L’objectif de cette thèse a été d’explorer l’association entre des variants génétiques impliqués dans la variabilité interindividuelle de la réponse au traitement par les opioïdes et la survenue d’effets secondaires. Nous avons recherché si des facteurs génétiques influençaient la réponse à la morphine dans le traitement de la douleur aigüe. L’allèle T du polymorphisme c.3435C>T d’ABCB1 est significativement associé aux doses de morphine et à la survenue de nausées dans une étude pilote chez des patients libanais en post-opératoire. Ensuite, l’étude de la réponse à la morphine chez des patients présentant une obésité morbide a montré que la fréquence de l’allèle 118G d’OPRM1 et le seuil de sensibilité à la douleur sont plus élevés que chez les patients à poids normal. La recherche des facteurs influençant la variabilité de la réponse à la méthadone chez des patients toxicomanes traités pour substitution a mis en évidence deux polymorphismes (TaqIA de DRD2/ANKK1 et c.118A>G d’OPRM1) significativement associés à la dose maximale de méthadone administrée. Trois facteurs sont associés au phénotype CYP3A, impliqué dans le métabolisme de la méthadone: la prise de benzodiazépines, l’infection par le VIH et un polymorphisme de POR, gène qui code une oxydoréductase. De plus, le travail mené sur les effets secondaires cardiaques de la méthadone a permis de mettre en évidence trois facteurs corrélés à l’allongement de l’espace QT : la dose, l’infection par le VIH et le polymorphisme p.Lys897Thr de KCNH2 codant pour le canal potassique hERG. Ces travaux contribuent à démontrer l’intérêt d’intégrer des données cliniques et génétiques dans la prescription personnalisée des opioïdes. / The main objective of this thesis was to explore the association between genetic variants potentially involved in inter-individual variability of opioids’ response and side effects in the treatment of pain and opiate substitution treatment. Initially, we investigated whether genetic factors influence the response to morphine in the treatment of acute pain. The T allele of the polymorphism ABCB1 c.3435C>T was significantly associated with doses of morphine and the outcome of nausea in a pilot study in Lebanese postoperative patients. Next, we examined the response to morphine in patients with morbid obesity (BMI>40); in these patients, the frequency of the 118G OPRM1 allele and the pain threshold appeared to be higher than in patients with normal BMI. The search for factors influencing the variability in response to methadone in patients treated for drug substitution showed that two polymorphisms (DRD2/ANKK1 TaqIA and OPRM1 c.118A>G) were significantly associated with the maximum doses of methadone. In addition, three factors were associated with the CYP3A phenotype, involved in the metabolism of methadone: the use of benzodiazepines, HIV infection and a polymorphism in POR gene, which encodes an oxidoreductase. Finally, the exploration of the cardiac side effects of methadone has highlighted three factors significantly correlated with QT prolongation: methadone doses, HIV infection and the polymorphism p.Lys897Thr in KCNH2 encoding a cardiac potassium ion channel. This work demonstrates the importance of integrating both clinical and genetic data in the personalized prescription of opioids.

The acute administration of an analgesic dose (1mg/kg) of morphine sulfate to conscious and hydrated rats produced a pronounced antidiuretic response. This was accompanied by an increase in urine osmolality and a decrease in free water clearance in the normal and Brattleboro rats heterozygous for the diabetes insipidus trait. The response was comparable to that of an exogenous dose of vasopressin. Antidiuresis was also observed in the homozygous D.I. rats. Since these rats are incapable of synthesizing vasopressin, the antidiuresis must be mediated by other mechanism(s). When arterial blood pressure was monitored, it was found that the mean arterial pressure decreased sharply immediately after the morphine injection in all animals. This can account for the antidiuresis in the homozygous D.I. rats. In the normal and heterozygous D.I. animals, it is quite possible that both morphine-mediated release of vasopressin and hypotension are responsible for the decrease in urinary flow. In addition, hypotension per se may act as a stimulus for vasopressin release. To study the chronic effects of morphine, rats were rendered tolerant and physically dependent by two means: multiple injections and pellet implantation of morphine sulfate. In contrast to the antidiuretic effects of acute morphine administration, chronic treatment resulted in polyuria. Using a vasopressin radioimmunoassay (RIA), it was found that rats implanted with a morphine pellet for 3 days had a significantly lower neurohypophyseal store of vasopressin (744.3 ± 27.9 ng, n=6) as compared to the placebo pellet implanted controls (1024. 1 ± 66.0 ng, n=6). This depletion was replenished as the animals developed tolerance to the drug.. abrupt withdrawal of the drug from physically dependent animals also produced a significant depletion of the neurohypophyseal vasopressin stores - from 902.4 ± 37.0 ng (n=6) to 638. 3 ± 36.0 ng (n=6). In contrast to rats implanted with morphine pellets, no significant changes in the neurohypophyseal vasopressin stores were observed in those injected daily with morphine for 2 weeks. Withdrawal from the drug in these animals also did not produce any detectable changes in the vasopressin stores. A withdrawal symptom, reduction in body weight, monitored after 24 hr of abstinence suggested that the degree of physical dependence in these animals is very light. ³H-naloxone binding performed on whole brain homogenate of rats injected with morphine for 2 weeks revealed no significant changes in the number of binding sites (q). The affinity constant (Kd) was augmented from a control value of 5.33 nM to 22.37 nM in the morphine-injected rats. The changes in g and Kd suggested the presence of morphine in the whole brain homogenates. Moreover, the Kd was restored to the control value in animals withdrawn from the drug for 24 hr. Vasopressin or oxytocin iid not have any direct effect on the ³H-naloxone binding. Thus, it is unlikely that the facilitation of morphine tolerance by the neurohypophyseal peptides is mediated by their direct action on the opiate receptor. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

Morphine-6-glucuronide is a minor metabolite. representing 5% of an administered dose of morphine. The metabolite has analgesic activity exceeding that of morphine and may contribute to analgesia following morphine administration. The aims of the study were to attempt to identify the reasons behind the improved activity of morphine-6-glucuronide over the parent compound and to examine a series of 6-substituted compounds, based on 6-substituted benzoate esters, as potential mimics of morphine-6-glucuronide. Morphine-6-glucuronide was seen to have similar affinity to morphine at l1-opioid receptors as assessed by ligand-binding assays in mouse brain homogenates. However a three-fold improved affinity at S-opioid receptor binding sites was observed and a ten-fold reduction in affinity at K-opioid sites. Using in vitro bioassay systems the glucuronide showed a two-fold improved potency over morphine in both the guinea-pig ileum and the mouse vas deferens preparations. Following in vivo (s.c.) administration in the mouse the glucuronide was seen to be equipotent with morphine in the tail-flick test, but was of much longer duration, lasting up to 9 hours. Exvivo binding assays confirmed that morphine-like material was still present in the central nervous system six hours after administration of the glucuronide, but was not observed at a similar time after morphine administration. Activity was retained if the hydroxyl groups of the sugar moiety of the glucuronide were protected as esters. In contrast the more prevalent morphine metabolite morphine-3-glucuronide was inactive in all in vitro and in vivo tests used and did not antagonise morphine in vitro or in vivo. A group of 3-substituted derivatives containing saturated and unsaturated substituents did show affinity for opioid receptors but no agonist activity of the compounds could be demonstrated in vitro. A series of synthetic 6-substituted compounds showed a variety of affinities for, and agonist potencies at, opioid receptors, though low affinity at Kopioid receptors was a general finding. For example, morphine-6- nitrobenzoate was l1-opioid receptor preferring, while morphine-6- phthalate had improved O-opioid receptor affinity and acted via Il-opioid receptors in the mouse vas deferens and in vivo. However the compounds were weaker than morphine and the duration of action in vivo was shorter than morphine-6-glucuronide. The conclusions from these studies are that morphine-6-glucuronide and morphine have similar in vitro affinities at the l1-receptor, although morphine-6-glucuronide has somewhat improved binding affinity for Il receptor sites, it has less affinity for K receptor sites. Pharmacokinetic reasons are probably responsible for the improved activity and duration of action of morphine-6-glucuronide over morphine. None of the synthetic compounds examined are potentially useful as direct mimics of the glucuronide because morphine-6-glucuronide is more potent and has a longer duration of action than the synthetic derivatives, though alteration at the 6-position of the morphine nucleus can lead to dramatic changes in selectivity and potency of ligands for the differing opioid receptors.

published_or_final_version / Pharmacology / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

The opiate alkaloid (-)-morphine and the Amaryllidaceae alkaloid (-)-galanthamine are well known for their analgesic and anticholinergic properties, respectively. The chemical feature that connects these two molecules is that they are both biosynthesized from an ortho-para phenolic oxidative coupling. Attempts to mimic this aesthetic chemistry in the laboratory for the practical production of these alkaloids have not resulted in good yields of these compounds and there is a lot of scope for improvement. Despite the enormous amount of work devoted to this area, the simple para-alkylation of an appropriately substituted phenol derivative to generate a cross conjugated 2, 5-cyclohexadienone has not been reported. This strategy would avoid the low-yielding phenolic oxidation reaction and the product would merely require a double reductive amination of the aromatic aldehyde and the latent aldehyde (in the acetal) to produce narwedine, the synthetic precursor to (-)-galanthamine. On the other hand, the same intermediate can be elaborated to (±)-morphine via a Henry reaction, followed by reduction and reductive amination. Following the aforementioned methodology, we have successfully completed the synthesis of both these alkaloids via the common intermediate, a 2, 5-cross-conjugated cyclohexadienone. A demonstration of the use of this methodology towards achieving an enantioselective synthesis of these compounds has also been made. The overall yield of the 8 step procedure for galanthamine proceeds in 65% yield, which is approximately five times the yield of the current manufacturing process for this molecule. The synthesis of (±)-morphine, for the first time, allows access to codeine without having to reduce codeinone and, with an overall yield of 20% for the 14 step process, makes this the shortest synthesis of morphine. / text

Thermoregulatory, analgesic and respiratory depressive responses as well as tolerance to morphine were investigated in young (3 to 5 months), mature (10 to 12 month) and senescent (26 to 28 month) male Fischer 344 rats. The thermoregulatory system of senescent rats was not able to maintain body temperature in hot and cold environments as well as the thermoregulatory system of young rats. Additionally, senescent rats had basal rectal temperatures which were approximately one degree lower than basal temperatures in young rats. Subcutaneous morphine caused biphasic effects on body temperature ie. hyperthermia at low doses and hypothermia at high doses. Senescent rats were less responsive to the hypothermic effects of subcutaneous morphine than young rats, but this was not due to decreased subcutaneous blood flow or inability to lose heat. Morphine injections intracerebroventricularly showed no age-related differences. A two site model for the actions of morphine on thermoregulation was proposed and it was suggested that the age-related differences are due to changes in a non periventricular site. Previously reported increased lethality of intravenous morphine in aged rodents was shown to be due to decreased respiratory reserve rather than increased sensitivity to respiratory depression. Senescent rats were also found to acquire tolerance to the thermic effects of morphine less readily than young rats regardless of the route of administration. Normal aging has been characterized as a decrease in adaptability, and it was suggested that senescent rats were less able to compensate for the thermic effects of morphine as well as young rats. In order to determine the mechanisms of decreased adaptability, neurotransmitters proposed to be involved in thermoregulation were injected intracerebroventricularly in morphine tolerant rats. The results suggested a shift from catecholaminergic to cholinergic transmitters with aging.

Chronic pain affects approximately 100 million Americans. Opioids are the mainstay therapy for the treatment of chronic pain. While physicians and patients alike are apprehensive about using opioids due to their side effects including respiratory depression and addiction, 259 million opioid prescriptions were written in 2012. Although opioids are the most efficacious available analgesics, they increase both positive and negative reinforcement, ultimately leading to addiction. The pro-nociceptive neurotransmitter, Substance P (SP) and its corresponding receptor (NK₁R), are not only found on pain pathways to promote pain but also found in the ventral tegmental area associated with dopamine neurons. Studies have shown that Substance P can potentiate positive reinforcement of opiates and may play a role in opioid reward. Here using in vivo microdialysis, we show that systemic morphine significantly increases SP release in the VTA, an effect mediated by ventral midbrain GABAergic neurons. Substance P administered to the VTA results in a significant increase in dopamine release in the nucleus accumbens (NAc). Using CRISPR-Cas9 knockdown of NK₁R in the VTA we prevent the induction of opiate reward as tested using a conditioned place preference paradigm (CPP). Finally, we developed a novel opioid agonist/NK₁R antagonist bifunctional compound, TY032, which inhibits acute and chronic pain in male rats. Importantly, TY032 microinjection into the VTA did not increase extracellular dopamine release in the NAc and did not produce a positive CPP score. These data indicate dual targeting of the dopamine reward circuitry and pain pathways with multifunctional opioid-NK₁R compounds may be an effective strategy in developing future analgesics that lack the potential for abuse.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Morphine is one of the most widely used drugs for the treatment of pain but its clinical efficacy is limited by adverse effects including persistent constipation and colonic inflammation. Morphine-induced colonic inflammation is facilitated by microbial dysbiosis and bacterial translocation. In this study, we demonstrate that secondary inflammation and persistent constipation are modulated by enteric glia. In chronic morphine treated mice (75 mg morphine pellet/5 days), ATP-induced currents were significantly enhanced in enteric glia isolated from the mouse colon myenteric plexus. Chronic morphine resulted in significant disruption of the colonic epithelium and increased Il-1β in the myenteric plexus. The increase in ATP-induced currents, IL-1β expression and ATP release were also observed in isolated glia treated with lipopolysaccharide (LPS) consistent with bacterial translocation as a potential mediator of chronic morphine-induced inflammation. These effects of LPS were reversed by carbenoxolone, a connexin43 hemichannel blocker. In-vivo treatment with carbenoxolone (25 mg/kg) prevented 1) ATP-induced currents in enteric glia, 2)the decrease in neuronal density, and 3) colonic inflammation in chronic morphine treated mice. Inhibition of connexin43 in enteric glia also reversed morphine mediated decrease in gastrointestinal transit. These findings indicate that bacterial translocation-induced enteric glial activation and inflammation is a significant modulator of morphine-related constipation.