Since long, research has been conducted in order to find and optimal painkiller which relieves selectively the pain without any side effects or alterating psychologycal function of the brain. Another urgent need is the discovery of the molecular mechanism of opioid tolerance and dependence.

OPIOIDS
Opioids can be defined as all the natural, synthetic, semi-synthetic morphine agonist, antagonist and morphomimetic peptides (Table 1.). The term opiate marks only the morphomimetic derivatives of the opium (poppy) alkaloids such as morphine and codein semi-synthetic derivatives (e.g. heroin).

Table 1. Endogenous and synthetic opioids.

In the body there are naturally syntethized endogen opioid molecules from their precursors (Figure 1.) such as proopiomelanocortin (POMC), proencephalin and prodinorphin. Endogen opioid peptides have an N-terminal message tetrapeptide sequence: Tyr-Gly-Gly-Phe and the first amino acid is essential for the physiological activity of the opioid peptides.
Absorbtion can be relalized by subcutan, intramuscular, nasal epithelium, gastrointestinal tract, transdermally (lipofil), epidurally, intrathecally. Per os, most of the compound are not so effective because they go throught first pass mechanism in the liver. They metabolise to polar compounds and the kidney clears off the body. Metabolits also have physiological effects e.g. heroin metabolises to morphine having analgetic effect. Entering the body they bind to plasm proteins (e.g. 1/3 of morphine, 8o% of fentanyl). Leaving the blood they accumulate in the lungs, the liver, the kidney, the spleen, the muscle and the fat tissue.

Figure 1. Precursors of endogenous opioid ligands. ACTH = adrenocortikotrop hormon, LPH = lipotrophin, MSH = melanocyte stimulating hormon

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SIGNAL TRANSDUCTION
1, Opioid receptors
Opioid ligands have different affinity to opioid receptors having seven transmembrane segments (G-protein coupled receptors) (Figure 2.) (affinity: the strength of interaction between the opioid ligands and the receptor expressed by the concentraction of the ligand that can bonds 50 % of all receptors.)
Many endogen opioid compounds produced by the body act on different types and subtypes of opioid receptors (mu, delta, kappa) causing various effects (Table 2.). Types and subtypes of receptors can be differentiated by using certain antagonists. For example, naloxazone blocks μ1 subtypes resulting in cessation of analgetic effect of morphine but cannot block the μ2 subtype having no effect on breathing depression.

Table 2. Opioid receptor types.
(*peptides)

Oioid receptors are abundant in those parts of the central nervous system which involved in pain transmission such as dorzal horn of spinal cord, periaqueductal area (PAG) and in the limbic system (thalamus, amygdala, hippocampus). In the periferial nervous system, we can find opioid receptors in the gastrointestinal plexus, vas deferens, adrenal medulla, heart and placenta. Localization of opioid receptors are limitid to synaptic areas and also can be find in organelles such as lysosomes, endoplasmatic reticulum and Golgi membrane.

2, G-proteins
Opioid receptors use G-proteins (guanine-nukleotide bindig regulator protein) associated to the intracellular surface of the cell membrane in order to trasfer the signal into the cell. G-proteins have four family (Gs, Gi, Gq, G12) constructed by different types of α, β, γ subunits. Opioid receptors activates Gi types. Upon ligand binding, the α subunit binds GTP and leave the lignad-receptor complex and also the βγ subunit. The α subunit stimulate the effectors then hydrolyze GTP regenerating the cycle (Figure 2).
Opioid receptors can be found in pre-synaptic and post-synaptic regions. Pre-synaptically, they decrease the firing of neurons by inhibition of Ca-channels and stimulations of K-channels resulting in the inhibition of the release of neurotransmitters and hormons such as acetil-choline, noradrenaline, dopamine, vazopressin and somatostatine. Post-synaptically, they inhibit adenylyl-cyclase which catalizes the transformation of ATP to cAMP regulating the activity of several cAMP-dependent protein kinase A resulting in altered metabolism and transcription (CREB dependent) of the cell.
Stimulation of G-proteins results in activation of phospholipse C (PLC) leading to formation of inozitol-1,4,5-triphosphate (IP3) and diacil-glicerol (DAG) which stimulate the release of calcium from the endoplasmatic reticulum and activate protein-kinase C (PKC), respectively. Opioids also have an effect of MAPK (mitogen-activated protein kinase) cascade with their βγ subunit by activation of ERK (extracellular signal regulated kinase).



Figure 2. Signal transduction of opioid receptors.

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PHYSIOLOGICAL EFFECTS
The effects include the most important analgetic effect mediated mostly by mu receptors. There are differences between drugs in their itrinsic activity (efficacy) which shows the maximal effect regardless of the dosage. (That drug has higher efficacy which has higher effect with the administration of the same dosage. This can be explained with that drugs with lower efficacy need to bind to more receptors for the same effect, and also different half-life of the molecule in the plasm can be a factor.)
The mechanism, these receptors alleviate pain can be realized by spinal and supraspinal level with either incrising the threshold of the pain stimuli or modulate the emotional reactions. These information based on the localization of opioid receptors in areas of the central nervous system which are parts of the monoaminerg analgetic pathways such as the dorsal horn of the spinal cord, thalamus, amygdala, hippocampus, periaquaeductal area and brain stem nuclei (nucleus raphe magnus, nucleus paragigantocellularis).
The descending monoaminerg analgetic pathway pulls out of the periaqueductal gray matter (PAG) and brainstem nuclei (nucleus raphe magnus, nucleus paragigantocellularis) and heading the dorsal horn of the spinal cord where it activates inhibitory opioid interneurons resulting in pre- or post-synaptic inhibiton of nociceptive neurons of the dorsal horn. This system is inhibited by GABAerg neurons in the PAG resulting in free pain transmission through the tractus spinothalamicus lateralis. This inhibitory effect can be inhibited by opiate sensitive inhibitory neruons resulting in analgezia (Figure 3.). .



Figure 3. Monoaminerg analgetic pathway. (Aα, Aβ, C : afferent fibres, (+): stimulation, (-): inhibition)


Other physiological effects include

• euphoria=”well-being”
• sedation
• slowness of mental functions=“mental clouding”
• breathing depression (decreased sensitivity of the medullar respiratory center to carbonic-dioxide) resulting in decreased frequency (3-4/min) and depth of pulmonary ventilation and increasing arterial pCO2.
• antitussive effect (depression of the medullar antitussive centrum)
• emesis (area postrema)
• pupillary constriction (stimulation of nucleus oculomotorius/ Edinger-Westfal)
• cardiovascularis effects: hypotony
• diuresis
• gastrointestinal effects: obstipatio (inhibition of acetil-choline release from the cholinergic neurons of the Auerbach and Meissner plexus; and partially central original effect)
• neuroendokrin effect (stimulation of prolaktin, adrenocorticotrop hormon, somatotrop hormon release; and inhibition of oxitocin, antidiuretic and lutheotropic hormon release)

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ADDICTION
Cronic usage of opioids leads to addiction such as tolerance and dependence.
Tolerance means that repeted (chronic) drug administration causes decreased analgetic effect that is higher dose of the drug needs to be introduced for the same effect. Development of tolerance depends on the dosage, the way of administration and the time between administrations. There is difference in the velocity and the extent of the development of tolerance between different opioid compounds, which can be explained with their specificity to different subtypes of opioid receptors. Cross-tolerance can be developed between opioid molecules that is, tolerance can develop to other drugs having similar pharmacological effects than the administered drug.
Dependence can be psychological and physical. Physical and behavioural signs of opiate withdrawal include rhinorrhea, lacrimation, salivation, chills, hyperventilation, midriasis (pupillary dilation), nausea, emesis, diarrhea and painful dysphoric state.
Most of the clinically used opioids (morphine, metadone, codein, fentanyl) bind to µ opioid receptors and have high abuse potential which is the ability of a drug to cause immediate well being (“high”).
The mechanism of above mentioned phenomenons are in the focus of research up to the present. The hypothesis include receptor desensitization (dissociation of receptor and G-protein), internalization (cell surface receptors internalized to microsomes) and down-regulation (number of receptors decreases). The mechanism of internalization can be caused by G-protein associated protein kinases phophorilating the C-terminal Ser and Thr amino acids of the receptor upon ligand binding resulting in association of arrestin and the formation of clathrin coated vesicles.

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DESIGN OF OPIOID PEPTIDES
High selectivity and affinity of a peptide can be achieved by decreasing their flexibility, so the binding of the compound to the different type of receptors become stronger or ceased, the selectivity and potency can be increased. In order to decrease the flexibility, the compound can be cyclize resulting in decreased mobility of the amino acids, so the secondary structure of the peptide is fixed. There are four ways to cyclize a peptide: conection/binding of the amino and carboxy terminals, conection of the amino or the carboxy terminal to a side chain and conection of two side chains. Another way to stabilize a molecule is the substitution of amino acids with modified amino acids. The non-natural amino acids decrease the flexibility of the Φ (Nα-Cα), Ψ (Cα-C(O)) and the χ (C(O)-NH) bonds and the side chains. There are several ways for the modification of an amino acid resulting in α-metil-amino acid, α-aminocicloalkan-carbonic acid (Acnc), Nα-Cα cyclized amino acid, Nαmetil-amino acid, β-and γ-amino-cicloalkan-carbonic acid (β- Acnc, γ-Acnc), α,β-unsaturated amino acid (dehydroamino acid), β,β-dimetil és β-metil amino acid, β-substituted-2,3-metano-amino acid, N-Cα és Cα-Cδ cyclized aromatic amino acid. Another way is constructing a molecule that mimicking the secondary structure of the biological active molecules.

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