ExP.ert
`Opinion
`
`1. Introduction
`
`2. The ubiquitin-proteasome
`pathway
`
`3. Proteasome inhibitors: a
`structurally diverse class of
`agents
`
`4.
`
`In vitro mechanisms of
`proteasome inhibitor-induced
`apoptosis
`
`5. Antitumour activity of
`proteasome inhibitors in vivo
`and in vitro
`
`6. Proteasome inhibitors in other
`disease mode ls
`
`7. Clinical experience with
`bortezomib in the treatment of
`cancer
`
`8. Expert opinion
`
`For reprint orders, please
`contact:
`reprints@ashley-pub. com
`
`A<hley Publk•a~< ~~11
`
`www.ashley-pub.com
`
`Review
`
`Proteasome inhibitors as
`therapeutic agents
`
`Julian Adams
`MillenniwnPhaiTJ7aceudca/s, Inc., 75 Sidney5treet, Cambridge, M4 02139. USA
`
`Highly regulated intracellular proteolysis is necessary for cell -cycl e progres(cid:173)
`sion and cell division . The ubiquitin-proteasome pathway (UPP) plays a cen(cid:173)
`tral role in the degradation of proteins and has therefore become an
`important, novel therapeutic target for diseases involving cell proliferation,
`most notably cancer. Proteasome inhibitors were initially used as research
`tools in cell biology to characterise the properties of the UPP. It was later
`determined that proteasome inhibition induced cell-cycle arrest and pro(cid:173)
`grammed cell death (apoptosis) in cancer cells in vitro and could inhibit
`tumour growth in animal xenograft models. Several classes of molecules that
`have proteasome-inhibiting characteristics have been studied. The dipeptidyl
`boronic acid bortezomib (Velcade™ Millennium Pharmaceuticals, Inc.), for(cid:173)
`merly known as PS-341 , LDP-341 and MLN-341 , is a potent and specific inhibi (cid:173)
`tor of the proteasome that holds promise as a potential human therapeutic.
`It is the first proteasome inhibitor to be examined in human clinical trials and
`according to preliminary Phase I and II data, the drug has exhibited both
`manageable toxicities and biological activity.
`
`Keywords: apoptosis. cancer, peptide boronate. proteasome inhibitor
`
`Expert Opin. Ther. Patents (2003) 13(1):45-57
`
`1. Introduction
`
`The 26S proteasome is a multisubunit proteolytic enzyme complex found in the
`nucleus and cytoplasm of all eukaryotic cells. It has tradit ionally been viewed as a cel(cid:173)
`lular recycler of damaged or misfold ed proteins but it has become increasingly ap par(cid:173)
`ent that this proteasome plays a regulatory role through the degradation of many
`types of proteins, including those that regulate cell-cycle progression Ill , transcription
`121. replication 131, tumour suppression 141 and apoptosis 151 . Coordinated degradation
`of various proteins by the proteasome may be required for normal progression of the
`cell cycle and also for accelerated and uncontrolled mitosis, which is characteristic of
`can cer develo pmen t and spread . In fact, researchers observed that the expression of
`proteasomal proteins is increased in cancer cells 161 and is found at elevated levels in
`patien ts with advanced cancer 171. Inhibition of the proteasome may therefore arrest
`growth by interfering with the ordered degradation of prote ins related to regu lation
`of the cell cycle 181. The inhibition of proteasomal function results in cell-cycle arrest
`and apoptosis in many cancer cell lines 19. 101. Pro teasome inhibitors have also been
`shown to inhibit tumour growt h in animal tumour models and to sensitise such
`tumours to traditional cancer therapies l ll -131 and radiation 11 4.1 51.
`The proteasome inhibitor bortezomib (Velcade™ Mill ennium Pharmaceu ticals,
`Inc.) , also known as PS -341 , MLN-341 and LDP-341 , is a first-in-class drug, now
`in Phase III clinical trials in patients with multiple myelo ma 1161. Another proteas(cid:173)
`ome inhibitor, PS-5 19 (Millennium Pharmaceuticals, Inc.) , is now being examined
`in Phase I clinical trials in patients with poststroke and myocardial infarction ischae(cid:173)
`r;n ic injuries. Preclinical and preliminary clinical data suggest that in hibi to rs of the
`ubiquitin- proteasom e pathway have the potential to be promising therapeutics
`aga inst a variety of human diseases, includ ing cancer.
`
`200 3 © Ash ley Publications Ltd ISSN 1354-3776
`
`45
`
`CFAD v. Anacor, IPR2015-01776
`ANACOR EX. 2013 - 1/13
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`

`
`Proteasome inhibitors as therapeutic agents
`
`208 Proteasome
`
`268 Proteasome
`
`198 Regulatory complex
`+ATP
`
`a
`
`b
`
`~5
`
`Figure 1. The 26S proteasorne. a) The 26S proteasome is a multi-protein complex, - 2000 kDa in size, comprised of a proteolytically
`active 20S core particle that is capped by one or two 19S regulatory particles. b) A cross-section of the 20S core shows the orentation of
`the three active sites within a single ~ subunit Note the binding of bortezomib to the chymotrypsin-like active site.
`CT: Chymotrypsin like: PGPH : Peptidyl glutamyl peptid e hyd rolase: T: Trypsin like.
`Reproduced w ith permission from Mi llennium Pharmaceut ica ls. In c .. Ca mbridge. M assachusetts. USA.
`
`~~~0~~
`0 y
`
`0
`
`1 ALLN
`
`2 MG-132 (Z·LLL)
`
`3 PSI
`
`Compounds 1 - 3.
`ALLN : N-acetyl-leucinyl -leucinyl -norleucinal: PSI: Carbobenzoxy -isoleucyl-y· t -bu tyl -g lutamyl -alanyl-leucinal: Z -LLL: Ca rbobenzoxy-leucinyl-leucinyl-leucina l.
`
`2. The ubiquitin-proteasome pathway
`
`Prior to pro tein degradation by the proteasome, target proteins
`are almost always tagged with ubiquitin. The series of events
`that initiate ubiquitylation and ultimately proteasome degrada(cid:173)
`tion is ca lled the ubiquitin- proteasome pathway (UPP) . A
`series of ubiquitin molecules are then attached to the target
`protein by ubiquitin ligases that ultimately create a polyubiqui(cid:173)
`tin 'flag', which is recognisable by the proteasome 1171. M any
`proteins that are to be degraded by the proteasome containing
`PEST and/or PEST-like sequences (containing proline, gluta(cid:173)
`mate, serine, and threonine), which are thought to confer pro(cid:173)
`tein instability due to their role in the targeting of proteins fo r
`degradation 11 81 . The phosphorylation of such amino acid
`sequences in response to cellular signals may mediate the initia(cid:173)
`tion of the ubiquitylation and degradation processes !191. These
`degradation sequences are recognised by ubiquitin ligases,
`
`which catalyse the attachment of a single ubiquitin molecule to
`target proteins 1201. Ubiquitin molecules are subsequently
`added to create a polyubiquitin chain on the targeted protein
`1171. This polyubiquitin chain marks the pro tein as primed for
`degradation and it is via the polyubiquitin chain that the 26S
`proteasome structu re recognises the targeted pro tein. There(cid:173)
`fore, the specificity of proteins targeted for degradation is con(cid:173)
`ferred largely by the enzymes involved in the ubiquitylation
`process , namely the ubiquitin ligating enzymes !1 71 .
`The 26S proteasome is a multi-protein complex co mprised
`of a 20S proteolytic core particle that is capped by two 19S
`regulatory complexes 12 11 (Figure 1) . The 19S subunit binds the
`polyubiquitin chain and cleaves it from the target protein ,
`which is then denatured . allowing access to the 20S subunit
`and subsequent proteolysis 11 71. The 20S proteasome subunit is
`part of a group of proteases called N -terminal hydrolases that
`use
`the side chains of N -terminal serines,
`threonines or
`
`46
`
`Expert Opin. Th er. Patents (200 3) 13(1 )
`
`ANACOR EX. 2013 - 2/13
`
`

`
`Adams
`
`Table 1. Proteasomal substrates.
`
`Cell division
`Pro- and anti-apoptosis factors
`
`Cell-cycle progression
`
`Transcriptional regulation
`
`Tumour suppression
`
`Protein name
`
`Topoisomerase I
`X lAP
`Bax
`Bcl-2
`Survivin
`Securin
`Cyclin E
`p27KIP1
`p21 CIP11WAF1
`
`Oestrogen receptor
`Androgen receptor
`Vitamin D receptor
`Myc
`Fos/Jun
`ld
`IKB-a
`p53
`Rb
`
`Protein function
`
`Ref.
`
`DNA unwinding
`Inhibitor of apoptosis
`Pro-apoptosis factor
`Inhibitor of apoptosis
`Inhibitor of apoptosis
`Anaphase promoting factor
`Kinase activator
`Cyclin-dependent kinase inhibitor
`Cyclin-dependent kinase inhibitor
`Transcription factor
`Transcription factor
`Transcription factor
`Transcription factor
`Transcription factor
`Transcription factor inhibitor
`Inhibitor of NF-KB
`Tumour suppressor/transcription factor
`Tumour suppressor
`
`[3]
`
`[29]
`
`[5]
`
`[30]
`
`[31]
`
`[32]
`
`[1]
`
`[33]
`
`[34]
`
`[35]
`
`[36]
`
`[37]
`
`[381
`
`[39]
`
`[21
`
`[40]
`
`[41
`
`[4 1]
`
`Bax: Bcl-2 associated X protein : NF -KB : Nuclear factor kappa B: Rb : Retinoblastoma : X lAP: X-linked inhibitor of apoptosis.
`
`cysteines to break peptide bonds [221. The proteasome is
`unique in this class , in that it has threonines at each of its cata(cid:173)
`lytic sites 1231- The 20S subunit is comprised of two ~ and two
`Ct. subunits. Each ~ subunit has three active sites: a chymot(cid:173)
`rypsin-like site , a trypsin -like site and a postacidic or caspase(cid:173)
`like site (traditionally termed peptidyl glutamyl peptide hydro(cid:173)
`lase or PGPH) 1241 . The chymotrypsin-like activity appears to
`be the most prevalent proteolytic activity in the 20S structure
`125.261. Proteins targeted for degradation by the proteasome are
`cleaved to generate peptides and ubiquitin molecules 127.281-
`The UPP is responsible for the highly regulated degradation
`of many cellular proteins including those involved in cell-cycle
`progression , tumour suppression , transcription , cell replication
`and apoptosis (Table 1) . The functional d iversity of proteaso(cid:173)
`mal substrates , therefore. suggests that the mechanism of cell(cid:173)
`cycle arrest and apoptosis induced by proteasome inhibition is
`probably multifactorial. Due to its integral part in cellular reg(cid:173)
`ulation and homeostasis, the proteasome has been identified as
`an important, novel target in a variety of cancer types 1101 .
`
`3. Proteasome inhibitors: a structurally diverse
`class of agents
`
`3.1 Peptide aldehydes
`Many of the first proteasome inhibitors were peptide alde(cid:173)
`hydes, which were structurally related to protease substrates.
`Among these include N -acetyl-leucinyl-leucinyl-norleucinal
`
`(ALLN ; calpain inhibitor I, compound 1) , carbobenzoxy(cid:173)
`leucinyl-leucinyl-leucinal (Z-LLL; MG -132 , compound 2)
`and carbobenzoxy - iso leucyl-y- t- bU:ty l -glutamyl~a lanyl - leuc i na l
`(PSI, compound 3) (Table 2) 1421 . Each of these compounds
`reversibly inhibits the chymotrypsin-like activity of the 20S
`proteasome l30.3 ll . They also have slow on-off binding rates
`1261. However, they are nonspecific, having inhibitory activities
`against cysteine proteases such as cathepsin B and the calpains
`and, without the necessary controls, it is not always possible to
`differentiate the cellular effect of proteasome inhibition from
`that of other cellular proteases. Furthermore, the aldehyde
`group displays poor metabolic stability 1261- Thus. despite rich
`preclinical experience with these molecules, none are being
`clinically evaluated , but some are available commercially and
`are often used for basic science purposes.
`
`3.2 lactacystin and derivatives
`Several proteasome inhibitors have been identified through the
`screening of molecular libraries. One such molecule, lactacys(cid:173)
`tin, is a fungal product that hydrolyses in aqueous solution to
`form clasto-lactacystin ~- lacto n e (~ - lactone , compound 4) ; the
`~ - lactone derivative inhibits all three proteolytic activities of
`the proteasome 1451. This inhibition occurs through a covalent
`modification of the trypsin -like and chymotrypsin-like protea(cid:173)
`somal activities 1461. Although the ~ - lactone is considered an
`irreversible inhibitor, its adduct with the proteasome is eventu(cid:173)
`ally hydrolysed in aqueous solution , resulting in the recovery
`
`Expert Opin. Ther. Patents (2003) 13(1)
`
`47
`
`ANACOR EX. 2013 - 3/13
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`

`
`Proteasome inhibitors as therapeutic agents
`
`Table 2. 20S proteasome inhibtitors [26.44].
`
`Type of molecule
`
`Compound
`
`Proteasome inhibitory activity Other significant
`inhibitory activities
`
`Reversible inhibitors Peptidyl boronate
`Peptidyl boronate
`Peptidyl aldehyde
`Peptidyl aldehyde
`Peptidyl aldehyde
`
`Irreversible inhibitors Lactacystin
`derivative*
`P-lactone derivative
`
`Bortezomib (PS-341) (6) Chymotrypsin-like site
`None found
`MG-262
`Chymotrypsin-like site
`None tested
`PSI (3)
`Chymotrypsin-like site
`Calpain; cathepsins
`MG-132 (2)
`Chymotrypsin-like site
`Calpain; cathepsins
`CEP1612
`Calpain; cathepsins
`Chymotrypsin-like site
`Omuralide; clasto(cid:173)
`Chymotrypsin-like site; trypsin-like Cathepsin A; TPPII
`lactacystin-p -lactone
`site; PGPH site
`PS-519
`Chymotrypsin-like site
`
`None found
`
`Epoxyketone
`
`Epoxomicin (5)
`
`Macrocyclic peptide TMC-95
`
`Chymotrypsin-like site; trypsin-like None found
`site; PGPH site
`Chymotrypsin-like site
`
`None found
`
`Peptidyl vinyl sulfone NLVS
`
`Chymotrypsin-like site; trypsin-like Cathepsins Sand B
`site: PGPH site
`' Alt hough the ~ - l actone is considered an irreversible inhibitor. its add uct w ith the proteasome is slowly hydrolysed (Ty, - 20 h) in aqueous solution. resulting in the
`eventual recovery of proteasomal proteolytic activities [261 .
`NLVS NiP-Leu3-vinyl sulfone: PGPH : Peptidyl glutamyl peptide hydrol ase: PSI: Carbobenzoxy-iso leucyl -y-t-butyl-glutamyl-alanyl -leucinal: TPPII Tripeptidyl protease II.
`
`of proteasomal proteolytic activities 1261 . In recent years,
`researchers have created and patented more efficient methods
`to produce the ~ - lacto ne derivative in vitro [20 11. D espite the
`improved specificity of ~ - lacto ne for the proteasome over his(cid:173)
`to ric peptide aldehydes, it exhibits some activity against lyso(cid:173)
`somal cathepsin A 1471. In addition , like the peptide aldehydes,
`the ~ - lacto n e derivative is unstable and in aqueous solution it
`becomes hydrolysed to an inactive metabolite 1451.
`such as
`H owever, other ~ - lactone- rel ated molecules,
`PS-519, have been engineered and examined in precl inical
`d isease models [48.491. It appears that in vitro, PS-519 is
`ap proximately 45 ,000 times more potent than ~ - lacto ne at
`inhib iting the chymotrypsin-like activity of the 20S proteas(cid:173)
`ome [481. Thus , this structural class of inhibitors holds prom(cid:173)
`ise as future therapeutic agents , and PS-519 is now being
`examined in Phase I human clinical trials.
`
`3.3 Peptide epoxyketones
`The epoxyketone-containing proteasome inhibitors, such as
`epoxomicin (co mpound 5) and eponemycin, are potent and
`irreversible proteasome inhibitors [50.511 and were discovered
`from the products of funga l organisms. Epoxomicin primarily
`inhibits the chymotrypsin-like activity of the proteasome but
`also inhibits, to a lesser extent, the two other proteolytic activi(cid:173)
`ties of the proteasome [SOJ. However, eponemyc in and its syn(cid:173)
`thetic analogue dihydroeponemycin inhibit chymotrypsin-like
`and PG PH activity at comparable rates [5 1[. These agents are
`extremely selective and potent proteasome inhibitors and have
`no known activities against any other cellular proteases 150.52 1.
`Other epoxyketone natural product inhibitors have also been
`identified . TMC-95 is a macrocyclic peptide with inhibitory
`
`properties toward the chymotrypsin-like active site [531 , and
`YU101 , a potent linear a . ~ -epoxy ketone, was also developed
`to maximise its binding to the chymotrypsin-like proteasome
`active site 154 1. Both of these molecules are highly selective for
`the proteasome. The future application of these drugs for the
`treatment of human diseases may be possible, although addi (cid:173)
`tional in vitro and in vivo studies are necessary to further char(cid:173)
`acterise their in vivo activities and potential toxicities.
`
`3.4 Peptide boronates
`The reference peptide boronate compound is dansyi-Phe-Leu(cid:173)
`boronate (D FLB , compound 7) . The peptide boronates are
`structurally similar to the peptide aldehyde proteasome inhib(cid:173)
`itors but contain a boronic acid in place of the terminal alde(cid:173)
`hyde moiety. These compounds are up to 1000-fold more
`potent than their aldehyde analogues and are selective for the
`proteasome over common serine proteases [421. The peptide
`boronic acids block the chymotrypsin-like activity of the pro(cid:173)
`teasom e in a reversible manner, but they dissociate from the
`proteasom e more slowly than the related peptide aldehyde
`inhibitors 126 [. Within this new class of peptide inhibitors , the
`dipeptidyl boronic acids offer the additional advantages of rel(cid:173)
`atively low molecular weight and simplicity of synthesis 1421.
`The most widely studied of these molecules is bortezomib
`(co mpound 6). It is a selective, co mpact, water-soluble and
`potent competitive inhibitor. It binds to the chymotrypsin(cid:173)
`like site with high affinity (K; = 0 .6 nM) and it dissociates
`slowly, conferring stable but reversible proteasome inhibition
`[4 21. These characteristics make it especially suitable for clini(cid:173)
`cal use and indeed , clinica l studies examining bortezomib for
`the treatment of a variety of can cers are now underway.
`
`48
`
`Expert Opin . Ther Patents (2003) 13(1)
`
`ANACOR EX. 2013 - 4/13
`
`

`
`Adams
`
`5 Epoxomicin
`
`S
`
`N
`H
`
`0
`
`4 C/asto-lactacystin- ~ -lactone
`
`· ~
`0
`~I
`(N:{
`~'-/r
`OH
`0 y
`I
`
`-....;:
`
`h
`
`N
`
`H
`
`-
`
`N
`
`6 Bortezomib
`
`/N
`
`I
`
`M~,:¢~~,:::(*0
`O 1-
`~~~ H
`
`~ ,...N:ca/'-..._ _....OH
`,..,.. ,,
`B
`I
`OH
`
`h-
`
`0
`
`0
`
`7 DFLB
`
`~
`
`I h-
`
`Compounds 4 - 7.
`DFLB: Oansyi-Phe-Leu-boronate: NLVS: NiP-Leu 3-vinyl sulfone.
`
`3.5 Other agents
`O ther metabolic derivatives have been identified as ZOS protea(cid:173)
`some inhibitors but only prelimi nary studies have addressed
`their activities in cells. Interestingly, it has been suggested that
`the cancer preven tative activities of Chinese green tea are due to
`the polyphenols it contains. One such molecule, epigallocate(cid:173)
`chin-3-galla te (EGCG), was shown to inhibit the chymot(cid:173)
`rypsin -like activity of the proteasome 155 1. The
`fungal
`epipolythiodioxpeperazine toxin , gliotoxin, was also shown to
`be an inhibitor of the chymotrypsin -like activity of the proteas(cid:173)
`ome and has previously been identified as a potent inhibitor of
`the transcription factor, nuclear factor kappa B (NF-KB) 156[.
`
`4. In vitro mechanisms of proteasome
`inhibitor-induced apoptosis
`
`Proteolysis by the 26S proteasome is a fundamental metabolic
`process and the blockade of proteasomal activity induces
`apoptosis in many types of cancer cells 11 01. Importantly,
`tumour ce lls seemed to be more sensitive to proteasome
`inhibitors than normal cells 157-591. For example, B cell chroni c
`lym phocytic leukaem ia (CLL) cells 1601 and patients' multiple
`myeloma cells 1611 were significantly more sensitive to the pro(cid:173)
`apoptotic effects of proteaso me inhibition than lymphocytes
`from healthy individua ls. Simi larly, myeloma cells isol ated
`from patients were ap proximately 1000 times more sensitive
`than patients' normal
`to bortezomib-induced apo ptosis
`plasma cells 1621.
`Proliferating cells were also more sensitive to proteasome inhi(cid:173)
`bition than quiescent or differentiated cells. Thus, human leu(cid:173)
`kaemia cells that were induced to differentiate were significantly
`less sensitive to the apoptotic effects of PSI than their rapidly
`proliferating precursors 1631. Likewise, quiescent endothelial cells
`were less sensitive to proteaso me inhibitor-induced apoptosis
`than were dividing cells 1641 . H owever, this phenomena was not
`
`exclusive to proteasome inhibitor-treated cells, as proliferat ing
`cells treated with adriamycin were more likely to undergo apop(cid:173)
`tosis than treated quiescent cells 1651. In contrast, slowly growing
`spheroid prostate cancer cells that have a very low growth frac (cid:173)
`tion were also sensitive to bortezomib-induced apoptosis 1661.
`which suggests that an accelerated growth rate is not necessary
`for proteasome inhibitor-induced apoptosis.
`In preclinical studies, proteasome inhibition also increased
`the sensitivity (or chemosensitivity) of cancer cells to tradi (cid:173)
`tional anticancer age nts, such as gemcitabine 1111, cisplati n
`167.681, paclitaxe l 1681, irinotecan 1131 and radiation 11•1.1 51, and
`proteasome inhibi tion alone may potentially overcome can(cid:173)
`cer cell chemoresistance 162.69.701. Similarly, higher doses of
`bortezomib induced apoptosis in MIA-PaCa-2 human pan(cid:173)
`creatic cancer ce lls, while low doses of the drug increased the
`cytotoxicity of gemcitab ine 11 11.
`The exact mechanisms which mediate proteasome inhibi (cid:173)
`tion -induced apoptosis are yet to be elucidated. The overex(cid:173)
`press ion of pro -apoptotic factors does not appear to block the
`induction of apoptosis by proteasome inhibition in some sys(cid:173)
`tems 171.721 but may in others 1731. Additionally, proteaso me
`inhib itor-induced apo ptosis may be dependent upon p5 3
`lines 110.74.751 but not in others
`expression in some cell
`110.13.62.70.761 . Although the stabilisation of p21 , p27 and p5 3
`are common responses to proteasome inhibition [10. 13.771 . the
`involvement of these regulatory proteins in apoptosis varies
`between cell types. Furt hermore, it is unknown whether these
`accumulated ubiquitinated proteins are active. However, there
`is some evidence that the ubiquitylation of proteins may be an
`activation or regulatory mechanism 1201 as well as a degrada(cid:173)
`tion marker for the proteasome.
`Interestingly, proteasome inhibition appears to repress the
`transcrip tio n-activating properties of N F-KB. Under normal
`circumstances in the cell , N F-KB is sequestered in the cyto(cid:173)
`plasm and is rendered inactive by the inhibitor protein IKB.
`
`Expert Opin. Ther. Patents (2003) 13(1)
`
`49
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`
`Proteasome inhibitors as therapeutic agents
`
`In times of cell stress however, IKB is degraded by the proteas(cid:173)
`ome and NF-KB translocates to the nucleus. There, NF-KB
`promotes cell survival by initiating the transcription of genes
`encoding stress-response enzymes, cell-adhesion molecules
`and antiapoptotic proteins such as Bcl-2, ciAP1 and ciAP2
`[78.79[. NF -KB is also important in the transcriptional activa(cid:173)
`tion of a number of growth factors including IL-6, IL-8 and
`VEGF [80.81[. Although NF-KB may be required for the
`induction of apoptosis in some instances [82 -84[, it is still
`thought that NF-KB
`is
`important in promoting
`widely
`growth in cancer cells [85-87[ . NF-KB is constitutively active in
`certain malignancies [881 and has been shown to promote
`tumour cell survival and reduce the effectiveness of anticancer
`therapy [50.59[. Furthermore, constitutive activity of NF-KB
`may also be correlated with in vitro drug resistance [89[. Pro(cid:173)
`teasome inhibition can block the chemotherapy-induced acti(cid:173)
`vation of NF-KB, resulting in enhanced chemosensitivity and
`increased apoptosis in cancer cells [13.90[.
`Similarly, inhibition of NF-KB activation by proteasome
`inhibitors
`increased
`radiation-induced
`apoptosis
`and
`enhanced the radiosensitivity of colorectal cancer cells in vitro
`and in vivo [14[. In some cases , however, inhibition of NF-KB
`alone did not produce radiosensitivity in cancer cells; the
`transduction of a 'super repressor' IKB mutant in both Hodg(cid:173)
`kin's cells and prostate cancer cells did not enhance their radi (cid:173)
`osensitivity compared with cells infected with the adenoviral
`control vector [91[. Likewise , the treatment of some cancer
`cells with lethal doses of proteasome inhibitors did not always
`result in the complete abrogation of NF-KB DNA binding
`[69 .9 1[. These findings suggest that, while NF-KB activity is
`associated with the promotion of cell survival, its inhibition is
`only one likely mechanism by which proteasome inhibition
`promotes apoptosis in cancer cells. Current theories of apop(cid:173)
`tosis suggest that the initiation of programmed cell death is
`not mediated by any one cellular pathway or factor but possi(cid:173)
`bly by the ratio of antiapoptotic to pro-apoptotic proteins
`within cancer cells [92[. Proteasome inhibition may therefore
`induce apoptosis or increase sensitivity to apoptosis, by dis(cid:173)
`turbing this balance [93[.
`
`s. Antitumour activity of proteasome
`inhibitors in vivo and in vitro
`
`Several proteasome inhibitors have demonstrated antitumour
`activity in vitro. Lactacystin and MG-132 potently induced
`apoptosis in B cells isolated from patients with CLL [94[ .
`These agents also induced apoptosis in cultured p53 defective
`leukaemia cells [951 and in several human malignant glioma
`lines [96 [. Similarly, MG-132 has been shown to induce apop(cid:173)
`tosis in HD -My-Z Hodgkin's cells and in p53-competent and
`p53 defective gastric cancer cells [76[.
`A National Cancer Institute (NCI , Washington, District
`of Columbia) cancer screen , examining 60 cancer cell lines
`derived from many types of human tumours , indicated
`that bortezomib has potent anticancer activity in vitro 1101.
`
`Bortezomib penetrated cancer cells and inhibited the pro(cid:173)
`teolysis of long-lived proteins. The drug potently inhibited
`the cells' growth; an average dose of 7 nM was required to
`achieve 50% growth inhibition across the 60-cell line
`panel. When the cytotoxicity profiles of bortezomib and
`12 other dipeptidyl boronic acids were compared with the
`profiles of 60.000 other compounds, they were found to be
`unique, with little similarity to standard chemotherapeutic
`agents or investigational agents [10[.
`Additional laboratory studies have subsequently confirmed
`the cytotoxicity of bortezomib in human MCF -7 breast carci (cid:173)
`noma cells 191 and BxPC3 pancreatic cancer cells [77/. Borte(cid:173)
`zomib was as effective or more effective at promoting
`apoptosis in multicellular spheroid cultures, as it was in mon(cid:173)
`olayer cultures and thus may exhibit activity against solid
`tumours with low growth fractions, such as prostate cancer
`[66 /. These seminal in vitro studies suggested that it would be
`relevant to examine the potential antitumour activity of pro(cid:173)
`teasome inhibitors in vivo.
`In 1998. Orlowski eta!. published the first xenograft ani(cid:173)
`mal tumour model study that examined the in vivo antitu(cid:173)
`mour activities of a proteasome inhibitor [57/. Researchers had
`found that the peptide aldehyde Z -LLF-CHO induced apop(cid:173)
`tosis in a dose-dependent fashion in a Burkitt's lymphoma
`(BL) cell line and that the BL cells were 40 times more suscep(cid:173)
`tible to drug-induced apoptosis than non-transformed human
`lymphoblasts. To examine the potential anticancer activity of
`the proteasome inhibitor, BL tumours were established in the
`hind flanks of severe combined immunodeficient (SCID)
`mice and treated with single doses of the peptide aldehyde
`(Z-LLF-CHO)
`or
`the
`equivalent
`peptide
`alcohol
`(Z-LLF-OH) , which has no proteasome-inhibitory or apop(cid:173)
`tosis-inducing properties . The aldehyde-treated mice demon(cid:173)
`strated a 42% tumour growth delay.
`Bortezomib is, however, the only proteasome inhibitor to
`have been extensively studied in xenograft models. Prelimi(cid:173)
`nary data indicate that bortezo mib has significant activity
`against human xenografts of multiple myeloma [97.981. mantle
`celllymphoma-xenografted SCID mice [77 /, pancreatic cancer
`[I ll. colon cancer [131. lung cancer 191. prostate cancer [101 and
`squamous cell carcinoma [991. with some study animals exhib(cid:173)
`iting complete tumour regression.
`The ability of bortezomib to synergise with other standard
`chemotherapies in vitro is in concordance with in vivo data
`from animal tumour models: bortezomib significantly inhib(cid:173)
`ited tumour growth in murine xenograft models of human
`pancreatic cancer (when administered in combination with
`gemcitabine [Ill or irinotecan 1771) and human colon cancer
`(when administered in combination with irinotecan therapy
`1131). It also significantly inhibited tumour growth of human
`colon cancer xenografts in mice receiving ionising radiation
`1141. Similarly, the drug enhanced the chemosensitivity of
`grafted murine prostate tumours treated with radiation [151
`and grafted murine mammary tumours treated with cisplatin,
`cyclophosphamide or radiation [9[.
`
`50
`
`Expert Opin. Ther. Patents (2003) 13(1)
`
`ANACOR EX. 2013 - 6/13
`
`

`
`Histological analys is of blood vessel density suggested that,
`in addition to direct cytotoxic effects, bortezomib had antian(cid:173)
`giogenic activity in tumours [99[. Studies of drug distribution
`fo llowing intravenous injection indicated that, wh ile most
`organs received a simi lar amount of the drug, bortezom ib was
`present at relatively low levels in the skin. Proteasome inhibi(cid:173)
`tion was not detected in CNS tissues, eyes or testes, suggest(cid:173)
`ing that bortezomib did not readily traverse tight endothelial
`cell junctions [I 0[.
`The class of proteasome-inhibiting molecules is structur(cid:173)
`ally diverse; however, it is largely unknown whether these
`agents are substrates for the efflux membrane glycoproteins
`that mediate ce llular mu ltiple drug resistance (MDR).
`Expression of P-glycoprotein was present in K562 cells,
`which when treated, effluxed the calpain and proteasome
`inhibitor ALLN , as well as the specific proteasome inhibitor
`lactacystin. However, treatment with the calpain inhibitors
`N-benzyloxycarbonyl-leuciny l-leucinal
`(zLLal)
`and
`MG -132 retarded P-glycoprotein degradation and lead to an
`accumu lation of ubiquitin ated protein on the surface of
`treated cells as well as in the cytoplasm 11001, suggesting that
`calpain may be involved in its endosomal/ lysosoma l degra(cid:173)
`dative pathway. While somewhat structurally similar to
`ALLN, bortezom ib was not bou nd by the effl ux proteins
`MRP3 or MRP5 in RPMI8226 cells [981 and although inves(cid:173)
`tigators developed a bortezomib-tolerant RPMI8226 line,
`the cells were still sensitive to proteasome inhibition by
`other agents and did not have enhanced tripeptidy l protease
`II
`(TPPII)
`activity. Furthermore, bortezo mib -tolerant
`tumours were sensit ive to bortezom ib treatment in vivo [98 [.
`Thus, it appears that at least in vitro, bortezomib tolerance
`was due to cellular mechanisms other than cellular efflux. To
`date, there has been no evidence of bortezomib to lerance in
`human clini cal trials (David Schenkein, MD, Millennium
`Pharmaceuticals, pers. commun.) .
`
`6. Proteasome inhibitors in other disease
`models
`
`The ability of proteasome inhibitors to modulate cellular pro (cid:173)
`tei n processing, inflammation and antigen presentation has
`prompted the exam ination of these agents in several animal
`disease models as well as ce ll cu lture models.
`
`6.1 Inflammatory disease
`An in vivo, allergen-induced eosinophilia asthma model was
`used to test the anti -inflammatory effects of the lactacystin
`derivative, PS-519 [•18[. Administration of the drug into rat
`lungs reduced leukocyte numbers and inhibited cellular infil(cid:173)
`tration in the lungs of sensitised rats after antigen challenge.
`Furthermore, in a mouse delayed-hypersensitivity model, the
`intravenous adm inistration of either PS-5 19 or ~ - lactone
`reduced ear swell ing to antigen challenge [48[.
`Epoxomicin has also been examined in an inflammation
`animal model [50[. BALB/c mice were injected intravenously
`
`Adams
`
`with the drug for 6 days (0.58 mg/kg) prior to an immu (cid:173)
`nological challenge to the ea rs. Contact sensitivity to epox(cid:173)
`omicin was measured 24 h later in immunised and non (cid:173)
`immunised an imals. A similar study was also performed
`with a sing le bolus dose (2.9 mg/kg) of drug followed by
`antigen challenge. Both experiments suggested that epox(cid:173)
`om icin had potent anti -inflammato ry properti es. Borte(cid:173)
`zo mib has also been shown to have anti-inflammatory
`effects in an arthritis model; rats receiving ora l drug had
`reduced symptoms of po lyarthritis after administration of
`streptococca l Type A ce ll wa ll proteoglycan [lOll.
`In addition to the asthma mode l study, PS-5 19 has been
`examined in an ischaemia and reperfusion injury model in
`isolated rat heart tissue; PS-5 19 reduced the accumulation of
`peripheral blood mononuclear cells (PBMCs) in ischaem ic
`myocardium and attenuated the expression of P-selecti n on
`coronary vascular endothelium [491 . In a rat focal cerebra l
`ischaem ia mod el, PS-5 19 decreased neutrophil infiltration of
`cortical and striatal infracted tissue [102[ . Also of interest was
`the fact that rats treated with the drug showed significant