**5.2.1 Proteasome inhibition and joint inflammation**

Proteasome inhibitors PS-341 and MG132 have been tested in different animal models of arthritis (Table 1) with pronounced anti-inflammatory effects. Here effects of proteasome inhibitor MG132 in adjuvant induced arthritis (AIA) rat model will be discussed in details. MG132 was administered subcutaneously daily at the onset of arthritis. Two weeks of administration significantly reduced signs of inflammation including swelling, redness and warmth in ankle joints compared to vehicle treated arthritis animals. Similar effects were observed in studies where proteasome inhibitor PS-341 was administered in different arthritis models. PS-341 significantly attenuated the arthritis severity and the clinical progression of the T cell dependent chronic phase of the disease. The chronic phase of arthritis was also associated with increased serum levels of NF-κB dependent proinflammatory factors such as IL-1, IL-6, and nitric oxide metabolites [Palombella et al., 1998]. The expression of TNF-α, IL-1β, IL-6, MMP-3, COX-2 and iNOS were decreased in PS-341 treated animals compared to untreated [Lee et al., 2009].

These are naturally product proteasome inhibitors isolated from actinomycete fermentation broths by screening for antitumor activity in mice. Examples of this class include Epoxomicin and eponemycin. These compounds inhibit the chymotrypsin like site only or the chymotrypsinlike and caspaselike, respectively. In contrast to previously mentioned inhibitors, epoxomicin initially forms a covalent bond between the proteasome's aminoterminal threonine hydroxyl at its C-terminal ketone carbonyl. This primary adduct formation is followed by formation of a stable six-membered ring adduct by a second attack

Proteasome inhibitors exhibit anti-inflammatory and anti-proliferative effects. Their use in diseases characterized by these processes is thought to be promising but the effects of proteasome inhibitors on the pathogenesis of inflammatory join disorder such as RA remain quite limited. To date the effects of proteasome inhibition have been studied only in animal models of arthritis; streptococcal cell wall induced polyarthritis in rats, collagen induced arthritis and adjuvant induced arthritis (Table 1). These animal models have several clinical and pathological similarities with human rheumatoid arthritis regarding inflammation, pain, swelling, synovial hyperplasia and destruction of cartilage and bone [Kannan et al.,

Animal models Proteasome inhibitor References

collagen induced arthritis in mice PS-341 Lee et al., 2009 Adjuvant induced arthritis in rat MG132 Ahmed et al, 2010 Adjuvant induced arthritis in rat PS-341 Yannaki et al., 2010

Table 1. List of proteasome inhibitors used in animal models of RA.

**5.2.1 Proteasome inhibition and joint inflammation** 

treated animals compared to untreated [Lee et al., 2009].

polyarthritis in rat PS-341 Palombella et al., 1998

Proteasome inhibitors PS-341 and MG132 have been tested in different animal models of arthritis (Table 1) with pronounced anti-inflammatory effects. Here effects of proteasome inhibitor MG132 in adjuvant induced arthritis (AIA) rat model will be discussed in details. MG132 was administered subcutaneously daily at the onset of arthritis. Two weeks of administration significantly reduced signs of inflammation including swelling, redness and warmth in ankle joints compared to vehicle treated arthritis animals. Similar effects were observed in studies where proteasome inhibitor PS-341 was administered in different arthritis models. PS-341 significantly attenuated the arthritis severity and the clinical progression of the T cell dependent chronic phase of the disease. The chronic phase of arthritis was also associated with increased serum levels of NF-κB dependent proinflammatory factors such as IL-1, IL-6, and nitric oxide metabolites [Palombella et al., 1998]. The expression of TNF-α, IL-1β, IL-6, MMP-3, COX-2 and iNOS were decreased in PS-341-

**5.1.4 Epoxyketones** 

2005].

by the terminal free amino group [Groll et al., 2000].

streptococcal cell wall induced

**5.2 Proteasome inhibition in animal model of arthritis** 

Fig. 7. Effects of proteasome inhibitor MG132 on (A) arthritis index; severity of arthritis was scored using a macroscopic scoring system according to changes in erythema and oedema in each paw (B) NF-κB and p50 activation in arthritic ankle joint of AIA rat. (a) autoradiograph of electrophoretic mobility shift assay. The upper two bands represent NF-κB and p50 homodimer complexes (indicated by arrows), (b) and (c) semi-quantification of the NF-κB and (p50)2 levels.

MG132 treatment significantly down-regulated the expression of NF-κB1 (p50) in inflamed ankle joints as well as the DNA binding activity of both NF-κB and p50 homodimer in arthritic ankle joints (Fig 7 A and B). These results indicate that MG132 hinders the nuclear localization of NF-ĸB by retaining them in the cytosol in an inactive form bound to the inhibitory protein IκB, and also blocks UPS-mediated processing of the p105 precursor to

Proteasome Targeted Therapies in Rheumatoid Arthritis 145

Fig. 8. Radiologic and histologic analysis of bone and cartilage destruction. A,

Ta; talus). Modified results from Ahmed et al, 2010.

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

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.
