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Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 198

The Impact of the (SP) Fragment SP1-7 on Chronic Neuropathic

ANNA JONSSON

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6192 ISBN 978-91-554-9206-9 UPPSALA urn:nbn:se:uu:diva-241637 2015 Dissertation presented at Uppsala University to be publicly examined in B21, BMC, Husargatan 3, Uppsala, Friday, 8 May 2015 at 09:15 for the degree of Doctor of Philosophy (Faculty of Pharmacy). The examination will be conducted in English. Faculty examiner: Professor of Medicine James Zadina (Tulane University School of Medicine, New Orleans, USA).

Abstract Jonsson, A. 2015. The Impact of the Neuropeptide Substance P (SP) Fragment SP1-7 on Chronic . Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 198. 64 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9206-9.

There is an unmet medical need for the efficient treatment of neuropathic pain, a condition that affects approximately 10% of the population worldwide. Current therapies need to be improved due to the associated side effects and lack of response in many patients. Moreover, neuropathic pain causes great to patients and puts an economical burden on society. The work presented in this thesis addresses SP1-7, (Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH), a major metabolite of the pronociceptive neuropeptide Substance P (SP). SP is released in the following a and binds to the NK1 . In contrast to SP, the degradation fragment SP1-7 is antinociceptive through binding to specific binding sites distinct from the NK1 receptor. The aim of this thesis was to investigate the impact of SP1-7 on neuropathic pain. To understand how SP1-7 exerts its effect, a series of N-truncated forms of the heptapeptide were biologically evaluated. A set of small high-affinity ligands was evaluated in animal models of neuropathic pain. To confirm a clinical relevance the levels of SP1-7 in neuropathic pain were assessed incerebrospinal fluid (CSF) collected from neuropathic pain patients. The results showed that SP1-7 could alleviate thermal as well as mechanical hypersensitivity in three different animal models of neuropathic pain. C-terminal amidation was connected with increased efficacy. N-terminal truncation of SP1-7 indicated a necessity of five amino acids in order to retain biological effect. One small high-affinity showed a significant anti-allodynic effect. CSF levels of SP1-7 in neuropathic pain patients were lower compared to controls. Taken together, these findings demonstrate that the formation of SP1-7 may be attenuated in neuropathic pain. C-terminal amidation and a majority of its amino acids are necessary for stability and permeability. Clearly, SP1-7 and SP1-7 mimetics with high affinity to the SP1-7 binding site ameliorate neuropathic pain-like behaviors in animal models of neuropathic pain. Overall, the findings presented in this thesis contribute to new knowledge regarding the role of SP1-7 and related analogues and fragments in neuropathic pain. In a future perspective, this could be essential for the development of efficient strategies for managing patients with neuropathic pain.

Keywords: neuropathic pain; substance P (SP); SP1-7; bioactive fragments; spared nerve injury; spinal cord injury; streptozotocin-induced diabetes; ; ; peptidomimetics; cerebrospinal fluid; spinal cord stimulation, radioimmunoassay

Anna Jonsson, Department of Pharmaceutical Biosciences, Box 591, Uppsala University, SE-75124 Uppsala, Sweden.

© Anna Jonsson 2015

ISSN 1651-6192 ISBN 978-91-554-9206-9 urn:nbn:se:uu:diva-241637 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-241637)

Till Daniel och Valentin

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Carlsson, A., Ohsawa, M., Hallberg, M., Nyberg, F., Kamei, J. (2010) Substance P1-7 induces antihyperalgesia in diabetic mice through a mechanism involving the -sensitive sigma recep- tors. Eur J Pharmacol, 626(2-3):250-255.

II Ohsawa, M., Carlsson, A., Asato, M., Koizumi, T., Nakanishi, Y., Fransson, R., Sandström, A., Hallberg, M., Nyberg, F., Kamei, J. (2011) The effect of substance P1-7 amide on nociceptive threshold in diabetic mice. , 32(1):93-98.

III Carlsson-Jonsson, A., Gao, T., Hao, J-X., Fransson, R., Sandström, A., Nyberg, F., Wiesenfeld-Hallin, Z., Xu, X-J. (2014) N-terminal truncations of substance P1-7 amide affect its action on spinal cord in- jury-induced mechanical allodynia in . Eur J Pharmacol, 738:319-325.

IV Jonsson, A., Fransson, R., Haramaki, Y., Skogh, A., Brolin, E., Watanabe, H., Nordvall, G., Hallberg, M., Sandström, A., Nyberg, F. (2015) Small constrained SP1-7 analogues bind to a unique site and promotes anti-allodynic effects after systemic injection in mice. Neuroscience, accepted manuscript

V Jonsson, A., Lind, A-L., Wadensten, H., Andrén, P.E., Hallberg, M., Nyberg, F., Gordh, T. (2015) Cerebrospinal fluid levels of substance P (SP) N-terminal fragment SP1-7 in patients with chronic neuro- pathic pain. Manuscript

Reprints were made with permission from Elsevier B.V./Inc.

Contents

Introduction ...... 11 Pain ...... 11 ...... 12 Peripheral neuropathic pain ...... 12 Central neuropathic pain ...... 13 Pain transmission ...... 13 Substance P ...... 14 Biological effects of Substance P ...... 15 NK1 receptor ...... 16 Substance P metabolism ...... 16 SP1-7 ...... 18 Biological effects of SP1-7 ...... 18 SP1-7 binding site ...... 19 peptides ...... 20 Sigma-1 receptor ...... 21 Treatment of chronic neuropathic pain ...... 22 Pharmacological treatment ...... 22 Spinal cord stimulation ...... 22 Drug development ...... 23 Development of SP1-7 analogues ...... 23 Methodological aspects ...... 25 Animal models of chronic pain ...... 25 Human Cerebrospinal Fluid ...... 26 Radioimmunoassay ...... 26 Aims ...... 28 Methods ...... 29 Animals ...... 29 Streptozotocin-induced diabetes ...... 29 Spared Nerve Injury (SNI) ...... 29 Spinal Cord Injury (SCI) ...... 30 Administration of SP, SP1-7 and SP1-7 related compounds ...... 30

Behavioral testing ...... 31 Thermal hyperalgesia in diabetic mice ...... 31 Mechanical allodynia in mice with SNI ...... 31 Mechanical allodynia in rats with SCI ...... 31 Acute in rats with SCI ...... 32 Motor tests in rats with SCI ...... 32 Receptor screen ...... 32 Patients and CSF sampling ...... 32 Radioimmunoassay ...... 33 Separation procedures ...... 33 Radioimmunoassay technique ...... 33 Statistics ...... 34 Results and discussion ...... 35 Effect of SP1-7 and SP1-7-NH2 on thermal hypersensitivity ...... 35 The effect of opioid and ligands on SP1-7-induced behavior ...... 36 Effect of SP1-7 and its amidated and truncated forms ...... 38 Effect of SP1-7 and small constrained SP1-7 analogues ...... 40 Conclusive remarks on the behavioral tests ...... 43 Screen for the SP1-7 binding site ...... 44 SP1-7 in human CSF ...... 44 Conclusions ...... 47 Svensk sammanfattning ...... 49 Acknowledgements ...... 51 References ...... 54

Abbreviations

(+)-PTZ (+)- ACE converting Arg; R Arginine CGRP gene-related CNS Central nervous system CSF Cerebrospinal fluid DAG diacyl glycerol DAMGO Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol DP-IV Dipeptidylpeptidase-IV EM Gln; Q Glutamine Gly; G HIV Human immunodeficiency virus i.p. Intraperitoneal i.t. Intrathecal IASP The International Association for the Study of Pain IP3 Inositol triphosphate Ki Equilibrium dissociation constant for inhibitor binding Leu; L Lys; K MAD Median absolute deviation Met; M MPE Maximum possible effect N/OFQ /orphanin FQ NAcc NEP Neutral endopeptidase NK Neurokinin NLX Naloxone NMDA N-Methyl-D-aspartate nNOS Neutral nitric oxide synthase Nor-BNI Nor- NTI PAG Phe; F

PLC Phospholipase C PNS Peripheral nervous system PPCE Post-proline cleaving enzyme PPT-A Preprotachykinin-A Pro; P Proline RIA Radioimmunoassay RVM Rostroventral medulla SCI Spinal cord injury SCS Spinal cord stimulation SEM Standard error of the mean Sigma-1 Sigma non-opioid intracellular receptor 1 SNI Spared nerve injury SNRI -noradrenaline reuptake inhibitor SP Substance P SPE Substance P endopeptidase STZ Streptozotocin TCA Tricyclic VAS Visula analog scale VTA Ventral tegmental area β-FNA β-funaltrexamine

Introduction

Pain is a subjective experience. The ability to feel pain is essential and pain plays a crucial role in protecting the body from potential harm and tissue damage. However, acute pain can become chronic and chronic pain is a ma- jor health problem that affects a large number of people worldwide. The underlying mechanisms behind chronic pain are not fully clarified and effec- tive pharmacological treatments are lacking. Thus, further knowledge re- garding the mechanisms behind chronic pain is necessary in order to develop new pharmacological therapies.

Pain Pain is defined by the International Association for the Study of Pain (IASP) as “An unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage”. Pain has an emotional as well as a psychological component and cannot be objective- ly measured. Some pain related terms of relevance for this thesis are defined in Table 1.

Table 1. Definition of pain terms as described in the latest version (2011) by the International Association for the Study of Pain (IASP). Term Definition Pain An unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage Allodynia Pain due to a stimulus that does not normally provoke pain Analgesia Absence of pain in response to stimulation that would normally be painful Central pain Pain initiated or caused by a primary lesion or dysfunction in the central nervous system Hyperalgesia Increased pain from a stimulus that normally provokes pain Neuropathic pain Pain caused by a lesion or disease of the somatosensory nervous system Neuropathy A disturbance of function or pathological change in a nerve: in one nerve, mononeuropathy; in several nerves, mononeuropathy multiplex; if diffuse and bilateral, polyneuropathy Central neuropathic pain Pain caused by a lesion or disease of the central somatosensory nervous system Peripheral neuropathic pain Pain caused by a lesion or disease of the peripheral somatosen- sory nervous system

11 Chronic pain Chronic pain is often defined as pain lasting more than three months and is a common health problem with a prevalence ranging from 12 to 30% in the general population (Breivik et al., 2006; Harker et al., 2012; Moulin et al., 2002; Schopflocher et al., 2011). In Sweden, 20% of the population are re- ported to suffer from chronic pain (SBU, 2006). Once the pain has become chronic, it is difficult to manage. The probability to recover from chronic pain is small and in a majority of the patients the pain is indeed persisting (Andersson, 2004; Breivik et al., 2006). Chronic pain is now recognized as a major health problem in Europe as it is connected to under-treatment, unsat- isfactory treatments that cause severe side effects and a reduced quality of life (Breivik et al., 2006). Pain is the most common reasons for people in Sweden to seek health care and the total public costs related to pain are esti- mated to be 9 billion EUR in 2003 (SBU, 2006). Chronic pain is a heterogenic condition, and can be of nociceptive and neuropathic character. Common types of chronic pain with mainly nocicep- tive characteristics include musculoskeletal pain, , and pain. One kind of pain that is common is low-, which can consist of both nociceptive and neuropathic components (Morlion, 2011). Neuropathic pain is defined by IASP as pain that originates from a lesion or disease of the somatosensory nervous system. It can originate from a trauma on the nervous system or as a consequence of disease. The preva- lence of neuropathic pain in the general population is estimated to be 7 – 10% (van Hecke et al., 2014) and is thus a common clinical problem. It is characterized by spontaneous pain, as well as allodynia and hyperalgesia (Woolf and Mannion, 1999) and a majority of neuropathic pain patients re- port a moderate to severe degree of pain (Perez et al., 2013). Neuropathic pain can be divided into central and peripheral neuropathic pain according to its origin, however, the presence of mixed neuropathic pain is not unusual (Perez et al., 2013).

Peripheral neuropathic pain Peripheral neuropathic pain is pain caused by a lesion or disease of the pe- ripheral somatosensory nervous system. The causes underlying peripheral neuropathic pain are diverse and include post-surgical pain, ranging from painful scars to amputation stump pain, and a wide range of pathologies such as postherpetic , human immunodeficiency virus (HIV) and diabe- tes (Scadding and Koltzenburg, 2006).

12 Central neuropathic pain Central pain can arise from a lesion or dysfunction in the central nervous system (CNS). It can be caused by many different kinds of lesions in the or spinal cord, such as multiple sclerosis or spinal cord injury (SCI). Central neuropathic pain, usually manifested at or below the site of injury (Rekand et al., 2012), causes suffering because of its constant and irritating character (Boivie, 2006). Chronic pain occurs in 26–96% of the SCI patients (Dijkers et al., 2009) and can be both nociceptive and neuropathic.

Pain transmission Pain signaling starts when detect a noxious stimulus. The noci- ceptors, i.e. primary afferents, are free nerve endings, which are found throughout the and internal organs, and are divided into Aδ and C fi- bers. Aδ fibers are thinly myelinated fibers with high velocity and thus give rise to the “first pain”, a sharp pain that is precisely localized. The Aδ fibers respond to mechanical and thermal stimuli. C-fibers are unmyelinated fibers, and are thus more slow-conducting compared to Aδ fibers. C fibers are pol- ymodal, meaning they respond to a variety of nociceptive stimuli, and they are responsible for transmission of the “second pain”, which can be de- scribed as burning, dull and long-lasting, and is poorly localized (Marken- son, 1996). In addition, there are myelinated Aß fibers that are large and rapidly respond to non-nociceptive mechanical stimuli (Basbaum et al., 2009). The primary afferents are pseudo-unipolar neurons with a cell body in the with that terminate in the periphery and in the dorsal horn of the spinal cord. In the spinal cord, the termination of the free nerve endings is highly organized. C-fibers terminate in lamina I and II whereas Aδ-fibers terminate in laminae I and V (Basbaum et al., 2009). The primary afferents synapse onto neurons that project from the spinal cord to the brain, where they terminate in several supraspinal sites. There are two main ascending pathways that project to the brain, the spinothalamic and the parabrachial pathway (Hunt and Mantyh, 2001). The neurons project to the , from where information is relayed to the cortex. This pathway is responsible for the discriminative and some affective components of pain. The parabrachial pathway mainly projects to the amyg- dala and , which mediate affective components of pain (Hunt and Mantyh, 2001; Woolf, 2004). Descending pathways are responsible for pain modulating and project from the brain to the spinal cord. The descending neurons mainly originate from the cortex, and the hypothalamus and project to the peria-

13 queductal gray (PAG), from which neurons project to the rostroventral me- dulla (RVM) and subsequently down to the spinal cord (Fields, 2004). The PAG and RVM are two important relay structures with a crucial role in descending pain modulation. In these structures, two distinct types of cells, ON-cells and OFF cells, project to the dorsal horn of the spinal cord. Activation of ON-cells contributes to enhanced nociception (enhanced re- sponse to noxious stimulation) whereas OFF-cell signaling inhibits nocicep- tive transmission (Fields, 2004; Porreca et al., 2002).

Substance P Several are shown to be involved in nociceptive transmission; one of them is the tachykinin substance P (SP), which is focused upon in this thesis. SP, H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (Chang et al., 1971) see Figure 1, was originally discovered by von Euler and Gad- dum in 1931. They found a substance that was particularly abundant in the brain and intestine of the horse that had a depressing effect on blood pressure by peripheral vasodilatation (V. Euler and Gaddum, 1931).

Figure 1. The neuropeptide Substance P

It was not until the 1980’s that other tachykinins were discovered. Four dif- ferent research groups discovered, independently of each other, (NKA) and (NKB) (Kangawa et al., 1983; Kimura et al., 1983; Minamino et al., 1984; Nawa et al., 1983). The tachykinins all have a similar C-terminal sequence, Phe-X-Leu-Met-NH2, where X is either an aromatic (Phe, Tyr) or aliphatic (Ile, Val) amino acid (Erspamer, 1981; Maggio, 1988). Although NKA and NKB have been known for several dec- ades, SP remains as the best characterized member of all the tachykinins. Biosynthesis of neuropeptides is a complex process that is different from the synthesis of classical . synthesis can take place throughout the neuron through enzymatic processes, whereas neu- ropeptide synthesis begins at the ribosomes (Hökfelt et al., 2003). SP is de-

14 rived from at least three different gene transcripts (α, β and γ TAC1), which originate from the tachykinin-1 TAC1 gene, previously known as preprota- chykinin-A (PPT-A) (Carter and Krause, 1990; Krause et al., 1987; Nawa et al., 1983). NKA can be formed from β and γ TAC1, whereas NKB is derived from a separate gene, the TAC3 gene. The gene transcripts encode for large , which are immature prepropeptides that are synthesized in the ribo- somes and packed in large dense core vesicles (Hökfelt et al., 2003). Follow- ing posttranslational conversion and modifications by convertases, carboxy- peptidases and an alfa-amidating enzyme, the mature peptide is released from the neuron (Harrison and Geppetti, 2001; Page, 2013). Amidation of the C-terminal contributes to the stability of SP, and is essential for biologi- cal activity (Eipper et al., 1992; Hanley et al., 1980).

Biological effects of Substance P Although SP was discovered for having a role in blood pressure and vasodi- latation (V. Euler and Gaddum, 1931), several other effects have been at- tributed to the undecapeptide. The discovery of the amino acid sequence of SP made it possible to synthesize SP (Tregear et al., 1971) and subsequently develop methods for biochemical, as well as pharmacological studies, of SP in order to further study its distribution and biological effects. SP is widely expressed both in the peripheral nervous system (PNS) as well as in the CNS. As early as 1953, Pernow published data on the distribu- tion of SP, showing a high concentration in the CNS, but also in the dorsal roots in the PNS (Pernow, 1953). Already at the time of his pioneering work on SP in 1953, Lembeck suggested that this peptide was a neurotransmitter in primary sensory neurons (Pernow, 1983). Subsequent studies showed that SP was present in unmyelinated fibers and that it could be released from free nerve endings in the periphery (Hökfelt et al., 1975) as well as in the spinal cord after nerve stimulation ex vivo (Otsuka and Konishi, 1976) and in vivo (Yaksh et al., 1980). High levels of SP were found in the spinal cord, espe- cially in laminae I and II of the dorsal horn (Hökfelt et al., 1975; Hökfelt et al., 1977), and SP-reactive cell bodies have been found in the dorsal horn (Hökfelt et al., 1977). This further strengthened the theory about the pres- ence of SP in primary afferents. Pearson et al. (1982) revealed that patients with Riley-Day’s syndrome, characterized by diminished pain sensitivity, were devoid of SP-containing nerve endings in the spinal cord. This gave additional support to the hypothesis that SP is involved in nociceptive signal- ing. Since then, numerous studies have confirmed the role of SP in nocicep- tive signaling. With the development of knock-out animals and a SP-conjugated toxin, it was possible to further study the mechanism of SP (Cao et al., 1998) and NK1 receptors (De Felipe et al., 1998) in nociceptive signaling. In addition, it also showed the importance of SP and the NK1 receptor in the maintenance

15 of neuropathic pain, because depletion of the NK1 receptor lamina I neurons resulted in an attenuated response to highly noxious stimuli as well as me- chanical and thermal hyperalgesia (Mantyh et al., 1997; Nichols et al., 1999). Activation of primary afferents results in the release of SP in the periph- ery. The release of SP together with other neuropeptides such as calcitonin gene-related peptide (CGRP) has been suggested to promote . When SP is released in response to a noxious stimulus, it stimulates the release of , which in turn results in plasma extrava- sation and vasodilatation (Foreman, 1987; Pedersen-Bjergaard et al., 1991). In addition to its pronociceptive and inflammatory effects, SP has been implicated in many diverse functions in the brain. As previously mentioned, SP and the NK1 receptor are present in several areas of the brain. SP has been detected in the ventral tegmental area (VTA), hypothalamus, amygdala, nucleus accumbens (NAcc), PAG, and substantia nigra (Hallberg et al., 2000). SP is also involved in and the rewarding effects of opi- oids (Murtra et al., 2000).

NK1 receptor SP and other tachykinins exert their effects through the tachykinin recep- tors. Three different receptors have been characterized and cloned, namely, NK1, NK2 and NK3 (Masu et al., 1987; Shigemoto et al., 1990; Yokota et al., 1989). Similar to most neuropeptide receptors, the NK receptors are G- coupled receptors. Activation of the Gq/11 coupled NK1 receptor, results in activation of phospholipase C (PLC), which in turn results in for- mation of (IP3) and diacylglycerol (DAG). The C-terminal is of importance for the tachykinins to bind to the NK receptors. Due to the similarities in the C-terminal for the tachykinins, SP and other tachykinins are able to bind and exert their effects at any NK receptor. However, SP binds preferentially to the NK1 receptor, whereas NKA and NKB have preferred binding to NK2 and NK3, respectively (Maggi, 1995; Regoli et al., 1994).

Substance P metabolism Neuropeptides do not have reuptake mechanisms like classic neurotransmit- ters, but are substrates for extracellular peptidases. Following its action on the NK1 receptor; SP is subject to enzymatic degradation. Several are involved in the metabolism of SP, the main enzymes are dipeptidylpepti- dase-IV (DP-IV), post-proline cleaving enzyme (PPCE), neutral endopepti- dase (NEP), angiotensin converting enzyme (ACE) and substance P endo- peptidase (SPE). DP-IV and PPCE act on the N-terminal part of the peptide, to yield C-terminal fragments SP3-11 and SP5-11 (Ahmad et al., 1992; Wang et al., 1991; Yoshimoto et al., 1983), whereas NEP and ACE cleave the peptide

16 in the middle or closer to the C-terminal. ACE cleaves mainly at the Phe7- Phe8 and Phe8-Gly9 bonds, and possibly also at the Gly9-Leu10 bond (Skidgel et al., 1984; Yokosawa et al., 1983). It may also subsequently act as a dipep- tidyl peptidase at the C-terminal of SP fragments (Yokosawa et al., 1983). NEP can metabolize SP at four different sites; Gln6-Phe7, Phe7-Phe8 and Phe8-Gly9 and Gly9-Leu10 (Karlsson et al., 1997; Matsas et al., 1983), where- as SPE acts at Phe7-Phe8 and Phe8-Gly9 bonds (Nyberg et al., 1984).

Figure 2. Processing of !-preprotachykinin to SP and subsequently SP1-7. Modified from (Nyberg, 2005)

Metabolism of neuropeptides generally results in inactive peptide fragments. However, it is also possible that enzymatic conversion can result in bioactive fragments with retained or modified biological activity. The latter has been shown to be the case for a number of neuropeptides in the CNS, including SP. Examples of other neuropeptides with bioactive metabolites are dy- norphin, CGRP and nociceptin/orphanin FQ (N/OFQ) (Hallberg, 2014; Hallberg and Nyberg, 2003), which mimic as well as counteract the effects of the mother peptides. In the case of SP, several N-terminal and C-terminal fragments are formed, many with retained biological activity. C-terminal fragments mimic the effect of SP, whereas N-terminal fragments in some cases mimic, but more often have opposing effects when compared with SP (Hallberg and Nyberg, 2003).

17 SP1-7

The N-terminal SP fragment SP1-7 (Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH) rep- resents one of the major bioactive fragments after SP degradation (Sakurada et al., 1999; Sakurada et al., 1985; Zhou et al., 1998) and has received par- ticular interest. Already in 1975 Benuck and Marks (1975) revealed an en- dopeptidase in the brain that was capable of metabolizing SP to yield SP1- 7 and SP1-8 and the corresponding C-terminal fragments SP8-11 and SP9-11. Later, it was recognized that the SP1-7 generating enzyme SPE, an enzyme acting specifically on SP, existed in the as well as cerebrospinal fluid (CSF) with an activity similar to that discovered by Benuck and Marks (Lee et al., 1981; Nyberg et al., 1984), suggesting the presence of SP1-7 in the human CNS. Over the years several new enzymes have been shown to be involved in the formation of SP1-7, including ACE, NEP and SPE, see Figure 2.

Biological effects of SP1-7 Soon after the discovery of the first SP cleaving enzyme, it was recognized that SP1-7 could exert biological effects, in many cases opposite to SP (Hall and Stewart, 1983). Central effects of peripherally administered SP1-7 were proposed based on the finding that SP1-7 was able to induce antinociception. The N-terminal fragment SP1-7 was found to exhibit antinociceptive activity, as opposed to SP and its C-terminal fragment SP5-11 (Stewart et al., 1982). The fragment was found to be abundant in the dorsal spinal cord and has been suggested to be a modulator of SP. In fact, the delayed antinociceptive effect seen from SP (Stewart et al., 1976) has later on been attributed to its N-terminal heptapeptide fragment. In addition to its antinociceptive effect, SP1-7 can potentiate analgesia (Komatsu et al., 2009). SP1-7 has been localized in several areas of the rat brain, including the VTA, hypothalamus, , amygdala, NAcc, PAG, striatum and substantia nigra (Hallberg et al., 2000; Sakurada et al., 1991). In addition to its antinociceptive effect, SP1-7 has been shown to reduce the development of morphine tolerance and morphine withdrawal (Kreeger and Larson, 1993, 1996; Zhou et al., 2009; Zhou et al., 2011), effects that are opposite to the biological effects of SP. Both SP and SP1-7 have been suggested to be in- volved in memory function, although the memory-promoting effect of SP is mediated by the N-terminal (Huston and Hasenöhrl, 1995; Tomaz and Nogueira, 1997). In addition to its opposing effects on opioid actions, SP1-7 has been shown to alleviate the actions of SP. For example, SP-induced bit- ing and scratching (Igwe et al., 1990a; Igwe et al., 1990b; Sakurada et al., 1999; Sakurada et al., 1988) and vasodilatation (Wiktelius et al., 2006) is inhibited by SP1-7. This has further strengthened the idea of SP1-7 as a modu- latory compound on opioid actions as well as on SP-induced effects. Alt-

18 hough SP1-7 has been shown to alleviate morphine-induced hyperalgesia (Sakurada et al., 2007), the intrinsic effects of SP1-7 on hyperalgesia and al- lodynia have not been investigated.

SP1-7 binding site

Several biological effects have been attributed to SP1-7, but still a molecular target is yet to be cloned. SP1-7 does not bind to the NK1 receptor (Geraghty and Burcher, 1993; Michael-Titus et al., 1999), which is most likely due to the missing amino acid sequence Gly-Leu-Met-NH2 in the C-terminal, a sequence crucial for binding to the NK1 receptor. The existence of a receptor that recognizes the N-terminal part of the receptor was initially proposed (Stewart et al., 1982), and thought to be responsible for the biological effects of the N-terminal SP fragments, including SP1-7. The effect could be blocked by the antagonist naloxone (Stewart et al., 1982; Wiktelius et al., 2006) and SP1-7 was believed to bind to or modulate the µ opioid receptor (Krumins et al., 1993; Krumins et al., 1989). However, binding sites for this heptapeptide have been found in the rat brain and spinal cord (Botros et al., 2006; Botros et al., 2008; Igwe et al., 1990a). Other SP fragments and na- loxone had low or negligible affinity for the binding site, as did ligands for the NK receptors, see Table 2 (Botros et al., 2006). Interestingly, the synthet- ic opioid ligand Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (DAMGO) and the highly potent endogenous endomorphin-2 (EM-2) possess high af- finity for the SP1-7 binding site in both the brain and spinal cord (Botros et al., 2006; Botros et al., 2008), see Table 2.

Table 2. Binding affinity of neurokinin and opioid ligands toward the SP1-7 binding site in the spinal cord. Data from (Botros et al., 2006).

Ligand Amino acid sequence Ki (nM)

SP1-7 Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH 0.75 SP1-6 Arg-Pro-Lys-Pro-Gln-Gln-OH 1830 SP1-8 Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-OH 74 SP Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 159 D- SP1-7 Arg-D-Pro-Lys-Pro-Gln-Gln-D-Phe-OH 1.78 9 11 [Sar , Met(O2) ]SP Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Sar-Leu-Met(O2)-NH2 814 R-396 Ac-Leu-Asp-Gln-Trp-Phe-Gly-NH2 >10000 Senktide Suc-Asp-Phe-N-Me-Phe-Gly-Leu-Met-NH2 >10000 DAMGO Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol 13.1 Endomorphin-1 Tyr-Pro-Trp-Phe-NH2 1030 Endomorphin-2 Tyr-Pro-Phe-Phe-NH2 7.5 Naloxone >10000

19 Opioid peptides The use of stretches back thousands of years, and there are early re- ports on the use of opium juice from the opium poppy , being used as an . The term opioid refers to all compounds related to opium, whereas are drugs derived from opium, including natural products such as morphine and , but also semisynthetic analogues thereof (Yaksh and Wallace, 2011). Early opioid research pointed towards central effects from . In 1973, specific receptors for the opioids were detected (Pert and Snyder, 1973; Simon et al., 1973; Terenius, 1973). Three different subtypes of opioid receptors were suggested (Martin et al., 1976) and were later identified and classified as the µ, δ and κ receptors. The existence of the opioid receptors led to the search for and discovery of the endogenous opioid ligands. Leu- and Met-enkephalin were the first endogenous opioids to be dis- covered (Hughes et al., 1975) and were followed by β-endorphin and the among others (Akil et al., 1984). The effect of opioids was shown to be naloxone-reversible, which became a hallmark for the opioids. β-endorphin binds preferentially to the µ and δ receptors, whereas the and dynorphins are δ and κ receptor preferring, respectively (Yaksh and Wallace, 2011). Later on, N/OFQ (Meunier et al., 1995; Reinscheid et al., 1995) and the corresponding NOP receptor (Mollereau et al., 1994) were discovered. The effect of N/OFQ differs from the classical opioid peptides and its role in pain processing is complex since it acts prono- ciceptive at a supraspinal level but is antinociceptive in the spinal cord (Zeilhofer and Calo, 2003). However, this peptide is not the focus of this thesis and will therefore not be discussed any further. Opioid peptides are involved in pain modulation and can act at both a spi- nal and supraspinal level. E.g., enkephalins are expressed in interneurons in the spinal cord and can modulate nociceptive signaling on a pre- as well as postsynaptic level (Hökfelt et al., 1977; Yaksh and Wallace, 2011). Release of opioids in the spinal cord prevents the release of SP from primary affer- ents (Brodin et al., 1983). Enkephalins are also able to modulate descending neurons at supraspinal sites, where they act mainly on the midbrain struc- tures PAG and possibly RVM through inhibition of the ON-cells and disin- hibition of the OFF-cells (Fields, 2004). Approximately 20 years after the discovery of the classical opioid pep- tides and their receptors, two endogenous opioid peptides with high affinity to the µ opioid receptor were discovered - endomorphin-1 (EM-1) and en- domorphin-2 (EM-2) (Zadina et al., 1997). EM-2 has been localized in pri- mary afferents and in laminae I and II of the spinal cord (Martin-Schild et al., 1998, 1999; Martin-Schild et al., 1997). As for EM-1, the highest density is in supraspinal sites, e.g. in the midbrain and brain stem (Martin-Schild et al., 1999). In contrast to the classical opioid peptides that are produced from

20 the large precursor proteins , and prody- norphin, the precursor for the remains unknown. Neverthe- less, they are the most potent endogenous ligands for the µ opioid receptor to which they bind and produce biological activity (Zadina et al., 1997).

Sigma-1 receptor In the work by Martin et al. (1976) attempting to find the different opioid receptors, the sigma receptor was discovered and was suggested to be an opioid receptor subtype, responsible for the paradoxical effects of opiates. It was soon identified that this was not the case. As opposed to opioid recep- tors, the sigma receptor is not a G-protein coupled receptor and neither does it bind naloxone. In fact, the sigma receptor sequence does not resemble any other protein (Su, 2015). The receptor that today is recognized as the sigma- 1 receptor was discovered in 1982, showing to bind the (+)-isomer of ben- zomorphans such as pentazocine (Su, 1982). Although two subtypes of sigma receptors exist, sigma-1 and sigma-2 re- ceptors (Quirion et al., 1992), the focus here will be the sigma-1 receptor. The sigma-1 receptor is an intracellular protein located at the endoplasmic 2+ reticulum where it regulates the flow of Ca through IP3 receptors (Hayashi and Su, 2007; Wu and Bowen, 2008). In addition, it has modulatory actions on several neurotransmitter receptors and ion channels (Cobos et al., 2008). From these actions it has received the current term sigma non-opioid intra- cellular receptor 1. The sigma-1 receptor has been implicated in a variety of diseases, includ- ing , Alzheimers disease, Parkinsons disease and addiction but also in pain (Maurice and Su, 2009). Sigma-1 receptors have been shown to possess anti-opioid actions; sigma-1 receptor attenuate the analgesic effects of opioids whereas antagonists increase the effect (Chien and Pasternak, 1993, 1994, 1995a, b). Moreover, several studies have suggested a role of the sigma receptor in neuropathic pain. Injection of sigma-1 ago- nists produced mechanical allodynia in mice (Yoon et al., 2009), and sigma- 1 receptors were up-regulated in animal models of chronic pain (Roh et al., 2008). In addition, the receptor appeared to be crucial for the development of allodynia and hyperalgesia (de la Puente et al., 2009). It is thus evident that sigma receptor system is of interest in order to understand the underlying mechanisms of, as well as developing new treatment strategies for, neuro- pathic pain.

21 Treatment of chronic neuropathic pain Treatment of chronic neuropathic pain is complicated. There are two main challenges associated with pain treatment. The first is to identify the under- lying cause or mechanisms of the pain, and the second is to find ways to normalize the hypersensitivity that has developed, and thus to prevent the hypersensitivity becoming permanent (Woolf, 2004). Once the pain has be- come chronic, it is difficult to treat and the pain relief achieved in neuro- pathic pain patients is generally only partial.

Pharmacological treatment Although different drugs are used based on the underlying condition, it has not been possible to show any distinct differences in treatment efficacy for most drugs on a specific condition. Tricyclic (TCA), seroto- nin-noradrenaline reuptake inhibitors (SNRI), and the antiepileptic drugs pregabalin and gabapentin have been proposed as first-line treatments (Finnerup et al., 2015). Gabapentin is one of the most commonly used drugs in the treatment of neuropathic pain and has been found to have several posi- tive effects. In addition, despite its adverse effects, it is well tolerated and may also improve sleep (Dworkin et al., 2007; Gordh et al., 2008; Rekand et al., 2012). Opioids have been seen as “the golden standard” when it comes to analgesia and are very effective in relieving acute pain. In neuropathic pain, opioids possess a similar efficiency compared to other treatment regimens (Dworkin et al., 2007). However, because they more frequently give rise to side effects such as sedation and respiratory depression, obstipation and long-term use increases the risk of tolerance and dependence, opioids are recommended as third-line treatment (Finnerup et al., 2015).

Spinal cord stimulation An alternative to pharmacological treatment is spinal cord stimulation (SCS). SCS is not a first-line choice for treatment of neuropathic pain, but an option when pharmacological treatments have been unsuccessful. SCS is becoming more and more common, with approximately 18000 new SCS systems implanted worldwide each year (Linderoth, 2009). SCS was first introduced in the late 1960’s and is based on the gate control theory pro- posed by Melzack and Wall (1965). By stimulating the non-nociceptive Aβ fibers, nociceptive input from Aδ and C fibers will be reduced and thereby reduce the ascending nociceptive signals. The mechanism by which SCS is not fully understood, but includes actions on a spinal as well as on a su- praspinal level (Wolter, 2014). An electrode is placed in the epidural space right above the affected region. Mild to moderate electrical pulses (50-60 Hz) are sent to the spinal cord, which give rise to paresthesia in the painful

22 region of the body (Guan, 2012). More recently, paresthesia free new SCS treatment modes have been developed (De Ridder et al., 2010; Van Buyten et al., 2013) The effects of SCS are intermediate, with half of the patients reporting at least 50% pain relief (Taylor et al., 2014).

Drug development Because pharmacological treatment regimens for neuropathic pain are unsat- isfactory, drugs that can alleviate chronic neuropathic pain are highly desira- ble. Due to the role of SP in nociception and inflammation, a lot of effort has been put into developing NK1 receptor antagonists as putative . With the finding of the genes encoding SP and the NK1 receptor, genetic approaches have been used in order to further understand the role of SP and its receptor. By deleting the TAC1 gene (Cao et al., 1998; Zimmer et al., 1998) or the TAC1R gene (De Felipe et al., 1998), which encode for SP and the NK1 receptor, respectively, it has become possible to further investigate their roles in e.g. pain signaling. Although some results from knock-out stud- ies indicate a role for SP and the NK1 receptor in inflammation as well as for acute and chronic pain, the results were mixed, and were only valid under some testing conditions (Berge, 2014). Clinical trials have indicated a weak antinociceptive effect of a NK1 on dental pain, equivalent to ibuprofen (Rupniak and Kramer, 1999). The only NK1 receptor antagonist available on the market is , which became available in 2003 for the treatment of chemotherapy-induced emesis (Hargreaves et al., 2011). To date, there are no clinical trials that indicate that classic NK1 receptor antag- onists could be used as analgesics (Berge, 2014). It is evident that the development of NK1 receptor antagonists as analge- sics did not turn out as expected. However, dual peptide ligands have been developed, with NK1 and opioid pharmacophores, some of which were found to attenuate neuropathic pain-like symptoms in rats (Guillemyn et al., 2015; Nair et al., 2013).

Development of SP1-7 analogues Since SP and its bioactive fragments seem to play an important role in pain signaling, alternative approaches to target the SP system in order to target pain still remains a topic of much interest. Due to the analgesic effects of SP1-7, it is tempting to draw attention to this heptapeptide fragment and the SP1-7 binding site. A medicinal chemistry program has been commenced in order to develop peptidomimetics, compounds with less peptidergic charac- ter but with retained binding affinity and biological activity, to the SP1-7 binding site. An Ala scan and a subsequent N-terminal truncation study of 7 SP1-7 revealed the importance of the Phe in the C-terminal for binding of the

23 heptapeptide (Fransson et al., 2008). In addition, it was identified that C- terminal amidation increases the binding affinity approximately 5-fold (Fransson et al., 2008). As previously mentioned, the endogenous opioid tetrapeptide EM-2 pos- sesses relatively high affinity for the SP1-7 binding site. A similar truncation study performed with EM-2 resulted in discovery of the dipeptide Phe-Phe- NH2, a small compound with high binding affinity (Fransson et al., 2010). Due to its intriguing pharmacological effects after intrathecal (i.t.) admin- istration (Ohsawa et al., 2011), Phe-Phe-NH2 became the starting point for the synthesis of small peptidomimetics. Small constrained Phe-Phe-NH2 analogues and phenylalanine based carbamates were primarily developed (Fransson et al., 2014; Fransson et al., 2013). Receptor binding studies and subsequent permeability and stability studies revealed one carbamate and one constrained Phe-Phe-NH2 analogue with favorable characteristics (Fransson et al., 2014; Fransson et al., 2013), which have been further evalu- ated in this thesis.

24

Figure 3. SP1-7, EM-2 and three of the most active lead compounds H-Phe-Phe-NH2 (1), Z-Phe-NH2 (2) and the constrained H-Phe-Phe-NH2 analogue (3) identified in an ongoing medicinal chemistry program aimed at developing small peptides and pep- tidomimetics targeting the SP1-7 binding site.

Methodological aspects Animal models of chronic pain The animal studies that have been used in the papers included in this thesis have been chosen in order to model some of the different kinds of pain that are represented in the clinic. Streptozotocin (STZ)-induced diabetes is an animal model of - dependent diabetes mellitus. Due to its selectivity for the glucose transporter 2, STZ can selectively enter the pancreatic cells and subsequently destroy the insulin-producing beta cells (Lenzen, 2008). This model is widely used for preclinical studies of diabetic neuropathic pain. The model was estab- lished in 1993 (Courteix et al., 1993) and is designed to explore neuropathic pain due to a specific disease. The diabetic animals display a hypersensitivi-

25 ty to thermal, mechanical and tactile stimuli (Courteix et al., 1993; Ohsawa and Kamei, 1999). A model of spinal cord injury (SCI) has been used in order to study neu- ropathic pain of central origin. The present SCI model has been developed at the Karolinska Institutet in Stockholm and is induced via ischaemic injury to the spinal cord. By using a laser towards the exposed spinal cord and simul- taneously injecting erythrosine B, a photochemical reaction occurs and is- chemia develops at the site of irradiation. This injury mimics the injury that can arise following i.e. stroke and with accompanying symptoms of tactile, mechanical and cold allodynia in areas around and below the level of injury (Hao et al., 2004; Hao et al., 1996; Xu et al., 1992). Spared nerve injury (SNI) is a model of peripheral neuropathic pain, orig- inally developed in rats (Decosterd and Woolf, 2000) and later transformed to mice (Bourquin et al., 2006). Two of three branches of the sciatic nerve, the tibial and common peroneal nerves, are ligated and subsequently resect- ed, whereas the sural nerve remain intact. This surgery results in an in- creased hypersensitivity to mechanical and cold stimuli in the lateral part of the ipsilateral paw.

Human Cerebrospinal Fluid CSF is produced in the choroid plexus of the brain and is in contact with the entire CNS. It is thus likely that the content of CSF reflects the ongoing neu- ropeptide activity in the brain and spinal cord (Nyberg, 1993). Since it is difficult, if not impossible, to study brain tissue from patients, the CSF would be the best mirror of the activity in the brain. In order to study dis- ease, which is likely to cause a change in the CNS, it is more probable to see a change in the CSF than in the blood circulation. In order to investigate the possible involvement of SP1-7 in human neuropathic pain, CSF was the first choice for peptide analysis.

Radioimmunoassay In the search for peptides in biological material, sensitivity and specificity are two important factors. Radioimmunoassay (RIA) is a method that has been used for this purpose for many years. In fact, this method dates back to the 1950’s, when the ability of measuring the levels of insulin in plasma was described (Yalow and Berson, 1959). The method is based on the interaction between an antigen and its antibody. A labeled antigen competes with the unlabeled antigen (the peptide of interest) to bind to the limited number of binding sites on an antibody. Free antigen is separated from the bound anti- gen with either a secondary antibody or with charcoal solution. The amount of labeled antigen bound to the antibody is measured, and the amount of unlabeled antigen can be calculated with a standard curve of known concen-

26 trations. The more unlabeled antigen in the sample, the less of the labeled antigen able to bind to the antibody. An important strength with this method is the sensitivity of the assay; it is, depending on the antibody, possible to measure down to the low femtomolar range. This is especially important when measuring CSF sam- ples, in which the levels of peptides in general are low. A drawback of the method is that cross-reactivity with similar peptides may occur. This can however be reduced with pre-separation procedures as well as antibodies that is very specific for its antigen. The SP1-7 antibody used in this assay shows less than 5% cross-reactivity towards SP and N- and C-terminal frag- ments (Eriksson et al., 1996). In addition, by doing an ion-exchange chroma- tography prior to the assay, peptides with different ion strength will be eluted in different fractions, which increases the specificity.

27 Aims

The overall aim of this thesis was to investigate the impact of the neuropep- tide SP fragment SP1-7 in chronic neuropathic pain.

The specific aims of this thesis were:

• To study the effects of SP1-7 in animal models of chronic neuropathic pain.

• To further characterize the binding properties of SP1-7 to its binding site.

• To study the pharmacological effects of SP1-7 related compounds in the search for new drugs to treat neuropathic pain.

• To study whether the CSF levels of SP1-7 are altered in neuropathic pain patients.

28 Methods

Animals ICR mice (paper I and II), NMRI mice (paper IV) and Sprague-Dawley rats (paper III) were used. Animals were housed 4-5 per cage in an animal room with a 12 hour light-dark cycle. Food and water was available ad libitum. The temperature was maintained at a constant level throughout each experi- ment. The experimenter was blinded to the drug treatments. All animal ex- periments were approved by a regional ethical committee and were carried out according to the Ethical Guidelines of IASP.

Streptozotocin-induced diabetes Male ICR mice (Tokyo Animal Laboratory Inc, Tokyo, Japan), weighing approximately 20 g, were rendered diabetic by an intravenous injection of STZ (200 mg/kg) prepared in a 0.1 N citrate buffer at pH 4.5. Age matched animals were injected with vehicle alone. Animals with a serum glucose level exceeding 400 mg/dL (22.2 mmol/L) were considered diabetic and were included in the study. Experiments were conducted two weeks after STZ injection.

Spared Nerve Injury (SNI) Male NMRI mice (Taconic, Denmark), weighing approximately 15 g were anesthetized with isoflurane (Abbott Scandinavia, Sweden) (4% v/v for in- duction and 3.5% v/v for maintenance during surgery). The surgical proce- dure was performed according to the protocol by Bourquin et al. (2006). Briefly, the animal was positioned on its right side with the left hindlimb immobilized. An incision was made at the mid-thigh level and the Biceps femoris muscle was separated at the mid-thigh level to expose the sciatic nerve. The common peroneal and the tibial nerves were ligated together with 6.0 silk thread (Vömel, Germany) and subsequently transected distal to the ligation. Great care was taken not to touch or stretch the sural nerve. After nerve transection, the muscles were placed back in the original position and the skin was closed with Reflex 7 mm wound clips. The animals were al- lowed to recover from surgery for at least one week before any post-surgical behavioral testing was performed.

29 Spinal Cord Injury (SCI) Female Sprague-Dawley rats (Möllegård, Denmark), weighing 250 g at the start of the experiment, were used. An ischemic SCI was induced photo- chemically as described previously (Hao et al., 2004; Hao et al., 1996; Xu et al., 1992). Briefly, animals were anaesthetized with 0.5 mg/kg intraperitone- al (i.p.) medetomidine (Domitor, Lääkefarmos, Finland) and 60 mg/kg i.p. (Ketalar, Parke-Davis, Sweden), and one jugular vein was cannu- lated. A midline incision was made on the skin overlying the vertebral seg- ments T12 – L1. The animals were positioned with the vertebral segment T12 or T13 (spinal segments L3-5) beneath the beam of a tunable argon laser (In- nova model 70, Coherent Laser Product Division, Palo Alto, CA; operating at 514 nm with an average power output of 160 mW) and irradiated for 10 min. Immediately prior to and 5 min after the start of the irradiation, erythro- sine B (Red N°3, Aldrich-Chemie, Steinheim, Germany) dissolved in 0.9 % (w/v) saline was injected intravenously at a dose of 32.5 mg/kg. The rat tem- perature was maintained at 37-38°C during the irradiation. The animals were allowed to recover from surgery for at least one week before any post- surgical behavioral testing was performed.

Administration of SP, SP1-7 and SP1-7 related compounds

In paper I and II, SP1-7 and SP1-7-NH2 were administered i.t. as described by Hylden and Wilcox (1980). The doses ranged between 0.5 and 4 pmol/mouse. The mice were manually restrained and the needle was inserted between the L5 and L6 vertebrae. This site is near the end of the spinal cord and minimizes the risk of spinal damage. In paper III and IV, SP1-7 and SP1-7 related compounds were administered i.p. In paper II, compounds were administered in cumulative doses, 1.85, 18.5 and 185 nmol/kg body weight, with doses adapted from (Stewart et al., 1982; Zhou et al., 2011). The results in paper III laid the foundations for the study design in paper IV, where one dose (185 nmol/kg) was administered i.p. SP was purchased from Bachem (Bubendorf, Switzerland) and SP1-7 was purchased from Polypeptide Group (Strasbourg, France). SP1-7-NH2 (Paper II-IV), truncated SP1-7 analogues (Paper III) were prepared as described pre- viously (Fransson et al., 2008) and constrained SP1-7 analogues (Paper IV) were prepared by standard Fmoc chemistry as previously reported (Fransson et al., 2014).

30 Behavioral testing Thermal hyperalgesia in diabetic mice The anti-hyperalgesic response was evaluated using a tail-flick apparatus (KN-205E Thermal analgesimeter, Natume, Tokyo, Japan) as described by D'amour and Smith (1941). Briefly, the mouse was gently placed in a re- strainer. The tail was positioned underneath a heat source that was focused at the tip of the tail. Latencies were determined as the mean of two trials. To study hyperalgesia, the voltage of a 50 W projection lamp was set to 50 V (Ohsawa and Kamei, 1999) giving a response from non-diabetic animals in 10-14 s. The cut-off time was set at 30 s to prevent injury of the tail. Tail- flick latencies were measured before and 5, 30, 60 and 90 min after i.t. injec- tion of SP1-7 or SP1-7-NH2.

Mechanical allodynia in mice with SNI Mechanical threshold was evaluated using the Von Frey test. Calibrated von Frey filaments (Bioseb, France) were applied according to the Chaplan “up- down paradigm” (Chaplan et al., 1994). Animals were placed in transparent chambers on an elevated mesh stand (IITC, USA). Prior to the testing, ani- mals were allowed to acclimatize to the environment for approximately 30- 60 min. The filament was applied for approximately three seconds to the medial plantar side of the paw. A positive response was registered if the mouse quickly withdrew the paw. If a positive response was registered, a thinner filament was applied. In the case of a negative response, a thicker hair was applied. The testing was performed until six responses around the threshold had been monitored. Two baseline values were obtained for each mouse before surgery. On the day of compound testing, a baseline value was obtained before drug injection and the mechanical threshold was evaluated at 15, 30, 45 and 60 min after injection.

Mechanical allodynia in rats with SCI Sensitivity to mechanical stimulation was tested by application of calibrated von Frey filaments (Stoelting, USA) onto the skin. The animal was gently restrained and the filaments were applied with increasing pressure on the back and flanks until they bent. Each filament was applied 5-10 times at a frequency of 1/s and the intensity of stimulation that induced vocalization at a > 75% response rate was considered the pain threshold.

31 Acute nociception in rats with SCI The reaction of the animals to an acute nociceptive stimulus was tested with the tail-flick test. The animal was gently restrained and placed on a glass surface with a heated light source underneath that was focused at the tip of the tail. The intensity of the light was set so that the animal responded in around 6 s. The time from the onset of the heat source to the withdrawal of the tail was registered. The cut-off time was set at 10 s to prevent injury to the tail.

Motor tests in rats with SCI A combined motor test of walking in an open field and a righting reflex to detect the potential motor and sedative effects of SP and its fragments in spinally injured rats was used, as described in Gao et al. (2013). The follow- ing scores were used in the tests. Walking: 0=Normal walking; 5=Walks with only mild deficit; 15=Hindlimb can support weight; 25=Frequent movement of hindlimb, no weight bearing; 40=Minor movement in hindlimb, no weight bearing; 45=No movement of hindlimb, no weight bear- ing. Righting: 0=Normal righting counter to direction of the roll; 5=Weakened attempt to righting; 10=Delayed attempt to righting; 15=No attempt to righting.

Receptor screen Radioligand binding assays and enzyme assays were performed by Ricerca Biosciences, LCC (Ohio, USA; www.ricerca.com). The SP1-7 analogue Phe- Phe-NH2 was screened towards two panels consisting of a total of 111 tar- gets; one core panel that included 96 common drug targets, and one pain panel that included 15 enzymes and receptors of interest with regard to pain. The concentration of the test compound was 10 µM. Assays were performed according to the standard assay protocols at Ricerca Biosciences.

Patients and CSF sampling CSF samples from 11 patients diagnosed with chronic neuropathic pain due to peripheral nerve or root injury and reporting satisfactory pain relieving effect from their SCS system were analyzed for levels of SP1-7 and compared with CSF from eleven voluntary patients (56±2 years, eight females and three males). Pain patients were recruited from a database of SCS users at the Multidisciplinary Pain Center at Uppsala University Hospital. For this study, each participant contributed one CSF sample. Prior to the sampling

32 procedure, the participants refrained from using their spinal cord stimulator for 48 hours. The patient’s pain sensation was rated on a (VAS) 0-10 (0 = no pain; 10 = worst possible pain) before the sample was collected. Control CSF was obtained from patients undergoing minor urological surgery under spinal anaesthesia (47±4 years, five females and six males). They had no known neurological disorder, nor any pronounced pain suffering, and samples were collected according to the same procedure as for the neuropathic pain patients. The samples were frozen at -70°C until analysis was performed.

Radioimmunoassay Separation procedures The frozen CSF samples (1 mL) were thawed on ice and purified by ion exchange chromatography as previously described (Nyberg and Hallberg, 2011). Briefly, each CSF sample was diluted with 3 mL buffer I (0.1M for- mic acid and 0.018M pyridine; pH 3.0) before being applied to separate col- umns packed with SP-Sephadex C-25 gel (total gel volume = 1 mL) pre- washed with 10 mL buffer I. After addition of the sample, and 2 mL of buff- er I, the columns were washed with 10 mL buffer II (0.1M formic acid and 0.1M pyridine). SP1-7 was subsequently eluted with 4 mL buffer IV (0.8M formic acid and 0.8M pyridine). The collected fractions were evaporated in a vacuum centrifuge (Savant, Hicksville, NY) and stored at -80°C until further analysis.

Radioimmunoassay technique The RIA was based on the charcoal adsorption technique as described in (Eriksson et al., 1996; Nyberg and Hallberg, 2011). Briefly, standards and samples were dissolved in MeOH: 0.1M HCl (1:1). Aliquots (25 µL) were incubated in incubation tubes together with 100 µL antibody and 100 µL iodinated peptide (approximately 5000 cpm/tube). The iodinated SP1-7 was 0 prepared from Tyr -SP1-7, see (Hallberg et al., 2000). The antibody and the iodinated peptides were diluted in gelatin buffer. The antibody for SP1-7 (89:2D) was raised in rabbits against the thyroglobulin conjugate and was used at a final dilution of 1:200 000. After 24 hours of incubation at 4°C, 200 µl dextran-coated charcoal (750 mg charcoal and 75 mg dextran in 200 mL 50 mM phosphate buffer) was added to each tube in order to separate the bound from the free peptides. After 10 min, the samples were centrifuged (Beckman GS-15R) for 2 min at 12 000 x g, 4°C. The supernatant (300 µL) was collected and subsequently analyzed in a gamma counter (1470 Wizard, Wallac, Turku, Finland). The detection limit was approximately 1 fmol/tube.

33 Statistics Statistics were performed using Graphpad Prism (Graphpad Software Inc., USA; Paper I, II, IV and V) and Statview software (SAS Institute Inc., USA; Paper III). The results are presented as the mean with standard error of the mean (SEM) or the median with median absolute deviation (MAD). In this thesis, results are also presented as % MPE (maximum possible effect). % MPE was calculated according to the following formula: (test latency-baseline latency) % MPE =100 × (30-pre-drug latency) Where 30 is the cut-off time in seconds. Data was analyzed by one-way or two-way ANOVA followed by Bonfer- roni’s or Dunn’s post hoc test. Paired data was analyzed with the paired t- test or the Wilcoxon signed rank test, whereas unpaired data was analyzed with the unpaired t-test or the Mann-Whitney U test. Correlation was calcu- lated using Pearson correlation. For all the statistics, p < 0.05 was considered significant.

34 Results and discussion

Due to the nature of chronic neuropathic pain and the lack of efficient phar- macological treatments, there is an unmet need for new treatment strategies based on knowledge on pain signaling and the endogenous analgesic system. In this thesis, the biological effect of SP1-7 was investigated in order to pro- vide further insights on the SP system in relation to chronic pain, and to bio- logically evaluate new SP1-7 related compounds in the search for novel anal- gesics. In addition, to further understand the role of SP fragments in neuro- pathic pain patients, human CSF was analyzed for the peptide fragment.

Effect of SP1-7 and SP1-7-NH2 on thermal hypersensitivity Thermal hypersensitivity was studied in STZ-induced diabetic mice and was evaluated using the tail-flick test. The effect of SP1-7 and its amidated ana- logue SP1-7–NH2 was investigated in Paper I and II, respectively. Diabetic mice displayed significant hypersensitivity to thermal stimulation, as as- sessed by the tail-flick test. After i.t. administration of SP1-7, a dose-dependent increase in tail-flick la- tency was seen for both diabetic and control mice, see Figure 4A. The effect was short-lived and peaked 5 min after administration. Although tail-flick latencies were affected in both diabetic and non-diabetic mice, the effect was more prominent in diabetic animals. A low dose of 0.5 pmol SP1-7 resulted in a significant increase in tail-flick latency in diabetic mice, whereas non- diabetic mice were devoid of an effect at that dose. This result may indicate that a low dose of SP1-7 can ameliorate thermal hypersensitivity induced by diabetes because control animals are unaffected.

35

Figure 4. Effect of A) SP1-7 and B) SP1-7-NH2 on tail-flick latency in diabetic and non-diabetic mice 5 min after i.t. administration. Data is expressed as the % MPE ± SEM. ** p <0.01, *** p<0.001 versus respective control group (One-way ANOVA followed by Dunnett’s multiple comparison test); n = 8-10 mice per group.

I.t. injection of SP1-7-NH2 seemed to yield a more prominent analgesic ef- fect than those of the native heptapeptide (see Figure 4B). A significant in- crease in tail-flick latency was found in diabetic animals as well as control animals. A reason for the potent effects of SP1-7-NH2 could be due to the higher binding affinity for SP1-7-NH2 towards the SP1-7 binding site (Fransson et al., 2008). Another plausible reason for these biological effects may lie in the stability, because C-terminal amidation of peptides occurs in order to increase stability, i.e. protect from enzymatic degradation (Eipper et al., 1992).

The effect of opioid and sigma receptor ligands on SP1-7-induced behavior In order to investigate the role of the opioid system on the prolonged tail- flick latency caused by SP1-7 (Paper I) and SP1-7-NH2 (Paper II), animals were pre-treated with opioid receptor antagonists. Data on SP1-7 is presented in Figure 5. The non-specific naloxone completely blocked the effect of SP1-7. This is in line with previous studies, in which SP1-7-induced antinociceptive and anti-inflammatory effects are reversed by naloxone (Stewart et al., 1982; Wiktelius et al., 2006). However, selective µ, δ, and κ opioid receptor block with β-funaltrexamine (β-FNA), naltrindole (NTI) or nor-binaltorphimine (nor-BNI), respectively, could not antagonize the effect of SP1-7. These results suggest that the SP1-7 binding site is distinct from the classical opioid receptors, which are in agreement with receptor binding studies, in which SP1-7 did not compete with specific opioid receptor

36 ligands (Botros et al., 2006; Igwe et al., 1990a). Nevertheless, the fact that naloxone could block the effect of SP1-7 still remains.

Figure 5. Effect of SP(1-7) (0.5 pmol, i.t.) on tail-flick latency after pretreatment with opioid receptor antagonists. NLX = naloxone; β-FNA = β-funaltrexamine; NTI = naltrindol, Nor-BNI = nor-binaltorphimine. * p<0.05, **p<0.01, ***p<0.001 ver- sus before treatment (Two-way ANOVA followed by Bonferroni test); n = 10 per group.

Because naloxone has been reported to bind to a sub-type of sigma receptors (Tsao and Su, 1997), and sigma-1 receptors have been suggested to possess anti-opioid actions (Chien and Pasternak, 1993), the next step was to investi- gate the role of the sigma receptor system in the biological effects of SP1-7 in diabetic animals. Animals were pre-treated with either the sigma-1 receptor (+)-pentazocine [(+)-PTZ] or the sigma-1 receptor antagonist BD1047 (see Figure 6). (+)-PTZ completely blocked the effect of SP1-7 in diabetic mice, whereas BD1047 did not affect the behavior induced by SP1-7. The effective blocking of the heptapeptide-induced effect by (+)-PTZ is indicative of the involvement of the sigma-1 receptor in the mediation of the SP1-7 effect. However, as no distinct interaction between the sigma-1 receptor agonist and the binding sites of SP1-7 could be confirmed it was suggested that the interaction with the sigma-1 receptor occurs downstream from the SP1-7 binding sites. Another plausible explanation could be that SP1-7 acts on a naloxone-sensitive sigma receptor-binding site, which would account for the biological effects seen from both naloxone as well as (+)-PTZ in the present work. However, this remains to be confirmed.

37

Figure 6. Effect of SP1-7 on tail-flick latency after pretreatment with the sigma-1 receptor agonist (+)-PTZ [(+)-pentazocine] and sigma-1 receptor antagonist BD1047. *** p<0.001 versus before treatment; ### p<0.001 vs SP1-7-control treated group (Two-way ANOVA followed by Bonferroni test); n = 6 (control) -10 per group.

Effect of SP1-7 and its amidated and truncated forms

In Paper III, SP1-7 was evaluated for its effect on mechanical allodynia, acute nociception and motor impairment in rats with SCI. SP1-7 was administered i.p. in cumulative doses of 1.85, 18.5 and 185 nmol/kg. SP1-7 could alleviate signs of mechanical allodynia in SCI rats see Figure 7. Although 18.5 nmol/kg tended to increase the mechanical threshold, a statistically signifi- cant difference was not detected until 15 and 30 min after the administration of 185 nmol/kg. SP1-7-NH2 was shown to be even more potent, with a notice- able anti-allodynic effect at 18.5 nmol/kg. An increased response after SP1-7- NH2 was expected according to the results in papers I and II, but also from a previous study on opioid withdrawal (Zhou et al., 2009). The increased effi- cacy of SP1-7-NH2 correlates well to its binding affinity, showing a five-fold higher potency compared with SP1-7 (Fransson et al., 2008). Similar to the results in papers I and II, the effect on mechanical hyper- sensitivity appears to increase in a dose-dependent manner. This is in con- trast to previous studies, which depict a bell-shaped dose-response curve for SP1-7 actions (Herrera-Marschitz et al., 1990; Stewart et al., 1982), with a peak in antinociceptive effect at 18.5 nmol/kg in mice (Stewart et al., 1982). Our results in paper III are more in line with the data presented in papers I and II, which also show a dose-dependent increase in analgesia for SP1-7, as well as SP1-7-NH2. Dose-dependent effects were also seen for the heptapep- tides on anti-inflammatory actions and opioid withdrawal in rats (Wiktelius et al., 2006; Zhou et al., 2009). However, detailed comparisons between the different studies are difficult due to the use of different rat and mouse strains, routes of administration and behavioral tests performed.

38

Figure 7. Effects of (A) substance P (SP) fragment SP1-7, (B) SP1-7-NH2, (C) SP2-7- NH2, (D) SP3-7-NH2, (E) SP, and (F) saline, on vocalization thresholds (mechanical hypersensitivity measured in the von Frey test) in spinal cord-injured (SCI) rats. Data were plotted as the mean ± S.E.M; n = 8 in all except B, where n = 6. * P < 0.05 versus baseline (time 0; Wilcoxon Signed Rank test). # P < 0.05 versus the vocalization threshold for saline-treated SCI rats at the corresponding time point (Mann-Whitney U-test).

Because the amidated heptapeptide appeared to be more efficacious than native SP1-7, and because C-terminal amidation is present in several endoge- nous neuropeptides (Eipper et al., 1992), it is tempting to speculate that ami- dation of the heptapeptide occurs in vivo. However, an amidated form of SP1- 7 has not yet been detected in vivo. 7 As previously mentioned, the amino acid Phe is essential for SP1-7 in or- der to bind to its binding site and to yield biological effects and N-terminal truncations down to a tripeptide have yielded compounds with retained bind- ing affinity to the SP1-7 binding sites (Fransson et al., 2008). In this study, we investigated the role of these amino acids on the biological activity in SCI rats. As shown in Figure 7, removal of Arg1 and Pro2 could be performed without losing the anti-allodynic effect, whereas further truncation resulted

39 in inactive compounds. One plausible explanation for the lack of activity of the shorter peptides, despite high affinity for the binding site, could be that they are more prone to enzymatic degradation. Another explanation could be altered permeability for the shorter peptides. A decrease in permeability would result in decreased levels of peptide at the site of action, which in turn could result in a decrease, if any, in biological activity. No compound showed any sign of sedation in the motor tests performed. This finding is of great interest; because several compounds that are effec- tive in this animal model also affect motor performance (Xu et al., 1992). None of the compounds included in this study had any acute antinociceptive effect. The results from this study are summarized in Table 3.

Table 3. Amino acid sequences and Ki values for SP1-7 radioligand binding to rat spinal cord membrane. Ki data from (Fransson et al., 2008), SP data from (Botros et al., 2006), and effects of the respective peptides on mechanical hypersensitivity (von Frey test), acute nociception (tail-flick test) and motor impairment. Statistically significant effect at (+) 185 nmol/kg; (++) 18.5 nmol/kg body weight.

Peptide Amino acid sequence Ki ± SEM Mechanical Acute Motor (nM) allodynia nociception impairment

SP RPKPQQFFGLM-NH2 159 ± 12 ------SP1-7 RPKPQQF 1.6 ± 0.06 + -- -- SP1-7-NH2 RPKPQQF- NH2 0.3 ± 0.02 ++ -- -- SP2-7-NH2 PKPQQF- NH2 2.8 ± 0.25 + -- -- SP3-7-NH2 KPQQF- NH2 4.4 ± 0.1 + -- -- SP4-7-NH2 PQQF- NH2 4.5 ± 0.3 ------SP5-7-NH2 QQF- NH2 1.9 ± 0.05 ------

Effect of SP1-7 and small constrained SP1-7 analogues

In paper IV, SP1-7 was evaluated for its effect on mechanical hypersensitivity in SNI mice. The dose 185 nmol/kg was chosen according to the results in paper III, in which a dose of 185 nmol/kg was required to detect an anti- allodynic effect with SP1-7. Although the effect was quite short-lived, the mechanical threshold showed a tendency to increase at 15 min after admin- istration and a significant effect was detected 30 min after administration of SP1-7. There was no significant difference compared with baseline after 45 min, see Figure 8A. A similar effect was seen for SP1-7-NH2, Figure 8B. Thus, exogenously administered SP1-7 and its amidated analogue appear to attenuate mechanical hypersensitivity in a similar fashion to what was re- ported in SCI rats (Paper III). Three small constrained SP1-7 analogues, lead compounds in the medicinal chemistry program, were evaluated for their effect on mechanical hypersen- sitivity. The same dose, 185 nmol/kg, was used for these compounds. The amidated dipeptide Phe-Phe-NH2 did not induce any anti-allodynic effect.

40 Although there was a tendency towards an increase in mechanical threshold, see Figure 8C, no statistically significant difference was seen. The lack of effect for Phe-Phe-NH2 was explained by a high in vivo clearance and the compound is most likely rapidly degraded in plasma (Fransson et al., 2014). While this compound is suggested to have poor drug-like properties (Fransson et al., 2014), its anti-allodynic effect was still of interest to this study. In fact, it has been shown to alleviate the signs of both thermal and mechanical hypersensitivity in diabetic mice after i.t. administration (Ohsawa et al., 2011).

41

Figure 8. Effect of A) SP1-7, B) SP1-7- NH2, C) Phe-Phe-NH2, D) Z-Phe-NH2 E) the constrained Phe-Phe-NH2 analogue and F) control on mechanical hypersensitivity in SNI mice. # p < 0.05, ## p < 0.01 compared to after surgery (baseline) (Wilcoxon signed rank test). * p < 0.05, ** p < 0.01 compared to baseline (Friedman test fol- lowed by Dunn’s multiple comparison test).

Z-Phe-NH2 in Figure 8D was the only analogue to induce a slight increase on the mechanical threshold; however, this increase was not statistically significant. There was great variation in response within the group, which most likely contributed to the fact that no statistical difference could be seen.

42 Similar to Phe-Phe-NH2, Z-Phe-NH2 had high plasma instability in rats with a clearance higher than the rat after i.v. administration. The compound was not measurable in rat plasma after oral administration because of exten- sive metabolic degradation (Fransson et al., 2014). Therefore, it is reasona- ble to believe that the plasma stability is poor in the mouse, which would explain the lack of efficacy. Interestingly, when Z-Phe-NH2 is infused in rats, it is capable of entering the CNS. Furthermore, the stability in human plasma is extremely high (Fransson et al., 2014), which makes this com- pound interesting to study further despite its instability in plasma from rats. The constrained Phe-Phe-NH2 had an anti-allodynic effect similar to the heptapeptides, with a significant effect 30 min after administration, see Fig- ure 8E. Although the significant effect was short lasting, the mechanical threshold did not revert to baseline levels after 60 min. A significant effect was expected with Z-Phe-NH2 because rigidification in the C-terminal yield- ed a compound with retained binding affinity for the SP1-7 binding site, to- gether with improved metabolic stability and cell permeability compared with Phe-Phe-NH2 (Fransson et al., 2014; Fransson et al., 2013). With the improved stability, it would have been expected that the effect would be more long lasting. However, it should be noted that this compound is subject to blood-brain barrier efflux (Fransson et al., 2014), which could account for a decreased anti-allodynic effect.

Conclusive remarks on the behavioral tests

SP1-7 and SP1-7-NH2 yield significant analgesic effects after spinal admin- istration in low picomolar doses. Our results talks in favor of the finding that specific binding sites are present in the mouse and rat spinal cord (Botros et al., 2006; Igwe et al., 1990a), to which SP1-7 and its synthetic analogues bind and exert anti-hyperalgesic-like effects. This is strengthened by previous studies, in which biological effects of spinally administered SP1-7 in picomo- lar doses have been detected (Komatsu et al., 2009; Sakurada et al., 2004; Sakurada et al., 2007). It was previously demonstrated that the SP fragment was antinociceptive when administered peripherally and that the mechanism of antinociception is central (Stewart et al., 1982). Studies on the effects of peripheral administered SP1-7 on e.g. opioid tolerance and memory (Huston and Hasenöhrl, 1995; Kreeger and Larson, 1993, 1996; Tomaz and Nogueira, 1997; Zhou et al., 2009; Zhou et al., 2011) are also in favor of a central mechanism of action for this peptide, even though local peripheral effects have been detected in the rat paw (Wiktelius et al., 2006). Interesting- ly, peripheral SP and NK1 receptors have been shown to have a role in neu- ropathic pain (Teodoro et al., 2012), suggesting that peripheral actions of the SP system may yield central effects. However, further studies need to be performed in order to verify this hypothesis.

43 Screen for the SP1-7 binding site

In paper IV, an attempt to further characterize and identify the SP1-7 binding site was performed. The high-affinity dipeptide Phe-Phe-NH2 was screened towards a number of enzymes and receptors that are involved in nociception, but also common drug targets. Results from the behavioral studies presented in this thesis indicate that SP1-7 and SP1-7 related compounds act on or down- stream to sites sensitive for naloxone and (+)-PTZ. SP1-7 has previously been suggested to interact with several receptor and enzyme systems, e.g. µ opioid receptors (Krumins et al., 1993; Krumins et al., 1989), NMDA receptors (Zhou et al., 2000), NK1 (Vigna, 2001; Yukhananov and Larson, 1994) do- pamine receptors (Zhou et al., 2003; Zhou et al., 2004) sigma receptors (Hornfeldt et al., 1996) and neuronal nitric oxide synthase (nNOS) (Kovacs et al., 2001). However, the screening results obtained revealed a low or neg- ligible binding of Phe-Phe-NH2 to the included targets. This is indicative of a poor ability for the targets to recognize the SP1-7 ligands. However, there is still a possibility that the SP1-7 binding site is located on a known receptor, but separate from the classical binding site.

Table 4. Binding affinity of the SP1-7 analogue Phe-Phe-NH2 (10 µM) to a selection of enzymes and receptors related to pain. For a complete list, see Paper IV.

Target % inhibition Glutamate, NMDA, agonism 16 Glutamate, NMDA, Glycine 12 Glutamate, NMDA, 1 nNOS 6 , δ 17 Opiate, κ -3 Opiate, µ -11 Sigma-1, σ1 -5 Tachykinin NK1 4

SP1-7 in human CSF

In paper V, human CSF was analyzed for SP1-7-like immunoreactivity using RIA. The mean level in CSF from 11 patients with severe neuropathic pain was significantly lower than that recorded in samples from controls, see Fig- ure 9. The level of SP1-7-like immunoreactivity in the neuropathic pain group was 11.7 ± 1.7 fmol/mL (range 3.4 – 22.4 fmol/mL) compared with the con- trols that had 18.1 ± 2.0 fmol/mL (range 8.7 – 34.5 fmol/mL). This is, to our knowledge, the first time SP1-7 has been measured in CSF from patients with neuropathic pain. The finding that SP levels are reduced in chronic pain pa-

44 tients has been presented previously in CSF (Almay et al., 1988; Nutt et al., 1980), as well as in saliva (Parris et al., 1990). In addition, another study has shown that SP levels are reduced supraspinally, as measured in ventricular fluid (Jost et al., 1991), which suggests decreased synthesis of SP, as well as SP release, not only at the spinal level. The levels of SP in lumbar CSF mainly reflect release from primary afferents (Nutt et al., 1980). Thus, our results revealing lower SP1-7 levels in neuropathic pain patients are probably due to low levels of the precursor SP in primary afferents.

Figure 9. Levels of SP1-7-like immunoreactivity in CSF from neuropathic pain patients and controls. n=11 in each group. * p<0.05 (unpaired t-test).

Because SP is known as a pain transmitter, it is plausible that the levels of SP, and subsequently SP1-7 levels, would increase in chronic pain patients. Instead, several studies have failed to show an increase of SP in CSF in pa- tients suffering from pain with a neuropathic character (Almay et al., 1988; Bäckryd et al., 2014; Lindh et al., 1997; Nutt et al., 1980). In the case of fibromyalgia and osteoarthritis, the SP levels are increased in CSF (Russell et al., 1994; Vaeroy et al., 1988). Thus, it seems that SP levels differ depend- ing on the etiology of the pain. In previous studies, correlations between pain score and SP- immunoreactivity have been observed in patients with osteoarthritis (Lindh et al., 1997) and with postoperative pain, peaking in their pain intensity (Sjöström et al., 1988). The patients included in this study had refrained to use SCS for 48 hours prior to CSF sampling. They did not use any other analgesics and suffered from severe pain. The possibility of finding an in- verse correlation between the VAS score and the SP1-7-like immunoreactivity was investigated. However, no correlation was found. This could be due to several reasons. Firstly, the number of samples was small, and secondly, the possibility of finding a correlation between SP1-7 levels and pain score is hypothesized based on knowledge about SP in neuropathic pain. As men- tioned previously, SP can be metabolized to SP1-7 by SPE, ACE and NEP (Hallberg and Nyberg, 2003). SPE-like activity is present in human CSF

45 (Nyberg et al., 1984), as well as in spinal cord tissue (Karlsson et al., 1997), and in various other areas of the brain (Zhou et al., 2001). A decrease in the activity of SPE-like enzymes would result in decreased conversion of SP to bioactive fragments, including SP1-7, and subsequently an increase of SP. It seems however less likely that the decrease can be explained by altered en- zymatic activity only, but it could possibly contribute. Although the underlying cause of a decrease in SP1-7 levels in CSF from neuropathic pain patients is not clear, it is an interesting finding, which we believe is mainly a consequence of a reduced amount of its precursor SP. This, together with previous studies on SP in different chronic pain condi- tions (Almay et al., 1988; Bäckryd et al., 2014; Lindh et al., 1997; Russell et al., 1994; Sjöström et al., 1988), suggest a possible role for the SP system, the native undecapeptide, as well as the N-terminal fragment SP1-7 in chronic neuropathic pain.

46 Conclusions

The studies presented in this thesis have identified that the endogenous SP N-terminal fragment SP1-7 has the ability to attenuate signs of thermal and mechanical hypersensitivity in three different animal models of neuropathic pain. In addition, the SP system, in particular SP1-7, appears to be affected in with neuropathic pain. Specifically, the conclusions were:

• The effect of i.t administered SP1-7, could reduce thermal hypersensi- tivity in diabetic mice and the effect increased in a dose-dependent manner. The effect was more pronounced in diabetic mice compared with controls, which suggests that the hypersensitivity is due to a dysregulation in the SP system. An anti-allodynic effect of SP1-7 was observed even after i.p. administration in SCI and SNI rodents.

• C-terminal amidation of SP1-7 led to an increase of analgesic effects, a result that can be attributed to the increased binding affinity to the binding site, but most likely also to increased stability. Similar to SP1-7, there was a dose-dependent effect on thermal hypersensitivity, and the biological effect was observed in animals with SNI and SCI.

• Blocking the effect of SP1-7 with the opioid receptor antagonist na- loxone, as well as the sigma-1 receptor agonist (+)-PTZ, could be indicative of an involvement of the opioid system in the mechanism by which SP1-7 exerts its effects. However, selective opioid receptor antagonists failed to mimic the effect of naloxone, and a large recep- tor screen revealed that neither sigma-1 nor the opioid receptors are the target for SP1-7. The characteristics of SP1-7 binding sites remain to be clarified, although it seems plausible that a down-stream effect of SP1-7 involves the opioid or the sigma receptor system.

• N-terminal truncation of SP1-7-NH2 indicated the necessity of at least five amino acids (Lys-Pro-Gln-Gln-Phe-NH2) in order to retain bio- logical activity. Further truncation led to inactive compounds, de- spite the fact that they still bind to the SP1-7 binding site with high affinity. This indicates that these five amino acids are necessary for the peptide to reach its binding sites. The binding sites are suggested to be located in the CNS, but most likely also in the periphery.

47 • In the evaluation of small SP1-7 analogues, one of the compounds yielded a significant anti-allodynic effect after i.p. administration. This was in agreement with previous in vitro studies on stability and permeability. These results can be useful in further modification studies of the lead compound Phe-Phe-NH2 in the search for novel compounds targeting the SP1-7 binding site.

• Measurement of the SP1-7-like immunoreactivity in CSF from neuro- pathic pain patients revealed a decrease when compared with that of control patients. This suggests that an imbalance in the SP system may contribute to neuropathic pain, and this finding might be im- portant in order to further understand the role of the SP sys- tem/bioactive SP fragments in chronic neuropathic pain.

Finally, although preclinical data are not easily transformed to humans, the finding that exogenously administered SP1-7 alleviates thermal and mechani- cal hypersensitivity, together with the fact that SP1-7 levels may be reduced in neuropathic pain patients, points toward a role of SP1-7 in chronic neuro- pathic pain. Overall, these results indicate that the SP1-7 binding site is a promising target for the development of peptidomimetics and drug-like mol- ecules as potential drugs with the aim to alleviate neuropathic pain.

48 Svensk sammanfattning

Kronisk smärta drabbar upp till 30% av Sveriges befolkning och är ett stort problem i dagens samhälle. Ungefär 10% av befolkningen har svårigheter att leva med sin smärta och livskvaliteten hos personer med långvarig smärta är lägre än vid många andra medicinska tillstånd. Dessutom är kronisk smärta kopplat till höga samhällskostnader. Neuropatisk smärta är en typ av kronisk smärta och definieras som en skada eller sjukdom i det perifera eller centrala nervsystemet och kan uppkomma av flera olika orsaker. Central neuropatisk smärta kan uppkomma till följd av ryggmärgsskada eller sjukdomar som stroke och multipel skleros. Perifer neuropatisk smärta kan uppkomma efter en fysisk skada på nerven, t ex vid operation, men även sjukdomar som dia- betes och HIV kan påverka nerverna och ge upphov till neuropatisk smärta. Den läkemedelsbehandling som finns att tillgå vid neuropatiska smärttill- stånd är i dagsläget inte tillfredsställande. Läkemedelsgruppen opioider, vilket inkluderar morfin, är effektiva vid akut smärta men är mindre effek- tiva vid neuropatiska smärttillstånd. Antidepressiva läkemedel och antiepi- leptika används också till viss del men behandlingen resulterar sällan i en fullständig smärtlindring och är ofta förknippad med kraftiga biverkningar. Det finns ett stort behov av nya läkemedel mot neuropatisk smärta. Substans P (SP) är en neuropeptid som finns i nervceller och som frisätts i ryggmärgen vid ett smärtsamt stimuli. Genom att binda till sin receptor, NK1 receptorn, på en närliggande nervcell bidrar SP bland annat till att smärtsig- nalen skickas vidare upp till hjärnan. Efter att SP har aktiverat sin receptor bryts den ner av enzymer till mindre peptidfragment. Ett av dessa fragment kallas SP1-7, vilken har studerats i detta avhandlingsarbete. SP1-7 består av sju aminosyror (Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH), binder till ett bindnings- ställe som skiljer sig från NK1 receptorn och har biologiska effekter som i många avseenden är motsatta till SP. Bland annat har det påvisats att SP1-7 har förmågan att dämpa akut smärta. Syftet med denna avhandling var att studera SP1-7:s förmåga att kunna motverka smärta av neuropatisk karaktär. SP1-7 och syntetiska SP1-7-analoger har studerats i olika modeller för mätning av smärta med avseende på deras förmåga att hämma just neuropatisk smärta. För att öka förståelsen om hur SP1-7 utövar sin effekt i kroppen, utvärderades effekten av peptider där man tagit bort en aminosyra i taget, s.k. trunkerade varianter av peptiden. Dessu- tom studerades små syntetiska SP1-7-analoger som binder specifikt till SP1-7:s bindningsställe och som tidigare utvärderats med avseende på deras stabilitet

49 i blodet och permeabilitet över biologiska membran. För att få en inblick i SP1-7:s roll vid kronisk neuropatisk smärta, mättes nivåerna av SP1-7 i rygg- märgsvätska från smärtpatienter. Resultaten visade att SP1-7 har förmåga att dämpa tecken på kronisk neu- ropatisk smärta i tre olika modeller. Effekten är tydlig, men kortvarig. Att effekten är kortvarig beror troligtvis på att den bryts ner av enzymer. Genom att byta ut hydroxylgruppen (-OH) mot en amid (-NH2) ökade effekten. Detta beror på att den binder hårdare till bindingsstället för SP1-7, men troligtvis också för att den blir mindre känslig för enzymatisk nedbrytning. N-terminal förkortning var möjlig ner till en tetrapeptid (Pro-Gln-Gln-Phe-NH2) men med ytterligare förkortningar uteblev effekten. Detta talar för att en majoritet av peptidens aminosyror behövs för bibehållen stabilitet och permeabilitet. I studien av de små modifierade molekylerna visade det sig att det gick att prediktera effekten utifrån tidigare studier på stabilitet och permeabilitet. En av de testade substanserna visade en särskilt tydlig effekt. Resultaten från den studien kan ligga till grund för syntes av ytterligare föreningar med po- tentiell effekt på neuropatisk smärta. Smärtpatienterna visade sig ha sänkta nivåer av SP1-7 i ryggmärgsvätska jämfört med kontroller, något som skulle kunna bero på en obalans i SP- systemet hos dessa personer. Dessa resultat bidrar till att öka förståelsen om att bioaktiva metaboliter av SP kan spela en roll i utveckling och ”main- tenance” av vissa typer av neuropatisk smärta. Sammantaget visar resultaten i denna avhandling att nivåerna av det bio- aktiva peptidfragmentet SP1-7 är sänkta hos en grupp patienter med svår kro- nisk neuropatisk smärta. Peptidomimetika som binder till samma bindnings- ställe som SP1-7 kan dämpa tecken på neuropatisk smärta av perifer och centralt ursprung, vilket är ett steg i rätt riktning i sökandet efter nya läke- medel mot kronisk neuropatisk smärta. Mer specifikt kan dessa resultat bidra till ökad kunskap för framtida utveckling av nya läkemedel mot smärta som verkar på samma ställe som SP1-7.

50 Acknowledgements

I have finally reached the time when I can thank all people that have helped me to reach to this point. The funding from Berzelii Technology Centre for Neurodiagnostics and Swedish Medical Research Council, and travel grants from the Swedish Academy of Pharmaceutical Sciences and Anna-Maria Lundins foundation at Smålands nation are greatly acknowledged.

Firstly, professor Fred Nyberg, my main supervisor, thank you for giving me the opportunity to go abroad to do my master thesis, and for accepting me as a PhD student at your lab! Thank you for believing in me at all times. You have taught me how to become an independent researcher and I value that you have had faith in me at all times, and how you have been able to express that when I’ve needed it the most. You are a kind and warm-hearted person, which I really appreciate.

Associate professor Mathias Hallberg, my co-supervisor, thank you for being an excellent mentor when it comes to teaching, it has been a great time with LOA teaching during these years. I also appreciate your valuable input on my work.

Professor Junzo Kamei, thank you for accepting me to come and do research in your lab. It was an instructive time, which gave me a great insight to re- search as well as Japanese culture. Masahiro Ohsawa, thank you for intro- ducing me to the field of pain research. Masa, you have excellent skills in the lab, and although I felt my progress was slow in the lab, your mantra “Practice, practice practice!” finally gave results J

I want to express a big thanks to all my co-authors. Anja Sandström, Rebec- ca Fransson and Anna Skogh, for great collaboration over the years, for great discussions and for all the enthusiasm you’ve put in to the SP1-7 project. Tor- sten Gordh and Anne-Li Lind, for bringing a clinical perspective to my re- search. Torsten, for your never-ending encouragement and enthusiasm. Anne-Li, for great discussions ranging from Berzelii project leadership and life as a PhD student to life outside BMC and commuting to Stockholm. Hiroyuki Watanabe, for excellent assistance in the lab and valuable input on experiments. Zsuzsanna Wiesenfeld-Hallin, Xiao-Jun Xu, Jing-Xia Hao and Tianle Gao for a fruitful collaboration. Tianle, for introducing me to the field

51 of spinal cord injury. Henrik Wadensten and Per Andrén for mass spectrom- etry studies. Henrik, for always being enthusiastic when I present my ideas.

Odd-Geir Berge, for giving me the opportunity to come to AstraZeneca in Södertälje in order to help me establish the SNI model in Uppsala. Carina Stenfors, Sandra Oerther and Helen Jongsma-Wallin for introducing me to SNI experiments and for valuable input on all SNI-related questions I had.

I would like to thank all past and present members of the research group, for creating a great working environment. Erika Brolin for your continuous sup- port. Jenny Johansson and Alfhild Grönbladh, for all the fun we’ve had at conferences, for the everyday chats, and your friendship. Yutaka Haramaki and Anna Lesniak, it’s been great to have some co-workers in the SP1-7 pro- ject. Yutaka, I had a great time doing experiments together with you. Anna, I’m so glad that you came to our lab and kept the SNI animal model as well as SP1-7 project running! Erik and Shanti, it’s great to have you in the group and you contribute with a lot of fun and laughter. Qin Zhou, for introducing me to animal experiments in the lab. Thanks also to all undergrad students. Britt-Marie, for having great source of knowledge about everything concern- ing laborative issues, which you happily shared. Agneta Bergström, for all the help with administrative matters.

All past and present PhD students at the department: Sara, Linnéa, Shima, Stina, Loudin, Helén for fun times at conferences, courses and interesting lunch discussions. Sadia, Lisa, Oskar, Rickard, Richard, Patrik, Inga and Annelie for creating a good atmosphere.

Ingrid Nylander, Lena Bergström and Anne-Li Svensson, for your valuable scientific knowledge and help in the lab. Erika Roman and Marita Berg for valuable input and discussions on animal handling, experiments and ethical permits. Lena Norgren and Raili Engdahl, for being helpful with all kinds of lab-related issues.

All past and present members of the department for contributing to the good atmosphere. Marina R, Elisabeth J, Agneta H, Marianne D, Kjell Å and Magnus E, Annika B, Mikaela A, Stina S, Johanna S, for helping with all administrative matters and making my life as a PhD student easier. Lova S, Sam R and Maria E, and for nice coffee breaks together. Magnus J and To- bias H, for helping me with all computer-related issues; there has been quite a few over the years.

Erika and Sara Palm, I am so happy to have two of my best friends at work. Erika, it’s been great to share room, experiments, conferences and all other work-related matters with you. You’ve added so much fun to my workdays!

52 Sara, thank you for giving me perspectives on work-related matters and helping me with all RIA related questions I have had.

Erika and Alfhild, for proof reading my thesis, and Shanti for excellent lan- guage review of my thesis. All spelling mistakes present are due to last mi- nute changes. Also, thanks to all three of you for being the supportive team I needed in order to finish up this thesis!

Det finns även många utanför BMC som jag vill tacka.

Frida, din vänskap har betytt så mycket för mig, ända sedan vi träffades på gymnasiet. Att sedan ha fått dela åren i Uppsala med dig har varit grymt. Monica, du är en fantastisk vän, och jag är så glad över att mitt första intryck av dig, första dagen på Farmis, stämde perfekt!

Apotekartjejerna i Uppsala, Sara, Maria, Karin, Annika och Stina, tack för er fina vänskap och alla roliga stunder och upptåg vi haft, både under apotekar- utbildningen och därefter. Farmisgänget Frida, Jenny, Kristine, Kristin och Erik för härliga kvällar av umgänge tillsammans med intressanta diskussion- er och för att ni har gett mig en inblick i arbetslivet utanför BMC.

Min bror Martin, tack för alla stunder av umgänge; tack Linda för att du förgyller dem och får dem att hända oftare. Er lille Bo är så härlig, storkusi- nen som Valentin verkligen ser upp till.

Mamma och pappa, tack för ert stöd i allt jag gjort. Ni har alltid trott på mig, vad jag än tagit mig för, och det har betytt så mycket för mig. Ni är fantas- tiska!

Daniel och Valentin – Daniel, tack för allt stöd under alla dessa år. Extra tack för allt du gjort de senaste månaderna när mitt fokus har varit på jobbet. Du kan alltid få mig att se på saker ur nya perspektiv, vilket jag uppskattar. Du är också en fantastisk pappa! Valentin, min lilla solstråle, du sprider så mycket glädje och du är helt underbar. Det är fantastiskt att mötas av ditt leende, även klockan fem på morgonen. Jag älskar er!!

Anna

Uppsala, March 2015

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Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 198 Editor: The Dean of the Faculty of Pharmacy

A doctoral dissertation from the Faculty of Pharmacy, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy. (Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy”.)

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