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Purpose: It is important that, when corticosteroids are used therapeutically, concentrations be reduced as much as possible to mitigate potential adverse events and side effects. This preliminary study compares the permeation for the delivery of a corticosteroid in a 1% hydrocortisone-supplemented topical cream containing anionic polar phospholipids (APP) in hydrogenated vegetable oil (triglyceride) versus a market-leading 1% hydrocortisone in a mineral hydrocarbon-based skin cream. Methods: Using the Franz diffusion cell method with cadaveric skin, the permeation of a 1% hydrocortisone-supplemented cream containing APP (test preparation) was compared with a commercially available 1% hydrocortisone cream (control preparation). The principal APP in the test preparation were phosphatidylinositol, phosphatidylserine and phosphatidylglycerol. Permeation was determined at 4 and 8 h time intervals. Results: The permeation values for the 1% hydrocortisone supplemental APP cream (test preparation) were comparatively very high 1180 ng/cm2 at 4 h and 2173 ng/cm2 at 8 h, in contrast to the 1% hydrocortisone cream (control preparation) values of 13 ng/cm2 at 4 h and 98 ng/cm2 at 8 h. Permeation of skin cream increased significantly from 0 to 4 and 8 h, when comparing the APP test preparation with the control preparation (p < 0.001). This translates, respectively, into the 90-fold greater and a 20-fold greater penetration of the test preparation APP cream over the 1% hydrocortisone cream at 4 h and 8 h time points. Conclusions: This preliminary study demonstrates the enhanced permeation of 1% hydrocortisone when applied topically to the skin in an APP skin cream vehicle. This enhanced permeation suggests the potential use of APP technology to deliver therapeutically effective hydrocortisone treatment to the skin at markedly reduced concentrations of steroid. As such, APP technology may offer an improved approach to the treatment of dermatoses associated with inflammatory diseases and conditions requiring prolonged topical corticosteroid therapy.

Controlled delivery of drugs has been achieved successfully by use of asymmetric membranes. In our study, we have evaluated the influence of various preparation parameters such as concentration of the polymer, concentration of the pore former and temperature of the precipitation bath on the permeability and the release characteristics of propranolol. Propranolol tablets were prepared by direct compression and were coated with varying concentrations of cellulose acetate and glycerin. The coat was precipitated in water, maintained at various temperatures, followed by air drying of the coat. Scanning Electron Microscopy (SEM) was used to characterize the asymmetric structure of the membrane. The influence of various preparation parameters on the release of propranolol from asymmetric coated tablets was evaluated. SEM confirmed the asymmetric nature of the membrane. A zero order release of propranolol was obtained from the coated tablets of propranolol. Various preparation parameters studied significantly affected (p<0.05) the release of propranolol hydrochloride from the asymmetric membrane coated tablets and the release was independent of the pH and the rate of agitation of the dissolution medium (p>0.05). Asymmetric membranes can be successfully utilized in the controlled delivery of highly water soluble drugs like propranolol and by modifying preparation parameters like polymer concentration, pore former concentration and temperature of the precipitation bath, desired release rates can be obtained.

A simple, rapid, and stability indicating reversed-phase high-performance liquid chromatography (HPLC) method of analysis for budesonide, a novel glucocorticoid prescribed for inflammatory bowel disease, was successfully developed. Budesonide is an epimeric mixture and both the epimers have similar anti-inflammatory activity. All the analytical methods reported in the literature are long and are based on separation of the epimers, thus our objective was to obtain a single sharp peak of the drug and to separate the drug peak from all the other degradation products. The method, was used to quantify budesonide in the developed formulation, employed a Kromasil C8, (150 mm x 4.6 mm) column with an isocratic mobile phase of acetonitrile-phosphate buffer (pH 3.2-0.025 M) (55:45 v/v), at a flow rate of 1.1 mL/min. Budesonide was detected by an ultraviolet detector at 244 nm. The method was validated for linearity, precision, repeatability, sensitivity, and selectivity. Selectivity was validated by subjecting stock solution of budesonide to acidic, basic, oxidative, and thermal degradation. The retention time of budesonide was about 4 min with symmetrical peaks. The method was linear over a concentration range 1-50 microg/mL (R2=0.9995). The limit of detection of budesonide was 0.1 microg/mL and the limit of quantitation was 0.25 microg/mL. The peaks of the degradation products did not interfere with the peak of budesonide. The developed method was used to quantify budesonide in budesonide-loaded micro-particles. Excipients present in the micro-particles did not interfere with the analysis and the recovery of budesonide from micro-particles was quantitative.

A simple, rapid and stability-indicating reverse-phase high-performance liquid chromatography (HPLC) method was developed for the assay of halofantrine (HF) base, 1,3-dichloro-alpha-[2-(dibutylamino)ethyl]-6-(trifluoromethyl)-9-phenanthrenemethanol. The HPLC method was validated for precision, accuracy, selectivity and linearity and range. It was used to assay for HF base in solid dispersions. Excellent linearity was observed between HF base concentration and the peak area (R(2)=0.9998). The limit of detection was 1 ng (with a signal-to-noise ratio of 2:1), and the limit of quantitation was 10 ng (with a signal-to-noise ratio of 10:1). The method proved to be selective. Selectivity was validated by subjecting a stock solution of HF to acidic, basic, oxidative and thermal degradations. The peaks of the degradation products did not interfere with the peak of HF. Excipients present in the solid dispersions did not interfere with the analysis.

Purpose: To compare the quantity of fluorescein delivered to the eye via fluorescein-impregnated paper strips of various sizes and surface areas and via various microliter volumes of fluorescein sodium using an in vitro assay., Methods: A commercially available fluorescein-impregnated strip (75 mm2) and three modified strips of reduced fluorescein-impregnated surface areas (10, 7.5, and 5.0 mm2) were used. The amount of fluorescein delivered to the eye for each of the four strips was approximated by applying each strip to a Whatman No. 1 filter paper under conditions simulating application of the strip to the eye, extracting the fluorescein from the filter paper in an aqueous solution, and performing spectrophotometric analysis at 484 nm. Similarly, this filter paper analytical system was calibrated using 1, 2, and 3 microl volumes of 2% w/v fluorescein delivered to the filter paper., Results: Using calibration curves, linearity was observed between absorbance and concentration of fluorescein sodium with an R2 value > or = 0.99. Using these calibration curves, the amount of fluorescein delivered to the eye for the four strips and the three fluorescein solution samples was determined. Fluorescein-impregnated strips with surface areas of 75, 10, and 5 mm2 delivered approximately the same quantity of fluorescein to the ocular surface as 3 microl, 1 microl, and 0.5 microl of fluorescein 2% solution, respectively., Conclusions: The surface area of the fluorescein-impregnated portion of the strip can be designed to control the amount of fluorescein delivered to the eye.

The low aqueous solubility of halofantrine (HF) and its low bioavailability from commercially available tablets (Halfan) suggested the formulation of solid dispersions (SDs) of HF to reduce its particle size and improve its wettability and aqueous solubility. Preformulation studies involved the development of a high performance liquid chromatography (HPLC) method for the analysis of HF. In addition, solubility studies were conducted on HF in aqueous solutions containing different concentrations of various carriers. Formulation studies included the preparation of SDs and physical mixtures (PMs) of HF with different carriers and their physicochemical characterization using differential scanning calorimetry (DSC), Fourier-Transform infra-red (FT-IR) spectroscopy and dissolution studies. A 3-month stability study at elevated temperatures was conducted on representative SDs of HF with selected carriers.

A simple, rapid, and stability-indicating high-performance liquid chromatographic (HPLC) method was developed and validated for the assay of propylthiouracil (PTU). The method was used to quantify PTU in topical formulations and in tablets. Excellent linearity was observed between PTU concentration and the peak area (R2= 0.999). The limit of detection was 1 ng, and the limit of quantitation was 1.2 ng. The method proved to be selective. Selectivity was validated by subjecting a stock solution of PTU to acidic, basic, and oxidative degradations. The peaks of the degradation products did not interfere with the peak of PTU. Excipients present in the dosage forms did not interfere with the analysis, and the recovery of PTU from each dosage form was quantitative.

A class of opioid receptor active derivatives of oxymorphone has been synthesized using a common enaminone intermediate. The derivatives have heterocyclic groups fused to the 6,7-positions of the morphinan system and all were synthesized in high yield. A pyrazolo derivative is an agonist for the mu and delta receptors and an antagonist for the kappa receptor.

Fluconazole suppositories were prepared in hydrophilic, lipophilic, and amphiphilic bases. In vitro evaluation was conducted to compare the effect of different bases on the release and permeation of fluconazole. Four types of suppository bases were evaluated; hydrophilic (polyethylene glycol, PEG), lipophilic (cocoa butter, CB; Witepsol W45 WW45), and amphiphilic (Suppocire AP SAP, a polyglycolized glyceride). The uniformity of dosage units prepared with each base was determined by ultraviolet (UV) spectroscopy. The influence of suppository base on the release of fluconazole was studied using USP dissolution apparatus I. Rate constants for each release pattern were determined and compared using a one-way analysis of variance (ANOVA) on ranks. The order of in vitro dissolution of fluconazole from the bases was as follows: PEG > (SAP = WW45) > CB. Results suggest that in vitro release of fluconazole is greater from a hydrophilic base (PEG). Preliminary permeation studies were conducted on each type of base using Franz diffusion cells. Permeation was studied through the rat rectal membrane, and normal saline was used as the receptor medium. A modified reverse-phase high-performance liquid chromatography (HPLC) method was used and validated for analyzing fluconazole. Flux values (microgram/cm2/hr) were calculated and compared using a one-way ANOVA (p < .001). The order of permeation was as follows: SAP > (PEG = WW45) > CB. The increased permeation characteristics seen with the SAP base are probably due to an alteration of the membrane characteristics due to the surface active properties of the base.

The effects of chronic administration of cocaine and its subsequent withdrawal on the binding of [3H]MK-801, an antagonist of the N-methyl-D-aspartate (NMDA) receptor, to brain regions and spinal cord of the mouse was determined. Chronic administration of cocaine (10 mg/kg, SC) twice a day for 7 days sensitized male Swiss-Weber mice to its locomotor activity, as evidenced by a greater locomotor activity in comparison with vehicle-injected controls. In nonwithdrawing mice, the binding of [3H]MK-801 was increased in the cerebellum and spinal cord but was decreased in the cortex and hypothalamus. On the other hand, during withdrawal from cocaine, a significant decrease in the binding of [3H]MK-801 was observed only in the cortex, but the changes in the cerebellum, spinal cord and hypothalamus were normalized. It is concluded that, in the mouse, sensitization to the locomotor activating action of cocaine is associated with differential changes in the NMDA receptors of the central nervous system.

The effects of ibogaine, an alkaloid isolated form the bark of the African shrub, Tabernathe iboga, and noribogaine, a metabolite of ibogaine, on morphine antinociception were determined in male Swiss-Webster mice. Mice were rendered tolerant to morphine by implanting them with a pellet containing 25 mg of morphine base for 3 days. Placebo pellet-implanted mice served as controls. The antinociception of morphine (10 mg/kg, s.c.) was determined alone or in combination with an appropriate dose of ibogaine or noribogaine. Tolerance to morphine developed as a result of morphine pellet implantation as evidenced by decreased antinociceptive response to morphine. Both ibogaine and noribogaine dose-dependently enhanced morphine antinociception in morphine-tolerant but not in morphine-naive mice. It is concluded that ibogaine and noribogaine enhance morphine antinociception in morphine-tolerant mice.

The effect of acute treatment with L-arginine, a substrate for nitric oxide synthase (NOS) that forms NO, an important second messenger, on morphine antinociception and distribution of morphine in central and peripheral tissues of male Swiss-Webster mice was determined. The antinociception activity of morphine (10 mg/kg, s.c.) was attenuated by 400 and 800 mg/kg doses of L-arginine, but a lower dose (200 mg/kg) had no effect. D-Arginine (200-800 mg/kg) did not modify morphine antinociception. The dose of L-arginine (200 mg/kg) that did not modify morphine antinociception also did not alter the distribution of morphine in brain regions and spinal cord. A dose of 800 mg/kg of L-arginine produced a significant decrease in the concentration of morphine in midbrain and spinal cord. The highest dose of L-arginine (800 mg/kg) also increased the concentration of morphine in spleen. None of the doses of L-arginine modified the concentration of morphine in serum or urine. The results suggests that acute activation of the NO system attenuates morphine antinociception possibly by inhibiting its uptake in central sites (midbrain and spinal cord) involved in antinociceptive actions.

The effects of chronic morphine administration on the kinetics of nitric oxide synthase (NOS) activity were determined in the mouse cerebral cortex and cerebellum. Male Swiss-Webster mice were implanted subcutaneously with morphine pellets each containing 25 mg of morphine base for 3 days. Mice implanted with placebo pellets served as controls. NOS activity was determined by measuring the rate of conversion of [3H]arginine to [3H]citrulline. Morphine pellet implantation increased NOS activity in the cerebral cortex and cerebellum. Analysis of the Eadie-Hofstee plot indicated that the Vmax of NOS in the cortex and cerebellum was 140 and 228 pmol [3H]citrulline formed/min/mg protein and the Km values were 9.3 and 10.1 mumol/l, respectively. Mice implanted with morphine pellets had higher Vmax values in both the cortex and cerebellum, but the Km values did not differ from those of control mice implanted with placebo pellets. It is concluded that chronic treatment with morphine increases NOS activity in the brain without modifying its substrate affinity.

1. Chronic administration of L-arginine (200 mg/kg, i.p) twice a day for 4 days decreased the antinociceptive response to subcutaneously, but not to intracerebroventricularly, administered morphine in male Swiss-Webster mice, as measured by the tail-flick test. 2. The decreased antinociceptive response to morphine was reversed by concurrent administration of NG-nitro-L-arginine (L-NNA) (5 mg/kg, IP), an inhibitor of nitric oxide synthase. 3. The concentrations of morphine in mice treated chronically with L-arginine and then given morphine (10 mg/kg, SC) were determined in the peripheral tissues. L-Arginine treatment significantly increased the concentration of morphine in spleen and lungs, did not modify it in liver, kidneys and urine. L-NNA by itself had no effect on the distribution of morphine in peripheral tissues but reversed the changes induced by chronic treatment with L-arginine. 4. Acute administration of L-arginine (200 mg/kg, IP) did not modify either the morphine antinociception or the morphine distribution in peripheral tissues. 5. Previous studies from this laboratory indicated that chronic treatment with L-arginine decreases the concentration of morphine in several brain regions and spinal cord of mice. 6. The facts that chronic treatment with L-arginine does not alter antinociception induced by ICV administered morphine and it increases the concentration of morphine in peripheral tissues while decreasing it in brain regions after peripheral administration of morphine suggest that the decreased antinociception induced by subcutaneously administered morphine may be related to its decreased entry into the brain.

The relationship between asthma and opioids has barely been investigated. This study examines whether active sensitization of rats changes the analgesic and thermic effects of intracerebroventricular morphine or the pharmacokinetics of the drug. Morphine (5, 10 and 20 microg) was given intracerebroventricularly to sensitized (active immunization to ovalbumin and Al(OH)3 then airway challenge with ovalbumin after 12 days) and normal (i.e. non-sensitized) male Sprague-Dawley rats. The tail-flick latencies and changes in colon temperature were determined before morphine injection and at 30 min intervals for a period of 300 min afterwards. Results were expressed as the area under the time-response curve. The analgesic and hyperthermic response to morphine for sensitized rats was less than that obtained for normal rats. Cerebrospinal fluid and blood samples were collected periodically for a period of 240 min and morphine levels were determined by a highly sensitive radioimmunoassay. The pharmacokinetic parameters half-life, terminal elimination rate constant and the mean residence time were determined in both cerebrospinal fluid and plasma by non-compartmental analysis. The area under the cerebrospinal fluid concentration-time curve from time zero to infinity was higher for sensitized rats than for normal rats for all three doses of morphine but these differences did not correspond with similar changes in pharmacological responses. In conclusion, the attenuated analgesic and thermic responses to intracerebroventricular morphine in the sensitized rats might be a result of pharmacodynamic alterations rather than to pharmacokinetic changes.

Twice daily injections of L-arginine (50, 100 or 200 mg/kg, i.p.) for 4 days dose-dependently, decreased morphine antinociception in male Swiss-Webster mice as measured by the tail-flick test. To determine the possible role of N-methyl-D-aspartate (NMDA) receptor in the action of L-arginine, the effects of MK-801, a noncompetitive antagonist of the NMDA receptor and of LY 235959, a competitive antagonist of the NMDA receptor on L-arginine-induced attenuation of morphine antinociception were determined. MK-801 (0.01-0.10 mg/kg, i.p.) or LY 235959 (1.0-4.0 mg/kg, i.p.) given 10 min before each injection of L-arginine (200 mg/kg, i.p.) reversed the action of the letter in a dose-dependent manner on morphine antinociception. It is concluded that NMDA receptors are involved in the action of L-arginine in attenuating morphine antinociception.

The effect of multiple intracerebroventricular (i.c.v.) injections of [D-Pen2, D-Pen5]enkephalin (DPDPE), a delta-opioid receptor agonist, on the activity of nitric oxide synthase (NOS) was determined in the brain regions and spinal cord of the mouse. Male Swiss Webster mice were injected twice daily with DPDPE (20 micrograms/mouse, i.c.v.) or its vehicle for 4 days. This procedure has previously been shown to induce tolerance to the antinociceptive actions of DPDPE in mice. On day 5, the animals treated with DPDPE were either sacrificed 20 min after an i.c.v. injection of DPDPE (tolerant) or without any injection (abstinent i.e., 16 h after the last injection of DPDPE). NOS activity in brain regions (cortex, striatum, hippocampus, midbrain, pons/medulla, hypothalamus and cerebellum) and spinal cord was determined by the rate of conversion of arginine into citrulline. Tolerance to DPDPE was associated increases in NOS activity in midbrain (49%) and pons/medulla (32%) and decreases in cerebellum (28%) and spinal cord (44%). However, NOS activity was unchanged in the cortex, corpus striatum, hippocampus and hypothalamus. On the other hand, during abstinence from DPDPE, NOS activity increased in midbrain (84%) and hypothalamus (35%) but decreased in cerebral cortex (27%) cerebellum (27%) and spinal cord (20%). NOS activity was unchanged in the corpus striatum, hippocampus and pons/medulla. Previous studies from this laboratory had demonstrated that chronic administration of mu- and kappa-opioid receptor agonists results in increases NOS activity in certain brain regions and that tolerance to mu- and kappa-, but not to delta-opioid receptor agonists, is attenuated by NOS inhibitors. The present studies, for the first time, demonstrate decreases in NOS activity in certain brain regions and spinal cord of mice treated chronically with delta-opioid receptor agonist. Furthermore, these findings may explain the inability of NOS inhibitors to attenuate tolerance to DPDPE in mice.

The effects of acute and chronic administration of cocaine on the antinociception and tolerance to the antinociceptive actions of mu-(morphine), kappa-(U-50,488H), and delta-([D-Pen2,D-Pen5]enkephalin; DPDPE), opioid receptor agonists were determined in male Swiss-Webster mice. Intraperitoneal injection of 40 mg/kg of cocaine by itself produced weak antinociceptive response as measured by the tail-fick test but the lower doses were ineffective. Administration of morphine (10 mg/kg, SC), U-50,488H (25 mg/kg, IP) or DPDPE (10 microg/mouse, ICV) produced antinociception in mice. Cocaine (20 mg/kg) potentiated the antinociceptive action of morphine and DPDPE but had no effect on U-50,488H-induced antinociception. Administration of morphine (20 mg/kg, SC), U-50,488H (25 mg/kg, IP) or DPDPE (20 microg/mouse, ICV) twice a day for 4 days resulted in the development of tolerance to their antinociceptive actions. Tolerance to the antinociceptive actions of morphine and U-50,488H was inhibited by concurrent treatment with 20 or 40 mg/kg doses of cocaine; however, tolerance to the antinociceptive action of DPDPE was not modified by cocaine. It is concluded that cocaine selectively potentiates the antinociceptive action of mu- and delta- but not of the kappa-opioid receptor agonist. On the other hand, cocaine inhibits the development of tolerance to the antinociceptive actions of mu- and kappa- but not of delta-opioid receptor agonists in mice.

Effects of multiple administrations of cocaine and subsequent cocaine withdrawal on the activity of nitric oxide synthase (NOS) in brain regions and spinal cord of male Swiss-Webster mice were determined. Chronic administration of cocaine resulted in the development of sensitization to its locomotor activity in the mouse. Chronic administration of cocaine was associated with increases in NOS activity in cerebral cortex, cerebellum, midbrain, hypothalamus, hippocampus, amygdala and spinal cord, but NOS activity was unaffected in pons/medulla and corpus striatum. Forty-eight hours after withdrawal from cocaine, NOS activity was increased only in the cerebral cortex. Recent studies have shown that cocaine-induced sensitization to behavioral effects can be inhibited by NOS inhibitors. The present studies provide the first evidence that chronic treatment with cocaine alters NOS activity in brain regions and spinal cord, and are consistent with behavioral studies with cocaine and NOS inhibitors.

BIM-44002, a pure competitive antagonist of parathyroid hormone (PTH), has a high affinity for the PTH/PTHrP receptor in vitro, and can completely inhibit the actions of a PTH agonist in rats in vivo. Toxicology studies in rats and dogs showed BIM-44002 to be devoid of any adverse effects. Therefore we undertook an investigation to evaluate the potential utility of BIM-44002 in lowering elevated serum calcium in three patients with primary hyperparathyroidism. BIM-44002 was administered by continuous intravenous infusion at dosages of 100 microg/hour (370 nmol/hour) for 12 hours, followed by 200 microg/hour for 12 hours, followed by 400 microg/hour for 12 hours. Vital signs and serum ionized and total calcium were monitored hourly and for 3 hours after cessation of the infusion. Blood for PTH determinations was obtained at the same time points. Serum calcium and PTH did not change during and after the infusion of the antagonist. No subject experienced any adverse reactions to the infusion of the antagonist. We conclude that although the PTH antagonist BIM-44002 was effective both in vitro and in vivo in animals, and it was safe in humans, it was not able to lower serum calcium in patients with hyperparathyroidism. Possible reasons for lack of clinical efficacy are discussed.

The effects of noribogaine, a metabolite of ibogaine, on the development of tolerance to the antinociception action of morphine was determined in male Swiss-Webster mice. Ibogaine is an alkaloid isolated from the bark of the African shrub, Tabernanthe iboga. Morphine tolerance in mice was developed by two different methods. Mice were rendered tolerant to morphine either by subcutaneous implantation of a pellet containing 25 mg morphine free base for 4 days or by injecting morphine (20 mg/kg, s.c.) twice a day for 4 days. Placebo pellet implanted mice or vehicle injected mice served as controls. To determine the effect of intraperitoneally administered noribogaine on tolerance development, the drug was injected in the appropriate dose twice a day. In pellet implanted mice, a dose of 20 mg/kg of noribogaine attenuated the tolerance to morphine whereas lower doses had no effect. Similarly, in mice given multiple injections of morphine, noribogaine attenuated tolerance development at 20 and 40 mg/kg doses. Previous studies from this laboratory had shown that ibogaine at 40 and 80 mg/kg doses inhibited tolerance to morphine. Because noribogaine could attenuate morphine tolerance at lower doses than ibogaine, it is concluded that the attenuating effect of ibogaine on morphine tolerance may be mediated by its conversion to noribogaine, a more active metabolite.

Chronic administration of L-arginine (200 mg/kg i.p.) but not of D-arginine (200 mg/kg i.p.) twice a day for 4 days decreased the antinociceptive response to subcutaneously administered morphine-6-beta-D-glucuronide (M6G), a potent metabolite of morphine, in male Swiss-Webster mice as measured by the tail-flick test. However, the antinociceptive response to intracerebroventricularly administered M6G was unaffected by chronic treatment with L-arginine. The decreased antinociceptive response to M6G (s.c.) was reversed by concurrent administration of NG-nitro-L-arginine (5 mg/kg i.p.), an inhibitor of nitric oxide synthase. Acute administration of L-arginine (200 mg/kg i.p.) had no effect on M6G-induced antinociception, but higher doses (400 and 800 mg/kg i.p.) decreased it. Since the antinociceptive response to centrally administered M6G was unaffected by chronic L-arginine treatment, the decreased antinociceptive response of peripherally administered M6G may be related to a decrease of M6G entry into brain structures responsible for antinociceptive action.

Male Swiss-Webster mice were injected intracerebroventricularly (i.c.v.) with [D-Pen2,D-Pen5]enkephalin (20 microg/mouse) twice a day for 2 days. This procedure resulted in down-regulation of binding sites for [3H][D-Pen2,D-Pen5]enkephalin as evidenced by a 52% decrease in the Bmax value. Twice daily injections of (+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]-cyclohepten-5,10-imine (MK-801) (0.1 mg/kg, i.p.) or [(-)3-SR,4a-RS,8a-SR-6-(phosphonomethyl)-1,2,3,4,4a,5,6,7,8,8a-decahy droisoquinoline-3-carboxylic acid] (LY 235959) (2 mg/kg, i.p.), the noncompetitive and competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor, respectively, for 2 days did not alter the Bmax or Kd value of [3H][D-Pen2,D-Pen5]enkephalin binding to the mouse brain. Concurrent treatment of MK-801, but not of LY 235959 with [D-Pen2,D-Pen5]enkephalin, reversed the decreases in Bmax value of [3H][D-Pen2,D-Pen5]enkephalin. Twice daily injections of [D-Ala2,Glu4] deltorphin II (20 microg/mouse) for 2 days caused an increase in the Kd value, but not the Bmax value of [3H][D-Pen2,D-Pen5]enkephalin to bind to brain membranes. Concurrent treatment of [D-Ala2,Glu4]deltorphin II with LY 235959 reversed the increase in Kd value of [3H][D-Pen2,D-Pen5]enkephalin binding induced by multiple injections of [D-Ala2,Glu4]deltorphin II, but MK-801 had no effect. The results suggest that multiple injections of delta1- and delta2-opioid receptor agonists down-regulate delta1-opioid receptors of the brain by modifying Bmax and Kd values of [3H][D-Pen2,D-Pen5]enkephalin binding, respectively. MK-801 and LY 235959 reverse delta1- and delta2-opioid receptor agonists-induced down-regulation of brain delta1-opioid receptor, respectively, apparently by different mechanisms. It is concluded that short term treatment of mice with delta1-opioid receptor agonist down-regulates brain delta1-opioid receptors by decreasing Bmax of the ligand which is partially reversed by concurrent treatment with MK-801 but not by LY 235959. On the other hand, short term treatment of mice with delta2-opioid receptor agonist down-regulates brain delta1-opioid receptors by increasing Kd of the ligand which is partially reversed by concurrent treatment with LY 235959 but not by MK-801.

1. The time course of the effects of multiple injections of U-50,488H, a kappa-opioid receptor agonist, and its subsequent termination on its analgesic action and nitric oxide synthase (NOS) activity was determined in the brain regions and spinal cord of the mouse. 2. Male Swiss-Webster mice were rendered tolerant to U-50,488H by twice-daily injections of the drug (25 mg/kg, IP) for 4 days. Vehicle-injected mice served as controls. 3. In tolerant mice, NOS activity was unchanged in brain regions and the spinal cord after treatment with U-50,488H. During abstinence from U-50,488H, NOS activity was found to be increased in the cortex and remainder of the brain, but no change was noted in the cerebellum, midbrain and spinal cord. 4. These studies demonstrate that withdrawal from the short-term treatment with U-50,488H in mice causes induction of NOS in certain brain regions. However, long-term treatment and withdrawal from U-50,488H are not associated with changes in the central NOS activity and indicate a possible adaptation in the NOS activity.

1. The time course of the effect of chronic administration of morphine on the activity of nitric oxide synthase (NOS) in the brain regions and spinal cord of the mouse was determined. The effect of naltrexone by itself on the NOS activity and that induced by morphine also were determined. 2. Male Swiss Webster mice were implanted subcutaneously with a pellet containing 25 mg of morphine free base for 4 days. Placebo pellet implanted mice served as controls. 3. Twenty-four hours after treatment with morphine, NOS activity decreased in the cerebellum, midbrain, cortex and remainder of the brain as well as in the spinal cord. Forty-eight and 72 hr after the treatment with morphine, NOS activity increased in the cerebellum and cortex, but no change was observed in the other brain regions and spinal cord. Twenty-four hours after morphine pellet removal (withdrawal), NOS activity in all brain regions and the spinal cord has returned to normal. 4. Implantation of a pellet containing 10 mg of naltrexone did not alter NOS activity in any brain region or spinal cord for 24, 48 and 72 hr or 24 hr after removal of the pellet. 5. Implantation of a naltrexone pellet in conjunction with a morphine pellet blocked the changes in NOS activity in the brain region and spinal cord induced by morphine. 6. It is concluded that the initial decrease in NOS activity by morphine may be related to enhanced motor activity, whereas the increase in NOS activity in certain brain regions may be associated with tolerance-physical dependence development. Additionally, the changes in central NOS activity by morphine appear to be mediated by opioid receptors because they were blocked by concurrent treatment with naltrexone.

The effects of twice-daily injections of L-arginine or D-arginine (200 mg/kg i.p.) for 4 days on morphine-induced antinociception, brain nitric oxide synthase activity and brain and serum distribution of morphine and brain mu-opioid receptors labeled with [3H][D-Ala2,MePhe4,Gly5-ol]enkephalin were determined in male Swiss-Webster mice. Chronic treatment with L-arginine, but not D-arginine, decreased the antinociceptive response to morphine in mice, increased the activity of nitric oxide synthase in the midbrain and decreased brain levels of morphine, compared with vehicle-injected controls. Significant decreases in morphine levels were observed in midbrain, pons and medulla, hippocampus, striatum and spinal cord of L-arginine-treated mice, in comparison with vehicle-injected mice. However, the levels of morphine in cortex, amygdala and hypothalamus of L-arginine- or D-arginine-treated mice did not differ from those of vehicle-injected controls. Acute treatment with L-arginine (200 mg/kg i.p.) or D-arginine (200 mg/kg i.p.) did not modify either morphine antinociception or morphine distribution in brain regions or the spinal cord. Chronic administration of L-arginine or D-arginine did not alter the Bmax or Kd values of [3H][D-Ala2,MePhe4,Gly5-ol]enkephalin binding to the mouse brain membranes. These results suggest that chronic treatment with L-arginine reduces the antinociceptive effect of morphine by increasing brain nitric oxide synthase activity and by decreasing the concentration of morphine in certain brain regions and spinal cord.

Ibogaine, an alkaloid isolated from the bark of the African shrub, Tabernanthe iboga, has been claimed to decrease the self-administration of drugs of abuse like morphine, cocaine and alcohol. To determine whether these effects are mediated via opioid receptor systems, the effects of ibogaine and its metabolite, noribogaine on the antinociceptive actions of morphine, U-50,488H and [D-Pen2,D-Pen5]enkephalin (DPDPE) which are mu- kappa- and delta-opioid receptor agonists, respectively, were determined in male Swiss-Webster mice. Administration of morphine (7 or 10 mg/kg, s.c.), U-50,488H (15 or 25 mg/kg, i.p.) or DPDPE (10 microg/mouse, i.c.v.) produced antinociception in mice as measured by the tail-flick test. Ibogaine (10, 20 or 40 mg/kg, i.p.) by itself did not alter the tail-flick latency. The same doses of ibogaine injected 10 min before the opioid drugs did not modify the antinociceptive actions of morphine, U-50,488H or DPDPE. Ibogaine administered 4 h or 24 h prior to morphine injection did not modify the antinociceptive action of the latter. A dose of 40 mg/kg (i.p.) of noribogaine enhanced the antinociceptive activity of morphine (10 mg/kg, s.c.). Similarly, the doses of 40 and 80 mg/kg of noribogaine enhanced the antinociception produced by a smaller dose of morphine (5 mg/kg, s.c.). However, antinociception induced by U-50,488H and DPDPE was not modified by noribogaine (10-40 mg/kg). It is concluded that ibogaine, which has been suggested to decrease the self-administration of cocaine and opiates like heroin in humans, does not produce such an action by interacting directly with multiple opioid receptors. However, the metabolite of ibogaine enhances the antinociception of morphine but not of U-50,488H or DPDPE. Thus, in vivo evidence has been provided for the possible interaction of ibogaine with mu-opioid receptor following its metabolism to noribogaine.

The effects of ibogaine, an alkaloid isolated from the bark of the African shrub, Tabernanthe iboga, on the development of tolerance to the antinociception action of morphine, U-50,488H and [D-Pen2,D-Pen5]enkephalin (DPDPE), which are mu-, kappa- and delta-opioid receptor agonists, respectively, were determined in male Swiss-Webster mice. Mice were rendered tolerant to opioid receptor agonists by injecting morphine (20 mg/kg, s.c.), U-50,488H (25 mg/kg, i.p.) or DPDPE (20 microg/mouse, i.c.v.) twice a day for 4 days. Ibogaine (20, 40 or 80 mg/kg, i.p.) given twice a day for 4 days did not alter the tail-flick latency. Ibogaine (40 or 80 mg/kg, i.p.) injected 10 min before each injection of morphine inhibited the development of tolerance to the antinociceptive action of morphine, however, the lower dose of ibogaine (20 mg/kg, i.p.) was ineffective. Ibogaine (20, 40 or 80 mg/kg, i.p.) given prior to the injection of U-50,488H or DPDPE did not modify the development of tolerance to their antinociceptive action. It is concluded that ibogaine inhibits selectively the development of tolerance to the antinociceptive action of mu- but not kappa- or delta-opioid receptor agonists in mice.

Twice daily injections of L-arginine (200 mg/kg, i.p.), a precursor for nitric oxide (NO), for 4 days decreased morphine antinociception in male Swiss-Webster mice. Chronic treatment with L-arginine also produced significant decreases in morphine levels in midbrain, pons and medulla, hippocampus, corpus striatum and spinal cord of mice following an injection of morphine (10 mg/kg, s.c.) in comparison to vehicle-injected mice. N(G)-nitro-L-arginine (L-NNA), an inhibitor of NO synthase (NOS), (5 mg/kg, i.p.) given prior to each injection of L-arginine reversed the effects of the latter on morphine antinociception and decreases in morphine levels in brain regions and spinal cord. Chronic injections of L-NNA alone did not modify either morphine antinociception or morphine distribution in brain regions and spinal cord of mice. These results suggest that decreases in morphine antinociception by chronic treatment with L-arginine is related to the decreases in the entry of morphine in the central sites. The reversal of L-arginine-induced effects by L-NNA suggests that NO-NOS system may be playing a critical role in the regulation of blood-brain barrier to morphine.

Male Sprague-Dawley rats were rendered tolerant to U-50,488H by twice-daily injections of the drug (25 mg/kg, i.p.) for 4 days. In tolerant rats, the binding of [3H]MK-801 was increased in pons and medulla and corpus striatum but decreased in midbrain and hippocampus and was due to changes in Bmax values. In U-50,488H-abstinent rats, the binding of [3H]MK-801 was increased in pons and medulla and hippocampus, and decreased in midbrain and amygdala. In hippocampus, the Bmax of [3H]MK-801 was increased but the Kd was decreased whereas in amygdala and pons and medulla, the changes were due to alterations in the Bmax values. Previous studies have shown that NMDA receptor antagonists block the tolerance to the analgesic action of U-50,488H in rodents. The present studies demonstrate differential changes in the NMDA receptors of brain regions of U-50,488H-tolerant and -abstinent rats.

The effects of the competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor, LY235959, were determined on the analgesic and hypothermic effects as well as on the development of tolerance to these effects of U-50,488H, a kappa-opioid receptor agonist in mice and rats. In the mouse, a single injection of LY235959 given 10 min prior to U-50,488H did not modify the analgesic action of the latter. Similarly, chronic administration of LY235959 twice a day for 4 days did not modify U-50,488H-induced analgesia in mice. Repeated pretreatment of mice with LY235959 dose-dependently attenuated the development of tolerance to the analgesic actions of U-50,488H. In the rat, LY235959 by itself produced a significant analgesia and prior treatment of rats with LY235959 enhanced the analgesic action of U-50,488H. Similar effects were seen with the hypothermic action. Pretreatment of rats with LY235959 attenuated the development of tolerance to the analgesic but not to the hypothermic action of U-50,488H. These results provide evidence that LY235959 produces differential actions on nociception and thermic responses by itself and when given acutely with U-50,488H in mice and rats. However, when the animals are pretreated with LY235959, similar inhibitory effects are observed on the development of tolerance to the analgesic action of U-50,488H in both the species. These studies demonstrate an involvement of the NMDA receptor in the development of kappa-opioid tolerance and suggest that the biochemical consequences of an opioid's interaction with the opioid receptor are not the only factors that contribute to the acute and chronic actions of opioid analgesic drugs.

Previous studies by our group have demonstrated that in vitro exposure to delta-opioid receptor agonists results in a significant immunostimulation, whereas in vitro exposure to non-peptidic delta-opioid receptor antagonists results in significant suppression of various immune functions. The present study assessed potential immunomodulation by the peptidic delta-opioid receptor antagonists TIPP, D-TIPP, and ICI 174864 using a panel of in vitro immune function assays. Splenocytes from female B6C3F1 mice were cultured with the peptides at concentrations of 0.00001-10 microM. B cell proliferation was quantified following cellular activation, T cell function was assessed by cytokine production following stimulation with anti-CD3 monoclonal antibody, natural immunity was assessed by quantitating natural killer (NK) cell activity following a 24-h exposure, and macrophage function was assessed by quantification of interleukin-6 (IL-6) production. None of the peptides examined significantly affected B cell proliferation. Production of IL-2 by T cells was not consistently affected by exposure to either TIPP or D-TIPP, but was significantly suppressed at 10 microM ICI 174864. Production of IL-4, however, was significantly suppressed by low concentrations of either TIPP or D-TIPP, and by 10 microM ICI 174864. IL-6 production by macrophages was unaffected except for sporadic incidents of enhanced production in cells exposed to ICI 174864. NK cell function exhibited a differential pattern of suppression, with the greatest degree of suppression observed following exposure to TIPP and only slight suppression in cells exposed to either D-TIPP or ICI 174864. These data suggest that peptidic delta-opioid receptor antagonists do not exhibit the same pattern or degree of immunosuppressive activity as the non-peptidic antagonists at equivalent in vitro concentrations.

Male Swiss-Webster mice were injected intracerebroventricularly (i.c.v.) with [D-Pen2,D-Pen5]enkephalin (DPDPE), a delta 1-opioid receptor agonist (20 micrograms per mouse) twice a day for either 2 or 4 days. Vehicle injected mice served as controls. Treatment of mice with DPDPE for 2 or 4 days decreased its analgesic response by 44 and 76%, respectively in comparison to vehicle injected mice. Treatment of mice with DPDPE for 2 or 4 days decreased density (Bmax) of [3H]DPDPE to bind to brain homogenates by 77 and 76%, respectively, in comparison to vehicle injected controls but the apparent dissociation constant (kd) values were not altered. The effects of i.c.v. injections of [D-Ala2,Glu4]deltorphin II (deltorphin II), a delta 2-opioid receptor agonist (20 micrograms per mouse) twice a day for either 2 or 4 days on its analgesic response as well as on brain receptors for [3H]DPDPE were also determined. The analgesic response to deltorphin II decreased by 51 and 78%, respectively in mice treated with deltorphin II for 2 or 4 days, respectively. Two or four days treatment with deltorphin II decreased the Bmax of [3H]DPDPE by 76 and 87%, respectively. The 2-day treatment also increased the Kd value by 58%, but the 4-day treatment with deltorphin II had no effect on the Kd of [3H]DPDPE to bind to brain membranes. Thus, multiple injections of delta 1- or delta 2-opioid receptor agonists results in the development of tolerance to their analgesic action and the intensity of tolerance increases with the duration of treatment. Both delta 1- and delta 2-opioid receptor agonist, on chronic administration, result in the down-regulation of delta 1-opioid receptors labeled with [3H]DPDPE.

The effects of chronic administration of [D-Pen2, D-Pen5]enkephalin and [D-Ala2, Glu4]deltorphin II, the selective agonists of the delta 1- and delta 2-opioid receptors, on the binding of [3H]MK-801, a noncompetitive antagonist of the N-methyl-D-aspartate receptor, were determined in several brain regions of the mouse. Male Swiss-Webster mice were injected intracerebroventricularly (i.c.v.) with [D-Pen2, D-Pen5]enkephalin or [D-Ala2, Glu4]deltorphin II (20 micrograms/mouse) twice a day for 4 days. Vehicle injected mice served as controls. Previously we have shown that the above treatment results in the development of tolerance to their analgesic activity. The binding of [3H]MK-801 was determined in brain regions (cortex, midbrain, pons and medulla, hippocampus, striatum, hypothalamus and amygdala). At 5 nM-concentration, the binding of [3H]MK-801 was increased in cerebral cortex, hippocampus, and pons and medulla of [D-Pen2, D-Pen5]enkephalin treated mice. In [D-Ala2, Glu4]deltorphin II treated mice, the binding of [3H]MK-801 was increased in cerebral cortex and hippocampus. The changes in the binding were due to increases in the Bmax value of [3H]MK-801. It is concluded that tolerance to delta 1- and delta 2-opioid receptor agonists is associated with up-regulation of brain N-methyl-D-aspartate receptors, however, some brain areas affected differ with the two treatments. The results are consistent with the recent observation from this laboratory that N-methyl-D-aspartate receptors antagonists block tolerance to the analgesic action of delta 1- and delta 2-opioid receptor agonists.

The effects of 7-nitroindazole (7-NI), an inhibitor of the neuronal nitric oxide synthase (nNOS) which does not increase blood pressure, on tolerance to the antinociceptive activity of mu-(morphine), kappa-(U-50,488H) and delta-([D-Pen2, D-Pen5]enkephalin, DPDPE) opioid receptor agonists were determined in mice. Male Swiss-Webster mice were made tolerant by twice daily injections of morphine (20 mg/kg, s.c.), U-50,488H (25 mg/kg, i.p.) or DPDPE (20 micrograms/mouse, i.c.v.) for 4 days. When tested on day 5, tolerance to their antinociceptive activity was evidenced by decreased response in chronic drug treated mice in comparison to vehicle-injected mice. Concurrent administration of 7-NI (20, 40 or 80 mg/kg, i.p.) with DPDPE did not modify the development of tolerance to the antinociceptive action of DPDPE. However, 7-NI (40 or 80 mg/kg, i.p.) inhibited the development of tolerance to the antinociceptive activity of morphine and U-50,488H but the lower dose of 7-NI (20 mg/kg, i.p.) was not effective. Chronic administration of 7-NI by itself did not modify the acute response to morphine, U-50,488H or DPDPE. It is concluded that a specific inhibitor of nNOS can inhibit tolerance to the antinociceptive activity of mu- and kappa- but not of delta-opioid receptor agonists in mice.

The effects of chronic administration and subsequent withdrawal of mu-, kappa- and delta-opioid receptor agonists on the levels of cyclic GMP in several brain regions and spinal cord of mice were determined in an attempt to further study the role of NO cascade in opioid actions. The agonists at mu-, kappa- and delta-opioid receptor included morphine, U-50,488H and DPDPE, respectively. Tolerance to morphine was associated with highly significant increases in cGMP levels in corpus striatum (41%), cortex (36%), midbrain (73%) and cerebellum (51%) relative to controls. Abstinence caused increases in cGMP levels in corpus striatum (61%) and pons and medulla (45%). Tolerance to U-50,488H resulted in increases in cGMP levels in midbrain (52%) whereas abstinence from U-50,488H increased the cGMP levels in pons and medulla (76%). Tolerance to DPDPE was associated with increases in cGMP levels in hypothalamus (12%) and pons and medulla (33%) but decreases in cerebellum (66%) and spinal cord (58%). Abstinence from DPDPE produced increases in cGMP levels in pons and medulla (14%) but decreases in cerebellum (67%) and spinal cord (50%). Overall treatment with morphine and U-50,488H produced increases in cGMP levels in brain regions whereas DPDPE produced decreases in brain regions and spinal cord. Previous studies have shown that chronic administration of mu- and kappa-opioid receptor agonists induce NO synthase (NOS) in certain brain regions and that the inhibitors of NO synthase attenuate tolerance to mu- and kappa- but not to delta-opioid receptors agonists. Since activation of NO increases the production of cGMP, the present results demonstrating alterations of cGMP levels by mu-, kappa- and delta-opioid receptor agonists are consistent with the behavioral results with NOS inhibitors on tolerance to mu-, kappa- and delta-opioid receptor agonists.

The role of endogenous opioid peptides in the regulation of bronchomotor tone, as well as in the pathophysiology of asthma is uncertain. We have studied the binding of highly selective [3H]labeled ligands of mu-([D-Ala2, MePhe4, Gly-ol5]enkephalin; DAMGO), delta ([D-Pen2, D-Pen5]enkephalin; DPDPE), and kappa-(U-69,593) opioid receptors to membranes of trachea, main bronchus, lung parenchyma and pulmonary artery obtained from normal (unsensitized) and actively IgE-sensitized rats acutely challenged with the specific antigen. [3H]DAMGO, [3H]DPDPE and [3H]U-69,593 bound to membranes of normal and sensitized tissues at a saturable, single high-affinity site. The rank order of receptor densities in normal tissues was delta- > or = kappa- > or = mu-, with lung parenchyma exhibiting the greatest binding capacity for delta- and mu- receptors compared to the other regions examined. The Kd values showed small differences between ligands and regions tested. The mu- and delta-opioid receptor densities were decreased in sensitized main bronchus and lung parenchyma, respectively, compared to normal tissues. By contrast, kappa-opioid receptor density was augmented in sensitized lung parenchyma but an increase in Kd values was also observed. These differential changes in the density and affinity of opioid receptor types may be related to alterations in endogenous opioid peptides during the process of sensitization.

The in vivo and functional effects of several 7-arylidene and 7-heteroarylidene morphinan-6-ones were determined at the mu-, delta-, and kappa-opioid receptors using the guinea pig brain membranes, guinea pig ileum (GPI), and mouse vas deferens (MVD). In vivo effects included the antagonism by these compounds given subcutaneously on the antinociceptive actions of intracerebroventricularly injected [D-Pen2, D-Pen5]enkephalin (DPDPE) and [D-Ala2, Glu4]deltorphin II (deltorphin II), the highly selective putative delta 1- and delta 2-opioid receptor agonists. Finally, the partition coefficients of these compounds were estimated (CLOGP) and determined experimentally at pH 7.4 in the 1-octanol/water system. Compared with E-7-benzylidenenaltrexone (BNTX), most compounds except for E-7-(4-chlorobenzylidene)naltrexone, were more potent at delta-opioid receptors than at the mu-opioid receptor, whereas, in comparison to the kappa-opioid receptor, the activities of the E-7-arylidene or E-7-heteroarylidene naltrexone derivatives at the delta-receptor were in the following order, where the 7-substituents were: 4-fluorobenzylidene- > benzylidene > 3-pyridylmethylene- > 4-pyridylmethylene- > 1-methyl-2-imidazolylmethylene > 4-chlorobenzylidene. In the MVD preparation, the potencies at the delta-opioid receptor, in comparison to BNTX, were in the following order, where the 7-substituents were: benzylidene = 1-methyl-2-imidazolylmethylene- > 4-fluorobenzylidene- = 3-pyridylmethylene- = 4-pyridylmethylene-. All compounds antagonized delta 1, and delta 2-opioid receptor agonist-induced analgesia. The antagonist potencies at the delta 1-opioid receptor were in the following order, where the 7-substituents were: benzylidene- > 4-chlorobenzylidene- > 4-fluorobenzylidene- > 3-pyridylmethylene- > 1-methyl-2-imidazolymethylene- approximately 4-pyridylmethylene-, whereas at the delta 2-opioid receptor, the order was benzylidene- > 4-chlorobenzylidene- > 4-fluorobenzylidene- > 3-pyridylmethylene- > 1-methyl-2-imidazolymethylene- > 4-pyridylmethylene. In general, all compounds exhibited greater potency at the delta 2- than delta 1-opioid receptor. The computed partition coefficients were, as expected, greater than the apparent log P values, which were determined experimentally. Generally, the lipophilicity values in decreasing order were: 4-chlorobenzylidene- > 4-fluorobenzylidene- > benzylidene > 3-pyridylmethylene- = 4-pyridylmethylene- > 1-methyl-2-imidazolylmethylene-. In general, the benzylidene and 4-pyridylmethylene derivatives, which have medium lipophilicities, were equally effective at the delta 1- and delta 2-receptors; the 3-pyridylmethylene and 1-methyl-2-imidazolylmethylene derivatives had lower lipophilicities and were more selective for the delta 2- than delta 1-receptor; the 4-chlorobenzylidene and 4-fluorobenzylidene derivatives were more lipophilic and had intermediate activity. The plot of pED50 values for the in vivo tests for the delta 1- and delta 2-receptors showed that the two receptors are not independent with respect to this series of compounds.

1. The effects of LY 235959, a competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor, on the analgesic and hypothermic actions of morphine, were determined in nontolerant and morphine-tolerant mice. 2. LY 235959 enhanced the analgesic and hypothermic action of morphine in nontolerant mice. 3. LY 235959 also enhanced the analgesic and hypothermic actions of morphine in morphine-tolerant mice. LY 235959, injected before saline injection, had no effect on the antinociception or the body temperature. 4. It is concluded that competitive antagonism of the NMDA receptor enhances the pharmacologic actions of morphine both in morphine-naive and morphine-tolerant mice and that these actions differ from the noncompetitive antagonism of the NMDA receptors.

The effects of chronic administration of 7-benzylidene-7-dehydronaltrexone, a delta 1-opioid receptor antagonist and naltriben, a delta 2-opioid receptor antagonist, on the antinociceptive responses to [D-Pen2, D-Pen5] enkephalin and [D-Ala2, Glu4]deltorphin II, delta 1- and delta 2-opioid receptor agonists, respectively, were determined in the mouse. Female B6C3F1 mice were given 7-benzylidene-7-dehydronaltrexone (3 mg/kg/day), naltriben (1 mg/kg/day) or the vehicle by subcutaneously implanted Alzet osmotic minipumps for 7 days. Both [D-Pen2, D-Pen5]enkephalin and [D-Ala2, Glu4]deltorphin II administered intracerebroventricularly (i.c.v.) produced antinociceptive as measured by the tail-flick test with ED50 values of 6.76 and 6.68 micrograms/mouse, respectively. Chronic administration of 7-benzylidene-7-dehydronaltrexone lowered the ED50 of [D-Pen2, D-Pen5]enkephalin but not of [D-Ala2, Glu4]deltorphin II. Chronic administration of naltriben lowered the ED50 of [D-Ala2, Glu4]deltorphin II but had no effect on the ED50 of [D-Pen2, D-Pen5]enkephalin. The binding of [3H][D-Pen2, D-Pen5]enkephalin to whole brain membranes of chronic 7-benzylidene-7-dehydronaltrexone-treated mice did not differ from chronic vehicle-treated mice. On the other hand, chronic administration of naltriben resulted in slight but reproducible elevation in the Bmax value of [3H][D-Pen2, D-Pen5]enkephalin to bind to whole brain membranes in comparison to vehicle-injected controls. The results suggest that chronic treatment with delta 1- and delta 2-opioid receptor antagonist cause behavioral supersensitivity to their agonists, respectively, and provides further evidence for the existence of delta-opioid receptor subtypes.

The effects of NG-nitro-L-arginine (NNA), an inhibitor of nitric oxide synthase (NOS) on the development of tolerance to the analgesic action of D-Pen2, D-Pen5 enkephalin (DPDPE), a delta 1 opioid receptor agonist, and morphine were determined in the mouse. Tolerance to DPDPE was induced in male Swiss-Webster mice by twice daily intracerebroventricular (i.c.v.) injections of the drug (20 micrograms/mouse) for 4 days. NNA was injected intraperitoneally (i.p.) 10 min before each injection of DPDPE. Chronic injections of DPDPE resulted in development of tolerance to its analgesic action. Multiple injections of NNA by itself did not modify the analgesic response to DPDPE. Concurrent injections of NNA did not affect the development of tolerance to the analgesic action of DPDPE. Twice daily injections of morphine (15 mg/kg s.c.) for 4 days resulted in the tolerance to its analgesic action. Concurrent administration of NNA with morphine attenuated the development of tolerance to morphine. It is concluded that NOS inhibition attenuates morphine, but not delta, opioid agonist-induced tolerance in the mouse.

Triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether) is a nonionic, broad spectrum, antimicrobial agent that, because of its favorable safety profile, has been incorporated into a variety of many personal care products, including deodorant soaps, underarm deodorants, shower gels, and health care personnel handwashes. Triclosan exhibits a moderate degree of substantivity to the skin, and, in many products, it imparts a remnant antimicrobial effect. Although direct contact with the material under exaggerated exposure conditions causes dermal irritation in laboratory animals, it has only rarely been associated with skin irritation or sensitization in human being in formulated products. Acute, subacute/subchronic, and chronic toxicity profiles have been established to determine that triclosan is neither an acute oral toxicant nor that it acts as a carcinogen, mutagen, or teratogen. A new application for triclosan is in oral dentifrices for plaque control. Currently under investigation in the United States, it is approved for oral care application in Canada and many European countries.

The effects of MK-801, a noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) receptor and of LY 235959, a competitive antagonist of the NMDA receptor on the analgesic action and development of tolerance to the analgesic action of D-Ala2, Glu4 deltorphin II (deltorphin II), a delta 2-opioid receptor agonist were determined in mice. Tolerance to deltorphin II was induced in male Swiss-Webster mice by twice daily intracerebroventricular (i.c.v.) injections of the drug (20 micrograms/mouse) for 4 days. To determine the effects of NMDA receptor antagonists, the drugs were injected intraperitoneally in appropriate doses 10 min before each injection of deltorphin II. The doses of MK-801 were 0.01, 0.03 or 0.10 mg/kg whereas those of LY 235959 were 1, 2 or 4 mg/kg. Chronic administration of NMDA receptor antagonists by themselves did not alter the tail-flick latency or the analgesic effect of deltorphin II. MK-801 given concurrently with deltorphin II inhibited the development of tolerance to deltorphin II, the significant effects were observed at 0.03 and 0.10 mg/kg doses. Similar inhibitory effect of development of tolerance to deltorphin II were observed with LY 235959 at 4 mg/kg dose. MK-801 and LY 235959 also antagonized the analgesic action of deltorphin II. It is concluded that antagonism of NMDA receptors attenuates the analgesic action as well as development of tolerance to the analgesic action of delta 2-opioid receptor agonist in the mouse.

The effects of tolerance to l-trans-delta 9-tetrahydrocannabinol (THC) following repeated administration and of subsequent abstinence following drug withdrawal on the cellular immune function were determined in female B6C3F1 mice. Mice were injected with THC (10 mg/kg s.c.) twice daily for 4 days. On day 5, analgesic response to various doses of THC was determined using the tail flick test. Similarly, hypothermic response to THC was also determined. Multiple injections of THC resulted in the development of tolerance to both the analgesic and hypothermic effects of THC. Immune function studies were performed on mice rendered tolerant to or abstinent from THC by these procedures. Neither tolerance nor abstinence subsequent to a 4-day THC administration had any effect on either body weight or thymus weight and cellularity, although both spleen weight and cellularity were decreased in THC-abstinent animals. Likewise, no significant effects on B cell proliferation were observed in either tolerant or abstinent mice. The production of the cytokine interleukin-2 by T helper cells was markedly suppressed in both tolerant and abstinent mice, whereas the production of interleukin-4 was significantly suppressed only in THC-abstinent mice. Significant suppression of tumor cell cytolysis mediated by cytotoxic T cells and natural killer cells was only observed in THC-abstinent mice. These results suggest that THC-mediated modulation of the immune response may result from a differential effect on cellular populations.

The effect of chronic administration of naltrexone, an opioid receptor antagonist, on the activity of the dopamine transporter in brain regions and spinal cord was determined. Male Sprague-Dawley rats were implanted with a pellet containing 10 mg naltrexone for 7 days. Rats serving as controls were implanted with a placebo pellet. Two groups of rats were used. In one, the pellets were left intact, and in the other they were removed 16 h prior to sacrificing. The dopamine transporter was labeled with [3H]GBR 12935. The binding of [3H]GBR 12935 in rats in which naltrexone pellets were left intact was decreased by 63 and 31% in corpus striatum and spinal cord, respectively, when compared to placebo pellet implanted controls. The decrease in binding in the striatum was due to changes in the Bmax value of [3H]GBR 12935; the Kd values did not differ. In hypothalamus, pons-medulla, hippocampus, midbrain, cortex, and amygdala of naltrexone and placebo pellet implanted rats, the binding of [3H]GBR 12935 did not differ. In naltrexone-treated rats from which pellets had been removed, the binding of [3H]GBR 12935 was decreased in hippocampus, amygdala, and spinal cord by 68, 77, and 61%, respectively, in comparison with tissues from the control rats. The results indicate that chronic blockade of opioid receptors results in downregulation of dopamine transporter in selected brain regions and spinal cord.

The specific binding of [3H]GBR 12935 to crude synaptosomal membranes of brain regions and spinal cord of morphine tolerant and abstinent rats was investigated. Male Sprague-Dawley rats were implanted with 6 morphine pellets each containing 75 mg of morphine base during a 7-day period. Placebo pellet implanted rats served as controls. Rats sacrificed without removal of the pellet were considered tolerant whereas those from which pellets were removed 16 hr prior to sacrificing were labeled abstinent. The binding of [3H]GBR 12935 was initially determined at a 1 nM concentration in all brain regions and spinal cord, which was followed by the determination of Bmax and Kd values in the corpus striatum, a highly enriched region for the dopamine transporter. In morphine tolerant rats, the binding of [3H]GBR 12935 was increased in the hypothalamus (182%) but was decreased in the corpus striatum (34%) and spinal cord (30%). The decrease in binding in the corpus striatum was due to an increase in the Kd value of [3H]GBR 12935. However, during morphine withdrawal, the binding of [3H]GBR 12935 was still higher in the hypothalamus (255%) but was decreased in the hippocampus (53%). Thus, chronic administration of morphine results in changes in the dopamine transporter function in selected brain regions and the spinal cord, and these changes are dependent upon whether or not the animals are undergoing the abstinence syndrome.

Two potent inhibitors of nitric oxide synthase (NOS), namely, NG-nitro-L-arginine (NNA) and NG-monomethyl-L-arginine (NMMA) were administered intracerebroventricularly (i.c.v.) in morphine-dependent mice to investigate their effects on abrupt withdrawal and naltrexone-precipitated abstinence signs. Male Swiss-Webster mice were rendered dependent on morphine by subcutaneous implantation of a morphine pellet containing 75 mg of morphine base. Mice implanted with placebo pellets served as controls. NMMA or NNA administered i.c.v. had minimal effects on body weight loss and hypothermia that occur during abrupt withdrawal of morphine. When administered i.c.v., both NNA or NMMA (0.1, 1 and 10 micrograms/mouse) dose-dependently inhibited naltrexone-induced stereotyped jumping behavior in mice. I.c.v. administration of NMMA also attenuated withdrawal induced fecal pellet formation. This effect, however, was not dose-dependent. In conclusion, these results suggest that brain NO plays an important role in the expression of behavioral signs of morphine withdrawal syndrome. In addition, these results support the idea that NOS inhibitors may be potentially useful in the treatment of opioid withdrawal syndrome.