ATP-gated P2X3 and P2X7 receptors : new mechanisms of anti-nociception by new ATP-analogues

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DISSERTATIONS | YEVHENIIA ISHCHENKO | ATP-GATED P2X3 AND P2X7 RECEPTORS: NEW... | No 438

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THE UNIVERSITY OF EASTERN FINLAND Dissertations in Health Sciences

ISBN 978-952-61-2647-0 ISSN 1798-5706

Dissertations in Health Sciences

YEVHENIIA ISHCHENKO

ATP-GATED P2X3 AND P2X7 RECEPTORS:

NEW MECHANISMS OF ANTI-NOCICEPTION BY NEW ATP-ANALOGUES

Chronic pain is one of the most debilitating condition, which strongly reduces quality of life and often is undertreated in clinic. Future development of new successful pain manage-

ment strategies require detailed exploration of the specific molecular mechanisms in pain production and perception. This thesis demon- strates the prospective analgesic action of novel ATP-analogues through the inhibition of

pro-nociceptive P2X3 receptors. Furthermore, we showed important functional properties of the 288 residue in the left flipper of the P2X7

receptor, which take important part in the inflammation and pain development.

YEVHENIIA ISHCHENKO

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ATP-gated P2X3 and P2X7 receptors: new mechanisms of anti-nociception by new

ATP-analogues

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YEVHENIIA ISHCHENKO

ATP-gated P2X3 and P2X7 receptors: new mechanisms of anti-nociception by new

ATP-analogues

To be presented by permission of the Faculty of Health Sciences, University of Eastern Finland for public examination in Mediteknia (MD100)., Kuopio, on Thursday, November

23^th 2017, at 13.00

Publications of the University of Eastern Finland Dissertations in Health Sciences

Number 438

A.I. Virtanen Institute for Molecular Sciences, Faculty of Health Sciences,

University of Eastern Finland Kuopio

2017

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Grano Oy Kuopio, 2017 Series Editors:

Professor Tomi Laitinen, M.D., Ph.D.

Institute of Clinical Medicine, Clinical Physiology and Nuclear Medicine Faculty of Health Sciences

Professor Hannele Turunen, Ph.D.

Department of Nursing Science Faculty of Health Sciences Professor Kai Kaarniranta, M.D., Ph.D.

Institute of Clinical Medicine, Ophthalmology Faculty of Health Sciences

Associate Professor (Tenure Track) Tarja Malm, Ph.D A.I. Virtanen Institute for Molecular Sciences

Faculty of Health Sciences

Lecturer Veli-Pekka Ranta, Ph.D. (pharmacy) School of Pharmacy

Faculty of Health Sciences Distributor:

University of Eastern Finland Kuopio Campus Library

P.O.Box 1627 FI-70211 Kuopio, Finland http://www.uef.fi/kirjasto ISBN (print): 978-952-61-2647-0

ISBN (pdf): 978-952-61-2648-7 ISSN (print): 1798-5706

ISSN (pdf): 1798-5714 ISSNL: 1798-5706

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Author’s address: A.I. Virtanen Institute for Molecular Sciences University of Eastern Finland

70211 KUOPIO FINLAND

Supervisors: Professor Rashid Giniatullin, M.D., Ph.D.

A.I. Virtanen Institute for Molecular Sciences University of Eastern Finland

KUOPIO FINLAND

Adjunct Professor Jorma Määttä, Ph.D.

Department of Cell Biology and Anatomy University of Turku

TURKU FINLAND

Professor Jukka Mönkkönen, Ph.D.

Department of Pharmaceutical Chemistry University of Eastern Finland

KUOPIO FINLAND

Reviewers: Docent Hanna Zemkova, MD, Ph.D.

Dept. Cellular and Molecular Neuroendocrinology Institute of Physiology ASCR

PRAHA

CZECH REPUBLIC Docent Sari Lauri, Ph.D.

Department of Biosciences University of Helsinki HELSINKI

FINLAD

Opponent: Professor Antti Pertovaara, M.D., Ph.D.

Dept. Physiology, Faculty of Medicine University of Helsinki

HELSINKI FINLAND

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Yevheniia Ishchenko

ATP-gated P2X3 and P2X7 receptors: new mechanisms of anti-nociception by new ATP-analogues.

University of Eastern Finland, Faculty of Health Sciences.

Publications of the University of Eastern Finland. Dissertations in Health Sciences Number 438. 2017, 66p

ISBN (print): 978-952-61-2647-0 ISBN (pdf): 978-952-61-2648-7 ISSN (print): 1798-5706 ISSN (pdf): 1798-5714 ISSNL: 1798-5706

ABSTRACT

Chronic pain is a very debilitating condition accompanying a wide range of etiologically different disorders. Unfortunately, chronic pain is often the clinical condition which remains unresolved. One of such conditions is a bone pain, which remains largely intractable due to specific bone morphology and complex nociceptive mechanisms. Detailed exploration of the specific molecular mechanisms in pain production and perception is a prerequisite for the development of new therapeutic strategies.

ATP-gated P2X3 receptors are primarily expressed in nociceptive neurons in various tissues. These receptors are considered as one of the most important players in pain transmission. In addition, pro- inflammatory P2X7 receptors, which are expressed in cells with an immune origin, are implicated in chronic pain processes due to their ability to regulate the release of multiple inflammatory agents.

Thus, both P2X3 and P2X7 receptors are considered as important therapeutic targets in pain relief. In the current study, a variety of methods were applied to investigate the receptor-mediated mechanisms of novel ATP-analogues as potential anti-nociceptive agents. In addition, we examined the basic functional properties of the P2X7 receptor with a special focus on the key region of this receptor called the left flipper.

Study I of this thesis explored the action of the two stable synthetic compounds, AppNHppA and AppCH2ppA, on pro-nociceptive P2X3 receptors. We found that these polyphosphates specifically and potently inhibited rat and human P2X3 receptors. An assessment of the good anti-nociceptive potency of these compounds provided a translational perspective. We also identified that the underlying molecular mechanisms in this inhibition involved a high-affinity desensitization of the receptor.

In study II, we elucidated the anti-nociceptive effects of the different type of polyphosphates and the nitrogen-containing bisphosphonates. We found that ApppI, which is an endogenous ATP-analogue, provided the most intense and specific inhibition of the P2X3 receptors with no effects on other P2X receptors. We also revealed that the inhibitory action of ApppI was calcium-dependent and particularly intense on human P2X3 receptors.

Study III provided a mechanistic explanation for the functional role of different polar and non-polar amino acids at position 288 in the left flipper region of the rat P2X7 receptor.

In summary, this thesis investigated the therapeutic potential of novel agents acting on pro- nociceptive P2X3 receptors as novel approach to the management of chronic pain. We characterized an important previously unknown role of the 288 residue in the left flipper of the pro-inflammatory P2X7 receptors.

National Library of Medicine Classification: QU 55.7, QU 57, QU 58, QV 285, WL 704.6 Medical Subject Headings: Receptors, Purinergic P2X3; Receptors, Purinergic P2X7; Adenosine

Triphosphate/analogs and derivatives; Polyphosphates; Diphosphonates; Nociception; Pain Management

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Yevheniia Ishchenko

ATP-ohjatut P2X3 ja P2X7 reseptorit: uusien ATP-analogien kipuaistimusta estävät mekanismit Itä-Suomen yliopisto, terveystieteiden tiedekunta

Publications of the University of Eastern Finland. Dissertations in Health Sciences Numero 438. 2017. 66 s.

ISBN (print): 978-952-61-2647-0 ISBN (pdf): 978-952-61-2648-7 ISSN (print): 1798-5706 ISSN (pdf): 1798-5714 ISSNL: 1798-5706

TIIVISTELMÄ

Hyvin monet etiologiset tekijät aiheuttavat potilaille kroonista kipua. Kipu voi olla hyvin vammauttavaa, ja valitettavan usein hoidot eivät ole riittävän tehokkaita. Tarvitaan siis lisää yksityiskohtaista tietoa kipuaistin toiminnasta ja aistimuksen synnystä keskushermostossa, jotta voitaisiin kehittää uusia hoitokeinoja. Ehkä tärkein kipuaistimuksen aloittava molekyyli on ATP- ohjattu P2X3-reseptori, jota ilmennetään lähinnä juuri kipuaistimusta välittävissä neuroneissa. Myös immuunijärjestelmän solujen ilmentävät P2X7-reseptorit osallistunevat kroonisen kipuaistimuksen syntyyn, sillä tämän reseptorin välittämät signaalit voimistavat tulehdusreaktioita ja tulehduksellisten välittäjäaineiden vapautumista kudoksiin. Niinpä sekä P2X3- ja P2X7-reseptoreita pidetään tärkeinä kipulääkekehityksen kohteina. Tässä tutkimuksessa tutkittiin useilla eri menetelmillä uusien mahdollisesti kipulääkeaihioina kehitettävien eri ATP-analogien toimintamekanismeja P2X3- ja P2X7-reseptoreissa. Lisäksi tutkimme P2X7 toimintamekanismia, erityisesti sen ns. "left flipper"-domeenin toimintaa.

Tämän väitöskirjan ensimmäisessä osatyössä tutkittiin kahden stabiilin synteettisen yhdisteen, AppNHppA ja AppCH2ppA, vaikutusta natiivin ja rekombinanttiproteiinina tuotetun P2X3- reseptorin toimintaan. Tässä työssä saatiin selville, että kumpikin näistä polyfosfaateista esti voimakkaasti ja spesifisesti sekä ihmisen että rotan P2X3-reseptorin toimintaa. Tällä perusteella olisi siis mahdollista tehdä translationaalisia tutkimuksia rottamalleilla. Inhibition vaikutusmekanismi osoittautui korkea-affiniteettiseksi reseptorin desensitisaatioksi.

Toisessa osatyössä tutkittiin useiden erilaisten polyfosfaattien, myös typpeä sisältävien bisfosfonaattien, mahdollista kykyä estää kipuaistimuksen syntyä. Sisäsyntyisen ATP-analogin ApppI:n todettiin olevan kaikkein spesifein ja voimakkain P2X3-reseptorin inhibiittorin. ApppI:lla ei ollut vaikutusta P2X2 ja P2X7-reseptorien toimintaan. Havaittiin myös, että ApppI vaikutti erityisen vahvasti ihmisen P2X3-reseptoriin, ja että tämä vaikutus oli riippuvainen ympäristön kalsiumionipitoisuudesta.

Kolmannessa osatyössä selvitettiin miten eri polaariset ja ei-polaariset aminohappotähteet rotan P2X7-reseptorin "left flipper"-osion peptidiketjukohdassa 288 vaikuttavat tämän reseptorin toimintaan.

Tässä väitöskirjatutkimuksessa selvitettiin siis, miten P2X3-reseptoriin vaikuttavat yhdisteet voisivat toimia kroonisen kivun hoitoon tähtäävinä kipulääkeaihioina. Tulehdusreaktiota voimistavan P2X7- reseptorin "left flipper"-osion 288. aminohappotähteellä osoitettiin olevan uusi aiemmin tuntematon tämän reseptorin epätyypillisiin ominaisuuksiin liittyvä merkitys.

Luokitus: QU 55.7, QU 57, QU 58, QV 285, WL 704.6

Yleinen suomalainen asiasanasto: reseptorit; adenosiinitrifosfaatti; kivunhoito; krooninen kipu

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Anybody who has been seriously engaged in scientific work of any kind realizes that over the entrance to the gates of the temple of science are written the words: 'Ye must have faith.'

-Max Planck-

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Acknowledgements

The present study was performed in the Laboratory of Molecular Pain Research, A.I. Virtanen Institute, Doctoral Program of Molecular Medicine in the Faculty of Health Sciences, University of Eastern Finland during 2013-2017.

This study was supported by CIMO (TM-13-8868, TM-14-9359) and Finnish Academy grants (277442).

First, I would like to thank my main supervisor, Professor Rashid Giniatullin, M.D, Ph.D., for his excellent mentoring and support during these research project. From the very first day, when faced with so many challenges, I enjoyed his constant support and encouragement.

Many thanks for all of your help, patience and trust in me, it played a major role in helping me to reach the summit of this Ph.D. mountain. Especially, I thank you for your great teaching and help in writing - from my first conference abstract up to this PhD thesis. I am very grateful to my second supervisor Adjunct Professor Jorma Määttä, Ph.D., for the excellent guidance and important advice during all of these years. In addition, I gratefully thank my supervisor, Professor Jukka Mönkkönen, Ph.D., for his unstinting support, despite his very tight working schedule.

I am especially grateful to Professor Antti Pertovaara, M.D., Ph.D., for acting as an opponent during the thesis defence. I want to acknowledge the reviewers Hana Zemkova, Ph.D., and Sari Lauri, Ph.D.; the criticism and comments they provided significantly helped to improve this thesis. I would also like to thank Ewen MacDonald, PhD, for the linguistic review of this work.

In addition, I would like to express my gratitude to my co-workers from Laboratory of the Molecular Pain Research for their help during this long journey. My dear colleagues, thank you very much, this achievement would have been an impossible task for me alone. Many, many thanks to Mrs. Raisa Giniatullina, M.D, Ph.D., for her kind support and valuable advice. I express my thanks to all co-authors of the articles we published during my PhD years, especially Andrey Skorinkin, M.D, Ph.D., for his significant contribution to data analysis and experiment planning. In addition, I am grateful for help and contributions from my project by Natalia Novosolova, Ph.D., and Kamil Kafisov, Ph.D. I would like to acknowledge my co-author Petri Turhanen, Ph.D. for the great collaboration and for synthetizing many of the compounds that I needed and doing it in such a short time.

My gratitude goes to my current group leader, Associate Professor Tarja Malm, Ph.D. for her patience and support while I simultaneously embarked on a new project while still finishing my Ph.D. thesis.

Many thanks to my friends Meike Keuters, Natalia Kolosowska and Evgeniia Marinina for support and guidance during my dark times, when struggling with personal and scientific problems. I also send a big thank you to my best friend and boyfriend, Eero Koponen, for his unwavering support and help to keep my attitude positive!

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I would like to use this opportunity to acknowledge the great support of my loving family!

Моя дорога родина, дякую вам за вашу нескінченнy підтримку і заохочення за yсі довгі роки мого навчання. Mамо, дякую за те що навчила ніколи не здаватись і була найкращим прикладом незламності та доброти. Тату, дякую за те що відкрили цікавість до навколишнього світу. Також моїм бабусі, сестрі та її родині за постійну підтримку.

Thank you all for creating an inspiring environment for work!

Kuopio, November 2017

Yevheniia Ishchenko

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List of the original publications

This dissertation is based on the following original publications:

I. Viatchenko-Karpinski V*, Novosolova N *, Ishchenko Y*, Azhar MA , Wright M, Tsintsadze V, Kamal A , Burnashev N, Miller AD, Voitenko N, Giniatullin R, and Lozovaya N. Stable, synthetic analogues of diadenosine tetraphosphate inhibit rat and human P2X3 receptors and inflammatory pain. Mol Pain 12:1–16, 2016

II. Ishchenko Y, Shakirzyanova A, Giniatullina R, Turhanen P, Määttä J, Mönkkönen J. and Giniatullin R. Selective calcium-dependent inhibition of ATP-gated P2X3 receptors by bisphosphonates-induced endogenous ATP-analogue ApppI.J Pharmacol Exp Ther 361(3): 472-481, 2017

III. Ishchenko Y, Novosolova N, Khafizov K, Bart G, Timonina A, Fayuk D, Skorinkin A and Giniatullin R. Reconstructed Serine288 in the Left Flipper Region of rat P2X7 Receptor Stabilizes Non-sensitized States.; Biochemistry 56 (26): 3394–3402, 2017

* Shared first authorship,authors with equal contribution.

The publications were adapted with the permission of the copyright owners.

Later in the text, references to these original studies are referred to as Studies I, II or III.

Other publications

Yegutkin GG, Guerrero-Toro C, Kilinc E, Koroleva K, Ishchenko Y, Abushik P, Giniatullina R, Fayuk D, Giniatullin R. Nucleotide homeostasis and purinergic nociceptive signaling in rat meninges in migraine-like conditions. Purinergic Signal. 12(3):561-74. 2016

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Abbreviations

ApppI 1-adenosin-5’-yl ester 3-(3- methylbut-3-enyl) ester

AppNHppA Diadenosine 5’,5’’’- P¹,P⁴-(β,-imido) tetraphosphate AppCH2ppA Diadenosine 5’,5’’’- P¹,P⁴-(β,-methylene)

tetraphosphate

Ap4A Diadenosine (tetra/n) phosphate

ASICs Acid-sensing ion channels ATP Adenosine triphosphate BPs Bisphosphonates

CGRP Calcitonin gene related peptide

CNS Central nervous system CFA Complete Freund's adjuvant c-Fos Proto-oncogene protein DRG Dorsal root ganglion

DMAPP Dimethylallyl

pyrophosphate

EC50 The concentration of a drug that achieves a half-maximal response

GDP Gross domestic product HAD High-affinity desensitization HHM Humoral hypercalcemia hP2X2 Human P2X2 receptor hP2X3 Human P2X3 receptor

hP2X7 Human P2X7 receptor IC50 The concentration of an inhibitor where the response is reduced by half

IPP Isopentenyl pyrophosphate

NBPs Nitrogen-containing bisphosphonates

NDG Nodose ganglion malignancy

NGF Neuronal growth factor Non-NBPs Bisphosphonates lacking nitrogen

NSAIDs Non-steroidal anti- inflammatory drugs

NTPDases Nucleoside

triphosphate

diphosphohydrolases

M-CSF Macrophage colony- stimulating factor

PAFs Primary afferent fibers PTH-rP Human parathyroid hormone-related protein

RANK Receptor Activator of Nuclear Factor κ B

RANKL Receptor activator of nuclear factor kappa-B ligand rP2X3 Rat P2X3 receptor

rP2X2 Rat P2X2 receptor rP2X7 Rat P2X7 receptor TG Trigeminal ganglion

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TNF-alpha Tumor necrosis factor TNP-ATP 2',3'-O-(2,4,6-

trinitrophenyl) adenosine 5'- triphosphate

TrkA Tropomyosin receptor kinase A TRPV Family of transient receptor potential cation channels

WT Wild type

zfP2X4 zebra fish P2X4 receptor TrkA+ Tropomyosin Receptor Kinase A

TRPV Family of transient receptor potential cation channels

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1 Introduction

Pain is familiar to all of us in different ways. Acute pain is primarily intended to warn about tissue damage or its possibility and can be considered as a beneficial signal. On the other hand, chronic pain markedly decreases the quality of life and represents a clinical, social and economic burden on the world’s population. Chronic pain is a prevalent and depleting medical condition affecting many European citizen, the numbers range from 12% (Spain) up to 40% (Italy, France and Ukraine) of the population. In Finland, it is estimated that about 19% of the population experience chronic pain (Breivik et al., 2013). Chronic pain ranks among the most expensive illnesses in terms of economic impact and lost productivity. The costs to the national health care budget associated with chronic pain in Europe are enormous, billions of euros annually, representing approximately 3–10% of the GDP (gross domestic product) (Breivik et al., 2013). Malignant bone pain is one of the most debilitating chronic pain conditions and one that often lacks any effective treatment (Smith and Mohsin, 2013).

Chronic pain, characterized by hyperalgesia and allodynia, results from sensitization of peripheral sensory nerves and central neurons (Perl, 2007; Woolf, 2011). Despite intensive research, few effective treatments for chronic pain have been developed. There is growing evidence pointing to the involvement of ion channels, including ATP-gated P2X receptors in pain transmission. During the last three decades, it has been appreciated that extracellular ATP is an important signaling messenger in the CNS, modulating neuro-glial communication and contributing to cellular responses to pain (Burnstock, 2006a). Several P2X receptor subtypes, including P2X3, P2X4, and P2X7, have been shown to play diverse roles in peripheral and central mechanisms of pain (Kuan and Shyu, 2016). Although there are several P2X receptors, the P2X3 receptor subtype is most abundantly expressed in sensory neurons (Burnstock, 2006b) suggesting that it has a prominent role in pain signaling. Notably, P2X2/3 heterotrimers, consisting of P2X3 and P2X2 subunits, are typically found in the nodose ganglion (NDG) (Burnstock, 2006b). During the last decade, P2X3 receptors have attracted much attention in the pain research field as promising therapeutic targets (North, 2003). Indeed, P2X3 receptor antagonists have been successfully tested in nociceptive and neuropathic pain models (Kennedy et al., 2003; North, 2003; Burnstock, 2013). Unfortunately, despite significant advances in the exploration of P2X3 receptors, potent and selective P2X3 antagonists with good pharmacokinetic and pharmacodynamic properties are still a rarity.

The knowledge accumulated in our group based on the description of functional properties of P2X3 receptors including their fast and profound desensitization properties combined with their exceptionally slow recovery suggested new mechanisms for modulation and inhibition of nociception (Giniatullin et al., 2003; Sokolova et al., 2006). In particular, the use- dependent high-affinity desensitization (HAD) of P2X3 receptors has been proposed as a way to inactivate this pro-nociceptive signaling without receptor activation (Fabbretti et al., 2004;

Sokolova et al., 2004, 2006).

These studies prompted us to search for promising new ligands to inhibit P2X3 receptors via the HAD mechanism; if identified, these would be useful pharmacological tools in the search

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for novel compounds with anti-nociceptive properties. In this study, we have examined the anti-nociceptive effects of several stable synthetic ATP-analogues among the ApnA (diadenosine (tetra/n) phosphate) compounds with specific long-lasting abilities to inhibit P2X3 receptors. ApⁿAs (n=3–7) are compounds derived from ATP, consisting of two adenosine moieties bridged by a chain of two or more phosphate residues attached at the 50- position of each ribose ring (Zamecnik, 1983; Boulos et al., 2016). It is known that endogenous ApⁿAs are present at high concentrations in various tissues including the CNS (Pintor et al., 1992). However, endogenous ApnAs undergo fast enzymatic cleavage in vivo that clearly reduces their therapeutic potential. Fortunately, this problem can be overcome by using synthetic methods and replacing one or more of the oxo-bridges in a polyphosphate chain with either aza- or carba-bridges. In the first part of this study, we describe the effects of two stable, synthetic Ap4A-analogues—AppCH2ppA (diadenosine 5’,5’’’-P¹,P⁴-(β,-methylene) tetraphosphate) and AppNHppA (diadenosine 5’,5’’’-P¹,P⁴-(β,-imido) tetraphosphate) in various in vitro tests. These tests are also designed to clarify the receptor-mediated mechanism of action and efficacy of these Ap⁴A compounds in order to control the nociceptive pain responses (study I).

Recently, bisphosphonates (BPs) have become widely used in the therapy of bone cancers and prevention of bone metastases. BPs, apart from their toxic effects on osteoclasts and cancer cells, also provide pain relief via still poorly understood mechanisms (Yuen et al., 2006; Haslbauer and Fiegl, 2009; Lopez-Olivo et al., 2012; Makhoul et al., 2015). It has been shown by the group of Professor J. Mönkkönen that the inhibition of farnesyl-diphosphate synthase by the nitrogen-containing BPs (NBP) induces formation of an endogenous ATP- analogue, ApppI (1-adenosin-5‘yl ester 3-(3-methylbut-3-enyl) triphosphoric acid diester).

ApppI may participate in a toxic effect of BPs on osteoclasts and tumor cells (Lehenkari et al., 2002; Green, 2004; Mönkkönen et al., 2006; Räikkönen et al., 2009, 2010, 2011). Moreover, since ApppI is structurally similar to ATP, it has also been suggested that it may act as a ligand for P2X receptors. However, the role of ApppI in BPs’ anti-nociceptive properties has not been studied. Since ApppI structurally resembles the anti-nociceptive Ap4A-analogues that we studied in the first part of this doctoral thesis (I), we investigated the effects of the NBP- induced derivative ApppI on the activity of P2X2, P2X3 and P2X7 receptors; the results are described in study II.

Neuron-glia crosstalk is largely based on ATP signaling of P2X7 receptors; these are the main triggers of the inflammatory responses (Di Virgilio et al., 2009; Toulme et al., 2010). P2X7 receptors are mostly expressed in immune cells and glia (Collo et al., 1997; Rassendren et al., 1997; Kuan and Shyu, 2016). P2X7 receptors are also important players in the development of chronic pain and they have been postulated as a potential target for pain therapies (Donnelly-Roberts and Jarvis, 2007; S. McGaraughty et al., 2007). Thus, treatment with P2X7 receptor antagonists was able to prevent the development of hyperalgesia and allodynia in neuropathic and inflammatory animal models (Chessell et al., 2005; Donnelly-Roberts and Jarvis, 2007; Kobayashi et al., 2011). In this regard, much effort has been expended to develop new, specific P2X7 antagonists, some of which have already been demonstrated to improve chronic pain treatments (Honore et al., 2006; S McGaraughty et al., 2007). However, in view of their complex properties as well as the not fully understood function of P2X7 receptors, the mechanisms of action for these tools are still poorly understood (Bhattacharya and Biber, 2016). For instance, P2X7 receptors are recognized by their intrinsic property to open ion

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pores in the cell membrane. However, this very common view was challenged in recent studies by Li et al. and Harkat et al. (Li et al., 2015; Harkat et al., 2017). To address these complicated issues, a molecular structure of the P2X receptors was actively sought, resulting in the recently published structure of the giant panda P2X7 (pP2X7) receptor (Karasawa and Kawate, 2016; Kasuya et al., 2017). However, many fundamental properties of these receptors, such as the desensitization and sensitization of P2X7 receptors are still not fully understood. Thus, in the third study of this thesis, we explored the role of the left flipper region of the P2X7 receptor focusing on the specific function of the key residue at position 288 in this receptor. Thus, we made a point mutation in this position of the left flipper, adding serine instead of natural phenylalanine at position 288 of the rat P2X7 (rP2X7) receptor subtype. By applying various complimentary methods of research, we explored the functional properties of the resulting mutant receptor. We further compared the hP2X7 wild type (WT) with mutants Y288S and Y288F and found that the latter shows rat-like deactivation kinetics. The results of this multidisciplinary approach combining electrophysiology, flow cytometry, kinetic and molecular modelling are presented in study III.

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2 Review of the Literature

2.1 TYPES OF PAIN. ACUTE AND CHRONIC PAIN

Pain plays an important role in our survival and protection from environmental threats. For instance, acute pain is a signal of danger and it serves our body as a protective mechanism.

However, excessive and long-lasting pain is a disturbing condition that eventually develops into a chronic pain syndrome. There are now more and more diseases that are accompanied by chronic pain. Such conditions are a result of long-lasting disturbances, even permanent damage, of the central or peripheral nervous system. The pain sensory symptoms are comorbid with behavioral disabilities, such as insomnia, anxiety and depression (Gambassi, 2009). Pain itself has been described by the International Association for the Study of Pain as an unpleasant sensory and emotional experience associated with actual or potential tissue damage (IASP 2017). However, it is important to differentiate pain and nociception in pain research. The term ‘nociception’ was first introduced by Charles Sherrington almost 100 years ago to describe nociceptive stimuli and the subsequent physiological responses to those stimuli (Sherrington, 1906). Nociception is the process of detection and transmission of the noxious stimuli from the external or/and internal environment generated by the nociceptors.

It involves both peripheral and central nervous system (CNS) neurons. Typically, noxious stimuli, including tissue injury or damage, are generated in peripheral nerve endings and transmit information to the dorsal horn in the spinal cord or to trigeminal nucleus caudalis.

Subsequently, this information is transmitted to the higher brain centers involved in perception of pain (National Research Council, 1992).

Pain can be classified in many ways, taking into account temporal, localization-based, intensity characteristics and/or physiological mechanisms of pain. For example, the pain can be classified as ‘chronic‘ when it has persisted for between 3 to 6 months (Schaible, 2006).

One of the most popular classification of chronic pain is based on pathophysiological mechanisms. Thus, there are two types of chronic pain: inflammatory and neuropathic pain.

Inflammatory nociceptive pain is elicited by the activation of specific nociceptors when the tissue has become inflamed or injured. It can occur without external stimulation or can present as hyperalgesia or/and allodynia (terms defined in Tab 1) (Schaible, 2006).

Neuropathic pain results from a lesion or damage to the peripheral sensory fibers or neurons in the CNS. Both of these pain types often lead to peripheral and central sensitization associated with allodynia and hyperalgesia (Millan, 1999).

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Table 1. Explanation of the most commonly used pain terminology Term name Description

noxious stimuli

potentially damaging or actually damaging stimuli that are able to activate a nociceptor

nociceptor a receptor that is localized in the periphery that can be activated by a noxious stimulus

analgesia (anti- nociception)

decrease in the pain sensation

hyperalgesia increase in the pain sensation in response to noxious stimuli, that also includes lowering of the normal pain threshold to noxious stimuli

allodynia pain sensation triggered by stimuli that in the normal condition would be below the pain threshold

peripheral sensitization

decrease of the excitatory threshold of the peripheral nociceptors that amplifies pain signals sent to the central nervous system

central sensitization

increase of excitability of the sensory neurons in the spinal cord dorsal horn that increases pain signalling to the upper pain centers, thus increasing pain sensation

It is noteworthy that this simple classification of chronic pain in patients is also suitable for pain research in experimental animals. However, in many cases, chronic pain in patients occurs as a complication of postsurgical, osteoporotic, migraine or bone cancer-related processes. Interestingly, migraine is the most common neurological disorder in the world, affecting over one billion people (C. Global Burden of Disease Study, 2015), with an elevated prevalence in women versus men of European ancestry (Stovner et al., 2006). Thus, new research will probably lead to a more diversified classification of pain e.g. one that takes into account both general and disease-specific neuronal mechanisms.

2.1.1 Neuronal pathways involved in chronic pain

The nociceptive system includes a complicated network of peripheral and central neurons.

Signals from the periphery detected by nociceptors are first generated in the ‘free nerve endings’ of the primary afferent fibers (PAFs, Fig 1). The free nerve endings of nociceptors terminate in skin, tendons, bones, joints, and in other body organs. It has been assumed that nociceptors of different tissues share most of their properties. Nevertheless, it has been claimed that there might be differences in the detailed morphological and functional properties of nociceptors in individual tissues (Schaible, 2006). For example, there is a difference in the mechanical threshold for activation in neurons supplying skin and bone, as demonstrated in Fig 2 (Castañeda-Corral et al., 2011). Aδ- and C- fibers are the typical nociceptors that preferably transduce noxious stimuli. In contrast, Aβ-fibers preferentially

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transduce innocuous stimuli such as vibration, touch and pressure, but not pain (Millan, 1999). PAFs are often classified based on their main characteristics such as diameter, structure and conduction velocity. Thus, they are: (i) thin C-fibers that are 0.4-1.2 μm in diameter, they are unmyelinated and are slowly propagating at a rate of 0.5-2.0 m/s; (ii), medium diameter Aδ-fibers have a diameter of 2-6 μm, they are myelinated to some extent, with a conduction rate of 12-30 m/s; (iii) Aβ-fibers have the large diameter (≥10 μm), they are heavily myelinated, with very fast conduction rate up to 30-100 m/s.

Figure 1. Schematic representation of input from PAFs into the spinal cord of dorsal horn laminas.

Aδ fibers preferably innervate lamina I, while the principal target of the C fibre input is lamina II0. Unmyelinated C fibers projecting from viscera, joints and muscle appear to preferentially innervate laminae I /IV/V/IV rather than lamina II. Sensory neurons (NS - nociceptive-specific, WDR – wide dynamic range and NON-N – non-nociceptive) are indicated. Adapted from Millan, 1999.

Most of the nociceptive neurons are multimodal as they can sense mechanical, thermal and chemical stimuli. Therefore, they are able to detect cutaneous, somatic and visceral pain (Belmonte and Cervero, 1996). Information from the peripheral nociceptors is transmitted to the neuronal bodies of the sensory afferents, which are located in the dorsal root ganglia (DRG, 31 pairs in humans) and its cranial analogue, the trigeminal ganglion (TG, one pair in humans). Further, DRG axons project to the spinal cord and form the synapses mostly in the substantia gelatinosa (lamina I-III) of the dorsal horn in the spinal cord as illustrated in Fig 1. Second order nociceptive neurons in the spinal cord form several ascending pathways to the higher pain centers in the CNS. Some of them project to the thalamo-cortical system that produces the conscious sensation of pain, whereas others, such as dorsal and ventral spinocerebellar tracts, can control motor functions (Willis, 2007). The other subset of sensory neurons projects to the motor nucleus of the spinal cord, to be involved in withdrawal reflexes as well in more complex pain avoiding behaviour (Schaible, 2006).

The macroscale morphology of the nociceptive system has been relatively well studied.

However, many fine mechanisms, especially those at the molecular level, are far from clear due to disease-specific pathophysiology diversities. Disease-specific diversity could be based on tissue specific prevalence of innervation by C-, Aδ- or Aβ-fibers, profile of pro-nociceptive

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endogenous agents and variable contribution of neuronal damage or peripheral inflammation (Goff et al., 1998).

2.2 DISTINCTIVE CHARACTERISTICS OF BONE PAIN 2.2.1 Bone innervation

The innervation of bone has been studied for over a century with a variety of methods, especially the now widely used technique of immunohistochemistry (Hurrell, 1937; Duncan and Shim, 1977). It is evident that bones are innervated by the sympathetic and somatic sensory nerves (Hukkanen et al., 1992; Serre et al., 1999; García-Castellano et al., 2000). Nerve fibers accompany the blood vessels in the bone which travel through all its tissue layers (Mach et al., 2002). Of all the bone tissue types, the periosteum is the most densely innervated.

It has been demonstrated that all bone tissues (mineralized bone, bone marrow and periosteum) are highly innervated by the primary nociceptive fibers (Mach et al., 2002; Falk et al., 2014; Mantyh, 2014). However, as the total volume of the periosteum is less than that of the mineralized bone and the bone marrow, the total number of sensory and sympathetic fibers in the latter is also high (Castañeda-Corral et al., 2011; Mantyh, 2014).

Figure 2. A schematic showing the approximate percentage of PAFs that innervate the skin and the bone. The skin is innervated by Aβ fibers - , thiny mielinated Aδ - and , peptide-rich C fibers - , and peptide-poor C fibers ( ). In contrast, the bone has been shown to be predominantly innervated by thinly myelinated Aδ, and , and peptide-rich C fibers ( ). In both, skin and bone, there is also a small proportion (< 5% of the total) of non-myelinated C-fibers (CGRP+, calcitonin gene related peptide positive; TrkA−, tropomyosin receptor kinase A negative fibers). (NF 200+, neurofilament 200 positive fibers) Adapted from Castañeda-Corral et al., 2011.

Notably, it has been shown that the bone is primarily innervated by the mildly myelinated Aδ- and unmyelinated C–fibers, which transmit noxious stimuli to the dorsal horn. These fibers express various neuropeptides such as neuropeptide Y (NPY), substance P (SP), vasoactive intestinal peptide (VIP) and calcitonin gene related peptide (CGRP) and some of these peptides are clearly pro-nociceptive. These fibers also display the major pro-nociceptive ATP-gated P2X3 receptors, acid sensing ion channels (ASIC) and several types of vanilloid receptors (TRPV1-4) (Bjurholm et al., n.d.; Fischer et al., 1996; Mach et al., 2002; Castañeda- Corral et al., 2011; Falk et al., 2014; Mantyh, 2014). However, most of the sensory fibers that

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innervate bone are mainly by thinly myelinated TrkA+ (Tropomyosin Receptor Kinase A) sensory nerve fibers (̴ 80%, belong to A- fibers), i.e. they are very different from the sensory fibers in skin (̴ 30% of Trka+, Fig 2)(Castañeda-Corral et al., 2011). Taken together, these data suggest that there is an abundance of the main pain transducing receptors in the bone and this has clear implications with respect to chronic bone pain.

2.2.2 Bone pain at a glance

Bone pain represents one of main chronic pain conditions. Bone pain shares many of the common characteristics of both inflammatory and neuropathic pain but it also has unique components due to the specific profile of cells in this tissue (Falk and Dickenson, 2014). The variety of bone disorders including malignant and non-malignant processes, contributes to the complex pathophysiology of bone pain. Pain associated with malignant bone processes is a complex condition involving spontaneous (background) pain and movement-evoked pain (Portenoy and Hagen, 1990; William and Macleod, 2008). Background pain is specified as dull and continuous pain, which increases with the progression of the main disease.

Usually, it can be reasonably well treated with traditional analgesics. Evoked pain is usually described as a breakthrough feeling, being less sensitive to common analgesics which effectively decrease background pain (William and Macleod, 2008). The treatment of this type of pain requires the combination of analgesics with the cancer- and bone-targeting specific therapies. This is especially important for patients with breast, prostate, kidney and lung cancers which preferably metastasize to the bone (Makhoul et al., 2015).

Until recently, we had very limited knowledge of tumor-induced bone pain. The popular view was that this kind of pain was caused by the compression of peripheral nerves or by vascular occlusion in the bone. In 1998, the first tumor-induced bone pain model was developed and published by Schwei et al. (Schwei et al., 1999). This animal model of femoral osteosarcoma provided the opportunity for researchers to study mechanisms underlying tumor-induced bone pain (Falk et al., 2014).

Notably, bone resorption and construction is permanent and very important processes. This processes of bone remodeling, bone formation and resorption are tightly coupled and directly influenced by interactions between osteoblasts and osteoclasts as illustrated in Fig 3 (John P. Bilezikian, Lawrence G. Raisz, 1996; Crockett JC1, Rogers MJ, Coxon FP, Hocking LJ et al., 2011). It is essential that there is an equilibrium in bone remodeling between resorption and formation in order to ensure the integrity of the bone structure. Therefore, an increase of the resorption by osteoclasts leads to osteoporosis, in contrast, excessive bone formation should result in disruption of calcium homeostasis. All of these conditions potentially could lead to bone pain, which is one of the most excruciating types of chronic pain.

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Figure 3. Bone remodeling and involved cell types (adapted from Weilbaecher 2011). Osteoclasts are mane players in resorption after they are maturation, they are multinucleated, polarized cells that adhere to the bone surface. RANKL – , RANK – , HSC – , MSC – , M-CSF – , specified in the abbreviation list.

Further improved models for bone pain helped to reveal the role of bone resorptive processes that were derived from osteoclast over-activation in bone malignancy disorders. Osteoclasts- induced resorption promotes a highly acidic environment in the resorption region. Osteoclast resorb the mineral bone matrix by secreting collagenases and proteases that demineralize and degrade proteins such as type I collagen of the bone (Weilbaecher et al., 2011; Florencio- Silva et al., 2015). During this resorption osteoclast release H⁺ (hydrogen ions) through the action of carbonic anhydrase through the ruffled border into the resorptive cavity what strongly increasing acidity in the actively resorptive regions, acidifying and aiding dissolution of hydroxyapatite mineral to Ca²⁺, H³PO⁴ and H²CO³(Currey, 2006). While normal remodeling this will not be noted, however during osteoclast over activated resorption, while osteoblast are in minor, this events directly lead to acidosis and first local and further humoral hypercalcemia (Fig 3 and 4). This directly activates the acid-sensitive TRPV1 and ASIC receptors expressed in nerve terminals innervating bone (Luger et al., 2005;

Nagae et al., 2007). Notably, it has been demonstrated that high Ca²⁺ concentrations can inhibit certain P2X receptors (Virginio et al., 1998). Intriguingly, detailed studies of the modulation by different cations of the P2X3 receptor subunit, revealed that unlike other P2X receptors, it is strongly facilitated by extracellular Ca²⁺ (Fig 5) that acts via specific sites in the ectodomain located next to the ATP binding pocket (Giniatullin et al., 2003; Petrenko et al., 2011; Giniatullin and Nistri, 2013). Furthermore, acidosis dramatically increases the expression of ASIC1b, ASIC1a and ASIC3 receptors in the DRG ganglion, resulting in peripheral sensitization and hyperalgesia, as presented in Fig 3 (Nagae et al., 2006, 2007). In addition, osteoclast induced hypercalcemia influences many processes in the whole body including the sensory system. Calcium is known as a strong modulator of nociceptive ATP- gated P2X3 receptors in sensory neurons (Giniatullin et al., 2003) being a co-activator of these P2X3 receptors (Fig 3). It can also affect the inhibitory activity of P2X3 antagonists (Giniatullin et al., 2003; Ishchenko et al., 2017). Notably, it has been shown in both vitro and in vivo models that bone cancer is associated with enhanced expression of P2X3 receptors in CGRP- positive sensory nerves, which are extensively present in the bone (Gilchrist et al., 2005; Wu et al., 2012, 2016). Thus acidosis and hypercalcemia, can lead to peripheral nerve sensitization

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and further, to central sensitization, the two main features of bone pain (Urch, 2004; Luger et al., 2005; Mantyh, 2014)

Figure 4. Scheme of the pathophysiology of malignant bone pain biology. (A) Representation of the peripheral and central sensitization. (B) Molecular mechanisms implicated into development of the bone pain and receptors involved in its transduction. Represented receptor and chemicals (P2X3, ASICs, TRPV3, TRPA1 and H+, IPP, ApppI, ATP, BPs, RANK, RANKL) see in main abbreviation list.

As sensory nerve fibers are distributed over all of the bone tissues, tumor proliferation associated with the secretion of the various pro-nociceptive compounds can strongly activate sensory nerve fibers in the bone (Luger et al., 2005). In addition, tumors are able to destabilize and reorganize the peripheral nerves, leading to either pathological sprouting or the destruction of distal sensory and sympathetic nerve fibers (Mach et al., 2002; Mantyh, 2014). Tumors and associated stromal cells can release high amounts of multiple algogens (i.e. endogenous pain-evoking compounds) such as NGF, pro-nociceptive cytokines, including tumor necrosis factor (TNF-alpha) along with extracellular ATP, which is the endogenous agonist of the P2X3 receptors, (Fig 4) (Urch, 2004; Goblirsch et al., 2006).

These destructive events and active pro-nociceptive compounds can induce peripheral and central sensitization and lead to pathological changes in the nociceptive system (Luger et al., 2005; Mantyh, 2006). This can contribute to osteoclast-induced resorption, mediating the release of the bone stored growth factors, further stimulating tumor proliferation and tumor growth (Fig 4) (Yoneda, 2013). The growing scientific interest in this field and the improved animal models have widened our understanding of the bone pain associated with malignant and non-malignant processes. However, the complexity of the bone pain processes still requires clarification.

2.2.3 Calcium metabolism and hypercalcemia

It is well known that the skeletal system is crucially involved in calcium metabolism. Both aging and a range of bone disorders are able to alter the calcium levels in the extracellular milieu and in blood, leading to either hypo- or hyper-calcemia. Recent evidence has revealed that the cancer-related bone pain is often associated with osteoclast activation and hypercalcemia as represented on Fig 4 (Luger et al., 2005; Nagae et al., 2006). In some pathological conditions, such as in parathyroid hormone-related crisis, the serum calcium levels are elevated from the normal ~2 mM to become hypercalcemic at 4.8 mM (Rahil and Khan, 2012). In addition, hypercalcemia has been reported to be a frequent (20 - 30 % of cases) complication in patients with cancer (Basso et al., 2011; Hu et al., 2013; Goldner, 2016; Tagiyev

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et al., 2016). Hypercalcemia, to even a mild degree, can dramatically worsening the patient’s condition, leading to a progressive mental impairment, as well as to renal failure and it carries a high risk of mortality. Even moderate hypercalcemia frequently results in marked neurologic dysfunction (Stewart, 2009). The two most frequent types of hypercalcemia are humoral hypercalcemia of malignancy (HHM) and osteolytic hypercalcemia. The HHM is often caused by the systemic secretion by tumor cells of the parathyroid hormone related protein (PTHrP). PTHrP promotes renal tubular calcium reabsorption, which elevates the serum calcium level (Ratcliffe et al., 1992). The second type of calcium growth is associated with a local osteolytic activity due to an increase in osteoclastic bone resorption in the areas surrounding the malignant cells. When compensatory mechanisms are exceeded, the serum calcium level rises causing hypercalcemia (Clines and Guise, 2005). There are also different, more specific cases of malignant hypercalcemia such as the production of ectopic calcitriol by malignant lymphocytes in multiple myelomas (Roodman, 1997) or direct PTH production ectopically by tumor cells (Inzucchi, 2004).

Notably, the neurological impairments in hypercalcemia often include pain and migraine (Malangone and Campen, 2015; Yin et al., 2016). Cancer patients diagnosed with hypercalcemia often suffer from headaches. Recent, clinical research based on 23,285 migraine patients and 95,425 controls revealed a direct association between the elevated serum calcium level and an increased risk of migraine (Yin et al., 2016). The current therapy of hypercalcemia, which aims to reduce the serum calcium concentration is focused on increased calciuresis, decreased bone resorption, and reduced intestinal absorption of calcium. It is important to understand the pathogenesis and treatment options for hypercalcemia associated with malignancy, in order that prompt treatment interventions can be administered.

However, there is still very little known about the molecular (receptor) mechanisms underlying malignant and non-malignant bone pain. However, data from our laboratory and from others already suggested some clues in elucidating the mechanism. Indeed, it has been demonstrated that extracellular calcium plays an important role in the signaling of P2X receptors, accelerating re-sensitization of the P2X3 receptor currents and making these pro- nociceptive receptors functionally more active (Cook and McCleskey, 1997; Cook et al., 1998) (Cook et al., 1998; Giniatullin et al., 2003). On the other hand, high calcium and magnesium levels can inhibit the activity of P2X7 receptors (Coddou, S. Stojilkovic, et al., 2011).

Thus, the role of hypercalcemia on development of pain should be clarified in order to identify new targets and develop analgesics able to combat these debilitating types of pain such as bone pain and migraine.

2.4 BISPHOSPHONATES AND CURRENT TREATMENTS OF BONE PAIN 2.4.1 Non-specific treatments for bone cancer

Majority of treatments applied for pain treatment in malignant and non-malignant bone disorders are similar to other pain disorders and their main differences are determined primarily by the severity of the disease. Common (non-specific) chronic pain treatment includes non-steroid anti-inflammatory drugs (NSAIDS) (Fu et al., 2015) and different opioids (Yaldo et al., 2016). Their efficacy is still limited by various adverse side effects and

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NSAIDS often provide insufficient pain relief. Thus, in many cases, bone pain cannot be resolved by common current therapies what strongly decrease quality of life (van den Beuken-van Everdingen et al., 2007, 2016; Coleman et al., 2014). In fact, currently, pain management of malignant bone disorders is considered as not satisfactory (Smith and Mohsin, 2013). Currently 85-95% of patients with bone cancer have significant malignancy- induced pain and 45% of these patients collide with an inadequate pain control therapy or unmanaged pain (Smith and Mohsin, 2013). Also, many NSAIDs treatments are not sufficient to decrease skeletal pain or have not strong enough effect in consideration of the personal side effect risks (Rasmussen-Barr et al., 2017). In addition, it is well known that NSAIDs have side effects including the gastrointestinal and cardiovascular complications (Sostres et al., 2010). In particular, they injure the upper and lower gut by depleting COX-1 derived prostaglandins causing the peptic ulcer (Sostres et al., 2010). Also, recent clinical meta- analysis found a direct link of NSAIDs treatment to the heart failure (Arfè et al., 2016).

Another major issue to consider is that some typical NSAIDs such as ibuprofen and Cox-2 inhibitors slow down fracture healing in animal models of bone fracture (O’Connor et al., 2009; Barry, 2010).

Therefore, new approaches to the chronic bone pain control are strongly needed. Ideally, if the analgesic effect is associated with promotion of the bone formation and with skeletal healing or, at least, the anti-nociception develops in the absence of bone healing inhibition.

2.4.2 Beneficial effects of the BPs for bone pain treatment

Currently, bisphosphonates (BPs) are the safest treatments for bone disorders associated with bone lesions. BPs are highly specific to the hard bone tissue containing hydroxyapatite. BPs are divided into two types: i) compounds lacking a nitrogen group (non-NBP) (Frith et al., 2001; Räikkönen et al., 2009; Rogers et al., 2011) and ii) nitrogen-containing compounds (NBP).

Interestingly, both type of BPs can induce the formation of endogenous ATP-analogues as represented on Fig 5. Thus, non-NBPs promote the synthesis of compounds with ApCp- groups, whereas NBPs induce IPP/DMAPP (Isopentenyl pyrophosphate/dimethylallyl pyrophosphate) and the ApppI (1-adenosin-5’-yl ester 3-(3-methylbut-3-enyl) ester). Since these are relatively recently discovered compounds, their roles in health and disease are still far from clear.

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Figure 5. Scheme of the cellular mechanism of BPs (top). Induction of the ATP analogues by BPs (bottom). Adopted from Russell, 2011.

Both non-NBPs and NBPs have anti-osteoclast and anti-cancer activity (Fig 5 and 6). They can interfere with mitochondrial ATP production or inhibit the mevalonate pathway and activate caspases (Fromigue et al., 2000; Oades et al., 2003; Green, 2004; Koshimune et al., 2007; Tanaka et al., 2013). Recently, evidence emerging from clinical studies has suggested that BPs, in combination with other treatment modalities, can diminish pain in cancer patients (Body et al., 2004; Tagiyev et al., 2016). Other clinical research findings have indicated that BPs can successfully reduce the level of persistent pain not only in bone disorders, but also in patients with chronic low back pain and complex regional pain syndrome type I (Haslbauer and Fiegl, 2009; Abe et al., 2011; Pappagallo et al., 2014).

In general, the anti-nociceptive effect of BPs can be either direct or indirect, mediated by production of endogenous ATP-analogues. For instance, there is a strong correlation between BPs’ anti-resorptive and anti-tumor effects and their ability to induce the formation of endogenous ATP-analogues (ApppI, IPP, AppCClp) in vivo (Ramanlal Chaudhari et al., 2012). Additionally, it has been proposed that BP-induced formation of ATP derivatives can participate in pain relief (Fromigue et al., 2000; Hadji et al., 2016). In particular, NBPs represent the most effective group, pointing to a possible involvement of ApppI and IPP in analgesia.

Notably, BPs induced analgesia could involve even more complicated pathways including modification of the immune response. Thus, two NBPs, zoledronate and residronate, activated human Vγ9Vδ2 T-cells, which have potent anti-tumor properties (Benzaïd et al.,

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2011, 2012). NBP pre-treated monocytes accumulate ApppI and IPP, contributing to the activation and proliferation of Vγ9Vδ2 T-cells (Roelofs et al., 2009). After NBP treatment, high levels of ApppI/IPP accumulate in cancer cells and these can be released during the apoptosis of tumor cells to activate surrounding bone cells and sensory nerve terminals. These findings suggest a novel, but largely unexplored, immune anti-tumor and anti-nociceptive effect for BPs.

Administration of BPs in patients with different bone cancers and metastases is intended not only to reduce tumor size and prevent bone fracture but also to provide pain relief (Yuen et al., 2006; Haslbauer and Fiegl, 2009; Lopez-Olivo et al., 2012). According to the Cochrane Reviews, there is evidence supporting the pain relief effectiveness of BPs in bone malignancy, although the analgesic effect of BPs is not so strong as obtained with morphine (Wong and Wiffen, 2002). However, when the other methods of pain relief are inadequate or strong opioids should be avoided, then treatment with BPs can be beneficial in patients with bone tumors (Wong and Wiffen, 2002). On the other hand, the clinical use of BPs for pain diseases in non-malignant bone disorders has achieved good results even without additional therapies. There was an improvement in the regional osteoporotic changes and reduced pain after administration of a low-dose of BPs in individuals with a complex regional pain syndrome type I (Abe et al., 2011). Likewise, in patients with chronic low back pain, these workers found evidence for the analgesic efficacy of the BP, pamidronate (Pappagallo et al., 2014).

In general, the analgesic efficiency of BPs in clinical research significantly increased the interest of scientists in investigating the molecular mechanisms behind the anti-nociceptive in conjunction with the pro-apoptotic effects of BPs on tumor cells. The latter could be due to the ability of BPs to inhibit the release of bone-stored growth factors, thus reducing the growth of the bone tumor. Interestingly, the pro-apoptotic action of BPs on tumor cells has been associated with the anti-apoptotic effects of these medicines on osteoblasts and osteocytes, a property that positively influences bone homeostasis (Bellido and Plotkin, 2011).

This important mechanism for bone survival is illustrated in Fig 6.

The decrease in the size of the lesions induced by BPs diminishes the acidic bone microenvironment, leading to less activation of the acid sensitive pro-nociceptive ASIC and TRP channels (Nagae et al., 2007). Thus, two non-NBPs, clodronate and etidronate, have been shown to reduce capsaicin-induced hyperalgesia in an inflammatory pain model and to decrease the number of c-Fos positive neurons in spinal cord (Kim et al., 2013). Similar results have been published for two NBPs, zoledronate and alendronate. Thus, zoledronate has decreased the mRNA expression of ASIC1a and ASIC1b as well as that of c-Fos in different pain models: (i) inflammatory pain (induced by injections in the hind-paw of the f PTH-rP or CFA) (Nagae et al., 2006) and (ii) a bone cancer model (Nagae et al., 2007). A recent study has demonstrated that alendronate effectively reduced the development of hyperalgesia in a rat model of ovariectomy-induced bone loss (Naito et al., 2017). Interestingly, a very unique direct effect on the pro-nociceptive ATP-gated receptors has been described forthe NBP, minodronic acid, which belongs to the third generation of these medicines. This NBP exerted a direct inhibitory effect on the pro-nociceptive P2X3 and P2X2/3 receptors and it showed evidence of analgesia in animal pain models (Kakimoto et al., 2008; Tanaka et al., 2017).

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Figure 6. Schematic representation of BPs-induced molecular mechanisms involved in analgesia, present in the environment of a bone malignancy.

Figure shows receptors (P2X3, ASICs, TRPV3, TRPA1) and pro-nociceptive endogenous compounds (H⁺, IPP, ApppI, ATP, BPs) implicated in bone pain (explained in the main abbreviation list).

Thus, accumulated evidence suggests that the very specific

involvement of the BPs themselves and the BPs-induced ATP derivatives are both involved in anti-nociception. Both types of BPs can evoke analgesic effects, but the exact mechanisms are not fully understood. These effects can vary depending on the BPs type and will need to be examined in further studies.

2.5 PURINERGIC MECHANISMS INVOLVED IN PAIN 2.5.1 Membrane receptors activated by extracellular ATP

It is nowadays generally accepted that ATP can act as a neurotransmitter and neuromodulator via specific membrane receptors called ‘purinergic receptors’ (Burnstock, 2009). ATP mediated signaling attracted scientific interest in pain research after the early observations that ATP itself was able to evoke pain (Bleehen et al., 1976; Khakh and North, 2012). There are two types of ATP sensing purinergic receptors: metabotropic P2Y and inotropic P2X receptors. There are seven distinct members of the P2X receptor family: P2X1- P2X7 receptors, which form trimeric, ligand-gated ion channels permeable to K⁺, Ca²⁺and Na⁺(North, 2016). These receptors can be organized either as homomeric and heteromeric channels composed by different subunits (Kawate et al., 2009).

ATP-gated P2X receptors are widely distributed in different tissues, being expressed in many cell types such as glial, neuronal and immune cells. These receptors are involved in fast synaptic transmission (Pankratov et al., 2002), modulatory processes in the peripheral and central nervous systems and in glia-neuron cross-talk (Irnich et al., 2002; Chen et al., 2012). It is significant that they have also been strongly implicated in nociception (Tewari and Seth, 2015). This diverse functionality makes P2X receptors important contributors to many key vital processes in health and disease.

The P2X receptor genes were identified in 1994 (Brake et al., 1994) and in the following two years, the whole family of P2X receptors was identified (Lewis et al., 1995; Buell et al., 1996;

Surprenant et al., 1996; Lê et al., 1997). During that exciting period, it became clear that P2X receptors represent a unique protein family. Later studies revealed the distinct functional properties of each homomeric P2X receptor (Fig 7, Tab 2). Furthermore, as the basic structure for this family was clarified, expression studies revealed that P2X receptors were much more extensively spread throughout various tissues than had been previously thought from functional studies. Notably, the predominant expression of P2X2, P2X4 and P2X6 subunits in

ATP-gated P2X3 and P2X7 receptors : new mechanisms of anti-nociception by new ATP-analogues

uef.fi

Dissertations in Health Sciences

YEVHENIIA ISHCHENKO

ATP-GATED P2X3 AND P2X7 RECEPTORS:

NEW MECHANISMS OF ANTI-NOCICEPTION BY NEW ATP-ANALOGUES

YEVHENIIA ISHCHENKO

ATP-gated P2X3 and P2X7 receptors: new mechanisms of anti-nociception by new

ATP-analogues

ATP-gated P2X3 and P2X7 receptors: new mechanisms of anti-nociception by new

ATP-analogues

Acknowledgements

List of the original publications

Contents

Abbreviations

1 Introduction

2 Review of the Literature