Effect of orally administered cisapride, bethanechol, and erythromycin on the apparent efficiency of colostral IgG absorption in neonatal Holstein-Friesian calves.

OBJECTIVE: To evaluate the effect of orally administered cisapride, bethanechol, and erythromycin on the absorption of colostral IgG in dairy calves. ANIMALS: Twenty-four healthy neonatal Holstein-Friesian calves. PROCEDURES: Calves were randomly assigned to one of the following treatments: 0.9% NaCl solution (2 mL, p.o.; negative control); erythromycin lactobionate (20 mg/kg BW, p.o.; anticipated to be a positive control); cisapride (0.5 mg/kg BW, p.o.); bethanechol chloride (0.5 mg/kg BW, p.o.). Calves were fed 3 L of pooled bovine colostrum containing acetaminophen (50 mg/kg) by suckling and oroesophageal intubation 30 minutes after each treatment was administered. Jugular venous blood samples were obtained periodically after the start of feeding and plasma total IgG, protein, acetaminophen, and glucose concentrations determined. Abomasal emptying rate was assessed by the time to maximal plasma acetaminophen concentration. RESULTS: Oral administration of cisapride facilitated the absorption of colostral IgG and protein. The effect of cisapride on abomasal emptying rate could not be evaluated because cisapride appeared to interfere with acetaminophen metabolism. Based on the total IgG and total protein concentration-time relationships, the beneficial effects of cisapride appeared to occur early after oral administration and were transient. CONCLUSIONS AND CLINICAL IMPORTANCE: Additional studies appear indicated to characterize the effect of cisapride dose on the magnitude and duration of its effect on facilitating the absorption of colostral IgG and protein. Identification of a nonantimicrobial method for increasing abomasal emptying rate, such as cisapride, will potentially provide a practical and effective method for facilitating transfer of passive immunity in colostrum-fed dairy calves.

An LC-MS/MS method for the simultaneous determination of lycorine and galanthamine in rat plasma and its application to pharmacokinetic study of Lycoris radiata extract in rats.

A rapid, sensitive, and selective liquid chromatography-tandem mass spectrometry was developed for the simultaneous determination of lycorine and galanthamine, two major constituents in Lycoris radiata extract, in rat plasma. Liquid-liquid extraction with ethyl ether was carried out using diphenhydramine as the internal standard. The two bioactive alkaloids were separated on a Zorbax SB-C18 reserved-phase column (150 mm × 4.6 mm, i.d., 5 µm) by gradient elution using a mobile phase consisting of methanol with 0.1% formic acid (A) and water with 0.1% formic acid (B) at a flow rate of 0.6 mL/min. All analytes showed good linearity over a wide concentration range (r (2)>0.99) and the lower limit of quantification was 3.00 ng/mL for each analyte. The average extraction recovery of the analytes from rat plasma was more than 82.15%, and the intra-day and inter-day accuracy and precision of the assay were less than 12.6%. The validated method was successfully applied to monitoring the concentrations and pharmacokinetic studies of two Amaryllidaceous alkaloids in rat plasma after an oral administration of Lycoris radiata extract.

Identification of carboxylesterases expressed in rat intestine and effects of their hydrolyzing activity in predicting first-pass metabolism of ester prodrugs.

Carboxylesterases (CESs) located in the intestine play an unique role in the absorption of many drugs especially ester prodrugs. In order to determine the expression and hydrolyzing activity of CESs isozymes (CES1 and CES2) located in rat intestine, the activities of CES1 and CES2 were evaluated by the intestinal S9 incubation with imidapril and irinotecan (CPT-11), the substrates of CES1 and CES2, respectively. The distribution characteristics of CES1, CES2, Pregnane X Receptor (PXR) and Constitutive Androstane Receptor were analyzed by real-time polymerase chain reaction (RT-PCR) or Western blot. Imidaprilat metabolized from imidapril by CES1 was too low to be detected in rat intestinal S9 fractions, while there was little and even no expression of CES1 mRNA in intestinal segments. In contrast, Vmax values for CPT-11 diminished gradually from proximal to distal segments within the rat intestine which was consistent with the mRNA expression level of CES2. These results indicated that CES2 represents the major CESs isoform in the rat complete intestine and decreased from duodenum to colon, whereas the expression of CES1 was too low to influence the metabolism of ester prodrugs. The expression of PXR and CAR decreased slightly along the entire intestine on both mRNA and protein levels which indicated that PXR and CAR may be one of the major factors which contribute to the expression of CES1 and CES2. Thus, the knowledge about the characteristic and site-specific expression of CES1 and CES2 in rat intestine will help to predict the oral bioavailability of ester prodrugs.

Cultivation and characterization of a bovine in vitro model of the cornea.

The aim of this study was to develop an in vitro model of the cornea of bovine cells, to characterise the model by histochemical methods and to investigate permeation of ophthalmic drugs through the model. As in the in vivo situation, an in vitro model of the cornea should consist of all three different types of cells. In the current study, the construction of the in vitro cornea was performed using cells prepared from primary cultures. To investigate the state of the cells in the cultures, growth curves were established. Immunocytochemical determination of keratin and vimentin was performed for all three isolated and sub-cultivated cell types of the bovine cornea. To further simulate the in vivo conditions, corneal epithelial cells were seeded onto the collagen-gel base containing the stromal cells with an underlying sheet of endothelium. Permeation experiments were performed with pilocarpine hydrochloride and timolol hydrogen maleate as model drugs and excised bovine cornea and the in vitro cornea as permeation barriers. The immunohistochemical investigations show that excised bovine cornea and the in vitro model of the cornea are comparable with respect to the expression of keratin K3, indicating that the primarily isolated cells correspond to the different cell types of the cornea. Culturing of the epithelial cells on the complex basis has led to the formation of a corneal epithelium with several layers, closely resembling the morphology of the in vivo epithelium. Although the permeation rates of the drug through the in vitro cornea were always higher, the sequence in which the drugs permeate through the two types of barriers was the same. The drug permeation through the in vitro cornea may therefore be a useful predictive tool to estimate the permeability coefficients of drugs through excised cornea.

Influence of hydroxypropyl beta-cyclodextrin on the corneal permeation of pilocarpine.

The influence of hydroxypropyl beta-cyclodextrin (HPbetaCD) on the corneal permeation of pilocarpine nitrate was investigated by an in vitro permeability study using isolated rabbit cornea. Pupillary-response pattern to pilocarpine nitrate with and without HPbetaCD was examined in rabbit eye. Corneal permeation of pilocarpine nitrate was found to be four times higher after adding HPbetaCD into the formulation. The reduction of pupil diameter (miosis) by pilocarpine nitrate was significantly increased as a result of HPbetaCD addition into the simple aqueous solution of the active substance. The highest miotic response was obtained with the formulation prepared in a vehicle of Carbopol 940. It is suggested that ocular bioavailability of pilocarpine nitrate could be improved by the addition of HPbetaCD.

A signal transduction pharmacodynamic model of the kinetics of the parasympathomimetic activity of low-dose scopolamine and atropine in rats.

We used a novel pharmacokinetic-pharmacodynamic (PK-PD) approach that had been applied for signal transduction kinetics to investigate the kinetics of the parasympathomimetic effect of scopolamine and atropine in rats. The parasympathetic tone was assessed by continuous measurement of the power of the high frequency band (HF) of electrocardiogram (ECG) R-R intervals obtained by power spectral analysis (PSA) of heart rate variability (HRV). To overcome the inherent noise of the HRV-HF data and to quantitatively identify temporal changes in the autonomic tone, a new approach of stepwise regression of the cumulative HF data was applied. The elevation of the parasympathetic tone occurred after a significant lag time (>70 min) following scopolamine administrations [0.25 and 0.5 mg/kg intravenous (iv) bolus or infusion over 100 min], followed by a gradual return to the baseline levels. A similar lag time in parasympathetic stimulation was observed following iv bolus administration of atropine (0.1 mg/kg). The plasma drug concentration versus time data were linked to the response versus time data using a signal transduction pharmacodynamic model that was fitted simultaneously to all four experimental data sets. This PK-PD model resolved the significant discrepancy between the concentration versus time and the response versus time patterns and successfully described the kinetics of the parasympathetic stimulation obtained for different drugs and different rates of administration. This work paves the way for further PK-PD preclinical investigations in this field.

Degradation kinetics of neostigmine in solution.

The degradation kinetics of neostigmine were studied in aqueous solutions with varied pH from 1.5 to 9.9 under accelerated storage conditions. The stability of neostigmine in solutions containing propylene glycol or polyethylene glycol 400 was also investigated. The reaction order of neostigmine in these aqueous and solvent systems followed pseudo-first-order degradation kinetics. The degradation rates of neostigmine under various buffer concentrations within the investigated pH range were obtained. They indicated that the degradation was independent of the species of buffering agent. Maximum stability of neostigmine was determined at pH 5.0 buffer species conditions. The activation energy could be estimated from the Arrhenius plot as 15.72 kcal/mole. The half-life of 883.7 days was estimated at room temperature in 0.1 M, pH 4.9 acetate buffer solution (mu = 0.5). Ultraviolet (UV) irradiation at 254 nm of the neostigmine solutions in pH 4.9 acetate buffer showed an accelerated degradation in comparison with light-protected samples. Incorporation of propylene glycol into the neostigmine solution at pH 4.9 enhanced the stability; however, an adverse effect on the stability of neostigmine was noted when a polyethylene glycol 400 solvent system was used.

Bioavailability of timolol and aceclidine after ocular instillation in the rabbit.

The bioavailability of timolol and aceclidine after the ocular instillation of each drug (timolol 0.5% or aceclidine 2%) or both combined (timolol 0.5% + aceclidine 2%) has been evaluated in rabbits. 15 male albino rabbits were treated by the instillation of timolol and aceclidine alone or combined in the conjunctival sac of the right eye. Timolol concentrations in humor aqueous were assayed at 10 min, 30 min, 1 hr, 2 hr, 4 hr and 6 hr after instillation by high-performance liquid chromatography (HPLC). Aceclidine was assayed by a pharmacodynamic method: pupillary diameter at the following time intervals 0 (basal value), 1 min, 5 min, 30 min, 1 hr, 2 hr, 4 hr, 6 hr after treatment. Our results demonstrated that no differences in timolol and aceclidine bioavailability were found between simple-drug preparations and their combination.

Pharmacokinetics of cisapride in horses after intravenous and rectal administration.

OBJECTIVES: To determine the i.v. pharmacokinetics of cisapride and measure systemic absorption after rectal administration. ANIMALS: 5 healthy adult mares (380 to 610 kg). PROCEDURE: Cisapride was administered, i.v., at a dosage of 0.1 mg/kg of body weight. In the same horses, after a 1-week washout period, cisapride was administered rectally at a dosage of 1 mg/kg by mixing crushed tablets with propylene glycol and administering the mixture into the rectum. After each drug administration, a series of blood samples were collected. Plasma was obtained and analyzed by high-performance liquid chromatography to determine cisapride concentration profiles after each drug administration. RESULTS: After i.v. administration, peak plasma concentration was 221.4 ng/ml and harmonic mean half-life was 1.9 hours. Rectal absorption of cisapride was negligible. Cisapride was detected in plasma from only 3 of 5 horses for which mean systemic availability was 1.23%. Mean maximal plasma concentration after rectal administration of cisapride was 13.5 ng/ml. CONCLUSION AND CLINICAL RELEVANCE: After i.v. administration of cisapride, plasma concentration is high for approximately 2 hours. Cisapride mixed with propylene glycol and administered rectally at a dosage of 1 mg/kg is poorly and incompletely absorbed. Thus, cisapride is not clinically useful for rectal administration in horses.

Prediction of species differences (rats, dogs, humans) in the in vivo metabolic clearance of YM796 by the liver from in vitro data.

The bioavailability after oral administration of (S)-(-)-2,8-dimethyl-3-methylene-1-oxa-8-azaspiro [4,5] decane-L-tartarate monohydrate (YM796), which is being developed as an antidementia drug, at a dose of 1 mg/kg was very low (3.4%) in rats, but considerably higher (16.1%) in dogs. The oral clearances (CLoral, Dose/AUCoral) in rats and dogs were, respectively, 300 and 18 times more than that already reported in humans. We have previously reported successful attempts to predict the in vivo hepatic metabolic clearance of YM796 from in vitro data in humans. In our study, the in vitro metabolism of YM796 was determined using liver microsomes prepared from both rats and dogs and we also investigated if the species difference observed in vivo could be quantitatively reproduced in vitro. In rats, total metabolite formation could be described by single component kinetics with a Km of 13.4 microM and a Vmax of 520 nmol/min/g liver. However, in dogs, total metabolite formation could be described by three components, as also reported for humans. The Km and Vmax values for the high-affinity, low-capacity component (Km1 and Vmax1) in dogs and humans were, respectively, 8.1 and 1.7 microM, and 10.9 and 1.2 nmol/min/g liver. The overall intrinsic metabolic clearances estimated from the in vitro studies (CLint,in vitro) for rats and dogs were 38.8 and 2.6 ml/min/g liver, respectively, being approximately 40 and 3 times more than that previously reported for humans (0.94 ml/min/g liver). The overall intrinsic hepatic clearances (CLint,in vivo) calculated from in vivo CLoral were 30.4, 3.4 and 0.73 ml/min/g liver for rats, dogs and humans, respectively, indicating that the in vivo hepatic clearance of YM796 can be predicted from in vitro metabolism data in each species. Thus, the pronounced species difference in the metabolic clearance observed in vivo can be quantitatively predicted from in vitro metabolic data using liver microsomes, and was predominantly due to the large difference in the Vmax values.

Prediction of in vivo hepatic metabolic clearance of YM796 from in vitro data by use of human liver microsomes and recombinant P-450 isozymes.

The metabolic rate of (S)-(-)-2,8-dimethyl-3-methylene-1-oxa-8-azaspiro [4,5] decane-L-tartarate monohydrate (YM796), an antidementia agent, was determined by use of 12 different human liver microsomal samples. The metabolism of YM796 was shown to consist of three components; one high-affinity (Km1 = 1.67 microM), one low-affinity (Km2 = 654 microM) and a nonsaturable component. Good correlations were observed between the individual CYP3A4 content in 12 different human liver microsomal samples and kinetic parameters such as CL(int, all), the high-affinity component clearance (Vmax1/Km1) and the low-affinity component clearance (Vmax2/Km2). Anti-human CYP3A4/5 antibodies inhibited the metabolism of YM796 at 1 microM by up to 75%. In addition, ketoconazole, an inhibitor of CYP3A4, inhibited YM796 metabolism by >90%. The metabolic clearance of YM796 in each of the 12 human liver microsomal samples was successfully predicted from the kinetic parameters obtained with the recombinant microsomes by taking into consideration the CYP3A4 content in each microsomal sample. Based on the CL(int, all) estimated from the in vitro experiments, the area under the plasma concentration-time curve after oral administration (AUC(oral)) of YM796 was also predicted by taking into account the hepatic blood flow rate (Qh), the unbound fraction of YM796 in human plasma (f(p)) and the fraction absorbed from the gut. In addition, AUC(oral) was determined in six healthy male volunteers. The predicted AUC(oral) was similar to the observed value in vivo, which suggests that the in vitro metabolism data obtained with human liver microsomes are useful for quantitatively predicting human liver metabolism in vivo and that recombinant microsomes are also available when the particular isozyme is almost completely responsible for the metabolism of the drug, the variation in P-450 content of human liver is known and the experimental conditions such as the amount of CYP reductase and cytochrome b5 are carefully optimized to mimic the activity found in native microsomes, as for YM796.

Double-blind, randomized study of the effect of cisapride on gastric emptying in critically ill patients.

OBJECTIVES: To investigate the absorption of the gastrokinetic drug, cisapride, and effect of cisapride on gastric emptying in critically ill patients; and to assess the usefulness of clinical signs of gastric emptying. DESIGN: Prospective, randomized, controlled study. SETTING: Medical/surgical/trauma intensive care unit (ICU) in a university hospital. PATIENTS: Twenty-seven consecutively enrolled patients, aged 18 to 65 yrs, with normal hepatic and renal biochemistry who were not receiving enteral nutrition and who had no contraindications to enteral nutrition. These patients were expected to stay in the ICU for at least 4 days. INTERVENTIONS: Patients were randomized to receive either placebo or rectal cisapride, 60 mg initially followed by two doses of 30 mg at 8-hr intervals. MEASUREMENTS AND MAIN RESULTS: Gastric emptying was estimated, using acetaminophen absorption on day 1 of the study. Placebo or cisapride was administered and a second acetaminophen absorption test for gastric emptying was carried out on day 2,24 hrs after the first test. Four patients were excluded because of incomplete data. Statistical analysis was performed, using the area under the acetaminophen absorption curve from 0 to 60 mins as the primary measure of gastric emptying. There was no significant change in the area under the acetaminophen absorption curve from 0 to 60 mins from day 1 to day 2 in patients who received placebo or cisapride. Using the combination of the time to maximum acetaminophen concentration (< or = 30 mins) with a maximum concentration (> 12 mg/L) to define "normal" emptying, on day 1, four of the 11 placebo patients had the "normal" gastric emptying, and by day 2, five patients fulfilled this criterion. Before administration of cisapride, four of the 12 patients fulfilled this criterion, whereas nine fulfilled the criterion after receiving cisapride. There was a large variation in gastric emptying from day 1 to day 2; a power calculation suggests that approximately 150 patients would have to be studied to determine the effect of cisapride. There was no correlation between gastric emptying and the volume of gastric aspirate or the presence of bowel sounds. Plasma cisapride concentrations 4 hrs after the third dose, during the second acetaminophen absorption test, averaged 53 ng/mL (range 20 to 111). CONCLUSIONS: Rectal cisapride in the dose given achieved average plasma concentrations similar to those concentrations achieved in healthy subjects after 30 mg of cisapride rectally. There is a large variation in gastric emptying from one day to the next and large numbers of patients are required to determine if cisapride administration improves early gastric emptying in critically ill patients. The volume of gastric aspirate and the presence of bowel sounds do not correlate with gastric emptying.

Enteral feeding associated gastroesophageal reflux and aspiration pneumonia: a review.

Hospital malnutrition continues to be a serious problem. Although enteral feeding of hospitalized patients is safe and less expensive than parenteral feeding, it is associated with side effects involving the gastrointestinal tract and respiratory systems.

Indirect parasympathomimetic activity of metoclopramide: reversible inhibition of cholinesterases from human central nervous system and blood.

Inhibitory effects of the dopamine D2-receptor antagonistic benzamide compound metoclopramide (MCP) on acetylcholinesterase (AChE; EC 3.1.1.7) isoenzymes of both erythrocytes and human caudate nucleus and on human serum cholinesterase (ChE; EC 3.1.1.8) were studied in vitro using a spectrophotometric assay with acetylthiocholine (ASCh) as substrate. MCP concentrations in the assays varied from 0.30 microM to 0.15 mM. All isoenzymes studied were inhibited by metoclopramide in a concentration-dependent manner. MCP inhibition of AChE and ChE isoenzymes was not time-dependent and of the reversible type. Double reciprocal plots of the reaction velocity against varying ASCh concentrations revealed that, for AChE isoenzymes of erythrocytes and of the caudate nucleus, MCP reduced both maximal reaction velocity (Vmax) and substrate affinity (apparent Michaelis constant, KM, increased). Thus, MCP inhibition of both AChE isoenzymes was of mixed competitive/non-competitive type. MCP constants for reversible competitive (Ki) and non-competitive (Ki) inhibition could be determined for erythrocyte AChE (Ki = 10 microM; Ki = 70 microM) and caudate nucleus AChE (Ki = 9.3 microM; Ki = 82 microM). In contrast to MCP inhibition of AChE isoenzymes, the type of reversible MCP inhibition of human serum ChE depended on substrate concentration. If substrate concentration exceeded 0.2 mM, MCP inhibition was of mixed competitive/non-competitive type (Ki = 0.19 microM; Ki = 1.4 microM). MCP inhibition was of uncompetitive type, if substrate concentration was below 0.2 mM (Ki(u) = 1.0 microM). The mixed-type MCP inhibition of cholinesterase isoenzymes, because of its non-competitive component, can only partially be overcome by increased concentrations of the cholinergic transmitter acetylcholine (ACh). Since, with intravenous infusions, peak MCP plasma concentrations in humans reach 4 microM, MCP inhibition of ACh hydrolysis in vivo may contribute both to prokinetic and anti-emetic actions of the substance and to its extrapyramidal side effects.

Determination of xanomeline in human plasma by ion-spray tandem mass spectrometry.

Xanomeline is a muscarinic receptor agonist currently in phase II clinical trials for the treatment of Alzheimer's disease. A fast, sensitive and specific assay has been developed to determine xanomeline plasma concentrations using ion-spray tandem mass spectrometry. Xanomeline and a structural analog, LY282122, were extracted from basifed plasma into hexane. The dried hexane extracts were reconstituted and injected onto a 10 x 1 mm C18 reversed-phase column. A mobile phase of 33 mM ammonium acetate and 0.33% acetic acid in 30/70 (v/v) water-acetonitrile was pumped through the column at 50 microliters min-1. The mobile phase eluant was introduced directly into the ion-spray interface. The mass spectrometer was operated in the positive ion mode for specific detection of the product ions of xanomeline and the internal standard. The method has a linear range of 0.075-5.0 ng xanomeline per milliliter of plasma. Sample run times were 2.5 min from one injection to the next.

Cisapride: a gastrointestinal prokinetic drug.

OBJECTIVE: To summarize the pharmacology, pharmacokinetics, efficacy, and safety of cisapride, and to evaluate its potential therapeutic role. DATA SOURCES: A computerized search of the MEDLINE database was used to identify English-language publications of cisapride data in humans. The MEDLINE search was supplemented by review article bibliographies. There was no attempt to limit the search to a specific gastrointestinal motility disorder. STUDY SELECTION: The MEDLINE search alone identified 165 citations. Because of the volume of available human cisapride data, the focus of the efficacy section is on complete published reports of controlled clinical studies. Abstracts and uncontrolled data are discussed only when other information is unavailable to address important aspects. DATA EXTRACTION: Information regarding study design, study population, results, and safety was recorded from each publication. The placebo response to gastrointestinal complaints in patients with motility disorders is high. Therefore, objective evidence of improvement was emphasized when documentation was available. DATA SYNTHESIS: Cisapride stimulates the motility of smooth muscle lining the esophagus, stomach, small intestine, and colon, and increases the tone of gut sphincters in vitro and in vivo. In controlled investigations, cisapride was superior to placebo in relieving symptoms associated with reflux esophagitis, nonulcer dyspepsia, and gastroparesis. Similar symptom and healing effects were observed with cisapride and histamine (H)2-antagonists in reflux esophagitis. Cisapride was either equal to or superior to metoclopramide in relieving reflux symptoms. However, metoclopramide was associated with significantly more central nervous system adverse effects. Cisapride was well tolerated, with adverse effects limited primarily to the gastrointestinal tract. CONCLUSIONS: Cisapride represents an attractive alternative to metoclopramide for the treatment of a variety of motility disorders. Because it addresses a primary underlying cause of reflux esophagitis, cisapride may also prove to be an effective alternative to acid suppressants in the management of this disorder.

Characterization of muscarinic receptors in guinea-pig uterus.

To characterize the muscarinic receptor present in guinea-pig uterus smooth muscle the affinities of a series of 27 muscarinic receptor antagonists for M1 (rat cortex), M2 (rat heart), M3 (rat submandibular gland), m4 (transfected in CHO cells) and muscarinic binding sites in guinea-pig uterus smooth muscle were determined in radioligand binding studies. In addition, functional experiments were performed to assess pKB values of the antagonist for muscarinic receptors in guinea-pig atrium and uterus. The results obtained are consistent with the presence of M2 receptors in the uterus through which the functional contractile response is mediated. Correlation coefficients of 0.98, 0.91 and 0.91 were calculated for the following linear regressions: pKi uterus vs. pKi M2, pKB uterus vs. pKi M2 and pKB uterus vs. pKB atrium. This study also revealed that the compounds dicyclomine, DAU 5884, DAU 6202 as well as AQ-RA 721 could distinguish m4 from M2 sites and are therefore important tools to characterize muscarinic receptor subtypes. In addition, DAU 5884 and DAU 6202 have been identified as highly potent M1 selective antagonists.

Characterization of the antinociception produced by intrathecally administered muscarinic agonists in rats.

The present study was designed to characterize the antinociception produced by the administration of a potent muscarinic agonist into the intrathecal space of the lumbar spinal cord of male Sprague-Dawley rats. Seven days after surgical implantation of intrathecal catheters, animals were injected with graded doses of (+)-cis-methyldioxolane. (+)-cis-Methyldioxolane produced hot-plate and tail-flick antinociception for up to 90 min, peaking 5 to 30 min after injection. The dose of (+)-cis-methyldioxolane that inhibited nociception by 50% was 12 nmol in both the hot-plate and tail-flick tests. This antinociception was not accompanied by a general depression of other spontaneous motor responses. The tissue concentration of (+)-cis-methyl-dioxolane in the lumbar spinal cord present at the time of maximal hot-plate and tail-flick antinociception was approximately 12 microM. In similarity to (+)-cis-methyldioxolane, intrathecally administered (+)-muscarine also produced strong hot-plate and tail-flick antinociceptive responses. In contrast, intrathecally administered N-methylcarbachol, a nicotinic agonist, had no effect on nociception. Five-minute pretreatment with graded doses of pirenzepine, methoctramine, idazoxan, LY53857, or S-(-)-zacopride each significantly antagonized hot-plate and tail-flick antinociception produced by 37.5 nmol of (+)-cis-methyldioxolane in a dose-related manner with median effective antagonist doses in the range of 0.4 to 2.2 nmol. Intrathecal pretreatment with graded doses of prazosin or naloxone enhanced the antinociception produced by (+)-cis-methyldioxolane in the tail-flick but not the hot-plate tests. Intrathecal vehicle, S(-)-propranolol or mecamylamine did not alter (+)-cis-methyldioxolane-induced antinociception. The data suggest that the antinociceptive responses produced by intrathecally administered (+)-cis-methyldioxolane involve the stimulation of muscarinic M1 and/or M2 cholinergic receptors, and may also involve activation of alpha-2 adrenergic, 5-hydroxytryptamine1c/2 and 5-hydroxytryptamine3 serotonergic receptor systems at the level of the lumbar cord.