Metaphit, a specific acylating agent for the [.sup.3 H] phencyclidine

Abstract

A derivative of phencyclidine (1) bearing an isothiocyanate moiety of the meta position of the aromatic ring (2; Metaphit) has been synthesized and identified as a rapid and specific site-directed acylating agent of the [.sup.3 H]-phencyclidine binding site in rat brain homogenates.

Description

##STR1## Additionally, the compound where X=NH.sub.2 is a known derivative of phencyclidine.

DESCRIPTION OF THE FIGURES

FIG. 1 shows examples of Scatchard plots of 3H-PCP binding in a crude membrane preparation from rats pretreated with saline (.cndot.), 1 umol/rat ( 0 ) or 2 umol/rat (.quadrature.) of Metaphit. A crude membrane preparation in 5 mM Tris-HCl, pH 7.4 (Tris buffer) was prepared from the brains of rats pretreated with saline or Metaphit administered i.c.v. 24 hours before sacrifice. Incubation tubes, prepared in triplicate, contained a varying amount of 3H-PCP (New England Nuclear) made up in Tris buffer, 100 ul of membrane preparation and enough Tris buffer to make a total volume of 500 ul. The tubes were incubated for 50 minutes on ice and filtered through Whatman GF/B filters, which were presoaked for at least 2 hours in 0.05% polyethylenimine. The test tubes were washed twice and the filters once more with 4 ml of ice-cold Tris buffer. The filters were placed into scintillation vials containing 10 ml of Aquassure and radioactivity counted by liquid scintillation spectrometry. Protein concentrations were determined by the method of Lowry et al, J. Biol. Chem., 193:254 (1951).

FIG. 2 shows PCP dose-response curves for (a) sterotypy and (b) ataxia determined in control rats (.cndot.), or 1 hour (.DELTA.) or 24 hours ( 0 ) after i.c.v administration of 1 umol/rat of Metaphit.

FIG. 3 shows PCP dose-response curves for (a) sterotypy and (b) ataxia determined in control rats (.cndot.) or 24 hours after i.c.v. administration of 1 umol/rat ( 0 ) or 2 umol/rat (.quadrature.) of Metaphit.

EXAMPLE 1

Even though Metaphit has been shown to acylate PCP receptors when added to brain homogenates in vitro, it is still necessary to determine whether Metaphit acylates PCP receptors when administered in vivo. This question was answered by kinetic analysis of 3H-PCP binding in saline and Metaphit-pretreated rats. Male Sprague-Dawley rats (Taconic Farms, Germantown, NY) weighing 200-250 g were used in all experiments. All rats were anesthetized lightly with ether before a 20-gauge needle was used to make a hole in the skull of rats for intracerebroventricular (i.c.v.) injection of drugs. The animals were allowed at least 1 day to recover before being used. The 3H-PCP binding assay was adapted from the method of Quirion et al, Peptides, in press (see description of FIG. 1). Specific binding was defined as total radioligand bound minus the amount of radioligand bound in the presence of 30 uM PCP. Kd and Bmax values were determined by least squares regression. The results are expressed as the mean .+-.S.E. of at least three experiments.

An example of Scatchard plots of 3H-PCP binding in membrane preparations from rats treated with saline or Metaphit 24 hours prior to sacrifice is shown in FIG. 1. There was no significant difference between the Kd values for 3H-PCP determined in rats pretreated with saline (Kd=237.+-.8 nM), 1 umol/rat (Kd=237.+-.8 nM) or 2 umol/rat of Metaphit (Kd=219.+-.24 nM). However, there was a 25% and 40% decrease in the Bmax of 3H-PCP determined in rats pretreated with 1 umol/rat (Bmax=1550.+-.70 pmol/mg) and 2 umol/rat (Bmax=1230.+-.50 pmol/mg) of Metaphit, respectively, as compared to control (Bmax=2030.+-.110 pmol/mg). These results indicate that Metaphit binds irreversibly to PCP receptors as administration of the drug changed only the number of binding sites without altering the affinity of binding in a dose-dependent manner and this effect was evident 24 hours after Metaphit pretreatment.

EXAMPLE 2

For the behavioral experiments concerning whether acylation of PCP receptors by Metaphit was associated with any changes in PCP's behavioral effects, rats were placed individually into 27.times.44.times.18 cm high plastic rat cages and allowed at least 1 hour to acclimate before testing began. PCP or Metaphit was administered by i.c.v. injection in 5 .mu.l of saline. Stereotypy and ataxia were measured at 5 min intervals using a PCP rating scale. A rating of 5 was considered a 100% response. Dose-response curves of PCP represent behavioral ratings determined 5 min after PCP administration, the time of peak effect. At least 21 rats were used to determine each dose-response curve and ED50. ED50 values and dose-response curves were evaluated using the Finney assay (Statistical Methods in Biological Assay, 2nd ed., Hafner Publishing Co., N.Y, 1964) with the aid of a computer.

Metaphit at a dose of 1 umol/rat did not produce any significant behavioral effects, but a dose of 2-8 umol/rat produced what appeared to be a calming effect as the rats generally remained stationary in a resting position in one spot, yet were not ataxic or catatonic and were generally alert. At these higher doses, a few rats exhibited qualitative changes similar to those exhibited by PCP treated rats, which were not quantitatively significant. The ability of PCP to induce stereotypy and ataxia was then evaluated 1 hour after 1 umol/rat of Metaphit. As illustrated in FIG. 2, PCP dose-response curves for stereotypy and ataxia were shifted significantly 3- and 2-fold, respectively, to the right of the control dose-response curves. This antagonism of PCP by 1 umol/rat of Metaphit was still evident 24 hours after Metaphit pretreatment, as the PCP dose-response curves for stereotypy and ataxia determined 24 hours after Metaphit pretreatment were not significantly different from those determined 1 hour after Metaphit pretreatment. The antagonism of PCP-induced stereotypy and ataxia was also dose-dependent (FIG. 3). Twenty-four hours after administration of 2 umol/rat of Metaphit, the PCP dose-response curves for stereotypy and ataxia were shifted 7- and 5-fold, respectively, to the right as compared to control.

These results show that acylation of PCP receptors by Metaphit results in antagonism of PCP-induced stereotypy and ataxia because doses of Metaphit that produced a significant decrease in the number of PCP receptors also antagonized the behavioral effects of PCP. In addition, these findings demonstrate that acylation of PCP receptors by Metaphit can be used to determine whether the effects of PCP are mediated by PCP receptors. The finding that Metaphit antagonized PCP-induced stereotypy is consistent with the reported correlation between PCP receptor binding and induction of stereotypy by PCP analogs. However, there are conflicting reports on the correlation between PCP receptor binding and induction of ataxia. The ability of Metaphit to antagonize PCP-induced ataxia shows that PCP receptors are involved in mediating PCP-induced ataxia but does not exclude the possibility that other mechanisms of action may be involved.

Dosage

The dosage of Metaphit or its related compounds noted in this application in relation to phencyclidine or ketamine is generally in the ratio of 1:2 to 2:1 effective weight percent with an optimum value of about 1:1 and the mode is by injection preferably i.p.

Claims

1. Metaphit, 1-(1-(3-isothiocyanatophenyl)-cyclohexyl)piperidine.