclic analogs of phencyclidine (PCP). Drug Metabolism and Disposition, 15, 648-652. BUDD,R. D., 1980, PHP, a new drug of abuse. New England Journal of Medicine, 4 September, p. 588. CHO,A. K., HALLSTROM, G., MATSUMOTO, R. M., and KAMMERER, R. C., 1983, The metabolism of the piperidine ring of phencyclidine, Phencyclidine and Related Arylcyclohexylamines: Present and Future Applications (Joint French- U.S. Seminar on the Chemistry, Pharmacology, Tosicology, Therapy and Drug Abuse Aspects of Arylcyclohexylamines, Montpellier), edited by Kamenka, J .-M., Domino, E. F. and Geneste, P. (Ann Arbor, MI, NPP Books), pp. 205-214. COHEN, L. S., GOSENFELD, L., WILKINS, J., KAMMERER, R. C., and TACHIKI, K., 1982,Demonstration of an amino acid metabolite of phencyclidine. New England Journal of Medicine, 306, 1427-1428. DIXON,W. J., 1988, B M D P Statistical Software Manual, (Los Angeles, CA: University of California Press), pp. 389-418. GOLE,D. J., PIRAT,J. L., and DOMINO, E. F., 1988, New aspects of phencyclidine (PCP) metabolism, Sigma and Phencyclidine-like Compounds as Molecular Probes in Biology, edited by Domino, E. F. and Kamenka, J. M. (Ann Arbor, MI, NPP Books), pp. 625442. HALLSTROM, G., KAMMERER, R. C., NGUYEN, C. H., SCHMITZ, D. A., DISTEFANO, E. W., and CHO,A. K., 1983, Phencyclidine metabolism in witro: the formation of a carbinolamine and its metabolites by rabbit liver preparations. Drug Metabolism and Disposition, 11,47-53. ROGAWSKI, M. A., THURKAUF, A,, YAMAGUCHI, S., RICE,K. C., JACOLSON, A. E., and MATTSON, M. V., 1989, Anticonvulsant activities of 1-phenylcyclohexylamineand its conformationally restricted analog 1,l-pentamethylenetrahydroisoquinoline.Journal of Pharmacology and Experimental Therapeutics, 249, 708-712. SCHENKAMN, J. B., REMMER, H., and ESTABROOK, R. W., 1967, Spectral studies of drug interaction with hepatic microsomal cytochrome. Molecular Pharmacology, 3, 113-123. SHANNON, H. E., 1981, Evaluation of phencyclidine analogs on the basis of the discriminative stimulus properties in the rat. Journal of Pharmacology and Experimental Therapeutics, 216, 543-551. SMIALEK, J . E., MONTFORTE, J. R.. GAULT, R., and SPITZ,W. U., 1979, Cyclohexamine (“Rocket Fuel”)phencyclidines potent analog. Journal of Analytical Toxicology, 3, 209-21 2. VAUPEL, D. B., MCCOUN, D., and CONE,E. J., 1984, Phencyclidine analogs and precursors: Rotarod and lethal dose studies in the mouse. Journal of Pharmacology and Experimental Therapeutics, 230, 20-27. WEBER, E., SONDERS,M., QUARUM, M., MCLEAN, S., Pow, S., and KJJANA, J. F. W., 1986, 1,3-Di(2-[5,3H]-toly)guanidine: a selective ligand that labels sigma-type receptors for psychotomimetic opiates and antipsychotic drugs. Proceedings of the National Academy of Sciences, U S A , 83,87848788. WONG,E. H. F., KNIGHT, A. R., and WOODRUFF, G. N., 1988,3H-MK801 labels a site on the N-methylD-aSpartate receptor channel complex in rat brain membranes. Journal of Neurochemistry, 50, 274-281.

Diversity of the pharmacological actions of some tolazoline analogues in human platelets and rat aorta.

Modulation by endothelium of the responses induced by endothelin-1 and by some of its analogues in rat isolated aorta.

Inhibition of endothelium-dependent vasorelaxation by arginine analogues: a pharmacological analysis of agonist and tissue dependence.

Immunosuppressive activity of antamanide and some of its analogues.

Polymorphism of carbon oxidation of drugs and clinical implications.

The synthesis and in vitro pharmacological evaluation of some novel enkephalin analogues [proceedings].

The pharmacological activity of some choline analogues and their acetylated derivatives [proceedings].

Pharmacological specificity of the phencyclidine discriminative stimulus in rats.

Branched-chain amino acid oxidation by isolated rat tissue preparations.

Analysis of amitriptyline and its analogues in body tissue.

Pharmacological properties of kryptopyrrole and its oxidation products on isolated sciatic nerve of rat and on guinea-pig ileum.

Inhibition of Mycobacterium tuberculosis dihydrodipicolinate synthase by alpha-ketopimelic acid and its other structural analogues.

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Effect of alpha-ketoglutarate and its structural analogues on hysteretic properties of alpha-ketoglutarate dehydrogenase.

Pharmacokinetic and pharmacodynamic properties of some phencyclidine analogs in rats.

Oxidation of carbon monoxide by perferrylmyoglobin.

adenylate cyclase system by glucose and some of its analogues.

Oxidation of one-carbon compounds to formate and carbon dioxide in rat liver mitochondria.

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Stereoselective inhibition by D- and L-alanine of phencyclidine-induced locomotor stimulation in the rat.

Hypothalamic polypeptides and analogues on corticotrophin production by rat adenohypophysial tissue in vitro [proceedings].

Chemistry and toxicology of pesticide chemicals V. Some pharmacological aspects of leptophos exposure in the rat.

Carbon monoxide oxidation by methanogenic bacteria.

Opioid properties of some isomeric derivatives of phencyclidine.