Chronic pain is a significant healthcare problem. It is disabling and diminishes quality of life. Opioids, such as morphine, remain a primary pharmacologic management for chronic pain. Opioids act at mu opioid receptors (MOPr) in the rostroventral medial medulla (RVM) to produce their analgesic effect. The RVM is a critical relay in pain inhibitory and facilitatory pathways of pain modulation. Furthermore, chronic inflammatory pain, produced by CFA hindpaw injection, leads to adaptive changes in the RVM that change the balance of these pathways and increase the potency of opioids. MOPr are known to produce their effects via Gi/o proteins. Pretreatment of several pain modulatory regions with pertussis toxin (PTX) effectively attenuates the antinociceptive effects of MOPr agonists, such as DAMGO. In the RVM, PTX effectively reduced DAMGO stimulated GTPãS binding in uninjured rats. However, despite their effective inactivation of Gi/o proteins, PTX did not diminish the antinociceptive effects of DAMGO in the RVM of uninjured rats. In contrast, in rats with a chronic inflammatory injury, PTX completely abolished the antinociceptive effects of DAMGO. These results suggest a transition from Gi/o independent to Gi/o dependent mechanisms following CFA treatment. In addition, the anti-hyperalgesic effects of DAMGO were not inhibited by PTX, suggesting that DAMGO produces anti-hyperalgesia and antinociception by different mechanisms. In the RVM, MOPr are present both postsynaptically and presynaptically. Postsynaptic MOPr are thought to produce antinociception by activating GIRK channels, resulting in hyperpolarization and inhibition of pain facilitatory neurons. Indeed, inhibition of GIRK channels in the RVM, via microinjection of tertiapin-Q, attenuated the antinociceptive effects of DAMGO in uninjured rats, providing the first behavioral evidence that MOPr agonists produce analgesia via this proposed mechanism. Interestingly, however, tertiapin-Q did not block the anti-hyperalgesic effects of DAMGO, nor did it diminish the antinociceptive effects of DAMGO in the contralateral hindpaw of CFA treated rats. Furthermore, these differential effects of tertiapin-Q in the uninjured and injured rats are not the result of transcriptional down regulation of GIRK channels in the RVM. Finally, tertiapin-Q alone in the RVM produced a modest antinociception in uninjured rats, providing the first evidence of constitutive GIRK channel activity in the RVM and demonstrating a role for these in pain modulation. Presynaptic MOPr are thought to produce antinociception by decreasing GABA release onto pain inhibitory neurons. Indeed, microdialysis studies demonstrated that levels of GABA release were decreased in response to DAMGO perfused into the RVM, as well as to high potassium after perfusion of DAMGO. However, they were not decreased in rats after CFA treatment. This suggests that chronic inflammatory injury alters the presynaptic actions of MOPr agonists in the RVM. Interestingly, levels of GLU release where not altered by DAMGO in uninjured or injured rats. Moreover, basal levels of GLU and GABA were also unaltered by CFA treatment. In conclusion, although MOPr mediate their antinociceptive effects in other pain modulatory regions via Gi/o proteins, this is not the case in the RVM during an uninjured state. However, MOPr-induced antinociception transitions from Gi/o independent to Gi/o dependent mechanisms after CFA treatment. Additionally, these results support both the presynaptic and the postsynaptic postulates by which MOPr agonists are thought to produce their analgesic effects. However, although CFA treatment alters the activity of neurons in the RVM and promotes changes that result in an enhanced anti-hyperalgesic and antinociceptive response to DAMGO in the RVM, neither the postsynaptic nor the presynaptic mechanism, in isolation, seem to account for this enhancement.

The use of nanocarriers is an intriguing approach in the development of efficacious treatment for brain disorders. The aim of the conducted research was to evaluate and quantify the impact of a liposomal nanocarrier formulation on the brain drug delivery. A novel approach for investigating the blood-brain barrier transport of liposomal DAMGO is presented, including in vivo microdialysis in rat, a high quality LC-MS/MS bioanalytical method and pharmacokinetic model analysis of the data. Factors limiting the brain distribution of the free peptide DAMGO were also investigated. Microdialysis, in combination with plasma sampling, made it possible to separate the released drug from the encapsulated and to quantify the active substance in both blood and brain interstitial fluid over time. The opioid peptide DAMGO entered the brain to a limited extent, with a clearance out of the brain 13 times higher than the clearance into the brain. The brain to blood ratio of unbound drug was not affected when the efflux transporter inhibitors cyclosporine A and elacridar were co-administered with DAMGO. Nor was the transport affected in the in vitro Caco-2 assay using the same inhibitors. This indicates that DAMGO is not transported by P-glycoprotein (Pgp) or breast cancer resistant protein (Bcrp). The blood-brain barrier transport was significantly increased for DAMGO when formulated in liposomes, resulting in 2-3 fold higher brain to blood ratio of unbound DAMGO. The increased brain delivery was seen both for glutathione tagged PEGylated liposomes, as well as for PEGyalted liposomes without specific brain targeting. The improvement in brain delivery was observed only when DAMGO was encapsulated into the liposomes, thus excluding any effect of the liposomes themselves on the integrity of the blood-brain barrier. Modeling of the data provided additional mechanistic understanding of the brain uptake, showing that endocytosis or transcytosis of intact liposomes across the endothelial cell membranes were unlikely. A model describing fusion of the liposomes with the luminal membrane described the experimental data the best. In conclusion, the studies presented in this thesis all contribute to an increased understanding of how to evaluate and improve brain delivery of CNS active drugs and contribute with important insights to the nanocarrier field.

The first nociceptive synapse in the spinal cord dorsal horn represents an important site, where nociceptive synaptic transmission can be modulated under pathological conditions. One of the modulatory mechanism involves activation of the transient receptor potential vanilloid 1 (TRPV1) that is expressed on central terminals of primary nociceptive neurons, where it regulates release of neurotransmitters and neuromodulators. Previous studies suggested that changes in TRPV1 activity may be related to effects of chemokine CCL2 (C-C motif ligand 2) and may be also involved in synaptic transmission modulation after µ-opioid receptors (MOP-R) activation. Because CCL2 receptors CCR2 often co-localize with TRPV1 and MOP-R, the goal of this work was to studypossible interactions of these receptors on the pre-synaptic endings of primaryafferents in the spinal cord dorsal horn and their role in nociceptive signalling under pathological conditions. The presented thesis focused on the effect of CCL2 during peripheral neuropathy and its interference with µ-opioid receptor activation. To studysynaptic transmission at the spinal cord level, patch-clamp recordings of excitatory post-synaptic currents (EPSC) in superficial spinal cord dorsal horn neurons in acute lumbar spinal cord slices from rats was used....

Drug addiction, opiates respectively, is a social problem which seriousness is currently on the rise. One of key elements causing addiction is tolerance to increasing doses of drug causing abstinence syndrome during withdrawal and craving. Opioid receptors are members of a large group of receptors coupled with heterotrimeric G-proteins (GPCR), whose properties can be investigated using agonist- stimulated binding [35 S] GTPγS. Many extracellular signals are transferred into a cell through GPCR. Opioid receptor agonists inhibit the activity of adenylyl cyclase and are coupled with G-protein group Gi/Go. This work is devoted to the study of changes in isolated plasma membranes of rat forebrain containing opioid receptors of healthy subjects with membranes acquired from morphine addicted subjects. The rats were long-term morphine treated in increasing doses, to develop the dependency. The comparison is done firstly by binding of [3 H]ouabain to Na,K-ATPase, which proves to be a negative standard of changes, secondly by binding [35 S]GTPγS to G-proteins, thereby providing the functional activity of G-protein in stimulating the binding by the agonist of δ-opioid receptors DADLE or agonist of µ-opioid receptors DAMGO. Furthermore, it has been studied the influence of prostaglandin E1 on binding [35...

-5- ABSTRACT A huge effort has been put in determining the mechanism of the development of tolerance and dependence in context of clinical use of morphine for treatment of severe pain. Understanding of this mechanism would help to design new and more efficient pharmaceuticals. This diploma paper discus the opiate receptors with a special focus on long-term effect of chronic morphine treatment, which was determined using a radioligand binding assays with a non-selective antagonist [3 H]Diprenorphine. One of the goals of this work was to create and optimise a method for preparation of pure plasma membranes from rat cortex using percoll gradient. There were five groups, which differed in the length of morphine treatment: ten days (M-10), twenty-eight days (M-28), ten days with seven days of regression (RM-10 twenty-eight days with seven days of regression (RM-28) and a control group (K). The loss of total opioid receptor number was noticeable after ten days and grew slightly during continuous morphine treatment and kept lowering in the period of regression. The total loss was approximately 30% of the control binding. The equilibrium dissociation constant (Kd), thus the affinity of [3 H]Diprenorphine wasn't significantly different among the groups. Morphine acts through µ-opioid receptor, that's why there was a...