**5.2.3 Proteasome inhibition and inflammatory pain**

Chronic pain is a major feature of RA and is maintained in part by long-lasting neuroplastic changes in the central and peripheral nervous system. Recent, pre-clinical studies demonstrated that the UPS is one of the systems involved in the maintenance of chronic pain by regulating proteins at pre- and post-synaptic levels [Speese, 2003; Mengual et al., 1996]. Effects of proteasome inhibitor MG132 on inflammatory pain was studied in the AIA animal model. Inflammation in joints significantly reduced the pain bearing capacity in arthritic animals as measured by the paw withdrawal threshold (PWT). Administration of MG132 significant increased PWT in arthritic animals compared to vehicle treated group (Fig 9).

Central and peripheral neuronal mechanisms are thought to play a critical role in inflammatory joint disorders, particularly with regard to inflammation and pain [Benrath, et al., 1995; Levine et al., 1985]. Sensory neuropeptides, substance P (SP) and calcitonin generelated peptide (CGRP), are shown to participate not only in pain modulation but also in inflammatory processes. An up-regulation in the SP and CGRP expression in ankle joints and their corresponding dorsal root ganglia was demonstrated in adjuvant arthritis [Ahmed et al., 1995]. The development and progress of joint inflammation in adjuvant arthritis was significantly attenuated by using the neurotoxin capsaicin, which specifically down regulates sensory innervation [Ahmed et al., 1995]. The beneficial effects of capsaicin on joint inflammation were correlated with reduced levels of SP and CGRP in the ankle joints and corresponding DRG. Methotrexate treatment has been shown to reduce the severity of joint inflammation and destruction, partly due to its inhibitory effect on sensory

mature p50 [Magnani et al., 2000], which is a subunit of mature NF-κB. Significantly lower levels of NF-κB dependent proinflammatory factors such as IL-1, IL-6, and nitric oxide metabolites were found in PS-341-treated animals than in control rats. Thus supporting the concept that the profound anti-inflammatory effects of PS-341 result, in part, from inhibition of NF-κB activity [Palombella et al., 1998]. Proteasome inhibitor MG132 and PS-341 treated animals gained significantly more body weight than the vehicle treated controls indicating that proteasome inhibitors given at therapeutically relevant doses were well tolerated.

Progressive destruction of bone and cartilage plays a pivotal role in the pathogenesis of RA. Effect of proteasome inhibitor MG132 on joint destruction was studied in AIA model [Ahmed et al., 2010]. The radiographic and histological analysis revealed that augmented cartilage and bone resorption, which is a characteristic feature of arthritis, was mitigated by

Bone resorption is a collective result of osteoclast stimulation and suppression of osteoblast precursors within the bone marrow. Previous studies have shown that NF-κB controls osteoclast activation through RANKL signalling [Soysa & Alles, 2009], while inhibition or deletion of RANKL prevents bone destruction [Zwerina et al., 2004; Pettit et al., 2001]. The protective effect of MG132 may be a consequence of with interfering osteoclast activation through the RANKL signalling pathway that is under control of NF-κB, or by enhancing the osteoblast activity. This assumption is supported by *in vitro* and *in vivo* studies indicating that the proteasome inhibitor bortezomib directly suppressed human osteoclast formation and promoted maturation of osteoblasts [Zangari et al., 2006; Mukharjee et al., 2008] and

reduced joint destruction and preserved bone density in CIA mice [Lee et al., 2009].

Chronic pain is a major feature of RA and is maintained in part by long-lasting neuroplastic changes in the central and peripheral nervous system. Recent, pre-clinical studies demonstrated that the UPS is one of the systems involved in the maintenance of chronic pain by regulating proteins at pre- and post-synaptic levels [Speese, 2003; Mengual et al., 1996]. Effects of proteasome inhibitor MG132 on inflammatory pain was studied in the AIA animal model. Inflammation in joints significantly reduced the pain bearing capacity in arthritic animals as measured by the paw withdrawal threshold (PWT). Administration of MG132 significant increased PWT in arthritic animals compared

Central and peripheral neuronal mechanisms are thought to play a critical role in inflammatory joint disorders, particularly with regard to inflammation and pain [Benrath, et al., 1995; Levine et al., 1985]. Sensory neuropeptides, substance P (SP) and calcitonin generelated peptide (CGRP), are shown to participate not only in pain modulation but also in inflammatory processes. An up-regulation in the SP and CGRP expression in ankle joints and their corresponding dorsal root ganglia was demonstrated in adjuvant arthritis [Ahmed et al., 1995]. The development and progress of joint inflammation in adjuvant arthritis was significantly attenuated by using the neurotoxin capsaicin, which specifically down regulates sensory innervation [Ahmed et al., 1995]. The beneficial effects of capsaicin on joint inflammation were correlated with reduced levels of SP and CGRP in the ankle joints and corresponding DRG. Methotrexate treatment has been shown to reduce the severity of joint inflammation and destruction, partly due to its inhibitory effect on sensory

**5.2.2 Proteasome inhibition and joint destruction** 

**5.2.3 Proteasome inhibition and inflammatory pain** 

to vehicle treated group (Fig 9).

the MG132 (Fig 8).

Fig. 8. Radiologic and histologic analysis of bone and cartilage destruction. A, Representative lateral view radiographs of ankle joint of (a) normal, and (b) vehicle- or (c) MG132-treated arthritic animals. B, Changes in the radiographic parameters of osteoporosis, bone erosion and joint space in ankle joints of arthritic animals treated with vehicle or MG132. C, Photomicrographs of haematoxylin and eosin stained ankle joints from (d) control rat; (e) vehicle-treated arthritic rat; and (f) MG132-treated arthritic rat. D, Changes in histologic parameters of cartilage and bone resorption and synovial infiltration in arthritic rats treated with vehicle or MG132. (c; articular cartilage, s; synovial membrane, Ti; tibia and Ta; talus). Modified results from Ahmed et al, 2010.

Proteasome Targeted Therapies in Rheumatoid Arthritis 147

B

Fig. 10. Immunofluorescence micrographs and semi-quantitative analysis of SP and CGRP in rat ankles. A, Nerve fibres positive to SP in the vehicle-treated control rats (a), and in the vehicle- (b) or MG132- (c) treated arthritis rats. B, Semi-quantitative analysis of SP immunoreactive nerve fibres (immunofluorescent area) in ankle joints of the control and arthritic rats treated with vehicle or MG132. C, Nerve fibres positive to CGRP in the vehicletreated control rats (d), and in the vehicle- (e) or MG132- (f) treated arthritis rats. D, Semiquantitative analysis of CGRP immunoreactive nerve fibres (immunofluorescent area) in ankle joints of the control and arthritic rats treated with vehicle or MG132. (s; synovial membrane, p; periosteum and v; blood vessel). Modified results from Ahmed et al, 2010.

Fig. 9. Hind paw withdrawal threshold (PWT) in control and arthritic groups treated with MG132 and vehicle.

neuropeptides [Ahmed et al., 1995]. In the AIA model, strong up-regulation of SP and CGRP in the periosteum and synovium structures, which are pain sensitive and prone to inflammation, was observed. This increased SP and CGRP expression coincided with decreased pain thresholds. Administration of MG132 resulted in the normalization of pain responses as well as significantly down-regulating the expression of SP and CGRP in arthritic ankle joints (Fig 10). Results also indicate that UPS regulates inflammation induced pain behaviour and that UPS-mediated protein degradation is involved in the peripheral sensitization.

Previously it has been shown that proteasome inhibitors MG132 and epoxomicin can prevent the development of behavioural signs of neuropathic pain and abolish abnormal pain induced by sustained morphine exposure [Ossipov et al., 2009; Moss et al., 2008]. These compounds inhibited the release of DYNA and CGRP and normalized molecular changes in the spinal cord contributing to central sensitization [Ossipov et al., 2009; Moss et al., 2008]. Although the cause and neurobiological mechanisms underlying neuropathic and inflammatory pain are different, the common mechanism for the effects of proteasome inhibitors in these pathological conditions is the similar central neuronal mechanism and the activation of neurotransmission mediated by the sensory neuropeptides including SP, CGRP and dynorphins.

The dorsal root ganglia (DRG) and the spinal cord actively participate in the peripheral and central sensitization. DRG neurons have very long t-shaped axons with one end forming a sensory terminal at the skin or joints and other end synapsing in the dorsal horn of the spinal cord. In the spinal cord these neurons project to the outermost region of the spinal dorsal horn (lamina I and outer lamina II) and terminate largely on spinal neurons that project to higher-order pain centers such as the cortex and the hypothalamus in the brain.

In AIA rats, a significant increase in the SP and CGRP expression has been reported in the DRG [Ahmed et al, 1995a]. In the spinal cord an enhanced release of SP and CGRP has been recorded in the lumber dorsal horn during inflammation [Garry & Hargreaves, 1992]. Upregulated SP expression in the DRG correlated with arthritis severity and nociceptive behavior of arthritic rats [Ahmed et al, 1995a]. This agrees with other observations that the altered expression of SP and CGRP is critical for the modulation of pain and inflammation (Ambalavanar et al., 2006; Hutchins et al., 2000). Moreover, it has been reported that

Fig. 9. Hind paw withdrawal threshold (PWT) in control and arthritic groups treated with

neuropeptides [Ahmed et al., 1995]. In the AIA model, strong up-regulation of SP and CGRP in the periosteum and synovium structures, which are pain sensitive and prone to inflammation, was observed. This increased SP and CGRP expression coincided with decreased pain thresholds. Administration of MG132 resulted in the normalization of pain responses as well as significantly down-regulating the expression of SP and CGRP in arthritic ankle joints (Fig 10). Results also indicate that UPS regulates inflammation induced pain behaviour and that UPS-mediated protein degradation is involved in the peripheral

Previously it has been shown that proteasome inhibitors MG132 and epoxomicin can prevent the development of behavioural signs of neuropathic pain and abolish abnormal pain induced by sustained morphine exposure [Ossipov et al., 2009; Moss et al., 2008]. These compounds inhibited the release of DYNA and CGRP and normalized molecular changes in the spinal cord contributing to central sensitization [Ossipov et al., 2009; Moss et al., 2008]. Although the cause and neurobiological mechanisms underlying neuropathic and inflammatory pain are different, the common mechanism for the effects of proteasome inhibitors in these pathological conditions is the similar central neuronal mechanism and the activation of neurotransmission mediated by the sensory neuropeptides including SP, CGRP

The dorsal root ganglia (DRG) and the spinal cord actively participate in the peripheral and central sensitization. DRG neurons have very long t-shaped axons with one end forming a sensory terminal at the skin or joints and other end synapsing in the dorsal horn of the spinal cord. In the spinal cord these neurons project to the outermost region of the spinal dorsal horn (lamina I and outer lamina II) and terminate largely on spinal neurons that project to higher-order pain centers such as the cortex and the hypothalamus in the brain. In AIA rats, a significant increase in the SP and CGRP expression has been reported in the DRG [Ahmed et al, 1995a]. In the spinal cord an enhanced release of SP and CGRP has been recorded in the lumber dorsal horn during inflammation [Garry & Hargreaves, 1992]. Upregulated SP expression in the DRG correlated with arthritis severity and nociceptive behavior of arthritic rats [Ahmed et al, 1995a]. This agrees with other observations that the altered expression of SP and CGRP is critical for the modulation of pain and inflammation (Ambalavanar et al., 2006; Hutchins et al., 2000). Moreover, it has been reported that

MG132 and vehicle.

sensitization.

and dynorphins.

Fig. 10. Immunofluorescence micrographs and semi-quantitative analysis of SP and CGRP in rat ankles. A, Nerve fibres positive to SP in the vehicle-treated control rats (a), and in the vehicle- (b) or MG132- (c) treated arthritis rats. B, Semi-quantitative analysis of SP immunoreactive nerve fibres (immunofluorescent area) in ankle joints of the control and arthritic rats treated with vehicle or MG132. C, Nerve fibres positive to CGRP in the vehicletreated control rats (d), and in the vehicle- (e) or MG132- (f) treated arthritis rats. D, Semiquantitative analysis of CGRP immunoreactive nerve fibres (immunofluorescent area) in ankle joints of the control and arthritic rats treated with vehicle or MG132. (s; synovial membrane, p; periosteum and v; blood vessel). Modified results from Ahmed et al, 2010.

Proteasome Targeted Therapies in Rheumatoid Arthritis 149

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11.

peripheral inflammation induces a dramatic up-regulation of PDYN biosynthesis in nociceptive neurons of the spinal dorsal horn (Przewlocki, 1987; Marvizon et al., 2009). As a future perspective it will be interesting to observe the effects of proteasome inhibition in the DRG and SC in inflammation. In the monosodium-induced model of osteoarthritis, which is a well-recognized model of osteoarthritis, MG132 treatment has normalized the upregulated expression of SP and CGRP in the inflamed knee joints and their corresponding DRG, with reduced pain behavior [Ahmed et al, unpublished data]**.** 
