Syed M. Nurulain

β-Caryophyllene, a CB2 receptor agonist produces multiple behavioral changes relevant to anxiety and depression in mice

Recent evidence suggests that the cannabinoid receptor subtype 2 (CB2) is implicated in anxiety and depression disorders, although few systematic studies in laboratory animals have been reported. The aim of the current experiments was to test the effects of the CB2 receptor potent-selective agonist β-caryophyllene (BCP) in animals subjected to models of anxiolytic- and antidepressant-like effects. Therefore effects of BCP (50mg/kg) on anxiety were assessed using the elevated plus maze (EPM), open field (OF), and marble burying test (MBT). However for depression, the novelty-suppressed feeding (NSF), tail suspension test (TST), and forced swim tests (FST) were used. Results indicated that adult mice receiving BCP showed amelioration of all the parameters observed in the EPM test. Also, BCP significantly increased the time spent in the center of the arena without altering the general motor activity in the OF test. This dose was also able to decrease the number of buried marbles and time spent digging in the MBT, suggesting an anti-compulsive-like effect. In addition, the systemic administration of BCP reduced immobility time in the TST and the FST. Finally, BCP treatment decreased feeding latency in the NSF test. Most importantly, pre-administration of the CB2 receptor antagonist AM630, fully abrogated the anxiolytic and the anti-depressant effects of BCP. Taken together, these preclinical results suggest that CB2 receptors may provide alternative therapeutic targets for the treatment of anxiety and depression. The possibility that BCP may ameliorate the symptoms of these mood disorders offers exciting prospects for future studies.

The cannabinoid receptor 2 agonist, β-caryophyllene, reduced voluntary alcohol intake and attenuated ethanol-induced place preference and sensitivity in mice.

Several recent studies have suggested that brain CB2 cannabinoid receptors play a major role in alcohol reward. In fact, the implication of cannabinoid neurotransmission in the reinforcing effects of ethanol (EtOH) is becoming increasingly evident. The CB2 receptor agonist, β-caryophyllene (BCP) was used to investigate the role of the CB2 receptors in mediating alcohol intake and ethanol-induced conditioned place preference (EtOH-CPP) and sensitivity in mice. The effect of BCP on alcohol intake was evaluated using the standard two-bottle choice drinking method. The mice were presented with increasing EtOH concentrations and its consumption was measured daily. Consumption of saccharin and quinine solutions was measured following the EtOH preference tests. Finally, the effect of BCP on alcohol reward and sensitivity was tested using an unbiased EtOH-CPP and loss of righting-reflex (LORR) procedures, respectively. BCP dose-dependently decreased alcohol consumption and preference. Additionally, BCP-injected mice did not show any difference from vehicle mice in total fluid intake in a 24-hour paradigm nor in their intake of graded concentrations of saccharin or quinine, suggesting that the CB2 receptor activation did not alter taste function. More importantly, BCP inhibited EtOH-CPP acquisition and exacerbated LORR duration. Interestingly, these effects were abrogated when mice were pre-injected with a selective CB2 receptor antagonist, AM630. Overall, the CB2 receptor system appears to be involved in alcohol dependence and sensitivity and may represent a potential pharmacological target for the treatment of alcoholism.

Eight new bispyridinium oximes in comparison with the conventional oximes pralidoxime and obidoxime: in vivo efficacy to protect from diisopropylfluorophosphate toxicity

In search for more efficacious reactivators of acetylcholinesterase (AChE) inhibited by organophosphorus compounds, experimental K‐oximes have been synthesized which show good in vitro efficacy. However, AChE inhibition by oximes themselves (as quantified by their intrinsic IC50) is the major cause of oxime toxicity and the dose‐limiting factor. To assess K‐oxime efficacy in vivo, the extent of protection from mortality induced by diisopropylfluorophosphate (DFP) was quantified by Cox survival analysis and compared with that of the clinically available oximes. Oximes were administered in an equitoxic dosage, i.e. half the LD01. Best protection was conferred by K‐27, reducing the relative risk of death (RR) to 16% of control RR (P ≤ 0.05), which was statistically significantly better (P ≤ 0.05) than all other tested oximes, except obidoxime, K‐53 and K‐75. The efficacy of obidoxime (RR = 0.19), K‐48 (RR = 0.28), K‐53 (RR = 0.22), K‐74 (RR = 0.38) and K‐75 (RR = 0.29) was significantly (P ≤ 0.05) better than that of 2‐PAM (RR = 0.62) and K‐113 (RR = 0.73). No significant protective effect was observed for K‐107 and K‐108. Our LD50 data show that K‐107, K‐108 and K‐113 (which strongly inhibit AChE in vitro) are in vivo markedly more toxic than all other oximes tested and can therefore only be safely administered at a low dosage which is insufficient to protect from DFP‐induced mortality. Dosage calculations based on in vitro IC50 measurements may therefore in future replace in vivo LD50 determinations, thereby reducing the number of animals required. Copyright

Mini review on blood–brain barrier penetration of pyridinium aldoximes

This paper reviews the blood–brain barrier (BBB) penetration of newly developed pyridinium aldoximes. Pyridinium aldoximes are highly charged hydrophilic compounds used in the treatment of subjects exposed to organophosphonates because they are effective as acetylcholinesterase reactivators. Pyridinium aldoximes have antidotal effects against poisoning with cholinesterase inhibitors, a frequent problem affecting people working with organophosphate‐based insecticides and pesticides. Toxic organophosphonate products such as sarin and tabun can be used by terrorists as chemical warfare agents. This poses a severe challenge to all innocent and peace‐loving people worldwide. This review gives a brief summary of BBB transporters and description of the current in vitro and in vivo methods for the characterization of BBB penetration of established and novel pyridinium aldoximes. The authors provide a putative mechanism of penetration, outline some future ways of formulation and discuss the possible advantages and disadvantages of increasing BBB penetration. Copyright

Pretreatment for acute exposure to diisopropylfluorophosphate: in vivo efficacy of various acetylcholinesterase inhibitors

Prophylactic administration of reversible acetylcholinesterase (AChE) inhibitors before exposure to organophosphorus compounds (OPCs) can reduce OPC‐induced mortality. Pyridostigmine is the only FDA‐approved substance for such use. The AChE‐inhibitory activity of known AChE inhibitors was quantified in vitro and their in vivo mortality‐reducing efficacy was compared, when given prophylactically before the exposure to the OPC diisopropylfluorophosphate (DFP). The IC50 was measured in vitro for the known AChE inhibitors pyridostigmine, physostigmine, ranitidine, tiapride, tacrine, 7‐methoxytacrine, amiloride, metoclopramide, methylene blue and the experimental oxime K‐27. Their in vivo efficacy, when given as pretreatment, to protect rats from DFP‐induced mortality was quantified by determining the relative risk of death (RR) by Cox analysis, with RR = 1 for animals given only DFP, but no pretreatment. Physostigmine was the strongest in vitro AChE‐inhibitor (IC50 = 0.012 µ m), followed by 7‐methoxytacrine, tacrine, pyridostigmine and methylene blue. Ranitidine (IC50 = 2.5 µ m), metoclopramide and amiloride were in the mid‐range. Tiapride (IC50 = 256 µ m) and K‐27 (IC50 = 414 µ m) only weakly inhibited RBC AChE activity. Best in vivo protection from DFP‐induced mortality was achieved when physostigmine (RR = 0.02) or tacrine (RR = 0.05) was given before DFP exposure, which was significantly superior to the pretreatment with all other tested compounds, except K‐27 (RR = 0.18). The mortality‐reducing effect of pyridostigmine, ranitidine and 7‐methoxytacrine was inferior, but still significant. Tiapride, methylene blue, metoclopramide and amiloride did not significantly improve DFP‐induced mortality. K‐27 may be a more efficacious alternative to pyridostigmine, when passage into the brain precludes administration of physostigmine or tacrine. Copyright

New K-Oximes (K-27 and K-48) in Comparison with Obidoxime (LuH-6), HI-6, Trimedoxime (TMB-4), and Pralidoxime (2-PAM): Survival in Rats Exposed IP to the Organophosphate Paraoxon

ABSTRACT Oximes are cholinesterase reactivators used in organophosphorus compound poisoning. The purpose of the study was to compare the protective effect of the K-oximes (K-27 and K-48) in male rats with that of obidoxime (LuH-6), trimedoxime (TMB-4), and HI-6, using paraoxon (POX) as a cholinesterase inhibitor. Pralidoxime (2-PAM) was also retested. Seven groups of six rats each were used. Group 1 (G1) received 1 μmol/rat POX (≈ LD75), the other groups (G2–7) received 1 μmol/rat POX + one of the six reactivators. The animals were monitored for 48 h and time of mortality was recorded. The procedure was repeated seven times. Subsequently, experiments as described were repeated using 10 and 15 μmol/rat POX. Mortality data were compared and hazards ratios (relative risks) ranked with the Cox proportional hazards model using the POX dose and group (reactivator) as time-independent covariables. K-27 followed by K-48 were the most potent reactivators. K-27 was statistically significantly superior to all other reactivators except K-48. The relative risk of death estimated by Cox analysis in K-27– and K-48–treated animals when compared with untreated animals, adjusted for the POX dose, was 0.22 (95% confidence interval [CI], 0.15 to 0.31) and 0.26 (95% CI, 0.18 to 0.37), respectively. We concluded that in the animal model used K-27 and K-48 are superior to older oximes in their ability to protect from paraoxon effects. They should be tested further using methyl- and propyl-organophosphates as toxic agents.

Antioxidants in Organophosphorus Compounds Poisoning

Oxidative stress has recently been implicated as a factor in the mortality and morbidity induced by organophosphorus (OP) compound poisoning. An overwhelming number of research papers are based on studying at the cellular and organ level. Such studies have concluded that antioxidants can be used as an adjunct compound in the treatment of both chronic as well as acute OP poisoning. Still, the role of antioxidants in reducing the mortality and morbidity induced by OP compounds has scarcely been verified, as well as their role as adjunct treatment compounds for both structurally and functionally different OP compounds. The present review of the literature was undertaken to establish the role of antioxidants in survival studies following acute exposure to OP compounds. The review found no substantial evidence that antioxidants demonstrate any positive effect following extremely toxic poisoning. However, for a more comprehensive and rational conclusion, further research needs to be conducted.

The dual-acting H3 receptor antagonist and AChE inhibitor UW-MD-71 dose-dependently enhances memory retrieval and reverses dizocilpine-induced memory impairment in rats.

Both the histamine H3 receptor (H3R) and acetylcholine esterase (AChE) are involved in the regulation of release and metabolism of acetylcholine and several other central neurotransmitters. Therefore, dual-active H3R antagonists and AChE inhibitors (AChEIs) have shown in several studies to hold promise to treat cognitive disorders like Alzheimers disease (AD). The novel dual-acting H3R antagonist and AChEI 7-(3-(piperidin-1-yl)propoxy)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline (UW-MD-71) with excellent selectivity profiles over both the three other HRs as well as the AChEs isoenzyme butyrylcholinesterase (BChE) shows high and balanced in vitro affinities at both H3R and AChE with IC50 of 33.9nM and hH3R antagonism with Ki of 76.2nM, respectively. In the present study, the effects of UW-MD-71 (1.25-5mg/kg, i.p.) on acquisition, consolidation, and retrieval in a one-trial inhibitory avoidance task in male rats were investigated applying donepezil (DOZ) and pitolisant (PIT) as reference drugs. Furthermore, the effects of UW-MD-71 on memory deficits induced by the non-competitive N-methyl-d-aspartate (NMDA) antagonist dizocilpine (DIZ) were tested. Our results indicate that administration of UW-MD-71 before the test session dose-dependently increased performance and enhanced procognitive effect on retrieval. However neither pre- nor post-training acute systemic administration of UW-MD-71 facilitated acquisition or consolidation. More importantly, UW-MD-71 (2.5mg/kg, i.p.) ameliorated the DIZ-induced amnesic effects. Furthermore, the procognitive activity of UW-MD-71 in retrieval was completely reversed and partly abrogated in DIZ-induced amnesia when rats were pretreated with the centrally-acting H2R antagonist zolantidine (ZOL), but not with the CNS penetrant H1R antagonist pyrilamine (PYR). These results demonstrate the procognitive effects of UW-MD-71 in two in vivo memory models, and are to our knowledge the first demonstration in vivo that a potent dual-acting H3R antagonist and AChEI is effective in improving retrieval processes in the one-trial inhibitory avoidance task and provide evidence to such compounds to treat cognitive disorders.

Efficacy of two new asymmetric bispyridinium oximes (K-27 and K-48) in rats exposed to diisopropylfluorophosphate: comparison with pralidoxime, obidoxime, trimedoxime, methoxime, and HI-6.

Introduction. The new K-oximes, K-27 [1-(4-hydroxyimino-methylpyridinium)-4-(4-carbamoylpyridinium) propane dibromide] and K-48 [1-(4-hydroxyimino-methylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide], show good in vitro efficacy in protecting acetylcholinesterase from inhibition by different organophosphorus compounds (OPCs), including nerve agents. To assess their efficacy in vivo, the extent of oxime-conferred protection from mortality induced by diisopropylfluorophosphate (DFP) was quantified and compared with that of five established oximes. Materials and Methods. Rats received DFP intraperitoneally in a dosage of 6, 8, or 10 μmol/rat and immediately thereafter intraperitoneal injections of K-27, K-48, pralidoxime, obidoxime, trimedoxime, methoxime, or HI-6. The relative risk (RR) of death over time (48 h) was estimated by Cox survival analysis, comparing results with the no-treatment group. Results. Best protection was observed when K-27 was used, reducing the RR of death to 19% of control RR (p ≤ 0.005), whereas obidoxime (RR = 26%, p ≤ 0.01), K-48 (RR = 29%, p ≤ 0.005) and methoxime (RR = 26%, p ≤ 0.005) were comparable. The RR of death was reduced only to about 35% of control by HI-6, to 45% by trimedoxime, and to 59% by 2-PAM (p ≤ 0.005). Whereas the differences between the best oximes (K-27, obidoxime, methoxime, and K-48) were not statistically significant; these four oximes were significantly more effective than 2-PAM (p ≤ 0.05). The efficacy of K-27 was also significantly higher than that of HI-6, trimedoxime, and 2-PAM (p ≤ 0.05). Conclusion. Our data provide further evidence that K-27 is a very promising candidate for the treatment of intoxication with a broad spectrum of OPCs.

Protective Drugs in Acute Large-Dose Exposure to Organophosphates: A Comparison of Metoclopramide and Tiapride with Pralidoxime in Rats

Weak and reversible inhibitors of cholinesterase(s), when coadministered in excess with a more potent inhibitor such as organophosphates, can act in a protective manner. The benzamide compound, metoclopramide, confers some protection (putatively via this mechanism) for cholinesterases against inhibition by paraoxon both in vitro and in vivo, after chronic small-dose exposure. Tiapride is a related benzamide. In this study, we compared the protection by metoclopramide and tiapride in rats acutely exposed to large doses of paraoxon with the therapeutic “gold standard,” pralidoxime. Group 1 received 1 &mgr;mol paraoxon (approximately 75% lethal dose), Group 2 received 50 &mgr;mol metoclopramide, Group 3 received 50 &mgr;mol tiapride, Group 4 received 50 &mgr;mol pralidoxime, Group 5 received 1 &mgr;mol paraoxon + 50 &mgr;mol metoclopramide, Group 6 1 &mgr;mol paraoxon + 50 &mgr;mol tiapride, and Group 7 1 &mgr;mol paraoxon + 50 &mgr;mol pralidoxime. All substances were administered intraperitoneally. The animals were monitored for 48 h and mortality was recorded at 30 min, 1, 2, 3, 4, 24, and 48 h. Blood was taken for red blood cell acetylcholinesterase measurements at baseline, 30 min, 24, and 48 h. With the exception of Group 7, in which some late mortality was observed, mortality occurred mainly in the first 30 min after paraoxon administration with minimal changes occurring thereafter. Mortality at 30 min was 0% in the metoclopramide, tiapride, and pralidoxime groups and 73 ± 20 (paraoxon), 65 ± 15 (paraoxon + metoclopramide), 38 ± 14 (paraoxon + tiapride), and 13 ± 19 (paraoxon + pralidoxime). Mortality at 48 h was 75 ± 18 (paraoxon), 67 ± 17 (paraoxon + metoclopramide), 42 ± 16 (paraoxon + tiapride), and 27 ± 24 (paraoxon + pralidoxime). Metoclopramide does not significantly influence mortality after acute large-dose paraoxon exposure. Both tiapride and pralidoxime significantly decreased mortality in our model. The protection conferred by tiapride was significantly less than that conferred by pralidoxime at 30 min, but was not significantly different at 24 and 48 h.