W badaniach przeprowadzonych w niniejszej rozprawie potwierdzono antyopioidowe działanie kryptein pochodzenia mysiego, oraz ich pegylowanych odpowiedników.
Potwierdzono również zmianę właściwości farmakodynamicznych pegylowanych peptydów poprzez zwiększoną odporność na działanie proteaz oraz wydłużenie czasu latencji w testach analgetycznych w wyniku podania, zarówno dokomorowego, jak i obwodowego.
W pracy przedstawiono po raz pierwszy przebieg procesu metabolizmu kryptein wywodzących się z prekursora NPFFA w homogenacie mózgu szczura oraz w rozdzielonych frakcjach. Zidentyfikowano także enzymy, które potencjalnie biorą w tym procesie udział.
Zbadano wpływ modyfikacji chemicznej kryptein polegającej na przyłączeniu do C- końca analizowanych peptydów różnej długości glikoli polietylenowych na przebieg procesu metabolizmu. Wykazano, że w wyniku proteolizy pegylowanych kryptein znacznie zmniejszono efektywność ich trawienia, w wyniku czego powstały tylko 2 produkty - N-końcowe tripeptydy.
Streszczenie
100
STRESZCZENIE
Jednym z ważniejszych wyzwań współczesnej medycyny jest efektywne leczenie bólu. Najczęściej stosowanymi związkami do leczenia bólu ostrego i przewlekłego są opioidy, głównie morfina. Ważną rolę w kontroli bólu i analgezji, poprzez interakcje z układem opioidowym, odgrywa także system NPFF. W ostatnim czasie wzrosło zainteresowanie peptydami wywodzącymi się z tego układu, ze względu na ich antyopioidowy charakter. Prekursor NPFFA zwiera kilka biologicznie aktywnych peptydów: NPFF, NPSF i NPAF. Ostatnio wykazano istnienie jeszcze jednej biologicznie aktywnej sekwencji – fragmentu 85-99. Krypteina ta, podobnie jak neuropeptyd FF, hamuje analgezję podanej obwodowo morfiny u myszy i szczurów, a zatem może być sklasyfikowana jako peptyd o działaniu antyopioidowym. Sekwencja ta może więc mieć istotne znaczenie w opracowaniu nowych terapii lekowych.
Celem pracy doktorskiej było zbadanie farmakologicznych funkcji kryptein w mechanizmach bólu. Dodatkowo, porównano działanie tych sekwencji z pegylowanymi analogami. W pracy zbadano również wpływ modyfikacji kryptein na ich właściwości biologiczne, poprzez przyłączenie do ich C-końca różnej długości glikoli polietylenowych. Przy użyciu metod in vitro określono wpływ długości łańcucha polimerowego na szybkość procesu hydrolizy enzymatycznej. Wykazano, że modyfikacja kryptein na C-końcu redukuje liczbę fragmentów peptydowych uwalnianych podczas proteolizy do dwóch N-końcowych tripeptydów.
Dowiedziono również, że mysie koniugaty NPSS-PEG3000, jak i NPSA-PEG3000 hamują analgezję wywołaną obwodowym podaniem morfiny w testach analgetycznych, oraz że efekt ten jest odwracany przez RF9 – selektywnego antagonistę receptora NPFF. Badania te porównano z niemodyfikowanymi krypteinami oraz peptydem NPFF. Antynocyceptywne właściwości peptydu NPSS-PEG3000 zbadano poprzez podanie związków zarówno dokomorowo, jak również obwodowo, za pomocą testu zanurzenia ogona. Ze względu na właściwości farmakodynamiczne, pegylowane peptydy stanowią obiecujące związki, które można wykorzystać podczas opracowywania nowych terapii lekowych.
Dalsza część projektu obejmowała proces degradacji kryptein w homogenacie mózgu szczura oraz zidentyfikowanie enzymów regulujących ten proces. Wykazano istnienie proteaz hydrolizujących szczurzą krypteinę do krótszych, ściśle określonych fragmentów. Za pomocą inhibitorów proteaz metodą in vitro oraz przy użyciu platformy proteomicznej,
101 zidentyfikowano również potencjalne enzymy, które mogą być zaangażowane w te procesy. Opisano 5 proteaz zaangażowanych w proces uwalniania metabolitów krypteiny.
Abstract
102
ABSTRACT
One of the major challenges for modern medicine is an effective pain treatment. The most popular compounds used for the treatment of acute and chronic pain are opioids, especially morphine. Recently, it was demonstrated that also NPFF system play an important role in the control of pain and analgesia by interaction with opioid system. The interest in peptides derived from this system has substantially increased, due to their antiopioid properties.
Rat precursor NPFFA, besides known peptides: NPFF, NPSF and NPAF, contains a yet another bioactive sequence, spanning between positions 85-99. This peptide possesses NPFF-like behavioral activity in rats and mice, and can be classified as an anti-opioid peptide. Thus, a novel cryptic peptide can provide new insights into acute and chronic pain development.
The main aim of this study was to investigate the pharmacological function of crypteins in the pain mechanisms.
The objective of this thesis was also to examine the effect of poly(ethylene glycol) (PEG) conjugation to C-terminal of crypteins, on their biological properties. For this purpose polyethylene glycols of various lengths were attached to the crypteins. The effect of PEG size on the resistance to proteolytic cleavage and pharmacological potency was investigated by in vitro methods. It was shown that modification of crypteins at their C-termini increases resistance to proteolytic attack and reduces the number of peptide fragments released.
It was also observed that both mouse peptides NPSS-PEG3000 and NPSA-PEG3000 inhibit morphine antinociception and this effect was reversed by RF9, a selective antagonist of NPFF receptor. The effects of both pegylated crypteins were compared with unmodified sequences and NPFF itself. The antinociceptive profile of NPSS-PEG3000 was determined via two routes of administration. Evaluations of their activity was performed using a tail immersion test. The pharmacodynamic properties of pegylated compounds make them a promising target that can be used for the rational design of pain treatment.
Further aims of this project were to investigate degradation of crypteins in the rat brain cortex homogenate and to indentify enzymes regulating this process. This study provide an evidence on the existence of proteases, converting crypteins to the shorter, well-defined, bioactive fragments. The possible enzymes involved in this process were identified by inhibitory studies in vitro and by proteomic tools. Finally 5 differential enzymes were described as potential proteases releasing cryptein metabolites.
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