Effects of anti-phencyclidine and anti-(+)-methamphetamine monoclonal antibodies alone and in combination on the discrimination of phencyclidine and (+)-methamphetamine by pigeons.

RATIONALE: Drug-specific monoclonal antibodies against phencyclidine (PCP) and (+)-methamphetamine [(+)-METH] should bind to these drugs to block their discriminative stimulus effects. OBJECTIVES: To determine if mouse monoclonal antibodies against PCP and (+)-METH can block the discriminative stimulus effects of the drugs in pigeons. MATERIALS AND METHODS: Pigeons were trained to discriminate among intramuscular injections of saline, 1 mg/kg PCP, and 2 mg/kg (+)-METH. After responding stabilized, cumulative dose-response curves were obtained for PCP and (+)-METH. Doses of an anti-PCP antibody at 620 mg/kg (anti-PCP mAb6B5) with a K (D) of 1.3 nM for PCP and no measurable affinity for (+)-METH and 1,000 mg/kg doses of anti-(+)-METH antibody (anti-METH mAb6H7) with a K (D) of 41 nM for (+)-METH and no measurable affinity for PCP were subsequently administered, first alone and later in combination after which the dose-response curves were redetermined. RESULTS: When the antibodies were given alone, the anti-PCP antibody blocked the discriminative stimulus effects of PCP, but not those of (+)-METH, and the anti-(+)-METH antibody blocked the discriminative stimulus effects of (+)-METH, but not those of PCP. The anti-PCP antibody shifted the PCP dose-response curve further to the right and for a longer time than the anti-(+)-METH antibody shifted the dose response curve for (+)-METH. When the anti-PCP and anti-(+)-METH antibodies were administered on the same day, the discriminative stimulus effects of both drugs were completely blocked 1 day after antibody administration. CONCLUSIONS: These experiments demonstrate the high specificity of the antibodies for the drugs to which they bind and show that monoclonal antibodies can be combined to antagonize the effects of more than one drug.

Effects of gonadal steroids on clearance of growth hormone at steady state in the rat.

Gonadal steroids have been implicated in the modulation of GH secretory patterns in the rat. We have studied the effects of testosterone (T) or estradiol (E2) on the steady state clearance (Clss) and plasma half-life (t1/2) of GH in male and female rats (n = 4-6/group). A femoral and a jugular cannula were surgically implanted into adult Sprague-Dawley rats. At the time of cannulation some rats were orchidectomized, and a Silastic capsule containing E2, T, or nothing was implanted sc. After recovery from surgery, either purified rat GH or a crude extract of rat pituitary was infused iv to attain steady state plasma GH concentrations. Blood samples were taken every 30 min for 4 h during the infusion, and nine samples were taken at 2.5-min intervals immediately after stopping the infusion. The mean Clss for GH in female rats were significantly (P less than or equal to 0.001) less than that in males, whereas the t1/2 did not differ between the two groups. Neither the Clss nor the t1/2 was affected by castration in males or females. The Clss of GH in E2-treated castrated males was significantly less (P less than 0.001) than that for intact males, but the t1/2 did not differ between the two groups. The Clss for GH was greater in T-treated ovariectomized rats than in intact females, but the t1/2 did not differ with T treatment. These results suggest that 1) the Clss for GH is lower in female rats than in males; 2) 4 weeks of gonadectomy has no effect on the Clss in males or females; 3) under experimental conditions, E2 decreases and T increases the Clss for GH; and 4) the t1/2 for GH is not different in males or females. The steroid-induced changes in Clss in the absence of detectable effects on t1/2 suggest that factors affecting the volume of distribution at steady state (i.e. plasma GH-binding proteins or GH heterogeneity) are involved in the effects of gonadal steroids on GH clearance in the rat.

Differential and region-specific activation of mitogen-activated protein kinases following chronic administration of phencyclidine in rat brain.

We have previously demonstrated elevation of the extracellular signal-regulated kinase (ERK) pathway in the cerebellum from patients with schizophrenia, an illness that may involve dysfunction of the N-methyl-D-aspartate (NMDA) receptor. Since the NMDA antagonist, phencyclidine (PCP), produces schizophrenic-like symptoms in humans, and abnormal behavior in animals, we examined the effects of chronic PCP administration in time- and dose-dependent manner on ERK and two other members of mitogen-activated protein kinase family, c-Jun N-terminal protein kinase (JNK) and p38, in rat brain. Osmotic pumps for PCP (18 mg/kg/day) and saline (controls) were implanted subcutaneously in rats for three, 10, and 20 days. Using Western blot analysis, we found no change at three days, but a significant increase in the phosphorylation of ERK1, ERK2 and MEK in the cerebellum at 10- and 20-days of continuous PCP infusion. For the experiments involving various doses of PCP, rats were infused with PCP at concentrations of 2.5, 10, 18, or 25 mg/kg/day, or saline for 10 days. We observed a dose-dependent elevation in the phosphorylation of ERK1 and ERK2 only in the cerebellum but not in brainstem, frontal cortex or hippocampus. The activities of JNK and p38 were unchanged in all investigated brain regions including cerebellum. These results demonstrate that chronic infusion of PCP in rats produces a differential and brain region-specific activation of MAP kinases, suggesting a role for the ERK signaling pathway in PCP abuse and perhaps in schizophrenia.

Relationship between plasma delta-9-tetrahydrocannabinol concentration and pharmacologic effects in man.

The relationship between each of two pharmacologic effects (tachycardia and psychological "high") of delta-9-tetrahydrocannabinol (THC) and plasma THC concentration was investigated in three male and three female experienced marihuana smokers. Each subject smoked 1% THC cigarette on two occasions separated by 2 h. Heart rate and subjective psychological self-rating were determined frequently throughout the 4 h study period. Data were analyzed by calculating the area under the parameter versus time curves, constructing hysteresis plots, and calculating the decay rate constants from pharmacologic effect versus time plots. In both males and females, dose inhaled and psychological response were apparently equivalent for the first and second cigarettes. While all subjects exhibited marked tachycardia in response to the first cigarette, heart rate in both male and female subjects was not increased as markedly during the second cigarette. Interestingly, female subjects had less tachycardiac response than males during the second cigarette. Hysteresis plots revealed that both heart rate and subjective psychological effect were elicited in an effect compartment which was "deep" relative to the reference plasma compartment. The time courses of tachycardiac and psychological responses lagged behind the plasma THC concentration-time profile. Zero-order decay rate constants for subjective psychological rating did not change substantially from the first to second cigarette. This study suggest that plasma THC concentration is a poor predictor of simultaneously occurring physiological and psychological pharmacologic effects.

Cytopathology and the management of early invasive cancer of the uterine cervix.

This study was designed to review the effectiveness of cytopathology as it entered into the evaluation of patients with possible microinvasive or early occult carcinoma of the uterine cervix. During the 7-year period of 1971 to 1977, 39 consecutive patients were found for whom either a cytopathologic diagnosis of early invasive carcinoma had been made or suggested, or a histopathologic diagnosis of early invasive carcinoma had been made. After review, 35 patients had an ample number of cytopathologic and histopathologic materials and clinical records to be included in the study. The results of these studies have shown that when cytopathology on review predicted a lesion more severe than carcinoma in situ, it was confirmed by histopathology in more than 78% of patients (22 of 28 cases). In those patients shown by histopathology to have microinvasive or occult invasive carcinoma, the cytopathology reflected it in 87% of patients (27 of 31 cases). In the cases of histologically proved microinvasive carcinoma, the corresponding genital smears either diagnosed or suggested invasive carcinoma in 81% of cases and carcinoma in situ in 19%. From these studies it has been concluded that diagnostic cytopathology is potentially a highly reliable tool when used in conjunction with other modern diagnostic modalities to aid the decision-making in cases of probable early cancer of the uterine cervix.

The clinical pharmacology and dynamics of marihuana cigarette smoking.

We have studied the dynamics of marihuana smoking, the plasma concentration of delta 9-tetrahydrocannabinol (THC), and the pharmacologic effects produced by the sequential smoking of two 1% marihuana cigarettes at a 2-hour interval. Three males and three females, experienced marihuana smokers, participated in the study. The results indicate that each subject smoked his or her two cigarettes at a similar rate. The THC plasma concentrations produced by the smoking of the second cigarette were slightly lower than those produced by the first cigarette. The levels of the psychologic "high" caused by the two cigarettes were similar. However, the first cigarette accelerated the heart twice as much as the second cigarette. Between males and females there were marked differences in the rate at which the cigarettes were smoked. In particular, males took more puffs, took them more often, and consumed the cigarettes more rapidly than females. The plasma concentrations of THC, the self-reported psychologic effects, and the heart rate acceleration produced by the smoking of the two cigarettes were identical between the sexes.

Brain microsomal metabolism of phencyclidine in male and female rats.

These studies examined the microsomal brain metabolism of phencyclidine (PCP) in male and female Sprague-Dawley rats. Several monohydroxylated metabolites of PCP were detected including cis- and trans-1-(1-phenyl-4-hydroxycyclohexyl)piperidine (c-PPC and t-PPC) and 1-(1-phenylcyclohexyl)-4-hydroxypiperidine (PCHP). The in vitro formation of these metabolites required NADPH and was inhibited by carbon monoxide. c-PPC was formed in the male and female brain microsomes at rates of 7.1 +/- 1.3 and 5.7 +/- 1.1 fmol/min per mg, respectively, while t-PPC was formed at rates of 16.2 +/- 3.3 and 16.5 +/- 4.2 fmol/min per mg. PCHP had the highest formation rate at 50.7 +/- 8.9 and 48.2 +/- 8.8 fmol/min per mg, respectively. Although previous studies with rat liver microsomes find higher levels of PCP metabolism in male rats and the formation of an irreversibly bound metabolite in male rats, the present study of brain metabolism found no sex differences in brain metabolism. The formation of PCP metabolites in male rat livers is at least partially mediated by the male-specific isozyme CYP2C11, and possibly CYP2D1. Nevertheless, the formation of the major brain metabolite, PCHP, was not inhibited by an anti-CYP2C11 or an anti-CYP2D6 antibody. However, PCHP formation was inhibited by drug inhibitors of CYP2D1-mediated metabolism, suggesting the involvement of a CYP2D isoform. These data indicate brain metabolism of PCP is significant, but unlike the liver it is not sexually dimorphic.

Intravenous (+)-methamphetamine causes complex dose-dependent physiologic changes in awake rats.

Hemodynamic and temperature dose-response relationships were characterized in freely moving rats following i.v. (+)-methamphetamine administration to mimic the rapid onset of effects experienced by many human users. Rats received saline and (+)-methamphetamine in a repeated-measures, mixed-sequence design at 22+/-1 degrees C. Significantly greater blood pressure and heart rate elevations were observed after 1.0 and 3.0 mg/kg (+)-methamphetamine vs. 0.1 and 0.3 mg/kg. The time to peak hemodynamic values and the duration of effects were significantly greater after 3.0 mg/kg vs. the lower doses. The time to peak temperatures was significantly longer after 1.0 mg/kg vs. the lower doses. Following 3.0 mg/kg, all rats experienced temperature decreases before having elevated temperatures. The duration and magnitude of the delayed temperature elevations were significantly greater after 3.0 mg/kg vs. the lower doses. In conclusion, the (+)-methamphetamine-induced hemodynamic and temperature effects were not temporally synchronized, and the complex responses were not linearly related to dose.

Generation of anti-(+)methamphetamine antibodies is not impeded by (+)methamphetamine administration during active immunization of rats.

The goal of these studies was to determine if chronic (+)methamphetamine ((+)METH) administration affects the production of anti-(+)METH antibodies during active immunization of rats. Active immunization for the treatment of chronic drug abuse has been proposed for drugs such as cocaine and nicotine. However, studies have not adequately addressed whether continual drug use during treatment would affect the development of an immune response. For the current studies, male Sprague-Dawley rats were immunized with either keyhole limpet hemocyanin (KLH; control group) or a (+)METH hapten ((+)METH with a six carbon spacer group at the para position of the ring structure)-KLH conjugate. The (+)METH-KLH animals were further divided into two groups. One group was immunized with no subsequent administration of (+)METH, while the other group was immunized and repeatedly challenged (twice a week throughout the study) with an i.p. dose of 3 mg/kg (+)METH. The results showed that the two groups of (+)METH-KLH immunized rats developed and maintained anti-(+)METH antibody titers. The anti-(+)METH immune responses of the two groups were not statistically different (P < 0.05) as measured by serum titers and the relative antibody affinities. These data suggest that repeated administration of (+)METH does not affect the generation of an anti-(+)METH antibody response in actively immunized rats.

Development of a liquid chromatography-tandem mass spectrometric method for the determination of methamphetamine and amphetamine using small volumes of rat serum.

The aim of this paper was to develop LC/MS/MS methodology for the determination of methamphetamine (METH) and amphetamine (AMP) using low microliter volumes (20-150 microl) of rat serum and demonstrate the use of this method for the study of serum pharmacokinetics in the rat. The analytes were extracted from rat serum using solid-phase extraction followed by an isocratic separation on a narrow-bore Hypersil C(18) column. Lower limits of quantitation for METH and AMP were 0.3 ng/ml using positive ion electrospray tandem mass spectrometry. The accuracy of the method was within 20% of the actual values over a wide range of serum concentrations. The within-day and between-day precision was better than 20% (R.S.D.). Ion-suppression matrix effects on electrospray ionization were evaluated for extracted rat serum. The LC/MS/MS method was further validated by comparing serum concentrations of METH and AMP to serum concentrations previously determined using an LC/[ (3)H]-METH assay with radiochemical detection. Finally, the LC/MS/MS method was used to study the pharmacokinetics of METH and AMP after a 1mg/kg intravenous bolus dose of METH to female Sprague-Dawley rats.

Phencyclidine pharmacokinetics and concentration-response relationships in the pigeon.

Phencyclidine (PCP) pharmacokinetics and drug discrimination were examined in pigeons (n = 6 in both groups) after intramuscular doses of 1.48 mg/kg. PCP absorption was rapid with maximum measured plasma concentrations ranging from 559 to 1450 ng/ml at 10-30 min after dosing, which corresponded to the time of maximum PCP stimulus effects in the drug discrimination studies. The terminal elimination half-life was 0.88 hr (harmonic mean). Average values for the volume of distribution and total body clearance were 1.6 l/kg and 18.2 ml/min/kg, respectively. In the behavioral studies, pigeons discriminated PCP-like effects from about 2 min to 2 hr after dosing. An average value for response on the PCP-appropriate key and for PCP concentration at each time point from 2 min to 2 hr was calculated from the individual subject data. Least-squares linear regression analysis of these data showed a highly significant relationship between the ability to discriminate PCP and log PCP concentration (y = 103x - 219, r2 = .810, p less than 0.005). This analysis suggests PCP concentration is a good predictor of behavioral efficacy.

Quantitative analysis of phencyclidine and metabolites by capillary column gas chromatography.

A sensitive capillary gas chromatography (GC) procedure was developed for the analysis of phencyclidine (PCP), two of its monohydroxy-metabolites, and a recently identified pentanoic acid metabolite. Two separate, but sequential, integrated extraction techniques were necessary to isolate all of the compounds from a 1.0-mL biological sample. Two GC techniques were necessary to analyze all the compounds; both methods used a capillary column and a nitrogen phosphorus detector (NPD). The first procedure permitted isolation and quantitation of PCP and two monohydroxy-metabolites. A second extraction of the biological samples permitted measurement of the pentanoic acid metabolite. The analytical methods illustrate good reproducibility based on within-day and day-to-day variation. Serum and urine samples were analyzed from a dog administered PCP to illustrate the utility of this procedure.

Chronic anti-phencyclidine monoclonal antibody therapy decreases phencyclidine-induced in utero fetal mortality in pregnant rats.

Illicit drug use during pregnancy is a serious social and public health problem inflicting an array of deleterious effects on both mother and offspring. We investigated the hypothesis that a murine anti-phencyclidine (PCP) monoclonal antibody (mAb6B5; K(D)=1.3 nM) can safely protect mother and fetus from PCP-induced adverse health effects in pregnant rats. Pregnant Sprague-Dawley rats (n=4-5) were intravenously administered bolus injections of PCP (1mg/kg) on multiple days during pregnancy. They were also chronically treated with anti-PCP mAb6B5 at 45 mg/kg as a PCP antagonist. This dose provided one mAb-PCP binding site for every four PCP molecules. Therapeutic and safety study endpoints included pregnancy outcome (litter size, number of live vs. dead pups), maternal hemodynamic status and locomotor activity. Maternal hemodynamic changes (i.e., blood pressure and heart rate) and locomotor activity were measured in dams from gestation days 6-21 (one day antepartum) using a radiotelemetry-tracking device with a femoral arterial pressure catheter. This mAb6B5 treatment regimen significantly (p=0.008) reduced the number of PCP-induced in utero fetal deaths (odds ratio=3.2; 95%CI 1.3 to 7.9) and significantly (p<0.05) reduced acute PCP-induced maternal locomotor effects in the second trimester. Maternal hemodynamic responses to PCP were not significantly affected by mAb6B5 treatment. In conclusion, these data suggest that anti-PCP mAb treatments administered during pregnancy can safely protect a mother and her fetus(es) from PCP-related morbidity and mortality even when the mAb dose is too low to significantly prevent other PCP-induced maternal pharmacological effects.

Anti-(+)-methamphetamine monoclonal antibody antagonists designed to prevent the progression of human diseases of addiction.

Anti-(+)-methamphetamine monoclonal antibodies (mAbs) have the potential to reduce the devastating behavioral and societal effects of the worldwide epidemic of (+)-methamphetamine (METH) addiction and transform the treatment paradigm for diseases of addiction. These novel, protein-based medications could play a vital role in helping patients to achieve sustainable abstinence from METH abuse by serving as an in vivo, around-the-clock sentry against a patient's vulnerability to relapse.

Phencyclidine dependence: the relationship of dose and serum concentrations to operant behavioral effects.

The dependence-producing properties of 10 days of chronic i.v. infusions of phencyclidine (PCP) and the relationship between PCP serum concentrations and behavioral effects were studied in Sprague-Dawley rats. For dependence studies, rats were trained to respond for food under a fixed-ratio 30 schedule during half-hour response periods every 6 hr. After training, implantation of jugular catheters, and restabilization of behavior, the rats were infused with PCP.HCl at 3.2, 5.6, 10.0 or 17.8 mg/kg/day (n = 5 or 6 per dose). The two higher doses initially decreased response rates, but tolerance developed within 4 to 5 days. When PCP infusions were terminated, dose-dependent decreases in session response rate occurred in the three highest dose groups (P less than .05). Mild, overt signs of abstinence were observed only in the highest dose group. Response rates returned to base line within 2 to 3 days after stopping PCP infusions. PCP serum concentrations in rats infused with 10 mg of PCP.HCl/kg/day for 10 days were stable from hour 24 to day 10 (mean steady-state concentration (+/- S.D.) = 97 (+/- 20) ng of PCP/ml; n = 4). The average terminal elimination half-life after stopping infusions on day 10 was 4.6 hr. Comparison of the average response rates with the average serum concentrations showed that during the first 24 hr of infusions, the rate of responding for food decreased as PCP concentrations increased; however, once the animals became tolerant to PCP there was no relationship. In contrast, during the first 24 hr after stopping infusions, response rates decreased as serum concentrations decreased.

Metabolism of phencyclidine by human liver microsomes.

These studies examined in vitro metabolism of phencyclidine (PCP) in a series of human liver microsomes (N = 10). Each sample was characterized for cytochrome P450 (CYP) content and for CYP1A, CYP2A, CYP2C, CYP2D, CYP2E, CYP3A, CYP4A, and lauric acid 11-hydroxylation metabolic activities. At least five PCP metabolites (c-PPC, t-PPC, PCHP, an unknown metabolite, and an irreversibly bound metabolite) were formed by the various human liver microsomes. Nevertheless, there was a large degree of inter-individual variation in the metabolite formation. For example, the irreversibly bound metabolite was formed in detectable amounts in only four of the ten samples. c-PPC, t-PPC and the irreversibly bound PCP metabolite formation rates significantly correlated with CYP3A activity. The CYP3A inhibitor troleandomycin was used to inhibit the formation of PCP metabolites. Troleandomycin inhibition was dose dependent with the highest dose producing complete inhibition of the formation of c-PPC, t-PPC, PCHP, and the irreversibly bound metabolite. In addition, PCP inhibited CYP3A-mediated testosterone 6 beta-hydroxylation by 50%. Furthermore, the relative intensity of CYP3A immunoreactive proteins significantly correlated with testosterone 6 beta-hydroxylation and with PCP metabolite formation (except for the unknown metabolite). PCHP formation also correlated with CYP1A activity, while the formation of the unknown PCP metabolite correlated with CYP2A activity. These studies suggest that several CYP isoforms contribute to PCP metabolism and that CYP3A plays a major role in PCP biotransformation in human liver microsomes.

Antiphencyclidine monoclonal antibody therapy significantly changes phencyclidine concentrations in brain and other tissues in rats.

These studies determined how high-affinity monoclonal antiphencyclidine (PCP) antigen binding fragments of immunoglobulin G (Fab) affects PCP tissue concentrations and serum protein binding in male rats. Animals received an i.v. bolus dose of 1.0 mg/kg of PCP, followed at 2 hr when distribution was complete (but about 70% of the dose remained) by either saline (for controls) or an equimolar dose of anti-PCP Fab. This dose of PCP was chosen because it produces behavioral effects and ataxia for about 40 min. The rats were sacrificed over the next 16 hr (n = 3 per time point) and blood, brain, fat, heart, kidney, liver, lung, muscle and testis were collected. After anti-PCP Fab treatment, serum PCP concentrations increased significantly (P < .05) for the duration of the experiment. This resulted in a decrease in the PCP volume of distribution and systemic clearance to 11 and 12% of controls, respectively. Because these parameters decreased to a similar degree, the terminal elimination half-life was unaltered after Fab treatment. The percentage of unbound PCP in serum averaged 47 +/- 15% (mean +/- S.D.) in controls and 3 +/- 2% in Fab-treated animals for the duration of sampling. The area under the tissue concentration vs. time curves after anti-PCP Fab administration were decreased substantially in the brain (23% of controls), fat (24%), heart (52%), lung (74%) and testis (12%), but increased in the liver (137%). Because of anti-PCP Fab renal elimination, kidney PCP concentrations were significantly increased at all time points after Fab treatment (P < .05), which resulted in an 18-fold increase in the PCP area under the curve. These studies show anti-PCP Fab can rapidly remove PCP from the brain and maintain it in a highly bound form for a significant time.