Effects of imidazoline I(2) receptor agonists and morphine on schedule-controlled responding in rats

Accumulating evidence indicates that imidazoline I₂ receptor agonists enhance the antinociceptive effects of opioids and therefore may be suitable for combination therapy with opioids for pain treatment. However, little is known of the effects of I₂ receptor agonists on other behavioral effects of opioids. This study used schedule-controlled responding and dose-addition analyses to examine interactions between the μ opioid receptor agonist morphine and two imidazoline I₂ receptor agonists, 2-BFI and BU224. In 8 rats responding under a fixed ratio 10 schedule of food presentation, morphine (3.2-17.8 mg/kg), 2-BFI (3.2-17.8 mg/kg), and BU224 (5.6-17.8 mg/kg) each dose-dependently decreased responding. The addition of fixed proportions of 2-BFI or BU224 shifted the morphine dose-effect curves leftward. The interactions between morphine and 2-BFI or BU224 were infra-additive when the same proportions of morphine and I₂ receptor agonists were mixed; however, the interaction between morphine and I₂ receptor agonists was additive when the drugs were mixed at other proportions. These results provide quantitative evidence that I₂ receptor agonists do not enhance the response rate-decreasing effect of morphine and suggest that the enhancement of morphine antinociception is selective. Together, these results further support the therapeutic potential of combining I₂ receptor agonists and opioids for pain control.     

The effect of two tetrahydrocannabinols, (Δ9-THC and Δ8-THC) on conditioned avoidance learning in rats and its transfer to normal state conditions

Rats trained in conditioned avoidance responding (CAR) after injections of either 7.5 mg/kg ⁹-THC (tetrahydrocannabinol) or 15 mg/kg ⁸-THC, showed no transfer when tested in the non-drugged state. Furthermore, these doses of the isomeric tetrahydrocannabinols exerted a disruptive effect on previously established CAR in rats, trained under normal conditions.Only the ⁹-THC-group showed an impairment of acquisition which was statistically significant compared to the control group.     

Neutralization of lethality and proteolytic activities of Malayan pit viper (Calloselasma rhodostoma) venom with North American Virginia opossum (Didelphis virginiana) serum

Malayan pit viper (Calloselasma rhodostoma) envenomation is a major health problem in South East Asia. During envenomation, venom components mainly affect the hemostatic system. The sera from the North American Virginia opossums (Didelphis virginiana) were able to neutralize the venom of the Malayan pit viper. These natural inhibitors could be explored as potential therapeutics against envenomations of a variety of venomous snake species in different geographical habitats.     

Comparative evaluation of acute toxicity of ivermectin by two methods after single subcutaneous administration in rats

Ivermectin was evaluated for its acute toxicity after single subcutaneous (s/c) administration by 'Acute Toxic Class' method as per OECD 423 and by conventional acute toxicity test using probit analysis in rats. 'Acute toxic class' method yielded LD(50) in category 2 i.e. between 5 and 50mg/kg which was comparable with conventional method where it was found to be 51.5mg/kg. Post mortem lesions were observed in the form of congestion of liver, which showed centrilobar necrosis and hemorrhages on histopathological analysis in both the methods. This study suggests, 'Acute Toxic Class' method may be used instead of conventional method to study acute toxicity of injectable preparations. Similarly the LD(50) of around 50mg/kg indicated a wide margin of safety (250x) considering therapeutic dose of ivermectin as 200microg/kg.     

Anorectic effect and brain concentrations of D-fenfluramine in the marmoset: relationship to the in vivo and in vitro effects on serotonergic mechanisms

Summary The present study investigated the anorectic activity of d-fenfluramine (d-F) and the relationship with brain levels of unchanged drug and its metabolite d-norfenfluramine (d-NF) in marmosets, relating them to neurochemical effects on the serotoninergic system. d-F and d-NF were equally active in reducing food intake (ED₅₀ about 3 mg/kg, p.o.). However, the brain concentrations of the metabolite required to reduce food intake after synthetic d-NF were more than twice those after d-F, indicating that d-NF contributes to but does not completely explain the anorectic effect of d-F. At this dose d-F did not appreciably modify the serotonin (5-HT) and 5-hydroxyindoleacetic (5-HIAA) contents of the brain regions examined, except for a slight enhancement of 5-HIAA in hippocampus. In vitro in brain cortical synaptosomes d-F inhibited [³H]5-HT uptake more potently than d-NF, as in other species. d-F and d-NF showed similar potency in stimulating [³H]5-HT release, in a Ca⁺⁺ dependent manner. The tritium released by d-F and d-NF appeared to be mainly unmetabolized [³H]5-HT.Like in other species the marmoset too has saturable and specific [³H]d-F binding sites, for which d-NF has lower affinity. d-F and d-NF have low affinities for 5-HT receptor subtypes, except that d-NF has appreciable affinity for 5-HT_1Cand 5-HT_1Dreceptors. Unlike in rodents but similarly to primates in the striatum the pharmacology of 5-HT receptors seems to correspond to the 5-HT_1D subtype. Brain concentrations of d-F and d-NF at anorectic doses exceeded the concentrations required in vitro to influence the serotoninergic system. Therefore the effect of d-F on food intake might possibly be explained by an interaction with the 5-HT system, particularly uptake and release mechanisms, and that of d-NF by an action on 5-HT_1C and 5-HT_1D receptor subtypes. Correspondence to S. Caccia at the above address     

Quantitative determination of the biological activity of botulinum toxin type A by measuring the compound muscle action potential (CMAP) in rats

Quantitative determination of the biological activity of botulinum toxin type A usually depends on the LD₅₀ method after intraperitoneal injection into mice. This method requires a large number of mice to determine the toxic activity at a high level of precision and 3–4 days to obtain the results. Techniques to replace the LD₅₀ method have been attempted at various institutes. As a substitute for this method, by directly measuring the inhibition of neuromuscular transmission after the administration of a toxin, a method to quantitatively assess the toxin's activity by determining the compound muscle action potential (CMAP) was examined.Toxin solutions were injected into the rat gastrocnemius muscle, and that of the CMAP amplitude was determined over time. The CMAP amplitude decreased over 4 days after the injection of the toxin, and then slowly recovered. A dose-response relationship was noted for each dose, and a linear relation was observed between 0.01 and 30 U on the 1st day. From these results, we propose the CMAP as a substitute for the LD₅₀ method to examine the activity of toxin products as it is simple and reliable, reduces the number of experimental animals required, and lowers pain levels.     

The pharmacology of the behavioral and hypothermic responses of rats to 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT)

The hypothermic and motor behavioural responses to 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) have been investigated in the rat. The dose-effect relationship showed that hypothermia appeared at a lower dose than a definite motor syndrome. The hypothermic response to 8-OH-DPAT was attenuated following depletion of 5-hydroxytryptamine (5-HT) by repeated intraperitoneal (IP) administration of parachlorophenylalanine (200 mg/kg) or by injection of 5,7-dihydroxytryptamine (5,7-DHT, 100 g) into the region of the third ventricle; the motor behavioural response produced simultaneously was not. Indeed, after 5,7-DHT, it was increased. Quipazine (1 mg/kg, IP) antagonised the hypothermic response and facilitated the motor behaviour. Clenbuterol (2.5 mg/kg, IP) increased both hypothermic and motor responses. (±)-propranolol was without effect on the simple hypermotility produced by 8-OH-DPAT, although it is known to antagonise the hypothermic and stereotyped motor responses. It is concluded that 8-OH-DPAT probably produces its hypothermic effects by actions at 5-HT receptors located presynaptically on 5-HT neurones, while the stereotyped components of the serotonin syndrome appear to be mediated by post-synaptic receptors.     

Characterization of pharmacological and behavioral differences to nicotine in C57Bl/6 and DBA/2 mice

Approximately 50-70% of the risk for developing nicotine dependence is attributed to genetics; therefore, it is of great significance to characterize the genetic mechanisms involved in nicotine reinforcement and dependence in hopes of generating better smoking cessation therapies. The overall goal of these studies was to characterize behavioral and pharmacological responses to nicotine in C57Bl/6 (B6) and DBA/2 (D2) mice, two inbred strains commonly used for genetic studies on behavioral traits. B6 and D2 mice where subjected to a battery of behavioral tests to measure nicotine's acute effects, calcium-mediated antinociceptive responses, tolerance to chronic treatment with osmotic mini pumps, and following three days of nicotine withdrawal. In general, D2 mice were less sensitive than B6 mice to the acute effects of nicotine, but were more sensitive to blockade of nicotine-induced antinociceptive responses by a calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor. B6, but not D2 mice, developed tolerance to nicotine and nicotine conditioned place preference (CPP). While B6 and D2 mice both expressed some physical withdrawal signs, affective withdrawal signs were not evident in D2 mice. These results provide a thorough, simultaneous evaluation of the pharmacological and behavioral differences to experimenter-administered nicotine as measured in several behavioral tests of aspects that contribute to smoking behavior. The B6 and D2 strains show wide phenotypic differences in their responses to acute or chronic nicotine. These results suggest that these strains may be useful progenitors for future genetic studies on nicotine behaviors across batteries of mouse lines such as the BXD recombinant inbred panel.     

Analgesic potency of sodium salicylate,indomethacin, and chlordiazepoxide as measured by the spatial preference technique in the rat

The analgesic potency of various doses of sodium salicylate (150, 200, 250, 300, 350 mg/kg), indomethacin (1.5, 2.0, 2.5, 3.0, 3.5, 5.0 mg/kg) and chlordiazepoxide (2.5, 5.0, 10.0, 20.0 mg/kg) were measured using the spatial preference technique. All three agents were active in a wide range of doses indicating that this technique is sensitive to the weak analgesics. The chlordiazepoxide data were interpreted to suggest that this tranquilizing agent may be able to impair the appreciation of the emotional (i.e., aversive) qualities of electric shock, thus reducing the animal's motivation to escape the noxious stimulus. A procedure for computing ED₅₀ estimates was also presented along with a summary of the ED₅₀ values for several standard narcotic and narcotic antagonist analgesics. Since this procedure is a reliable and sensitive index of drug-induced analgesia, it should be useful as a screening procedure in evaluating the analgesic potency of a wide variety of chemical agents.     

A new ELISA for determination of potency in snake antivenoms.

A competitive ELISA for potency determination of bothropic equine antivenom was developed and compared to the conventional in vivo ED(50) assay, with the aim of partially substituting the in vivo assay in the monitoring of antivenom immunoglobulin levels. On this purpose, blood samples were taken at different times during and after the immunization protocol of the lot of horses used for production of snake antivenom at the Instituto de Higiene, Uruguay. Both the competitive ELISA and the ED(50) assay were performed on those samples. In addition, a group of five commercial pepsin-digested antivenoms were tested by both methods. A significant (P<0.001) correlation (Pearson's r=0.957) was found between the ELISA titres and the corresponding ED(50) values, indicating that the in vitro test can estimate the neutralizing antibody capacity of the sera as well as the in vivo assay. By means of this new ELISA, it was found that the immunized animals maintained good venom antibody titres, in the order of 20-50% of the maximum achieved, even 10 month after the end of the immunization schedule. The main advantage of our ELISA design is its ability to correctly estimate the neutralization capacity of crude hyperimmune plasma and antivenom sera independently of their antibody composition in terms of whole IgG or F(ab')(2) fragment.     

Antagonists of platelet fibrinogen receptor are less effective in carriers of Pl(A2) polymorphism of beta(3) integrin

Barbiturates have three different effects on the GABA(A) receptor channels: coactivation, direct activation, and blockage. We investigated the activation and blockage of the GABA(A) receptor channels by pentobarbital using the alpha(1)beta(2)gamma(2S) GABA(A) receptor channels transiently expressed in HEK293 cells in combination with the ultrafast application of agonists. The peak current amplitude of the pentobarbital activated ionic current proportionally increased to the first power of the pentobarbital concentration (Hill coefficient approximately 0.7), indicating that one binding step of pentobarbital at alpha(1)beta(2)gamma(2S) GABA(A) receptor channels can describe the experimental dose-response relation. The maximum peak current amplitude occurred at 1 mM pentobarbital and decreased at higher concentrations due to an open channel block. After the end of the pentobarbital pulses, rebound currents due to transition from the open-blocked to the open state of the receptor were observed. A kinetic scheme was constructed allowing the quantitative analysis of the pentobarbital activated ionic currents through GABA(A) receptor channels.     

Effects of d-amphetamine on temporal and spatial discrimination in rats

Rats were trained to lever-press for food reinforcers on a multiple schedule that had a fixed-interval (FI) and a differential reinforcement of low rate (DRL) component. Illumination of a stimulus light above the right-hand lever indicated that responses on this lever would be reinforced according to a FI 60-s schedule while responses on the left-hand lever were without programmed consequences. However, when the light above the left-hand lever was illuminated only responses on this lever were reinforced according to a DRL 15-s schedule. When the behaviour of the subjects had been brought under schedule control so that characteristic patterns of FI and DRL responding were emitted and there were relatively few responses on the incorrect levers, the effects of several doses of d-amphetamine (0.25, 0.5, 1.0, and 2.0 mg/kg) were assessed. The drug increased preference for responding on the right-hand lever. Thus, as dosage increased performance tended towards a constant high rate of responding on the right-hand lever throughout a session, with a much lower response rate on the left-hand lever. This result emphasises that the behavioural effects of drugs depend not only on patterns of ongoing behaviour but also on the context in which this behaviour occurs.     

Aripiprazole (OPC-14597) fully substitutes for the 5-HT<Subscript>1A</Subscript> receptor agonist LY293284 in the drug discrimination assay in rats

Rationale Aripiprazole (OPC-14597) is a novel atypical antipsychotic drug with a low incidence of side effects. The therapeutic action of aripiprazole has been attributed to its unique agonist/antagonist effects at D₂ dopamine receptors; however, aripiprazole also has significant in vitro affinity at 5-HT_1A receptors.Objectives The 5-HT_1A agonist property of aripiprazole has so far not been evaluated in any in vivo assay.Methods Thirteen male Sprague Dawley rats trained to discriminate the 5-HT_1A agonist LY 293284 (75 nmol/kg) from saline, using a fixed ratio (FR) 50 schedule of food-reinforcement in a two-lever operant-conditioning task, were used to evaluate the behavioral effect of aripiprazole at 5-HT_1A receptors.Results Aripiprazole fully mimicked LY 293284 in a drug-discrimination assay with an ED₅₀ of 1.39 mol/kg (0.62 mg/kg). In combination tests, aripiprazole did not block the LY 293284 cue but at 8.92 mol/kg (4 mg/kg) significantly reduced the response rate by lowering the threshold for induction of the 5-HT syndrome produced by the training dose of LY 293284. Moreover, the selective 5HT_1A receptor antagonist WAY 100635 was able to block the substitution of aripiprazole in LY-293284 trained rats.Conclusion Although the efficacy of aripiprazole against the positive symptoms of schizophrenia may be related to its dopamine receptor interactions, it seems possible that its atypical profile may derive, at least in part, from its 5-HT_1A agonist effect, rather than from unusual D₂ receptor properties.     

Delta-9-tetrahydrocannabinol (THC) increases brain prostaglandins in the rat

Intraperitoneal administration of 9-trans-delta-tetrahydrocannabinol (THC), the major psychoactive component of cannabis, in doses of 0.5, 1.0 and 2.0 mg/kg, produced a dose-related increase in the brain concentrations of prostaglandin (PG) E₂ and PGF₂ in male rats 4 h after THC administration, as assessed by radioimmunoassay. A time-course investigation indicated that THC (2 mg/kg, IP) induced maximal increases in rat brain concentration of both PGs 2 and 4 h after administration; PG levels declined appreciably by 8 h and were normal by 24 h.A time-course study on the hexobarbitone (100 mg/kg, IP) -induced hypnosis potentiating effect of THC (2.0 mg/kg, IP) in male rats revealed that this pharmacological action of the cannabinoid correlated well with the time-course of the THC-induced increase in rat brain PG concentrations.The present study lends support to earlier reports contending that PGs may mediate some of the central actions of THC.     

A global model to define the behavior of partial agonists (bell-shaped dose-response inducers) in pharmacological evaluation of activity in the presence of the full agonist

The dose-response models for full agonists and for a particular type of partial agonist can be described by sigmoidal curves and bell-shaped curves, respectively. The methods currently used to evaluate the interaction of a full agonist and a partial agonist require a large number of experimental units and base their analysis on nonlinear regression analysis, which may not be statistically appropriate. We propose an appropriate design and a global nonlinear model to evaluate such interactions. The new model allows us to estimate the interaction parameters and the parameters that characterize the individual partial agonist curve and the full agonist curve.     

Effects of Δ9-Tetrahydrocannabinol (THC) and chlordiazepoxide (CDP) on state-dependent learning: evidence for asymmetrical dissociation

⁹-Tetrahydrocannabinol (THC) and chlordiazepoxide (CDP) were studied for their effects upon acquisition, performance, state-dependent learning and reciprocal substitution in mice. Over a wide dose range, CDP had no effect while THC had a biphasic effect (depression at low doses and facilitation at high doses) on avoidance acquisition. Both agents elicited evidence for state-dependent learning; mice trained under drugged conditions failed to transfer learning to the non-drugged state. In contrast, performance decrements occurred only after high doses (40 mg/kg) of each were given to avoidance trained mice. Administration of CDP facilitated avoidance performance in drug naive mice after doses of 1.25 mg/kg and above. THC failed to prevent learning deficits in CDP-trained subjects. In contrast, CDP prevented avoidance deficits after doses of 5 mg/kg and above in THC-trained subjects. These results suggest that an asymmetrical dissociation exists between THC and CDP or between either agent and saline.Presented in part at 5th International Congress on Pharmacology, July 23–28, 1972, San Francisco, California.     

Acute and Subchronic Toxicity Study of Tamra Bhasma (Incinerated Copper) prepared from Ashodhita (Unpurified) and Shodhita (Purified) Tamra in Rats

The use of metals in traditional medicines is very often seen as matter of concern these days, especially the Bhasma preparations which are always under stringent observations for containing highly reactive inorganic elements such as lead, mercury, arsenic and others. One of the Bhasma extensively used in routine Ayurvedic practice is Tamra (copper) bhasma. If it is not prepared properly or Shodhana procedure is not done properly, it acts as a poison. To indicate its toxic potential, Ashtamahadoshas (eight major ill effects) have been quoted in classics and due emphasis have been given to its Shodhana procedure. In the present study, Tamra bhasma prepared from Shodhita and Ashodhita Tamra was subjected to oral toxicity study to ascertain the role of Shodhana process on safety profile of Tamra bhasma on subchronic administration to albino rats. Both the samples were administered to rats for 45 consecutive days at the doses of 5.5, 27.5, and 55 mg/kg. Animals were sacrificed on 46^th day and parameters like hematological, serum biochemical, and histopathology of various organs were studied. Results showed that Tamra bhasma prepared from Ashodhita Tamra has pathological implications on different hematological, serum biochemical and cytoarchitecture of different organs even at therapeutic dose level (5.5 mg/kg). Whereas, Tamra bhasma prepared from Shodhita Tamra is safe even at five-fold to therapeutic equivalent doses (27.5 mg/kg). These observations emphasize the role of Shodhana and importance of dose in expression of toxicity of the medicinal preparations.     

Anandamide mediates hyperdynamic circulation in cirrhotic rats via CB(1) and VR(1) receptors

BACKGROUND AND PURPOSE: Hyperdynamic circulation and mesenteric hyperaemia are found in cirrhosis. To delineate the role of endocannabinoids in these changes, we examined the cardiovascular effects of anandamide, AM251 (CB(1) antagonist), AM630 (CB(2) antagonist) and capsazepine (VR1 antagonist), in a rat model of cirrhosis. EXPERIMENTAL APPROACH: Cirrhosis was induced by bile duct ligation. Controls underwent sham operation. Four weeks later, diameters of mesenteric arteriole and venule (intravital microscopy), arterial pressure, cardiac output, systemic vascular resistance and superior mesenteric artery (SMA) flow were measured after anandamide, AM251 (with or without anandamide), AM630 and capsazepine administration. CB(1), CB(2) and VR1 receptor expression in SMA was assessed by western blot and RT-PCR. KEY RESULTS: Anandamide increased mesenteric vessel diameter and flow, and cardiac output in cirrhotic rats, but did not affect controls. Anandamide induced a triphasic arterial pressure response in controls, but this pattern differed markedly in cirrhotic rats. Pre-administration of AM251 blocked the effects of anandamide. AM251 (without anandamide) increased arterial pressure and systemic vascular resistance, constricted mesenteric arterioles, decreased SMA flow and changed cardiac output in a time-dependent fashion in cirrhotic rats. Capsazepine decreased cardiac output and mesenteric arteriolar diameter and flow, and increased systemic vascular resistance in cirrhotic rats, but lacked effect in controls. Expression of CB(1) and VR1 receptor proteins were increased in cirrhotic rats. AM630 did not affect any cardiovascular parameter in either group. CONCLUSIONS AND IMPLICATIONS: These data suggest that endocannabinoids contribute to hyperdynamic circulation and mesenteric hyperaemia in cirrhosis, via CB(1)- and VR1-mediated mechanisms.     

Regulation of intravenous cocaine self-administration in rats selectively bred for high (HiS) and low (LoS) saccharin intake

Rationale Rats selectively bred for high saccharin (HiS) intake consume more alcohol and acquire intravenous (i.v.) cocaine self-administration more rapidly than their low saccharin (LoS)-consuming counterparts. The present experiment was designed to determine whether HiS and LoS rats differed in other aspects of drug abuse. Objective The purpose of the present experiment was to use a two-lever dose self-selection procedure to investigate the regulation/dysregulation of i.v. cocaine self-administration in female HiS and LoS rats. Materials and methods HiS and LoS rats were trained to self-administer eight different doses of cocaine during daily 5-h sessions, with the cocaine doses ranging from 0.2 to 1.6 mg/kg in steps of 0.2 mg/kg. The dose size increased after a response on one lever (infusion duration lengthened by 3 s) and decreased after a response on the other lever (infusion duration shortened by 3 s), with a lower limit of 0 s and with an upper limit of 24 s (a corresponding range of 0 to 1.6 mg/kg); the animals increased or decreased their self-administered dose in nine discrete steps. Results The HiS rats showed less precise regulation of their postinfusion interval (PII) than LoS rats based on the size of the previously self-administered cocaine dose. Correlations between these variables were lower for HiS than for LoS rats during the acquisition and maintenance phases, and HiS rats had lower PIIs than LoS rats after many of the cocaine doses. The HiS rats also self-administered significantly more cocaine infusions during the maintenance phase than the LoS rats, especially at the highest dose. Conclusions These data indicate that HiS rats are more likely to self-administer more cocaine infusions, at higher doses, and with less precise dose-time regulation than LoS rats. Thus, rats selectively bred for HiS showed less inhibitory control over their cocaine intake than LoS rats, suggesting that a genetic predisposition for saccharin preference is related to the rewarding effects of i.v. cocaine.     

Development of quantitative structure-activity relationship (QSAR) models to predict the carcinogenic potency of chemicals: I. Alternative toxicity measures as an estimator of carcinogenic potency

Determining the carcinogenicity and carcinogenic potency of new chemicals is both a labor-intensive and time-consuming process. In order to expedite the screening process, there is a need to identify alternative toxicity measures that may be used as surrogates for carcinogenic potency. Alternative toxicity measures for carcinogenic potency currently being used in the literature include lethal dose (dose that kills 50% of a study population [LD₅₀]), lowest-observed-adverse-effect-level (LOAEL) and maximum tolerated dose (MTD). The purpose of this study was to investigate the correlation between tumor dose (TD₅₀) and three alternative toxicity measures as an estimator of carcinogenic potency. A second aim of this study was to develop a Classification and Regression Tree (CART) between TD₅₀ and estimated/experimental predictor variables to predict the carcinogenic potency of new chemicals. Rat TD₅₀s of 590 structurally diverse chemicals were obtained from the Cancer Potency Database, and the three alternative toxicity measures considered in this study were estimated using TOPKAT^®, a toxicity estimation software. Though poor correlations were obtained between carcinogenic potency and the three alternative toxicity (both experimental and TOPKAT) measures for the CPDB chemicals, a CART developed using experimental data with no missing values as predictor variables provided reasonable estimates of TD₅₀ for nine chemicals that were part of an external validation set. However, if experimental values for the three alternative measures, mutagenicity and logP are not available in the literature, then either the CART developed using missing experimental values or estimated values may be used for making a prediction.