A Single Compound Alternative to a Buprenorphine/Naltrexone Combination

Relapse to drug taking is a major factor contributing to the low success rate of opioid addiction treatment programmes. Recently, studies have revealed a buprenorphine/naltrexone combination had successfully increased the treatment retention rate (compared to naltrexone alone) among heroin addicts (with history of cocaine abuse) who had undergone detoxification. However, buprenorphine and naltrexone could not be administered as a single formulation due to their different bioavailability, which could create compliance issues. Therefore, in this project, we aimed to synthesise a series of ligands each having the pharmacological profile of the buprenorphine/naltrexone combination (partial agonist (ORL-1 receptors), antagonist (u- and x-opioid receptors)). Based on the group's previous work, this profile can be achieved within the orvinols series. Compound BU127, a buprenorphine analogue with phenyl substituent (C20) is very close to the desired profile. Therefore, in order to optimize BU127's profile, we designed and synthesised a series of aromatic analogues, including analogues with a small group attached to the aromatic system to increase the ORL-1 receptor efficacy, while retaining the low efficacy / antagonist activity at the u-opioid receptor and antagonist activity at x-opioid receptor. However, [35S]GTPyS screening has shown a sudden increase of x-opioid receptor efficacy with these modifications. The related compound BU10119, having a Cv-methyl, met the desired profile at all targeted receptors in the [35S]GTPyS screen. A few analogues were selected for further evaluation in functional assays in the isolated tissue preparations (rat vas deferens (for the ORL-1 and u-opioid receptors) and mouse vas deferens (for the K-opioid receptor)) to estimate their binding affinity (Ks) and potency (pA2) of the compounds relative to buprenorphine, using Schild analysis and Schild equation. Of the analogues synthesised, only compounds BU127 and BU1 0119 have met the desired profile at the targeted receptors (competitive reversible at the ORL-1 and u-opioid receptors) and having binding affinity at each receptor similar to buprenorphine (ORL-1, ~- and K-opioid receptors). Based on these results, at this point, the optimum features of buprenorphine analogues in order to achieve the targeted profiles are having a small group at Cy and a 6-membered aromatic substituent at C . 20 Without any substituent group attached to the aromatic ring.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

The crucial role of nociceptin opioid peptide (NOP) receptor in attenuating relapse to morphine/methamphetamine (poly-drug) addiction in mice

A combination of buprenorphine/naltrexone treatment had been proven to successfully attenuate relapse (reinstatement) to morphine/methamphetamine (poly-drug) addiction in mice using a conditioned place preference (CPP) model. This treatment combination exhibits a mixed opioid receptor pharmacological activities (mu/kappa antagonist and partial NOP agonist). Thus, this recent study aimed to investigate the independent role of the NOP receptor in attenuating relapse to this poly-drug addiction. The male Swiss albino mice were made dependent on 7.5 mg/kg morphine/1.0 mg/kg methamphetamine (poly-drug). The mice were injected with SB 612111 (a selective NOP antagonist) during reinstatement phase, 30 minutes before the administration of0.3 mg/kg buprenorphine/1.0 mg/kg naltrexone combination. The priming dose of poly-drug (2.5 mg/kg morphine/1.0 mg/kg methamphetamine) were given 10 minutes later. The percentage of time spent at the drug-paired compartment was compared with the control group (did not receive SB 612111 treatment). The results revealed that the relapse to poly-drug addiction happened in the group of mice that received SB 612111 prior to buprenorphine/naltrexone (53.81 ± 11.23 %, n = 6). This result was significantly different compared to its own baseline (before the mice were made dependent on poly-drug [-5.37 ± 6.42 %, n = 9, p < 0.05]). In contrast, relapse was significantly attenuated (reduced) in the group of mice that did not receive SB 612111 prior to buprenorphine/naltrexone (19.14 ± 16.89 %, n = 5). The percentage of time spent at the drug-paired compartment was found to be not significantly different compared to its own baseline (-16.14 ± 4.81 %, n = 13). The result suggests that an agonist activity at the NOP receptor is crucial in attenuating relapse to morphine/methamphetamine (poly-drug) addiction. Hence, further investigation needs to be done to evaluate the involvement of this receptor at the brain level

Rapid analysis of alpha-glucosidase inhibitory activity of Psychotria Malayana jack leaf applying infrared fingerprinting

Psychotria malayana Jack is the plant belongs to Rubiaceae family and known in Malaysia as “meroyan sakat/salung”. Despite its potential use as an antidiabetic agent, a rapid analytical approach for the quality control of this plant has not been developed. Thus, the objective of this study was to establish a validated analytical method for the prediction for the alpha-glucosidase inhibitory activity of the leaves of P. malayana through implementation of Fourier Transformation Infrared Spectroscopy-fingerprinting utilizing a multivariate statistical calculation, orthogonal partial least square. The dried extracts prepared with different solvents ratios of methanol-water (0, 25, 50, 75, and 100% v/v), were evaluated for the bio-activity and analyzed via infrared spectroscopy. Orthogonal partial least square was accomplished through correlating the bioactivity and infrared spectra of every extract. The 100% methanol extract possessed the highest inhibitory activity against the alpha-glucosidase (IC50 2.83 ± 0.32 μg/mL). The loading plot from the statistical calculation revealed several functional groups, including hydroxyl (O-H), alkenyl (C=C), methylene (C-H), carbonyl (C=O), and secondary amine (N-H) groups, which actively induced the α-glucosidase inhibitory activity. The established validated model can be utilized in predicting the inhibitory activity of new set of P. malayana Jack leaves and can also be used as an assessment tool in the quality control of this plant

Kappa antagonist to prevent drug relapse: does stage in addiction cycle matter?

Introduction: An upregulation of the kappa opioid receptor (KOR) system during preoccupation stage in addiction cycle will cause dysphoria among addicts which can lead to relapse. Therefore, KOR antagonism might hold the key to prevent relapse. In this study, we aim to identify the exact addiction's stage in which KOR antagonist can be given. Methods: Using a conditioned place preference (CPP) model, adult Swiss albino mice were divided into two major groups. The first group received treatment at the initial stage of morphine withdrawal (7.5 mg/kg. i.p) while the second group received treatment after complete abstinence was achieved. Each major groups were further divided into two treatment groups (n=8-12), either received a functional KOR antagonist (0.3 mg/kg buprenorphine/ 1mg/kg naltrexone combination, i.p) or a selective KOR antagonist (10 mg/kg nor-BNI, i.p) prior to morphine priming (2.5 mg/kg, i.p). All data were analyzed using paired sample t-test. Results: The results showed that relapse was successfully attenuated in the groups that received KOR antagonists only after complete abstinence was successfully achieved (not significantly different from their baseline). However, the mice developed unusual sign of behavior sensitization (intermittent freezing, licking) when buprenorphine/naltrexone combination was given at initial stage of withdrawal. Conclusion: Our initial findings suggest that KOR antagonism might be beneficial only after the addicts achieved complete abstinence to prevent future drug-induced relapse. Brain study should be conducted to explain the unusual behavior seen when the drug intervention is given at an earlier stage of withdrawal

The distinct role of kappa opioid receptor in attenuating relapse to morphine/methamphetamine (polydrug) dependence in mice

A Combination of 0.3mg/kg buprenorphine and 1.0 mg/kg naltrexone treatment shows a promising result due to its ability to attenuate reinstatement (relapse) in morphine/methamphetamine (polydrug)-dependent mice in a conditioned place preference (CPP) model. This prompted us to identify which opioid receptor that contributes to its anti-relapse activity. Using the same CPP model, 10 mg/kg nor- BNI (a selective kappa opioid receptor [KOR] antagonist) was used to evaluate the involvement of KOR in mediating relapse to polydrug dependence. By applying the immunohistochemistry (IHC) technique, the investigation was extended to the mice brain using KOR antibody (EPR18881), focusing on the brain regions that are abundant in KOR density. The results showed that nor-BNI alone failed to attenuate relapse to polydrug dependence. However, the IHC results proved that the number of KOR significantly increased in the striatum during reinstatement compared to post-conditioning (p <0.05). The KOR was significantly suppressed in the treatment group which strengthens the findings from previous studies proving that the KOR plays an important role in mediating relapse to polydrug dependence

The crucial roles of nociceptin opioid peptide (NOP) receptor in attenuating relapse in morphine/methamphetamine (poly-drug) dependence model

BACKGROUND: Effective treatment for relapse prevention to poly-drug is still not yet available although this public health issue is alarming. Our previous study has proved that a combination of buprenorphine/naltrexone treatment successfully attenuated relapse (reinstatement) in morphine/methamphetamine (poly-drug) dependent mice using a conditioned place preference (CPP) model. The net pharmacological effects of buprenorphine/naltrexone treatment combination are agonism at the kappa opioid receptor and a partial agonist at the NOP receptor. Thus, this recent study aimed to investigate if the NOP receptor is the key opioid receptor that is responsible in attenuating relapse to this poly-drug addiction. METHOD: The previous CPP protocol was maintained. The male Swill albino mice were injected with 6.0 mg/kg SB 612111 (a selective NOP antagonist) 30 minutes prior to the administration of the same dose of buprenorphine/naltrexone combination (0.3 mg/kg morphine and 1.0 mg/kg, respectively). The priming doses of poly-drug (2.5 mg/kg morphine/1.0 mg/kg methamphetamine) were given 10 minutes after. The percentage of time spent at the drug-paired compartment was compared between groups. RESULTS: The results revealed that the priming doses of poly-drug induced relapse in the group of mice that received SB 612111 prior to buprenorphine/naltrexone treatment (53.81 ± 11.23 %, n = 6) which was significantly different compared to its baseline (-5.37 ± 6.41 %, n = 9, p < 0.05). In contrast, relapse was significantly attenuated in the group of mice that only received buprenorphine/naltrexone treatment prior to drug priming (19.14 ± 16.89 %, n = 5) which was not significantly different from its baseline (-16.14 ± 4.81 %, n = 13). CONCLUSION: From these results, it is suggested that an agonist activity at the NOP receptor is crucial in preventing relapse to morphine/methamphetamine (poly-drug) dependence. Hence, further investigation needs to be done to evaluate the involvement of this receptor at the brain level

A single compound alternative to A Buprenorphine/Naltrexone combination to prevent relapse to drug addiction

Recent studies suggest the combination of buprenorphine and naltrexone is a promising drug lead to prevent relapse to drug taking [1]. However it has proven to be a challenge to design a single formulation of this combination due to different routes of administration needed in the clinical setting. In this project, we targeted buprenorphine analogues as single compound mimics of the combination, having lower efficacy at mu opioid receptors (MOR) than buprenorphine, but retaining buprenorphine's activity at kappa (KOR) and ORL-1 receptors. Novel buprenorphine analogues were synthesized and screened initially using [35S]GTPgS and radioligand binding assays and a number of candidate compounds (BU127, BU10101, BU10136, BU10112 and BU10119) were then more fully studied in isolated tissue assays. Rats (male, 300-325 g), Sprague Dawley strain) were killed using CO2, vasa deferentia were excised and suspended in a tissue bath (3 ml volume) under 1 g tension in standard Krebs-bicarbonate solution at 37◦C (95%O2/5%CO2). Nerve-evoked muscle contractions were induced with single square pulses (0.1 ms duration, 0.1 Hz) and recorded isometrically. To study the effects of candidate compounds on ORL-1 receptors, cumulative concentration response curves for the ORL-1 receptor agonist nociceptin (1 mcM-30 mcM) were constructed in the absence and presence of increasing concentrations of buprenorphine, BU127, BU10101, BU10136, BU10112 and BU10119 (1 mcM-30 mcM, n - 4-8 tissues per compound). The antagonist potency for these compounds at ORL-1 receptor were compared against the standard ORL-1 antagonist SB612111 (10 nM-100 nM, n = 8). To study the effects of candidate compounds on MORs, similar experiments were performed using the MOR agonist DAMGO (10 nM-100 mcM) in the presence of increasing concentrations of buprenorphine, BU127, BU10101 and BU10119 (0.1 nM-30 nM, n = 4-6 tissues per compound) and the standard MOR antagonist naltrexone (1 nM-30 nM, n = 5). All test compounds were added to the organ bath 15-20 min prior to agonist treatment. A single high concentration of each candidate compound (30 - 100 mcM) confirmed their lack of ORL-1 or MOR agonist activity in this isolated organ preparation. In ORL-1 studies, increasing concentrations of buprenorphine, BU127, BU10101, BU10112, BU10119 and Bu10136 produced rightward shifts in the concentration-response curve for nociceptin. Schild plots were constructed to determine the pA2 values. For the standard ORL-1 antagonist, buprenorphine and some test compounds, competitive reversible antagonism was observed with the following pA2 values: SB612111 (8.08, 95% CI 7.99, 8.17), buprenorphine (6.02, 95% CI 5.85, 6.19), BU127 (5.88, 95% CI 5.73, 6.04) and BU10119 (5.87, 95% CI 5.79, 5.96). The Gaddum equation was used to estimate the PA2 value compounds showing pseudo-irreversible or non-competitive antagonism and the estimated pA2 values (mean ± SEM) were 5.97 ± 0.13 for BU10101, 5.65 ± 0.18 for BU10136 and 5.14 ± 0.10 for BU10112. In the MOR studies, a similar approach was taken. Naltrexone, BU127 and BU10119 showed competitive reversible MOR antagonism with the following pA2 values: naltrexone (8.90, 95% CI 8.68, 9.12), BU127 (10.24, 95% CI 10.07, 10.41), BU10119 (10.24, 95% CI 10.14, 10.33). The other compounds tested displayed pseudo-irreversible or non-competitive antagonism, with estimated pA2 values of: buprenorphine (9.66 ± 0.10), BU10101 (9.79 ± 0.03). Studies into the activity of these compounds at KOR are ongoing, but these data suggesr a range of novel compounds with mixed antagonist activity at MOR and ORL-1 receptors, which may mimic a combination of buprenorphine and naltrexone

The effect of dose on the intensity of dependence in morphine-dependent mice

Introduction: Animal behaviour research is an important tool to study the addiction properties of addictive substances. The Conditioned Place Preference (CPP)paradigm is among the model used to achieve this purpose. It can be used to investigate a wide range of addiction related behaviour including intoxication and relapse potential. Since it involves a long cycle experiment, it is important to optimise the experimental conditions, but minimising any unnecessity. Therefore, in this study we aim to investigate the effects of different morphine dose (3.75 - 10 mg/kg) towards intensity of morphine dependence in adult Swiss albino mice. Methods: The mice were divided into four group (n=10-12 for each group), where each group received different doses of morphine and saline on alternate day for 6 days during conditioning phase. The intensity of dependence was measured during post-conditioning phase where the preference at the drug-paired compartment was calculated during a 15 minutes observation. Results: The result has shown that three out of four groups significantly developed dependence (P < 0.05). However, the group of mice that received highest morphine dose (10 mg/kg) did not develop dependence. When baseline was compared, it was found that the mice that received the lowest morphine treatment (3.75 mg/kg) shows the highest increment from their baseline (164.7±33.3 seconds), while the group that received 10 mg/kg morphine shows the lowest increment (119.0±31.6 seconds). The trend of increment at the drug-paired compartment shows a declining trend when the dose of morphine is increased. Conclusion: Therefore, it is concluded that the optimum dose of morphine that should be administered to the adult Swiss albino mice is around 3.75 mg/kg, provided that all other experimental conditions and protocols are maintained

Synthesis, mechanistic and biological evalution of novel bis-sydnone styrylketone as antiproliferative and antioxidant agents

Various literature sources have documented that sydnones are important bioactive molecules with a wide spectrum of activities involving the anti-proliferative and anti-inflammatory actions. Phenyl styrylketones and their derivatives as members of the chalcone family have also been reported as significant biological agents. The current study was initiated to evaluate in vitro cytotoxic and anti-inflammatory activity of sydnone-based compounds including some novel sydnone-styrylketone hybrids. The classical cyclodehydration of N-nitroso amino acids was applied in the preparation of the sydnone ring. Aldol condensation was utilized to join two sydnone rings by a styrylketone linker. Compounds identity was confirmed using FTIR, NMR and MS spectroscopy. MTT assay was used to evaluate the cytotoxicity of the synthesized compound. ELISA test was performed to investigate the COX-1/COX-2 inhibitory activity. The binding of sydnones with COX enzymes was examined by Glide docking and the Molecular Mechanics-Generalized Born Surface Area (MM-GBSA). The drug-likeness scores and membrane permeability of the compounds were also in silico predicted. Twenty-five sydnone-containing compounds were synthesized. Compounds 46-48 and 56-58 were reported as new sydnone derivatives. Compounds 61-63 were synthesized as novel structures containing two sydnone rings linked via α,β-unsaturated ketone. The structure of the synthesized compounds was confirmed by FTIR, 1H NMR, 13C NMR and ToF-MS analyses. All compounds exhibited low cytotoxicity especially against normal cell lines (IC50 in the range of mM). Only compound 45 had a significant antiproliferative activity against prostate (IC50 = 42 μM) and breast (IC50 = 63 μM) cancer cell lines. The in vitro COX inhibition assay showed varied activity. Compounds 47, 51, 58 and 63 showed the most potent COX inhibitory effect at a concentration of 200 μM. Selectivity index showed that only compound 63 was a selective COX-2 inhibitor. Acetylation of the sydnone ring at C4 was detrimental to the cytotoxic activity while prolific for the anti-inflammatory effects (COX inhibition). Docking analysis showed that COX-2 selectivity was due to a favorable positive charged interaction between the sydnone ring of 63 and Arg513 in the catalytic region of COX-2. Compound 51 was hydrogen bonded to the guanidinium group of Arg513. The low inhibitory effect of 63 against COX-1 was due to an unfavorable polar interaction with His513 in the binding pocket of COX-1. Drug-likeness prediction disclosed that the compounds comply with Lipinski’s rule and CMC-like rule. Similarity search delineated that sydnone-styrylketone hybrids had common structural features with known anti-inflammatory agents. Prediction of permeability through the physiological membrane revealed a good pharmacokinetic profile with intestinal absorption more than 80% and a potential BBB penetration. In conclusion, the compounds were successfully synthesized and characterized. However, only two compounds 59 and 60 were not successfully prepared. The structure of 3-(4-chloro-3-nitrophenyl)sydnone 45 could be a lead molecule in designing potent chemotherapeutic agents. Compound 63 shared architecture and pharmacophoric characters with known selective COX-2 inhibitors (coxib family) making it a good candidate for designing selective and safe NSAID