Signal Transduction Pathway Targets for Anticancer Drug Discovery
`
`Current Pharmaceutical Design, 2000, 6, 361-378
`
`361
`
`Alex A. Adjei*
`
`Mayo Clinic, Division of Medical Oncology, 200 First Street S. W., Rochester, MN 55905,
`USA
`
`Abstract: There are currently over 80 agents officially approved for the treatment
`of cancer world-wide. However, the most common epithelial cancers, which cause
`greater than 75% of cancer deaths, remain incurable. Most drugs have been developed
`empirically by testing large numbers of chemicals on rapidly growing transplantable
`rodent tumors, and more recently, human tumor xenografts. This approach has
`identified prodeminantly DNA-active drugs that are considerably toxic and have
`limited efficacy. Novel molecular targets, which are selective for neoplastic cells, are needed for
`chemotherapeutic agents to improve cure rates of epithelial malignancies, with acceptable toxicity. In
`recent years, agents inhibiting signal transduction pathway molecules have entered clinical trials. These
`include antibodies and small molecules, which inhibit growth factor receptors and their receptor tyrosine
`kinases, inhibitors of cytoplasmic second messengers such as ras, raf and MEK, inhibitors of protein
`trafficking, and inhibitors of protein degradation.
`
`INTRODUCTION
`
`Cell proliferation and differentiation are
`regulated by a number of hormones, growth factors
`and cytokines. These molecules interact with
`cellular receptors and communicate with the nucleus
`of the cell through a network of intracellular
`signaling pathways (Fig 1). In cancer cells, key
`components of these pathways may be altered by
`oncogenes through over-expression or mutation,
`leading to disregulated cell signaling and cell
`proliferation. The components of these abnormal
`signaling pathways, which are specific to neoplastic
`cells, represent potential selective targets for new
`anticancer therapies. These potential targets include
`ligands (typically growth factors), cellular receptors,
`intracellular second messengers and nuclear
`transcription factors. A detailed description of all
`these possible targets is beyond the scope of this
`review. The interested reader is referred to several
`recent, excellent reviews [1-5]. This discussion will
`focus exclusively on
`the
`targets for which
`promising anti-neoplastic agents are in clinical
`trials.
`
`INHIBITION OF GROWTH FACTOR
`RECEPTOR BINDING
`
`The first, obvious point of intervention in a
`signaling cascade is the neutralization of ligands
`
`*Address correspondence to this author at the Division of Medical
`Oncology, Mayo Clinic, 200 First St. SW, Rochester, MN 55905, USA;
`Telephone:
`507-538-0548;
`Fax:
`507-284-1803;
`E-mail:
`adjei.alex@mayo.edu
`
`before they can associate with their receptors. This
`approach has been investigated, initially through the
`use of antibodies, which interact with growth
`factors. Small cell
`lung cancer, and other
`malignancies secrete bombesin-like peptides, such
`as gastrin releasing peptide. A recently published
`phase I trial established that repeated doses of
`monoclonal antibody 2A11, which binds to the
`bombesin-like peptide, GRP with high affinity,
`could be given safely to SCLC patients, and
`sustained plasma levels could be achieved on a 1-
`week schedule of antibody administration [6]. This
`approach is limited by the need to prospectively
`identify patients whose tumors express the receptor,
`and whose plasma contains significant amounts of
`circulating growth factor. Non-specific inhibition of
`several growth factors was explored through the use
`of the polysulfonated naphthylurea, suramin [7].
`This agent neutralized a number of growth factors,
`but presented challenging dosing problems because
`of an extremely long half-life and significant
`toxicities. In spite of evidence of modest activity in
`prostate cancer,
`its development has been
`discontinued, because of toxicity problems.
`
`The second approach to abrogating signaling
`pathways is the prevention of the binding of growth
`factors to their receptors. Several strategies have
`been under development in an attempt to block
`growth factor receptors. The most successful
`approach to date has been the development of
`monoclonal antibodies which bind to receptors and
`by so doing prevent the binding of the endogenous
`ligands.
`
`1381-6128/00 $19.00+.00
`
`© 2000 Bentham Science Publishers B.V.
`
`NOVARTIS EXHIBIT 2014
`Breckenridge v. Novartis, IPR 2017-01592
`Page 1 of 18
`
`

`

`362 Current Pharmaceutical Design, 2000, Vol. 6, No. 4
`
`Alex A. Adjei
`
`(1). Schematic representation of some important signal transduction pathways in cancer cell proliferation. DAG,
`Fig.
`diacylglecerol; IP3, inositide triphosphate; PI3K, phosphoinositide-3-kinase; PLC, phospholipase C; PIP2, phosphoinositide
`diphosphate; PKC, protein kinase C; MEK, mitogen-activated protein kinase kinase; MAP kinase, mitogen-activated protein
`kinase.
`
`The Epidermal Growth Factor Receptor
`(EGFR) Family
`
`The epidermal growth factor (EGF) receptor was
`cloned in 1984 by Ullrich et al. [8]. This receptor
`has two cysteine-rich regions in the extracellular
`domain and a single kinase domain. Three other
`members of this family, HER-2, HER-3 and HER-4
`(referred to also as erbB2, erbB3 and erbB4) are
`known. The designation HER-2 stands for Human
`Epidermal growth factor-like Receptor type 2.
`Members of the EGF receptor family and their
`ligands are overexpressed or expressed as an
`autocrine loop in a number of tumor types,
`including pancreatic, lung, ovarian, renal cell, gastric,
`hepatocellular and breast cancers [10-12].
`
`Anti-EGFR Antibodies
`
`Because over-expression of EGFR has been
`associated with a more aggressive disease and a
`poor prognosis, the blockade of EGFR activation
`has been proposed as a target for anticancer
`therapy. The most promising agent is the human-
`mouse chimeric monoclonal antibody 225 (C225),
`which inhibits activation of the EGFR receptor
`tyrosine kinase. This inhibition of EGFR activation
`
`causes cell cycle arrest in G1. The mechanism of
`growth inhibition has been shown to involve an
`elevation in the levels of p27KIP1 and inhibition of
`cyclin-dependent
`kinase-2
`activity
`[13].
`Preclinically, C225 in combination with several
`chemotherapy
`agents,
`including
`cisplatin,
`doxorubicin and paclitaxel exhibited synergistic
`antitumor activity, with successful eradication of
`well-established
`tumor xenografts
`that were
`resistant to treatment with either C225 or drug alone
`[14]. Phase I clinical trials have established the
`safety of repeated administration of single-agent
`C225 at concentrations that maintain receptor-
`saturating blood levels for up to 3 months [15].
`Phase I
`trials exploring C225
`treatment in
`combination with
`the chemotherapy agents
`mentioned above, are ongoing [16], and single-
`agent phase II trials are in progress. Preliminary
`data indicate that antitumor activity is significantly
`augmented when the antibody
`is utilized
`in
`combination with cytotoxic chemotherapy.
`
`Anti-HER-2 Antibodies
`
`The proto-oncogene HER-2/neu is localized to
`chromosome 17q, and encodes a 185kDA
`
`NOVARTIS EXHIBIT 2014
`Breckenridge v. Novartis, IPR 2017-01592
`Page 2 of 18
`
`

`

`Signal Transduction Pathway Targets
`
`transmembrane glycoprotein receptor with intrinsic
`tyrosine kinase activity. No endogenous ligands for
`the HER-2/neu protein are known. When HER-
`2/neu protein is activated, it can interact with many
`different cellular proteins such as shc, PLC, GAP
`and the ras MAP kinase pathway [17]. The HER-2
`receptor can also form heterodimers with other
`members of the EGFR family. Amplification of the
`HER-2/neu gene or over-expression of the HER-
`2/neu protein has been identified in 10-34% of
`breast cancers. Possible techniques for evaluating
`HER-2/neu status in breast cancer cells include
`gene-based assays such as polymerase chain
`reaction methods and in-situ hybridization utilizing
`both fluorescent (FISH) and non-fluorescent
`approaches. Qualitative protein measurements are
`the
`most
`common
`techniques
`used.
`Immunohistochemistry,
`typically on archival
`tissues
`is utilized. A significant discordance
`between HER-2/neu detection methods has been
`reported [18]. The discordance has been between
`immunohistochemistry methods and between
`immunohistochemistry and gene-based assays [19].
`A number of clinical studies involving over 10,000
`women have examined the relationship of HER-
`2/neu gene and/or protein abnormalities and breast
`cancer outcome. Results of these studies have not
`been uniform. Several studies including the original
`study published by Slamon et al. [20] found that
`HER-2/neu
`over-expression
`independently
`predicted poor overall survival and disease-free
`survival. Some immunohistochemical studies have
`found significant correlation between HER-2/neu
`protein immunoreactivity and disease outcome in
`univariate, but no independent predictive status in
`multivariate analysis [21]. A few studies have
`found no correlation whatsoever with disease
`outcome [22.23]. Compared to the prognostic
`information outlined above, there are fewer studies
`correlating the expression of HER-2/neu protein
`with response to therapy. Several studies have
`found HER-2/neu over-expressing tumors to be
`resistant to tamoxifen therapy [24, 25]. One large
`study in 200 patients, however, failed to show
`resistance
`to
`tamoxifen
`in HER-2/neu over-
`expressing tumors [26]. While HER-2/neu over-
`expression has been associated with enhanced
`response to chemotherapy regimens containing
`doxorubicin in clinical samples, a poor response to
`CMF
`(cyclophosphamide, methotrexate, 5-
`fluorouracil) therapy has been found in the same
`population of patients [27]. In cultured breast
`cancer cell
`lines, HER-2/neu expression
`is
`associated with resistance to paclitaxel [28].
`However, another study
`indicated a 3-fold
`increased response to paclitaxel in the same
`population [29]. The preponderance of evidence
`
`Current Pharmaceutical Design, 2000, Vol. 6, No. 4 363
`
`would suggest a poorer prognosis and poor
`response to some therapeutic agents in patients with
`HER-2/neu expressing tumors. The conflict in the
`data may be explained in part, by the different
`methods used to document HER-2/neu expression.
`
`HER-2,
`
`(rhuMAb
`
`Trastuzumab
`Herceptin(cid:210) )
`is a humanized monoclonal
`Trastuzumab
`antibody that targets the HER-2 receptor with
`demonstrated activity in metastatic breast cancer.
`This is the first monoclonal antibody to be
`approved for the treatment of a solid tumor. In
`cultured cells that express high levels of HER2,
`trastuzumab causes growth arrest in the G0/G1
`phase of the cell cycle [30]. The growth inhibitory
`effects have been explained by a marked induction
`of the cyclin dependent kinase-2 kinase inhibitor,
`p27, as well as the retinoblastoma-related protein
`p130 [30]. These data suggest that treatment of
`HER-2 overexpressing cells is antiproliferative, and
`that cytostasis may result from an inhibition of cell
`cycle progression.
`
`In early preclinical studies, Slamon et al.
`demonstrated interactions between trastuzumab and
`several anticancer agents [31], using in vitro
`clonogenic studies. Synergistic
`interactions at
`clinically relevant drug concentrations were
`observed for trastuzumab in combination with
`cisplatin, thiotepa and etoposide. Additive cytotoxic
`effects were observed with trastuzumab plus
`doxorubicin,
`paclitaxel, methotrexate
`and
`vinblastine. 5-fluorouracil, was found to be less
`than additive with trastuzumab. Studies were
`further
`conducted with
`drug/trastuzumab
`combinations in HER-2/neu-transfected, MCF-7
`human breast cancer xenografts in athymic mice.
`Combinations
`of
`trastuzumab
`and
`cyclophosphamide, doxorubicin
`, paclitaxel,
`methotrexate, etoposide, and vinblastine in vivo
`resulted in a significant reduction in xenograft
`volume compared
`to
`chemotherapy
`alone.
`Combinations of trastuzumab and 5-fluorouracil
`yielded equivalent results to those achieved by 5-
`fluorouracil alone. The 5-FU
`results were
`consistent with the sub-additive effects observed
`with this combination in vitro. The synergistic
`interaction of trastuzumab with alkylating agents,
`platinum analogs and topoisomerase II inhibitors,
`as well as the additive interaction with taxanes,
`anthracyclines and some antimetabolites in HER-
`2/neu over-expressing breast cancer cells guided the
`choice of combination studies in clinical trials.
`Currently, data from six breast cancer trials have
`
`NOVARTIS EXHIBIT 2014
`Breckenridge v. Novartis, IPR 2017-01592
`Page 3 of 18
`
`

`

`364 Current Pharmaceutical Design, 2000, Vol. 6, No. 4
`
`Alex A. Adjei
`
`been published. Three studies have evaluated
`trastuzumab alone for the treatment of metastatic
`breast cancer. Two studies evaluated trastuzumab in
`refractory breast cancer [32]. Response rates were
`11.6 and 15%, respectively [33]. The antibody was
`well tolerated. The third trial evaluated trastuzumab
`as first-line treatment of metastatic breast cancer. A
`preliminary response rate of 24% has been reported
`[34]. The pivotal multinational phase III study
`reported by Slamon et al. evaluated trastuzumab –
`paclitaxel, or doxorubicin/cyclophosphamide in 469
`first-line metastatic breast cancer patients. Overall,
`the median time to progression was improved from
`4.6 to 7.6 months by the addition of trastuzumab,
`with an improvement in overall response from 32%
`to 48%. Toxicity was generally mild, consisting of
`febrile
`episodes
`and mildly
`increased
`myelossuppression. An increased incidence of
`symptomatic cardiac toxicity was noted when
`trastuzumab was added to anthracycline-based
`chemotherapy (19% vs. 3%). The etiology of this
`increased cardiotoxicity is unclear, but continues to
`be investigated [35]. Trastuzumab plus cisplatin was
`evaluated in patients with refractory breast cancer,
`yielding a response rate of 24% [36].
`
`INTRACELLULAR
`PATHWAYS
`
`SIGNALING
`
`The intracellular signaling pathways that are
`activated after growth factors and other cell-
`proliferation-associated
`ligands bind
`to
`their
`receptors are complex and incompletely understood.
`Major components of these effector systems such
`as the protein tyrosine kinases, protein kinase C and
`the ras/MAP kinase pathway have, however, been
`identified.
`
`PROTEIN TYROSINE KINASES
`
`Protein tyrosine kinases (PTKs) catalyze the
`phosphorylation of tyrosine residues on target
`proteins [37]. Two major groups of PTKs have
`been described to date, receptor and non-receptor
`tyrosine kinases. Non-receptor tyrosine kinases are
`cytoplasmic proteins which transduce extracellular
`signals to downstream intermediates in pathways
`that
`regulate
`cell growth,
`activation
`and
`differentiation. Many non-receptor tyrosine kinases
`are linked to transmembrane receptors including
`those for peptide hormones and cytokines. Unlike
`receptor tyrosine kinases, they lack transmembrane
`domains and ligand binding. They are activated by
`ligand binding to their associated receptors or
`events such as cell adhesion, calcium influx or cell
`cycle progression [38]. More than 30 members are
`
`classified in 10 families including src, abl, JAK,
`MKK2, FES [38].
`
`Receptor tyrosine kinases (RTKs) share several
`structural
`features. They
`are glycoproteins
`possessing an extracellular ligand-binding domain,
`which conveys ligand specificity, and a single
`hydrophobic
`transmembrane domain, which
`anchors the receptor to the membrane. Intracellular
`sequences typically contain regulatory regions in
`addition to the catalytic domain. Ligand binding
`induces activation of the intracellular tyrosine kinase
`domain leading to the initiation of signaling events
`specific for the receptor. The RTKs have been
`organized
`into families based on
`sequence
`homology, structural characteristics and distinct
`motifs in the extracellular domain. There are
`currently 19 known families in vertebrates. The
`various subfamilies include receptors for epidermal
`growth factor (EGFR), platelet derived growth
`factor (PDGF), vascular endothelial growth factor
`(VEGF), fibroblast growth factor (FGF) and
`hepatocyte growth factor (HGF). Ligand binding to
`a RTK
`induces
`receptor dimerization with
`conformational changes that result in intermolecular
`phosphorylation at tyrosine residues at multiple
`sites. Receptor heterodimerization can also occur, as
`reported with transforming growth factor alpha
`interaction with receptor heterodimers comprising
`HER-2 and EGFR [39]. In malignant tumors, a
`number of these receptors are over-expressed or
`mutated, leading to abnormal cell proliferation.
`
`PLATELET DERIVED GROWTH FACTOR
`(PDGF)
`
`PDGF is a major mitogen for endothelial cells,
`fibroblasts, smooth muscle cells and glial cells.
`PDGF exists as disulfide-linked homodimers and
`heterodimers of A and B chains, resulting in 3
`isoforms (PDGF-AA, PDGF-BB, PDGF-AB).
`PDGF and its receptors are expressed in a wide
`variety of cultured neoplastic cells including breast,
`prostate and colon cancers. Expression has also
`been documented in tumor biopsies of ovarian
`cancer and gliomas [40].
`
`The tyrphostins are synthetic protein tyrosine
`kinase inhibitors derived from erbstatin, a natural
`product with broad spectrum activity against protein
`tyrosine kinases and protein kinase C. SU101 is a
`tyrphostin derivative, which predominantly inhibits
`the PDGF receptor tyrosine kinases. In a completed
`phase I study, the most common toxicities were
`mild to moderate nausea, vomiting, and fever.
`Neutropenia was uncommon, and occurred only at
`
`NOVARTIS EXHIBIT 2014
`Breckenridge v. Novartis, IPR 2017-01592
`Page 4 of 18
`
`

`

`Signal Transduction Pathway Targets
`
`the highest dose levels [41]. Phase III studies are
`ongoing in recurrent gliomas, and phase II studies
`have been completed in lung, prostate and ovarian
`cancers [42].
`
`VASCULAR ENDOTHELIAL GROWTH
`FACTOR (VEGF)
`
`VEGF has 5 isoforms, which are splice variants
`and exist as disulfide-linked homodimers, with
`some structural similarities to PDGFs. These
`isoforms bind with high affinity to 2 receptors,
`fms-like tyrosine kinase (flt-1) and fetal liver kinase
`(flk-1). The biologic significance of these multiple
`VEGF receptor forms is not understood. VEGF
`stimulates the growth of endothelial cells during the
`process of angiogenesis, but has also been
`identified as a vascular permeability factor. A
`recombinant humanized monoclonal antibody,
`rhuMAb VEGF, has been developed to inhibit the
`effects of VEGF in the treatment of solid tumors.
`Phase II trials have been completed in lung and
`colon cancer. Results are awaited with interest A
`small molecule inhibitor of VEGF tyrosine kinase,
`SU5416 is undergoing phase I testing, and a phase
`I/II study in AIDS-related Kaposi's sarcoma is
`ongoing [43].
`
`A second generation broad spectrum RTK
`inhibitor, SU6668 is currently undergoing phase I
`testing. This agent is a small organic molecule, that
`possesses anti-angiogenic and anti-proliferative
`properties. It inhibits the autophosphorylation of
`three distinct
`tyrosine kinases, Flk-1/KDR;
`PDGFR, and FGFR with IC50 values of 0.2 m M,
`0.2 m M, and 4.1 m M, respectively. EGFR kinase
`activity remains uninhibited (IC50 > 100 m M). In
`vitro kinetic analyses demonstrate that SU6668 is a
`competitive
`inhibitor of ATP binding. This
`mechanism is similar to that of the bioflavonoids
`such as genistein and quercetin, and distinct from
`the
`tyrphostins and
`their derivatives, which
`compete for the substrate binding site of RTKs
`[44].
`
`Current Pharmaceutical Design, 2000, Vol. 6, No. 4 365
`
`GROWTH
`EPIDERMAL
`RECEPTOR (EGFR)
`
`FACTOR
`
`EGFR differs from the other receptor tyrosine
`kinases in that there is a single isoform, from a
`single 26 exon gene located across 110kb on
`chromosome 7p11-13. It serves as the predominant
`receptor for multiple distinct ligands, including
`EGF, TGF-a
`, amphiregulin and HB-EGF (Table
`1). EGFR interacts with most members of the
`EGFR (c-erbB) family of RTKs. As previously
`mentioned, the ligand for c-erbB2 is unknown,
`while erbB3 and erbB4 serve as heregulin and
`neuregulin receptors. The major function of these
`other receptors appears to be as downstream
`effectors of each other. They cross-phosphorylate
`and modulate signaling from each other in specific
`pairs. EGFR interacts with HER-2 and HER-3 but
`not HER-4. HER-4 pairs with HER-2.
`
`It is noteworthy that HER-3 lacks kinase
`activity, but serves as a docking protein to recruit a
`broader spectrum of downstream effectors after
`phosphorylation by EGFR or HER-2
`[45].
`Currently, 3 EGFR tyrosine kinase inhibitors are
`undergoing phase I clinical testing. One of these is
`ZD1839, which is an orally active, and selective
`inhibitor of the EGFR (HER-1) tyrosine kinase. In
`preclinical studies, administration of this agent daily
`for 4 months resulted in significant tumor growth
`delay in rodents bearing human xenografts. Ex vivo
`examination of xenograft tissues revealed a time
`and dose-dependent decrease in c-fos mRNA, a
`marker for EGFR signaling [46]. An ongoing
`phase I trial has enrolled 38 patients. A partial
`response has been noted in NSCLC, with minor
`responses in head and neck and renal cell cancers.
`The most common toxicity is a skin rash [47, 48].
`The second agent in this class, CP-358774 is also a
`selective EGFR tyrosine kinase inhibitor. An
`acneform skin rash, diarrhea and mild hepatic
`transaminase elevations were the most common
`toxicities [48]. The third agent in this class, CI-
`1033 is a non-specific inhibitor of the EGFR family
`(HER-1, HER-2, HER-4) tyrosine kinases [49].
`
`Table 1. Members of the Human Epidermal Growth Factor Gene Family
`
`Gene
`
`HER-1 (c-erbB-1)
`
`HER-2 (c-erbB-2)
`
`HER-3 (c-erbB-3)
`
`HER-4 (c-erbB-4)
`
`Ligand
`
`EGF, TGFa
`
`, Beta cellulin, Amphiregulin, Heparin binding growth factor
`
`? Heregulin
`
`Heregulin, neu differentiation factor 1+2
`
`Heregulin, neu differentiation factor 1+2
`
`NOVARTIS EXHIBIT 2014
`Breckenridge v. Novartis, IPR 2017-01592
`Page 5 of 18
`
`

`

`366 Current Pharmaceutical Design, 2000, Vol. 6, No. 4
`
`Alex A. Adjei
`
`This agent demonstrates an in vitro IC 50 in the low
`nanomolar range with all four RTKs. Results of
`phase I studies with this agent are awaited with
`interest.
`
`PROTEIN KINASE C INHIBITORS
`
`is a family of
`Protein kinase C (PKC)
`serine/threonine directed protein kinases consisting
`of a group of at least 10 related isoenzymes that are
`involved in the transduction of signals for cell
`proliferation and differentiation. PKC has therefore
`been considered a suitable
`target for novel
`antineoplastic drugs. The PKC family is divided
`into Ca2+-responsive
`and Ca2+-unresponsive
`subtypes [50, 51]. The enzyme consists of an N-
`terminal regulatory domain, which binds and
`inhibits the C-terminal catalytic domain.
`
`PKC modulatory agents, which affect the
`catalytic domain, are the staurosporine congeners.
`Staurosporine is the parent compound derived from
`microbial sources two decades ago, and has poor
`selectivity. It is inhibitory towards tyrosine kinases
`and cAMP-dependent protein kinases as well as
`PKC at similar concentrations [52]. Its analogs 7-
`hydroxy-staurosporine (UCN-01) and CGP 41251
`have greater selectivity, and effectively arrest the
`growth of several human-derived tumor cell lines in
`vitro. They also possess antineoplastic activity in
`vivo in human tumors grown as xenografts in nude
`mice. CGP 41251 reverses the multidrug-resistance
`phenotype of cancer cells [53]. Both agents are
`currently under clinical evaluation as potential
`antitumor drugs. These agents are also potent
`modulators of the cyclin dependent kinase system,
`which determines the progression of cells through
`the cell cycle. The nature of this interaction is
`complex. UCN-01 blocks cells in GI phase by
`promoting accumulation of dephosphorylated
`retinoblastoma protein as a consequence of
`inhibition of the activity of certain cyclin-dependent
`kinases, down-regulation of their partner cyclins
`and an increase in the expression of cyclin-
`dependent
`kinase
`inhibitor
`proteins
`[54].
`Preliminary results of early clinical trials suggest
`that UCN-01 and CGP41251 are without
`remarkable toxicity but display high binding to
`human plasma protein [55].
`
`Several studies indicate a role for PKC in the
`regulation of the multi-drug resistance (MDR)
`phenotype, since several PKC inhibitors are able to
`partially reverse MDR and inhibit P-glycoprotein
`(Pgp) phosphorylation. The MDR phenotype is
`also associated with variation in PKC iso-enzyme
`
` over-
`in particular with PKC-
`content,
`expression. Based on these results, it has been
`hypothesized that PKC inhibitors may synergize
`with cytotoxic agents through modulation of MDR.
`However, other potential mechanisms of PKC
`interaction with anticancer drugs exist and have
`been documented, such as the enhancement of
`chemotherapy-induced apoptosis by safingol, a
`specific PKC inhibitor [56]. Safingol is undergoing
`a phase I clinical trial in combination with
`doxorubicin. While no final data are presently
`available, it appears that plasma levels of safingol
`approach those associated with potentiation of
`chemotherapy in animals, and no pharmacokinetic
`interaction between the two drugs exists. Drugs
`targeting PKC are therefore felt to possess potential
`as modulators of cytotoxic agents.
`
`Inhibitors of the regulatory domain of PKC
`include the ether lipids and bryostatins. Bryostatin
`1, a macrocyclic lactone isolated from the marine
`organism Bugula nerutina, is a partial PKC agonist,
`and has shown potent antineoplastic properties in
`in vivo. Bryostatin 1 has both
`vitro and
`antineoplastic and immune-stimulatory properties,
`including the induction of cytokine release and
`expansion
`of
`tumor-specific
`lymphocyte
`populations. Bryostatin
`I has demonstrated
`antitumor activity in phase I trials in patients with
`malignant melanoma,
`lymphoma and ovarian
`carcinoma and is undergoing broad phase II testing,
`both as a single agent, and in combination with
`standard cytotoxic agents [57, 58]. The dose-
`limiting toxicity is myalgia. In a completed phase II
`study, 16 previously treated patients with malignant
`melanoma were treated with bryostatin at 25 m g/m2
`weekly for three courses followed by a rest week.
`The principal toxicities were myalgia, phlebitis,
`fatigue and vomiting. Of 15 patients evaluable for
`tumor response, 14 developed progressive disease.
`One patient developed stable disease for 9 months
`after bryostatin treatment. The authors concluded
`that single-agent bryostatin was ineffective in the
`treatment of metastatic melanoma in patients
`previously
`treated with
`chemotherapy, but
`suggested that it should, however, be investigated
`further in previously untreated patients [59].
`
`antisense
`an
`641A),
`(ISI
`3521
`ISIS
`phosphorothioate oligonucleotide to protein kinase
`C-a
`, has been studied in a phase I trial. The
`antisense approach involves targeting specific
`ribonucleotide (RNA) sequences in order to block
`translation of the RNA message into protein. Drug-
`related toxicities included mild to moderate nausea,
`vomiting, fever, chills, and fatigue, hematologic
`toxicity was
`limited
`to mild
`to moderate
`
`NOVARTIS EXHIBIT 2014
`Breckenridge v. Novartis, IPR 2017-01592
`Page 6 of 18
`
`a
`

`

`Signal Transduction Pathway Targets
`
`Current Pharmaceutical Design, 2000, Vol. 6, No. 4 367
`
`thrombocytopenia. Dose escalation on this study
`was discontinued on attaining peak plasma
`concentrations which approached that associated
`with complement activation in primates. No dose
`limiting toxicity was identified, and clinical activity
`was observed in two patients with non-Hodgkin's
`lymphoma who achieved complete responses [60].
`
`mutations are found in bladder, kidney and thyroid
`carcinomas; and N-ras mutations are found in
`melanoma,
`hepatocellular
`carcinoma
`and
`hematologic malignancies [67]. Association of ras
`with
`the plasma membrane, and consequent
`activation require prenylation, i.e. the attachment of
`a farnesyl group, at the carboxy-terminus.
`
`ras/MITOGEN
`THE
`PROTEIN KINASE PATHWAY
`
`ACTIVATED
`
`ras INHIBITORS
`
`As previously mentioned, effector molecules
`recruited by receptor tyrosine kinases (RTKs)
`include phospholipase C (PLC), phosphoinositide-
`3-kinase (PI3-kinase) and ras. The ras pathway is
`fairly well understood. The effector molecules bind
`to phosphorylated tyrosine residues on the RTKs
`via src homology 2 (SH2) domains or an adapter
`protein, Grb2/SOS in the case of ras. Activated ras
`activates raf, which is a serine/threonine kinase.
`Raf activates mitogen activated protein kinase kinase
`(MEK, MAPKK) which
`in
`turn activates
`mitogen activated protein (MAP) kinase. MAP
`kinase activation results in phosphorylation and
`activation of ribosomal S6 kinase and transcription
`factors such as c-jun, c-myc and c-fos, resulting in
`the switching on of a number of genes associated
`with proliferation [61-63]. Signaling by
`this
`pathway is more complex than outlined above,
`since ras and raf-independent pathways for MAP
`kinase activation exist. Secondly, a subfamily of 54
`kDa MAP kinases which function independently of
`MEK has been identified [64].
`
`The ras protein plays an essential role in signal
`transduction,
`as
`already
`described. Three
`mammalian ras proto-oncogenes encode four
`related and highly conserved proteins, H-ras, N-
`ras, and the splice variants K-ras 4A, and K-ras 4B
`[65]. The p21ras proteins bind guanosine
`triphosphate (GTP) and guanosine diphosphate
`(GDP) with high avidity. When GTP is bound, the
`ras protein is in an “active” state. When GDP is
`bound in the resting state, p21ras is inactive. One
`means of negative regulation of p21ras activity is via
`the GTPase-activating protein (GAP). Single point
`mutations of the ras gene can lead to its constitutive
`activation. These mutated forms have impaired
`GTPase activity. Although they bind GAP, there is
`no “off” sign since GTPase is not activated. This
`results
`in continuous stimulation of cellular
`proliferation, and inhibition of apoptosis [66].
`Oncogenic ras mutations have been identified in
`approximately 30% of human cancers [67]. K-ras
`mutations are frequent in non-small cell lung,
`colorectal and pancreatic carcinomas; H-ras
`
`Because of the high percentage of human tumors
`harboring oncogenic ras mutants, interrupting the
`ras signaling pathway has been a major focus of
`new drug development efforts. The current
`promising approaches taken are i) the inhibition of
`ras
`protein
`expression
`through
`antisense
`oligonucleotides, ii) the prevention of membrane
`localization of ras, and iii) the inhibition of ras
`function through inhibition of downstream ras
`effectors.
`
`Antisense Oligonucleotides
`
`Oligonucleotides that are complementary to
`messenger RNA (mRNA)
`transcripts of
`the
`activated ras oncogene, have been utilized to
`decrease
`ras
`protein
`expression.
`These
`oligonucleotides hybridize
`to complementary
`mRNA sequences and decrease protein expression
`through a variety of mechanisms, including RNase
`H-mediated cleavage of hybridized ras mRNA.
`Roth and co-workers developed a retroviral
`antisense K-ras expression vector, which has been
`shown to eliminate the expression of human ras
`protein in lung cancer cells [68]. This K-ras
`retroviral construct was successfully administered
`intratracheally to nude mice bearing implanted
`human lung cancers. Significant activity was
`observed with 87% of treated mice being tumor-
`free compared to 10% of control mice [69]. A
`persistent problem had been the inability to deliver
`intact antisense RNA agents into tumor cells. In
`recent years, the distribution of phosphorothioate
`oligodeoxynucleotides, presumably by endocytosis
`in rodent tissues after intravenous administration
`has been demonstrated [70]. An investigational
`phosphorothioate antisense oligodeoxynucleotide,
`ISIS 2503 designed to hybridize to the 5’-
`untranslated region of human H-ras mRNA, has
`completed testing in phase I clinical trials. Interim
`results indicate a tolerable toxicity profile, with
`moderate thrombocytopenia and fatigue as the only
`adverse events. An indication of biologic activity
`has been seen, with mild tumor shrinkage in a
`patient with sarcoma [71]. The schedule that has
`
`NOVARTIS EXHIBIT 2014
`Breckenridge v. Novartis, IPR 2017-01592
`Page 7 of 18
`
`

`

`368 Current Pharmaceutical Design, 2000, Vol. 6, No. 4
`
`Alex A. Adjei
`
`Fig. (2). Simplified scheme of signal transduction pathways, with an indication of the molecules to which clinical agents are
`targeted.
`
`1) anti-receptor antibodies such as trastuzumab, receptor tyrosine kinase inhibitors such as monoclonal antibody C225,
`ZD1839, CP258774 and CI-1033
`
`2) farnesyltransferase inhibitors (SCH66336, R115177, L748259, BMS214662); antisense oligonucleotides (ISIS 2503)
`
`3) antisense oligonucleotides (ISIS 5132), HSP90 inhibitor (17AAG)
`
`4) MEK inhibitor (PD 184322)
`
`5) PKC inhibitors (bryostatin, UCN-01, CGP41251) antisense oligonucleotide (ISIS 3521)
`
`6) CCI-779
`
`been selected for furth