Effects of narcotic antagonists on fluid intake in the rat

The fluid intake (sweetened Enfamil) of rats that had been deprived of food and water for 24 hours was measured following the subcutaneous administration of eight narcotic antagonists and agonists and $\text{d}$-amphetamine. Drugs were tested over at least a 30-fold range of doses. Fluid intake was depressed by the highest dose of each drug, but only the narcotic antagonists naloxone, naltrexone and nalorphine produced dose-related decreases in fluid intake that were not associated with gross disturbances of behavior. The anorexigenic activity of these drugs in the rat does not appear to be related to the drugs' narcotic antagonist properties.     

The effects of morphine and various narcotic antagonist type analgesics on body temperature in rats

ED50s were determined for morphine, nalorphine, butorphanol and pentazocine induced hyperthermia in rats. Morphine produced a significant hyperthermia with the doses of 5–160 mg.kg in rats. The peak hyperthermic effect was found 1 hr after 5–20 mg/kg doses of morphine. Nalorphine, butorphanol and pentazocine produced biphasic effects on rectal temperature. Initially they produced a dose-dependent hyperthermia and later hypothermia. In a comparison of the hyperthermic ED50's of morphine, nalorphine, butorphanol and pentazocine it was found that butorphanol is more active than the others (ED50s were 4.7, 4.3, 0.54 and 11.5 mg/kg respectively). The narcotic antagonist naloxone significantly inhibited both morphine and antagonist type analgesic induced hyperthermia. These results suggests that a different mechanism(s) is involved in the hyperthermic actions of antagonist type analgesics and agonist drugs.     

Effects of the intracerebroventricular injection of antinociceptive doses of acetylcholine on the stereospecific binding of 3H-dihydromorphine

The antinociceptive effects of intraventricularly administered acetylcholine (ACh) and its congeners have been demonstrated by previous investigators. The opiate receptor binding concept was used in this study to investigate possible correlations between ACh antinociception and its effects on opiate stereospecific binding. ACh $\text{in vitro}$ decreased the stereospecific binding of ³H-dihydromorphine in mouse brain homogenates. Such decrease was also observed in the brain homogenates of mice which had been treated with ACh intracerebroventricularly (i.v.t.). The decrease in the stereospecific binding of ³H-dihydromorphine induced by (i.v.t.) acetylcholine was inhibited by naloxone, atropine, cyclazocine and pentazocine. The $\text{d}$-isomers of cyclazocine and pentazocine were more potent than the $\text{l}$-isomers in antagonizing the inhibitory effects of i.v.t. acetylcholine upon the stereospecific binding of ³H-dihydromorphine to mouse brain homogenates. The same stereospecificity of these two narcotic analgesics in blocking acetylcholine had been previously observed in the tail-flick test. It is suggested that the antinociceptive effects of acetylcholine are related to the inhibition of opiate stereospecific binding, the mechanism of which is yet to be understood.     

Evidence that opiate receptors mediate suppression of hypertonic saline-induced drinking in the mouse by narcotic antagonists

The effects of naloxone, its $\text{dextro}$-stereisomer, and five other narcotic antagonists were determined on water intake induced by intracellular dehydration in the mouse. The intraperitoneal administration of a 2M sodium chloride solution served as the model for intracellular dehydration. $\text{1}$-Naloxone (0.01-10 mg/kg) reduced drinking in a dose-dependent fashion with an ED₅₀ of 0.55 mg/kg. In contrast, $\text{d}$-naloxone failed to suppress water consumption at doses up to 10 mg/kg. The other narcotic antagonists tested --- naltrexone, diprenorphine, levallorphan, oxilorphan, and nalorphine --- also produced dose-dependent decreases in water consumption. The order of potency of these narcotic antagonists in suppressing water intake was highly correlated with their orders of potency in other procedures involving the opiate receptor. The stereoselectivity and order of potency suggest that the suppressant effects of the narcotic antagonists on drinking induced by hypertonic saline administration in the mouse are mediated through an opiate receptor-dependent mechanism.     

Lactic/glycolic acid polymers as narcotic antagonist delivery systems.

Lactic/glycolic acid polymers of several compositions were evaluated as the vehicle material for long term, controlled delivery of narcotic antagonists. L(+)-lactic, and glycolic acids-designated L(+), and G, respectively-were converted to polymers with weight ratios of 75 L(+)/25 G, 90 L(+)/10 G, and 100 L(+). Naltrexone base and naltrexone pamoate were incorporated into these polymers as a physical blend at several drug/polymer mass ratios. The mixtures were formed into small cylinders and spheres which were suitable for subcutaneous implantation by means of a trochar. $\text{In vitro}$ screening was carried out followed by $\text{in vivo}$ testing in mice. Radioactive assay and direct challenge with morphine using the tail-flick test were used to evaluate the drug release. The release rates approximated zero-order kinetics for most of the release period and the narcotic antagonist response to a challenge dose of morphine was maintained from one month to over six months depending on the formulation tested. Factors affecting narcotic antagonist delivery system design were polymer composition, narcotic antagonist solubility, drug loading level of the dosage form, use of a pure polymer coating around the drug/polymer matrix, and the surface area/unit volume of the dosage form.     

Stereospecific aversive property of narcotic antagonists in morphine-free rats

If endogenous, morphine-like substances have physiological functions, narcotic antagonists should have effects $\text{in vivo}$ even in the absence of exogenous, narcotic agonists. This hypothesis was supported by studies of taste aversions conditioned with narcotic antagonists; rats drank smaller amounts of distinctively flavoured solutions when their consumption on previous occasions preceded injections of naloxone (1–10 mg/kg), naltrexone (3.2 mg/kg), Mr 1452 (10 mg/kg) or (-)-BC-2860 (10 mg/kg). Stereoisomers (i.e. Mr 1453, (+)-BC-2860) which were inactive as narcotic antagonists did not induce significant taste aversions. It was suggested that the consistency and stereospecificity of aversion with the antagonists gave some support to interpretations in terms of antagonist actions at receptors for endogenous opioids.     

Naloxone-precipitated jumping in mice pretreated with acute injections of opioids.

Naloxone induced jumping was examined in mice pretreated with single dose of narcotic agonist (morphine, heroin, LAAM, methadone), mixed agonist-antagonist (pentazocine, cyclazocine, buprenorphine), or an enkephalin analog (D-met², pro⁵)-enkephalinamide. Acute sensitization to naloxone, as demonstrated by jumping, was observed after pretreatment with the narcotics, the enkephalin analog, and to a lesser degree after cyclazocine and pentazocine. Mice pretreated with buprenorphine did not jump in response to naloxone. This procedure may be of value in the rapid identification of drugs with a propensity to produce morphine-like physical dependence.     

Enkephalin analogues and naloxone modulate the release of growth hormone and prolactin - evidence for regulation by an endogenous opioid peptide in brain

Met⁵-enkephalin amide, D-Ala²-Met⁵-enkephalin amide, D-Ala²-Leu⁵-enkephalin amide, morphine sulfate and naloxone hydrochloride were examined for their effects on growth hormone and prolactin release $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. Subcutaneous injection of D-Ala²-Met⁵ enkephalin amide^a, D-Ala²-Leu⁵ enkephalin amide^b and morphine sulfate, but not Met⁵-enkephalin and amide^c, resulted in significant elevations in the serum growth hormone and prolactin of immature female rats. Naloxone blocked the hormone-stimulatory effect of the opioid receptor agonists and when administered alone significantly reduced serum growth hormone and prolactin concentrations. None of the drugs demonstrated a direct action on anterior pituitary tissue growth hormone or prolactin release $\text{in}$$\text{vitro}$.     

Inhibition of ganglionic transmission by narcotic analgesics; an effect antagonized by naloxone, nalorphine and calcium

Methadone, azidomorphine, oxycodone and fentanyl inhibit synaptic transmission in isolated sympathetic ganglia of the frog and rat, just as did morphine and pethidine in our previous investigations. This inhibitory effect can be antagonized not only by naloxone and nalorphine but also by increasing calcium concentration of the perfusion fluid of the ganglia. The inhibitory effect on transmission of narcotic analgesics takes place on specific opiate receptors of the peripheral ganglia.     

Interactions of thyrotropin releasing hormone and morphine sulfate in rodents.

Thyrotopin releasing hormone (TRH) produces “wet dog shakes” in rats similar to those observed during morphine withdrawal. The shaking behavior precipitated by morphine abstinence can be exacerbated by TRH administration while the other components of the morphine withdrawal syndrome remain unchanged. Morphine, chlorpromazine, apomorphine, and Δ⁹-tetrahydrocannabinol effectively block shakes induced by either TRH administration or morphine withdrawal. These results suggest the possibility that endogenous TRH may be associated with the “wet dog shakes” observed as a portion of morphine's abstinence syndrome in rats. However, TRH is unable to alter the stereospecific binding of morphine $\text{in}$$\text{vivo}$ or $\text{in}$$\text{vitro}$, and naloxone fails to potentiate the number of TRH-induced shakes. TRH has no antinociceptive properties, and it cannot alter those of morphine. These data suggest that more than one neuromechanism may be responsible for shaking behavior in rats.     

Quaternary narcotic antagonists' relative ability to prevent antinociception and gastrointestinal transit inhibition in morphine-treated rats as an index of peripheral selectivity

Single doses of naloxone (0.025 to 0.5 mg/kg) or of one of four quaternary narcotic antagonists (i.e. nalorphine allobromide, nalorphine methobromide, naloxone methobromide or naltrexone methobromide, 1 to 60 mg/kg) were given s.c. to rats before morphine, 5 mg/kg i.v. In the absence of antagonists morphine reduced G.I. transit of a charcoal meal to about 15% of drug-free controls and consistently delayed nociceptive reactions (55°C hot plate) in all animals. Doses of antagonists slightly reducing morphine antinociception (centrally effective = A) and restoring G.I. transit to about 50% of drug-free rats (peripherally effective = B) were estimated. The A:B ratio, indicating peripheral selectivity, was at least 8 for any of the quaternary antagonists given 10 min before morphine, but prolonging this interval may have resulted in a lower figure (i.e. less peripheral selectivity) because of reduced A and increased B. This was definitely so for naltrexone methobromide (A:B, > 60 at 10 min, about 1 at 80 min) and was not apparent for nalorphine methobromide according to available data, which for nalorphine allobromide and to a lesser extent for naloxone methobromide showed only an increase in B at intervals longer than 10 min. Both morphine-induced antinociception and inhibition of G.I. transit were reduced by naloxone at the lower doses tested and were fully prevented at the higher. These findings indicate that, unlike naloxone, the investigated quaternary narcotic antagonists are interesting prototype drugs for selective blockade of opiate receptors outside the CNS, although certain critical aspects, possibly biological N-dealkylation to the corresponding tertiary antagonists, condition peripheral selectivity.     

The estrogenic activity of DDT: Invivo and invitro induction of a specific estrogen inducible uterine protein by o,p'-DDT

The pesticide o,p'-DDT stimulates the production of a specific uterine protein, the so-called induced protein or IP, normally associated with an estrogenic response of the uterus. $\text{In}$$\text{vivo}$ stimulation of IP production is observed 1 hour after the administration of 250 mg/kg of o,p'-DDT to immature rats. $\text{In}$$\text{vitro}$ stimulation of IP production is observed after a 1 hour incubation of uteri with 100 μM o,p'-DDT. This $\text{in}$$\text{vitro}$ response is blocked by Actinomycin D. In contrast to o,p'-DDT, which binds to the cytoplasmic estrogen receptor and stimulates IP production, p,p'-DDT which does not bind well to the estrogen receptor does not stimulate IP production $\text{in}$$\text{vitro}$. These findings represent the first report of an estrogenic effect of o,p'-DDT in a completely $\text{in}$$\text{vitro}$ system.     

Altered levels of β-endorphin fragments after chronic morphine treatment of guinea-pig ileum invitro and invivo

The isolated myenteric plexus-longitudinal muscle of the guinea-pig ilem (GPI) was used as testsystem to study the influence of chronic morphine treatment on the levels of enkephalins, β-endorphin and some of its fragments. The peptides were assayed by means of a combination of high pressure liquid chromatography and radioimmunoassays. It was found that the levels of methionine- and leucine-enkephalin and β-endorphin were not altered by chronic morphine treatment of guinea-pigs $\text{in}$$\text{vivo}$ nor in GPI exposed to morphine $\text{in}$$\text{vitro}$. However, the levels of some β-endorphin fragments i.c. γ-endorphin and des-tyrosine-γ-endorphin were elevated after morphine treatment $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$ respectively. It is suggested that β-endorphin and its fragments are involved in homeostatic processes during development of opiate tolerance.     

Effect of morphine sulfate on adenylate cyclase and phosphodiesterase activities in rat corpus striatum.

The effect of morphine sulfate (MS) on adenylate cyclase (AC) and phosphodiesterase (PDE) activities in the rat striatum was investigated. MS produced a dose-dependent increase in basal AC activity and did not alter sodium fluoride-induced stimulation both $\text{in}$$\text{vivo}$ (7.5–30 mg/kg, 1 hr pretreatment, i.p.) and $\text{in}$$\text{vitro}$ (1–100μM). $\text{in}$$\text{vitro}$, when submaximal effective concentrations of dopamine and MS were combined, there was an additive effect. However, administration of MS $\text{in}$$\text{vivo}$ did not alter dopamine-induced stimulation of AC activity. MS, $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$ inhibited PDE activity in a dose-dependent manner only with the high substrate concentration (3.3 × 10⁻³M cyclic AMP). Preliminary results from this study indicate that morphine affects the cyclic AMP system.     

III - Prostaglandin hyperalgesia: Relevance of the peripheral effect for the analgesic action of opioid-antagonists

Morphine injected into the rat cerebral ventricles had a marked analgesic effect, while no effect was observed with pentazocine and naloxone or nalorphine caused a strong hyperalgesia. Administered systemically (IP) naloxone and nalorphine caused a transitory analgesia followed by a long lasting hyperalgesic effect; morphine and pentazocine showed only an analgesic effect. It was concluded that the site of analgesic action of opioid-antagonists is peripheral rather than central. The peptidase-resistant enkephalin-analog, BW 180c, which does not cross the blood brain barrier, caused a marked analgesia by IP administration to paws made hyperalgesic by PGE₂ or carrageenin. It is suggested that agents derived from morphine, morphine-antagonists, enkephalins or cGMP devoid of central effect but having a strong peripheral effect may constitute a new class of safer analgesics.     

Humoral endorphin: A new endogenous factor with opiate-like activity

Humoral (H) endorphin, a novel endogenous opioid ligand detected in brain, blood and cerebrospinal fluid was tested in a series of opiate sensitive assays. H-endorphin displaced radiolabeled enkephalin from its specific bindings sites and inhibited the electrically evoked contraction of the guinea pig ileum and mouse vas deferens. When injected to unanesthesized animals, humoral endorphin induced analgesia in rats and mydriasis in mice. The activity of H-endorphin both $\text{in}\text{vitro}$ and $\text{in}\text{vivo}$ attests to its opioid nature. However, while its antinociceptive effect was blocked by naloxone, mydriasis induced by H-endorphin was resistant to the effect of the opiate antagonist. Similarly, intermediate concentrations of naloxone inhibited the effect of H-endorphin on the guinea pig ileum while its effect on the mouse vas deferens was completely refractory to naloxone. The physiological function of humoral endorphin in various naturally occuring states that show similar paradoxical interactions with naloxone is discussed.     

Inhibition of drug metabolism by chronically administered naltrexone

Naltrexone, a long-acting narcotic antagonist, was administered to mice via aong-term delivery system of 1.5 mm beads containing 2 mg of naltrexone in a 90/10 polylactic/glycolic acid copolymer (Dynatech R/D Comp.). A single bead implanted subcutaneously antagonized the analgesic action of interimittently administered morphine sulfate (20 mg/kg, i.p.) for 25–35 days. During this 4–5 week period during which the naltrexone was pharmacologically active, the activities of the hepatic, microsomal mixed function oxidases aminopyrine N-demethylase and aniline hydroxylase were depressed to 30–50% of the levels seen in sham-implanted controls. Hexobarbital sleeping time and zoxazolamine paralysis time were significantly prolonged, and the blood half-lives of ¹⁴C-pentobarbital and ¹⁴H-amphetamine were lengthened when the monoxygenase activities were inhibited. Sleeping time following administration of ethanol was unaffected. $\text{In}$$\text{vitro}$, both naltrexone and its major metabolite, ß-naltrexol, were found to be inhibitory of the activities of aminopyrine N-demethylase and aniline hydroxylase, although the parent compound was the more potent inhibitor of both activities by a factor of 2–3.     

The influence of mitochondria on ribosomes. Cycloheximide resistance of ribosomes from petite mutants of saccharomyces cerevisiae

The influence of cycloheximide on amino acid incorporation into protein of standard strain ? + and cytoplasmic mutant ? ? of $\text{S.}\text{cerevisiae}$ was determined $\text{in}\text{vivo}$ and $\text{in}\text{vitro}$. $\text{In}\text{vivo}$ cycloheximide at the concentration which inhibits protein synthesis in ? + strain by over 60% has litle or no effect in mutant ? ? strain. $\text{In}\text{vitro}$ cycloheximide in the range of 0.05 to 0.3 ug/ml of incubation medium inhibits polyphenylalanine synthesis in ? + strain by 50% and in ? ? strain by less than 10%. Similar resistance to this antibiotic are shown in standard strain grown in anaerobic conditions. It has been found that the resistance to cycloheximide is associated with changes in cytoplasmic ribosomes and may depend on the integrity of mitochondrial system.     

The effect of sodium ion on antinociception and opiate binding in vivo.

Large quantities of NaCl and CaCl₂ but not KCl given intrapertoneally decreased the antinociceptive activity of morphine. NaCl also antagonized the effect of morphine on the stereo-specific binding of opiates. This high dose of NaCl doubled the level of sodium in the brain but did not alter the specific gravity of brain tissue. These $\text{in}$$\text{vivo}$ effects of NaCl confirm the antagonistic effects of NaCl $\text{in}$$\text{vitro}$ that have been reported.     

Location and characterization of opiate receptors regulating pituitary secretion

The site at which opiate agonists and antagonists act to alter secretion of prolactin, growth hormone and luteinizing hormone as well as the pharmacological specificity of the opiate receptors mediating these effects were examined in rats. Injection of β-endorphin but not a 10 fold higher dose of the non opiate peptide β-endorphin, increased release of prolactin and growth hormone in male rats while inhibiting luteinizing hormone release in ovariectomized, estrogen primed female rats. Prior treatment with naltrexone i.p. blocked these responses. Injection of naltrexone into the hypothalamus lowered prolactin release. In rats with a surgically formed hypothalamic island systemic administration of morphine or naltrexone altered prolactin release in the same manner as was observed in intact animals. In contrast no effects of β-endorphin or naltrexone were observed on the spontaneous secretion of prolactin $\text{in}$$\text{vitro}$. In addition β-endorphin did not alter the inhibition of prolactin release produced by apomorphine $\text{in}$$\text{vitro}$. The ED₅₀ for stimulation of prolactin release following intraventricular administration of β-endorphin or the synthetic enkephalin analog FK 33-824 was the same, approximately 0.1 ng/rat. However FK 33-824 at 0.2 ng/rat was able to produce much greater analgesia and catatonia than β-endorphin. The metabolism and distribution of β-endorphin was examined but did not account for these differential effects. These results indicate that opiate agonists and antagonists can act at the hypothalamic but not the anterior pituitary level to alter release of prolactin, growth hormone and luteinizing hormone. In addition the data suggest that the opiate receptors mediating release of prolactin may have a different pharmacological specificity from those involved with analgesia and catatonia.