Anti-Endothelin Drugs in Solid Tumors

Review

Anti-endothelin drugs in solid tumors

† Antonio Russo , Giuseppe Bronte, Sergio Rizzo, Daniele Fanale, Francesca Di Gaudio, Nicola Gebbia & Viviana Bazan † 1. Background Universita` di Palermo, Section of Medical Oncology, Department of Surgical and Oncological 2. Medical need Sciences, Via del Vespro 129, 90127 Palermo, Italy 3. Existing treatment Importance of the field: The endothelin (ET) axis, which includes the biolog- 4. Current research goals ical functions of ETs and their receptors, has played a physiological role in 5. Scientific rationale normal tissue, acting as a modulator of vasomotor tone, tissue differentiation 6. Competitive environment and development, cell proliferation and hormone production. Interestingly, it 7. Conclusions also functions in the growth and progression of various tumors. Several researchers have identified the blockade of the ET-1 receptor as a promising 8. Expert opinion therapeutic approach. Areas covered in this review: The clinical investigation of an orally bioavailable ET antagonist, atrasentan, in prostate cancer, is encouraging. In this neoplasia, it has shown antitumor activity, bone metastasis control and amelioration of cancer-related pain but improvement in time to progression and overall survival has still not been demonstrated. The clinical trials of other ET antagonists are reported. Literature research was performed by Pubmed and Pharmaprojects. What the reader will gain: A comprehensive view about the use of atrasentan in the treatment of castration-resistant prostate cancer (CRPC) is provided together with the scientific rationale based on the function of ET and its receptor in various cancer development mechanisms. Take home message: Atrasentan seems to be active in CRPC, although

For personal use only. strong scientiﬁc evidence is still to be found. Interesting clinical ﬁndings regard zibotentan.

Keywords: atrasentan, endothelins, prostate cancer, zibotentan

Expert Opin. Emerging Drugs (2010) 15(1):27-40

1. Background

The endothelins (ETs), discovered by Yanagisawa in 1988, are a family of small peptides, well conserved from the evolutionary aspect, with multiple roles in a variety of tissues [1]. Initially, ET was thought to be important in cardiovascular

Expert Opin. Emerging Drugs Downloaded from informahealthcare.com by Eli Lilly and Company on 02/18/15 homeostasis; ET receptor antagonists and endothelin-converting enzyme (ECE) inhibitors have, therefore, been developed mostly for the treatment of cardiovascular diseases. Molecular biology has also provided valuable information about ET, including evidence that the ET system plays an important role in the early development of the neural crest and, thus, in the formation of organs [2]. At present, the ET system, which comprises ETs, their receptors and the enzymes of ET biosynthesis and degradation, is thought to have a role in the pathophysiology of many tumor types, including prostatic, pulmonary, cervical, endometrial, ovarian, colorectal, breast, bladder, renal and brain tumors, Kaposi’s sarcoma, bone metastases and melanoma [3-5]. In mammals, the ETs comprise a family of three 21 amino- acid peptides, ET-1, ET-2 and ET-3, whose structure consists of a single a-helix and two intramolecular disulﬁde bonds. Whereas ET-1 and ET-2 have similar structures, ET-3 differs in structure at 6 of 21 amino acids. ET-1 is not organ-speciﬁc and is expressed primarily by endothelial cells, whereas ET-2 is mainly present in the

intestine and kidney, and ET-3 is mainly localized in the brain 2. Medical need and to a lesser extent in gastrointestinal stromal cells and lung epithelial cells [4]. Nearly all patients with advanced prostate cancer are sen- The ETs are encoded by distinct genes and are regulated at sitive to androgen ablation therapy. However, the effect of the level of mRNA transcription and post-transcription [1]. this treatment on disease progression is temporary. These The primary translation product of the mRNA of ET-1 gene is patients ultimately become resistant to androgen ablation the 212-aa prepro-ET-1. Active ET-1 is synthesized from a and are then classified as having castration-resistant prostate biologically inactive 38-aa precursor, Big ET-1, by an unusual cancer (CRPC). To date, chemotherapy is the main option hydrolysis of the Trp21–Val22 bond by a metalloprotease for this disease. Docetaxel represents the best tolerated and with intracellular and membrane bound isoforms, known as effective agent of treating it. the ECE-1 [6,7]. The mature peptide is continuously released There has been great interest regarding the possibility of from vascular endothelial cells by the constitutive pathway, using this drug for the treatment of even elderly patients, producing intense constriction of the underlying smooth because prostate cancer apparently occurs more frequently in muscle and contributing to the maintenance of endogenous older men. An intravenously delivered chemotherapeutic vascular tone. Several studies of various malignancies have agent with high incidence of hematological toxicity, even revealed that members of the ET family are produced by if better tolerated than other drugs with the same clinical several epithelial tumors, in which they act as autocrine and/or indication, such as docetaxel, may hinder the accessibility of paracrine growth factors. ET-1 is the most common comorbid patients. For several years, oncologists have been circulating form of ET [8]. trying to find new agents with minimal toxicity to be ETs are powerful vasoconstrictive peptides that exert their administered per os. effects through two specific heptahelical GPCR subtypes: the Another hurdle for these patients is the development of endothelin A receptor (ETAR) specific for ET-1 and ET-2 and resistance to docetaxel. When CRPC patients progress after the nonspecific endothelin B receptor (ETBR) [1,9,10]. The ET first-line therapy, there are only a few options available for receptors share about 59% similarity in the primary structure. their treatment. New types of therapy are required in this The ETAR selectively binds ET-1 and ET-2, whereas the patient setting. The effect of ET receptor antagonists on ETBR binds all three ET forms with similar affinity. While the angiogenesis might be exploited to overcome taxane resistance. ETAR primarily mediates vaso-/bronchoconstriction, mito- genesis, antiapoptosis, matrix formation, acute and neuro- 3. Existing treatment pathic pain, the ETBR mediates inflammatory pain and

For personal use only. vasodilatation and has further been proposed to contribute Treatment with hormone therapy or chemotherapy is the to clearance of ET as well as to autoinduction of ET-1 [11]. standard option for patients with CRPC. After progression on Although the ETBR may also mediate vasoconstriction, this both a luteinizing-hormone-releasing hormone agonist and subtype, which is primarily situated at the plasma membrane antiandrogen, the withdrawal of antiandrogen therapy results of endothelial cells, is ﬁrst of all considered causing transient in an antiandrogen withdrawal response in 25 – 50% of NO-mediated vasodilatation. The ETBR shares several intra- patients. Secondary hormone medications, including ketoco- cellular signaling pathways with the ETAR; however, unlike nazole and hydrocortisone, could induce a clinical response in ~ ETA receptor-dependent effects, ETBR-dependent phenom- 25% of patients. This beneﬁt lasts for only a short time, with ena are shorter lasting and reversible. ET receptor interacts a mean duration of 4 months. For this reason, chemotherapy with heterotrimeric G proteins. After ligand binding, ETARs is considered the main choice for such patients. The devel- become internalized and undergo receptor recycling followed opment of novel therapeutic agents for the treatment of CRPC by relocation to the cell membrane, whereas ETBRs can be is still to be explored in this patient population.

Expert Opin. Emerging Drugs Downloaded from informahealthcare.com by Eli Lilly and Company on 02/18/15 translocated to the lysosomal compartment for degradation [12] Combination chemotherapy regimens involving agents that or translocated to nuclear membranes for further signaling affect microtubule integrity appear to have activity with events [13]. ETs and ET receptors are ubiquitously expressed tolerable adverse effects. In particular, combination regimens and regulate a wide range of physiological and pathophysio- including taxanes seem the most promising treatment, achiev- logical functions, such as cardiovascular, mitogenic and neu- ing > 50% response rates. However, the median duration of roregulatory events, hormone production and placental response is limited to ~ 6 months. Additional survival beneﬁts development [14]. reported in Phase II trials have not yet been conﬁrmed in ET-1 production is stimulated by cytokines (IL-1h), growth Phase III randomized trials. factors (TNF-a, TGF-h and platelet-derived growth factor Because of the increasing proportion of patients included in (PDGF)) and major signals of cardiovascular stress, such as earlier application of taxane-based chemotherapy, there is an vocative agents (angiogenesis II, norepinephrine, vasopressin ever enlarging subset of patients with disease progression who and bradykinin), thrombin, mechanical stress and hypoxia. remain viable candidates for additional therapy. To date, we Prostacyclin, NO and atrial natriuretic peptide are the lack enough data from clinical trials to guide decision making, predominant effectors of negative feedback mechanisms [15]. although clinical investigation is ongoing for this subset of

28 Expert Opin. Emerging Drugs (2010) 15(1) Russo, Bronte, Rizzo, Fanale, Di Gaudio, Gebbia & Bazan

patients. Usually, CRPC patients previously treated with and activates a G protein that triggers a parallel activation of first-line chemotherapy are classified into two groups: several signal transducing pathways. This interaction can in mitoxantrone-treated patients, for whom the utility of taxanes fact activate multiple signal transduction pathways including as second-line therapy should be considered, and taxane- phospholipase C activity with a consequent increase in treated patients, for whom the activity of retreating with intracellular Ca2+ levels, PKC, phosphati-dylinositol 3-kinase taxanes, mitoxantrone or other microtubular targeted agents and MAPK [21].ETBR-mediated coupling in choriocarcinoma is under investigation. cell lines leads to the activation of the Ras/Raf-MAPK path- ET receptor antagonists studied for cancer treatment are way [22]. ET-1 causes EGFR transactivation which is partly not yet marketed. responsible for MAPK activation by a ligand-dependent A previous US registration submission as Xinlay for the mechanism involving a non-receptor tyrosine kinase, such treatment of metastatic CRPC received a non-approvable as Src. Recent work has reported that a specific ETAR letter (Scrip Daily Online, 17 October 2005, S00899072). antagonist can reduce the EGFR transactivation [14,23-25]. Abbott decided not to pursue further registration of astrasen- The phenotypic change of vascular smooth muscle cells in tan for this indication (direct communication, Abbott, culture is concommitantly associated with a change in the ET 24 August 2007). It had US fast-track status for prostate receptor subtype which potentiates mitogenic activity. This cancer (press conference, Abbott, 6 February 2001). suggests that the switching of the ET receptor subtype from A Regarding Zibotentan, European and US filings for the to B during phenotypic change may contribute to a certain treatment of CRPC are expected in 2011 (company pipeline, extent to the development of vascular lesions [26]. AstraZeneca, 31 January 2008). ET-1 promotes DNA synthesis and cell proliferation in various epithelial tumor cells, including prostate, cervical and 4. Current research goals ovarian cancer cells. Synergistic interactions with other growth factors, including EGF, basic fibroblast growth factor (bFGF), The main goals of treatment with ET receptor antagonists for insulin, IGF, PDGF, TGFs and IL-6, intensify mitogenic CRPC patients include: activity [27]. . Availability of agents with minimal or manageable toxicity and orally bioavailable to extend applicability of anticancer 5.2 Role of ET-1 in angiogenesis treatment even for elderly and comorbid patients. Angiogenesis, the formation of new vessels from existing . Efficacy on cancer-related pain and bone metastases com- vasculature, is an important early event in tumor progression plications, which are the most frequent factors impairing

For personal use only. which begins in premalignant lesions. VEGF and bFGF are quality of life. the principal regulators of neovascularization. During the . Applicability also in nonmetastatic CRPC to improve formation of new blood vessels, endothelial cells are stimu- both progression-free survival and overall survival and lated to release proteases, such as MMP-2, in order to migrate, to delay major complications deriving from clinically proliferate and invade surrounding tissue and form capillaries. evident metastases. Initiation of angiogenesis is controlled by different regulators The great hope of the researchers regards the prospect of including local hypoxia, which activates the expression of using ET receptor antagonists even in other malignancies. angiogenic factors that can stimulate endothelial cell Until now, no clear evidence has been provided for activity of growth [28]. Activation of ETAR by ET-1 promotes tumor these agents in different cancers. A better understanding of the growth and progression by stimulating the production of the biological mechanisms of their action will make it possible to key angiogenic factor VEGF in response to hypoxia. ET-1 investigate them in a more suitable patient setting. regulates various stages of neovascularization, including endo-

Expert Opin. Emerging Drugs Downloaded from informahealthcare.com by Eli Lilly and Company on 02/18/15 thelial cell proliferation, migration, invasion, protease 5. Scientiﬁc rationale production and tube formation, and also stimulates neovascularization in vivo [29]. ET-1 increases VEGF mRNA expres- 5.1 Role of ET-1 in cell growth sion and VEGF protein levels in a dose- and time-dependent ET-1 is a powerful vasoconstrictor with mitogenic or co- manner, and does so to a greater extent under hypoxia. ET-1 mitogenic properties, which stimulates proliferation in vitro of stimulates VEGF production through the hypoxia-inducible ﬁbroblasts, renal mesangial cells, smooth muscle and factor HIF-1a and this mechanism might be responsible for several tumor cell lines, including colorectal cancer [16-20]. increasing tumor angiogenesis. Degradation of HIF-1a is in Studies involving colorectal, ovarian and prostate cancers fact reduced in ET-1-treated ovarian carcinoma cells under suggest that the receptor responsible for the ET-1 mitogenic both hypoxic and normoxic conditions, indicating that the a action is ETA, which is probably upregulated. Activation of induction of HIF-1 protein production by ET-1 is due to a the ETAR by ET-1 mediates a signaling cascade, which enhanced HIF-1 stability. After ETAR activation by ET-1, promotes tumor cell growth, synergizing with other growth HIF-1a protein levels are increased, the HIF-1 transcription factors to cause cell proliferation (Figure 1). ETA interacts with complex is formed and binds to the hypoxia-responsive

Expert Opin. Emerging Drugs (2010) 15(1) 29 Anti-endothelin drugs in solid tumors

Prepro-ETs

ECE

ET-1 ET-2 ET-3

NEP

G-protein ETARETBR

For personal use only. PLC PKC PI3K MAPK

Cell Cell Autoinduction ET clearance proliferation migration of ET-1

Evasion Inflammatory Angiogenesis Angiogenesis of apoptosis pain

Figure 1. Molecular mechanisms of endothelin axis. Expert Opin. Emerging Drugs Downloaded from informahealthcare.com by Eli Lilly and Company on 02/18/15

element binding site [3]. Furthermore, not only does ET-1 COX-2 promoter activity and prostaglandin PGE2 produc- a stabilize HIF-1 under hypoxic and normoxic conditions, tion. PGE2 promotes angiogenesis and this effect is mediated inducing the transcription of angiogenic genes, but HIF-1a is by VEGF. The use of EP agonists and antagonists indicates also able to modulate transcription of ET-1 in various cell that ET-1 and PGE2 stimulate VEGF production, principally types. Moreover, the ETAR blockade exerts an antiangiogenic via two receptors, EP2 and EP4. effect by completely inhibiting growth and neoangiogenesis in different tumors [30]. 5.3 Role of ET-1 in apoptosis Prostaglandins (PG) and their rate-limiting enzymes Apoptosis is an active cell death process that takes place in a COX-1 and -2 are involved in the progression of a variety wide spectrum of physiologic situations such as normal cell of malignancies in association with elevated levels of turnover, embryogenesis and endocrine-dependent tissue proangiogenic factors [31-33]. atrophy. Under normoxic condition, ET-1 signiﬁcantly stimulates Recently, investigations into the role of the ET axis in the expression of COX-1 and -2 at mRNA and protein levels, apoptosis inhibition have provided evidence of the importance

30 Expert Opin. Emerging Drugs (2010) 15(1) Russo, Bronte, Rizzo, Fanale, Di Gaudio, Gebbia & Bazan

of the ET-1 axis in cell survival. Previous studies have junctions and this might serve as a basis to further evaluate indicated a potential function for ET-1 in cell proliferation the cell–cell metabolic uncoupling and the cell detachment or as a cell survival factor. that occurs during tumor progression [40]. In epithelial cancer, Interaction between the Fas receptor (CD95/APO-1), a the loss of the epithelial features and the gain of a mesenchy- member of the TNF-receptor superfamily, and the Fas ligand mal phenotype, a process known as epithelial to mesenchymal (FasL) triggers a pathway to cell death involving caspase-8 transition (EMT), determine the acquisition of tumor inva- activity. However, despite expression of Fas and caspase, death siveness. A primary event regulating EMT is the disruption of signals are frequently interrupted by also anti-apoptotic mod- the E-cadherin-mediated stable interactions between the cells. ulators, including caspase inhibitory proteins such as FLIP [34]. In ovarian carcinoma cells, activation of the ETAR pathway by In human glioblastoma cell lines, ET-1, acting as survival ET-1 contributes to disruption of normal host–tumor inter- factor, probably acts through ERK phosphorylation and the actions by downregulating the expression of E-cadherin and PKC pathway, determining a stabilization of the short form associated b-catenin adhesion proteins [41]. ET-1 induces of FLIP protein [35]. The binding of ET-1 to high-affinity sites expression of the transcription factor Snail, which has been inhibits FasL-induced apoptosis, while the binding of either identified as a potent repressor of E-cadherin expression. ET-1 or receptor antagonists to low-affinity sites promotes FasL-induced apoptosis. Thus, ET signaling pathways do not 5.5 Role of ET-1 in osteogenesis induce human cancer cell proliferation, but are survival signals The ET axis seems to have a central role in osteoblastic bone controlling resistance to apoptosis [36]. metastasis. Alkaline phosphatase, a marker of new bone As these effects are mediated by ETAR, activation of ETB by formation, is elevated in the presence of exogenous ET-1 [42]. ET-1 conversely induces cell death via apoptosis in human ETA and ETB appear to regulate tumor progression by a cancer cells [37]. mechanism involving bone deposition in skeletal metastases In ovarian carcinoma cells, the addition of ET-1 signif- through the activation of osteoblasts. It is thought that ET-1 icantly inhibits paclitaxel-induced apoptosis in a dose- production by metastatic cancer cells located in the bone is dependent fashion as a result of Bcl-2 phosphorylation stimulated by osteoblast- and endothelial cell-secreted IL-1, suppression. ET-1 also stimulates Akt activation in a TNF-a and TGF-h. ET-1, in turn, would stimulate mitotic PI3K-dependent manner. Interestingly, the use of specific activity in osteoblasts, decreasing both osteoclastic bone ETAR antagonists has demonstrated that ET-1 contributes resorption and osteoclast motility [43,44]. Several experiments to paclitaxel resistance through ET R binding via activation A conducted in human prostate cancer cell lines have shown that of anti-apoptotic signaling pathways [21]. osteoclastic bone resorption is significantly blocked by the For personal use only. presence of ET-1 in a dose-dependent fashion. In prostate and breast cancer models, selective ET R inhibition, but not 5.4 Role of ET-1 in tumor invasiveness A ET R modulation, suppressed ET-1-induced osteoblastic Tumor invasion is characterized by changes in MMPs, con- B response [45,46]. It seems that a calcineurin/FAT (nuclear factor nexin, integrin and cadherin expression. The presence of high of activated T cells) pathway is involved in the molecular levels of ET-1 in ascitic fluids of ovarian cancer patients has mechanism of ET-1-mediated osteoblast stimulation. shown that ET-1 may increase the secretion of extracellular matrix-degrading proteinases and metastatization process. In fact, ETAR activation induces matrix-degrading enzymes, such 5.6 ET receptor antagonists’ preclinical development as MMPs and urokinase plasminogen activator, which play ET-1 has shown pleiotropic mechanisms in different pathways important roles in tissue remodeling and tumor metastasis [38]. of cancer development and progression. For this reason, many The ET-1 system acts by enhancing the invasive potential researchers have hypothesized a role of the ET-1 axis targeting

Expert Opin. Emerging Drugs Downloaded from informahealthcare.com by Eli Lilly and Company on 02/18/15 of tumor cells. ET-1, in fact, activates MMP-2, MMP-3, for cancer treatment. Various approaches have been identiﬁed MMP-7, MMP-9 and MMP-13. EP2 and EP4 receptor in order to impair ET biological function in tumors, for antagonists block MMP activity and cell invasion, dem- example, inhibition of ET biosynthesis, blockade of ET onstrating that these receptors are the principal PGE2 production from preproETs, promotion of ET degradation, receptors involved in these processes [39]. Furthermore, selective blockade of ETAR/ETBR activation and enhance- ET-1 stimulates focal adhesion kinase (FAK) and paxillin ment of tumor perfusion by ETBR activation to potentiate phosphorylation through the ETAR, which directly corre- efﬁcacy of antineoplastic drugs. lates with tumor cell migration and invasion [24]. This indi- Red wine polyphenols (i.e., quercetin, resveratrol, cathe- cates that ETAR antagonists can inhibit cell migration and chins, etc.) can suppress ET-1 release and the transcription of possibly other FAK-associated processes. prepro-ET-1 gene [47]. Similarly, green tea polyphenol epi- Defects in regulation of intercellular communication, such gallocatechin-3-gallate in ovarian carcinoma cells and xeno- as dysregulated or reduced expression of connexins, allow grafts have inhibited both ETAR and ET-1 expression and cancer cells to escape microenvironmental control from the reduced basal and ET-1-induced cell proliferation and normal surrounding tissue. ET-1 is able to disrupt gap invasion [48]. ET synthesis might be blocked by inhibition

Expert Opin. Emerging Drugs (2010) 15(1) 31 Anti-endothelin drugs in solid tumors

of the enzyme which converts preproETs into ETs. ECE atrasentan depends on ETAR expression. In this type of tumor, inhibitors have been studied for various cardiovascular, atrasentan has also induced more evident apoptotic effects renal, pulmonary and neurological diseases. They may be when combined with cytotoxic drugs such as cisplatin and selective or may also inhibit the ACE and neutral endo- 5-fluorouracil in xenograft models. The xenografts of meta- peptidase [49]. ET degradation has been achieved by means static tumors have demonstrated a role for atrasentan in of neutral endopeptidase transfection in ovarian carcinoma reducing metastases to the lung from both nasopharyngeal cells. NEP is a cell surface aminopeptidase with a ubiquitous and bladder cancers [56,57]. expression and is able to degrade various bioactive peptides, Several other ET receptor antagonists have also been including ET-1 [50]. studied in preclinical cancer models. Among all these options, the ET receptor blockade has Zibotentan (ZD-4054) is an orally active specific ETAR reached the most advanced phases of preclinical and clinical antagonist. ETAR blockade by zibotentan inhibits ET-1- drug development. Several small molecules functioning as induced mitogenic effects, while the ETBR antagonist, BQ ETAR antagonists have provided a clearer understanding of 788, is ineffective. Zibotentan is able to inhibit the prolifer- the physiologic role of ET-1 and its effects on ET receptor- ative activity of ET-1 in ovarian carcinoma cells [58]. The mediated signal transduction in tumor development and combination of zibotentan and paclitaxel produces additive progression. Atrasentan and ZD-4054 are the most potent effects on the reduction of ETAR-driven angiogenesis and and selective ETAR antagonists. They are orally bioavailable. invasive mediators, such as VEGF, COX-1/2 and MMP. For this reason, they are well qualified for clinical development Forty percent of mice bearing human ovarian cancer remained in cancer treatment. tumor-free, suggesting a possible clinical role of zibotentan as Atrasentan is able to inhibit effectively cell proliferation and monotherapy or in combination with cytotoxic drugs [59].In VEGF secretion both in ovarian carcinoma cell lines and ovarian carcinoma xenografts, zibotentan has enhanced the primary cultures. This action of atrasentan can be translated efficacy of gefitinib, achieving partial (82%) or complete into biological effects such as reduction of microvessel density, tumor regression. A cross-talk between the EGFR and VEGF and MMP-2 expression and increase in the percentage ETAR pathways has been documented together with the of apoptotic tumor cells. evidence of decreased vascularization, Ki-67, MMP-2, Its combination with paclitaxel seems to be synergistic VEGF, MAPK and EGFR, and enhanced E-cadherin because of the enhancement of antitumor, proapoptotic expression [60]. and antiangiogenic effects. These phenomena may be Bosentan, a dual ET receptor antagonist targeting ETAR explained by the sensitization to paclitaxel-induced apoptosis and ETBR, has therapeutic indications for cardiovascular

For personal use only. by the ETAR blockade [51]. Moreover, the synergistic action of diseases. In a breast carcinoma cell metastasis model, bosentan chemotherapy and ETAR antagonists is supported by the has shown inhibition of tumor growth, neovascularization and ﬁnding of speciﬁc higher expression of ETAR gene in post- bone metastasis. In melanoma xenografts, bosentan has chemotherapy samples of primary ovarian tumors [52].In enhanced the effects of alkylating agents, such as dacarbazine, breast carcinoma, the overexpression of ETAR has induced when a combination treatment was delivered [61]. resistance to chemotherapy. Some authors have suggested the A-182086 is another dual antagonist. It can inhibit tumor use of the evaluation of ETAR expression levels to predict the growth by blocking proliferation not only of tumor cells but responsiveness of cancer patients to chemotherapy [53]. also of endothelial cells expressing ETBR. It has shown a Atrasentan has induced tumor growth inhibition in xeno- potential role in Kaposi sarcoma treatment because of simul- grafts of cervical carcinoma cells. This effect has been observed taneous interference with cell proliferation, invasiveness and with just two cycles of treatment [54]. The additive effects of angiogenesis [62,63]. paclitaxel when combined with atrasentan have been obtained BQ788 is a peptide ETBR antagonist which inhibits growth

Expert Opin. Emerging Drugs Downloaded from informahealthcare.com by Eli Lilly and Company on 02/18/15 both in cervical carcinoma models [30] and in ETAR- and induced death of melanoma cells in vitro and in vivo [64]. overexpressing prostate cancer cells [43]. They are associated A-192621 is a specific ETBR antagonist studied in xeno- with a lower cell viability and a higher apoptotic rate. grafts of human melanoma cells. It suppresses HIF-1a accu- In prostate cancer, the proliferative action of exogenous mulation, tumor growth, neovascularization and VEGF and ET-1 is blocked by ETAR antagonists. This observation has MMP-2 expression [65]. been explained by the finding of bone–tumor interaction impairment through the direct inhibition of osteoblasts. 6. Competitive environment The role of ET receptor antagonists in prostate cancer is also supported by the finding of a high expression of ETAR, The pharmaceutical development of ET receptor antagonists both in primary and metastatic tumors, which is associated has derived from the observation of the prominent role of the with advanced tumor stage and grade. ET-1 axis in cardiovascular diseases. ABT-627 is an agent ETAR expression is also correlated with survival and might developed by Abbott Laboratories; it is also known as atra- predict distant metastases in nasopharyngeal carcinoma sentan and marketed under the brand name of Xinlay. It is (NPC) [55]. The inhibition of NPC cell proliferation by one of the most potent and selective ETAR antagonists. Its

32 Expert Opin. Emerging Drugs (2010) 15(1) Russo, Bronte, Rizzo, Fanale, Di Gaudio, Gebbia & Bazan

effects on the ET-1 blockade were discovered in in vitro and patients treated with 10 mg atrasentan. In the same group, a in vivo models for cardiovascular diseases and malignancies. statistical significance was observed in the improvement of Atrasentan seems qualified for clinical use because it is orally ‘pain interference with relations with other people’ (p = 0.031) bioavailable and has a half-life of 25 h, thus, allowing daily and ‘worst pain in the last 24 h’ (p = 0.030). This finding delivery. It has produced mild adverse effects in healthy suggests a possible specific role of atrasentan to increase the volunteers [66], for example, transient headache, rhinitis quality of life [73]. and nausea, which appear to be associated with atrasentan A subsequent Phase II trial investigated the antitumor action on the inhibition of the function of ET-1 as a activity as identified through PSA reductions observed in vasoconstrictor. However, no liver or hematological toxicity previous Phase I studies. This second trial, double-blind has been reported. and placebo-controlled, evaluated the effect on time-to- Atrasentan is able to increase plasma levels of ET-1 due progression prolongation. Asymptomatic hormone-refrac- to the blockade of ETB. Zibotentan is the only ET receptor tory metastatic prostate cancer patients were divided into antagonist in clinical development that targets ETAR three arms, those treated with a placebo, with 2.5 mg and without inhibition of ETBR at doses under clinical with 10 mg atrasentan. In the evaluable set, but not in the investigation [67]. Table 1 reports a description of the intent-to-treat analysis, a statistically significant difference main features of the agents of this class studied for in the delay of disease progression was seen between placebo anticancer treatment. and 10 mg atrasentan groups (p = 0.021). Prostate cancer patients have been identified as the best Overall survival analysis in this study found no significant clinical setting for testing the safety and efficacy of ET advantage in the atrasentan arms versus placebo. This result receptor antagonists. Preclinical findings, in fact, have seems to be due to the confounding effect of the crossover already shown a clear role of ET-1 in the development toward atrasentan treatment administered to placebo group and progression of prostate carcinoma. patients. Survival advantage proved to be significant when atrasentan treatment groups were combined into a single arm achieving HR = 0.69 [68]. 6.1 Atrasentan Tolerabilty profile included mild toxicity with headache, Two Phase I clinical trials were designed to treat hormone rhinitis and edema as the most common side effects. refractory prostate cancer patients with escalating doses of The markers of bone deposition (alkaline phosphatase, atrasentan ranging between 2.5 and 95 mg orally for 28 days. bone alkaline phosphatase) and bone resorption (N-telo- In one of the two studies, headache was found to be the dose- peptides, C-telopeptides and deoxypyridinoline) were eval-

For personal use only. limiting toxicity obtained at 75 mg, while in the other study uated in the same Phase II trial with atrasentan, which the maximum tolerated dose was not reached. As previously showed a dose-dependent effect on the stabilization of these found in healthy subjects, headache, rhinitis and peripheral markers and a clear trend toward the improvement of the edema were the most common side effects [68,69]. Even though bone scan index was found in the 10 mg arm [74].These prostate specific antigen (PSA) response was not the aim of the findings agree with preclinical data regarding the role of the study, 68% (15/22) of patients achieved a reduction of PSA ET-1 axis in bone formation. Osteoblasts have shown the levels ranging between 5 and 95%. Doses of atrasentan expression of high affinity ETAR receptors and strong showed no correlation with PSA declines. response to ET-1 stimulation leading to the osteoblastic Ten patients in the same study were affected by pain which proliferation associated with metastatic breast and prostate needed treatment with opioids. In all, 70% of them experi- cancers [44,75]. In vivo models of bone metastasis have enced pain reduction evaluated by VAS. Preclinical findings clarified the role of ETAR in osteogenesis by means of indicated the particular effect of ET-1 in inducing hyperalge- the use of ETARantagonists[46].

Expert Opin. Emerging Drugs Downloaded from informahealthcare.com by Eli Lilly and Company on 02/18/15 sia and pain through cutaneous injection [70,71]. This phe- On the basis of the clinical findings of a Phase II trial nomenon is mediated by the excitation of ETAR-mediated involving disease progression delay, a Phase III clinical study nociceptors. On the other hand, ETBR activation induces (M00-211) was designed to verify the ability of atrasentan to analgesia by the release of b-endorphin and the activation of duplicate time to progression in asymptomatic hormone- the opioid pool [72]. refractory metastatic prostate cancer patients. These were These effects of atrasentan in controlling cancer pain grouped into two arms, placebo and 10 mg atrasentan. were studied in one Phase II clinical trial, double-blind and Secondary end points were PSA response rate, markers of placebo-controlled; for hormone-refractory metastatic pros- bone metabolism and quality of life. As in the Phase II tate cancer, patients were affected by pain needing opioid M96-594 trial, time to progression was significantly delayed supplementation. The primary end point of this particular in the evaluable set analysis but not in the intent-to-treat study was to evaluate the rate of pain relief after 12 weeks of analysis (p = 0.007 vs 0.091). All the secondary end points treatment. No statistically significant difference was observed. showed a significant favor trend for the 10 mg atrasentan arm. A trend toward cancer pain amelioration with control of Moreover, in the 10 mg atrasentan group, a stratification of analgesic consumption was obtained only in the group of this trial data made it possible to observe a significant

Expert Opin. Emerging Drugs (2010) 15(1) 33 Anti-endothelin drugs in solid tumors

Table 1. Endothelin receptor antagonists studied in clinical trials for cancer treatment.

Compound Company Mechanism of action Indication Stage of Side effects development

Atrasentan Abbott, USA ETAR antagonist Castration-resistant Phase III trial Headache, (ABT-627, Reduction of microvessel density, prostate cancer rhinitis and Xinlay) VEGF and nausea MMP-2 expression and increase of the percentage of apoptotic tumor cells

Zibotentan AstraZeneca, UK ETAR antagonist Castration-resistant Phase III trial Headache, (ZD-4054) Inhibits ET-1-induced mitogenic prostate cancer peripheral effects edema, fatigue, The combination with paclitaxel nasal congestion produces additive effects on and nausea the reduction of ETAR-driven angiogenesis and invasive mediators Bosentan Hoffmann-La Dual ET receptor antagonist Melanoma Phase II trial Headache, Roche, targeting fatigue, nausea, Switzerland ETAR and ETBR back pain and Inhibits tumor growth, abnormal neovascularization and bone hepatic function metastasis

ET: Endothelin; ETAR: Endothelin A receptor; ETBR: Endothelin B receptor.

prolongation of time to progression in the subgroup of of docetaxel at doses ranging from 60 to 75 mg/m2 every patients with bone metastases independently of soft tissue 21 days, with daily oral atrasentan 10 mg starting on day 3 metastases [76]. This ﬁnding suggests interesting implications until evidence of disease progression or unacceptable toxicity.

For personal use only. regarding the clinical use of atrasentan in CRPC due to the The maximum tolerated dose of docetaxel in this regimen is large number of patients (85%) with bone metastasis. 70 – 75 mg/m2. Overall survival and progression-free survival Subsequently, another Phase III trial was performed in are similar to that normally obtained with docetaxel and nonmetastatic hormone-refractory prostate cancer patients prednisone. The PSA response rates are slightly lower than with no radiographic evidence of metastases but rising PSA expected. A Phase III trial to compare this combination with levels. These were randomized to receive 10 mg atrasentan or docetaxel and prednisone had already been designed and is still placebo. The primary end point, time to progression deﬁned ongoing [79]. as the time to the onset of metastases, was not achieved. In advanced NSCLC, 10 mg/day atrasentan plus standard A favoring trend for atrasentan was highlighted through the paclitaxel–carboplatin combination have been investigated for evaluation of the secondary end points: time to PSA progres- safety and pharmacokinetics. They appear to be well tolerated, sion, change in bone alkaline phosphatase levels, PSA dou- without pharmacokinetic interaction of paclitaxel and atra- bling time and overall survival. Large geographic differences in sentan. Efﬁcacy and survival have proved to be similar to those

Expert Opin. Emerging Drugs Downloaded from informahealthcare.com by Eli Lilly and Company on 02/18/15 the median time to progression were observed, which implies already observed in studies involving the use of chemotherapy that trial conduct may have inﬂuenced the results. A more alone in advanced NSCLC patients [80]. Phase III trials and adequate study design should, therefore, be considered [77]. other combination regimen trials should also be designed. Atrasentan has also been studied in clinical trials involving the combination with other antineoplastic agents. A Phase II study for the comparison between atrasentan alone and its 6.2 Zibotentan combination with zoledronic acid was designed to evaluate Zibotentan, when studied in asymptomatic or mildly symp- bone turnover markers in men with bone metastases from tomatic hormone-refractory prostate cancer patients with prostate cancer. There is no evidence for additive or synergistic bone metastases, obtained similar results. Primary end point effects of combination treatment on these markers [78]. (time to progression) was not achieved, but advantage in Preclinical data regarding the combination therapy with overall survival was seen in both 10 and 15 mg zibotentan taxanes and atrasentan have already suggested a potential role arms [81]. in the treatment of refractory malignancies. In metastatic A subsequent Phase I study evaluated escalating doses of CRPC patients, a Phase I/II trial investigated the feasibility zibotentan ranging between 10 and 200 mg/day p.o. in

34 Expert Opin. Emerging Drugs (2010) 15(1) Russo, Bronte, Rizzo, Fanale, Di Gaudio, Gebbia & Bazan

Table 2. Clinical studies with ETAR antagonists.

Citation Phase Patients Agents Schedule Results

Atrasentan Zonnenberg I Metastatic CRPC Atrasentan A 2.5 – 95 mg/day No dose-limiting adverse 2003 [69] p.o. events observed up to 95 mg The most common adverse events were rhinitis, headache and peripheral edema Armstrong I/II Metastatic CRPC Atrasentan A 10 mg/day p.o. Overall survival and 2008 [79] + docetaxel + D every 3 weeks progression-free survival i.v. 60 – 75 mg/m2 are comparable to that seen with docetaxel and prednisone, whereas the rates of PSA decline are slightly lower than expected Drug-related grade 3 – 4 toxicities included neutropenia (50 – 63%) and febrile neutropenia (16 – 25%) Chiappori I/II Advanced NSCLC Atrasentan + A 10 mg/day p.o. Atrasentan plus 2008 [80] carboplatin + + C every 3 weeks paclitaxel–carboplatin was paclitaxel i.v. AUC6 + P every safe and well tolerated, 3 weeks i.v. with no apparent 225 mg/m2 paclitaxel–atrasentan pharmacokinetic interaction Efﬁcacy and survival in advanced NSCLC were

For personal use only. comparable with studies of chemotherapy alone Carducci II Metastatic CRPC Atrasentan A 2.5 mg/day p.o. Median TTP prolongation 2003 [68] vs placebo A 10 mg/day p.o. Headache, peripheral Placebo edema and rhinitis were primary side effects Michaelson II Prostate cancer Atrasentan + A 10 mg/day p.o. No evidence for additive or 2006 [78] with bone zoledronic acid A 10 mg/day p.o. + synergistic effects of metastases ZA every 4 weeks combination therapy with i.v. atrasentan and zoledronic acid on bone turnover markers Commonly observed adverse effects were

Expert Opin. Emerging Drugs Downloaded from informahealthcare.com by Eli Lilly and Company on 02/18/15 edema, rhinitis, fatigue and shortness of breath Carducci III Metastatic CRPC Atrasentan vs A 10 mg/day p.o. Atrasentan did not delay 2007 [76] placebo Placebo disease progression in men with metastatic CRPC despite evidence of biologic effects on markers of disease burden

A: Atrasentan; B: Bosentan; b.i.d.: Twice a day; C: Carboplatin; CRPC: Castration-resistant prostate cancer; D: Docetaxel; ETAR: Endothelin A receptor; i.v.: Intravenous; MWTD: Maximum well-tolerated dose; P: Paclitaxel; p.o.: Per os; PSA: Prostate speciﬁc antigen ; TTP: Time to progression; Z: Zibotentan; ZA: Zoledronic acid.

Expert Opin. Emerging Drugs (2010) 15(1) 35 Anti-endothelin drugs in solid tumors

Table 2. Clinical studies with ETAR antagonists (continued).

Citation Phase Patients Agents Schedule Results

Nelson III Nonmetastatic CRPC Atrasentan vs A 10 mg/day p.o. Although the primary end 2008 [77] placebo Placebo point was not achieved, large regional differences in TTP were noted, suggesting that trial conduct may have inﬂuenced the results Zibotentan James II Metastatic CRPC Zibotentan vs Z 10 mg/day p.o. The primary end point of 2009 [81] placebo Z 15 mg/day p.o. TTP was not achieved in Placebo this study, but an improvement was seen in overall survival in both active treatment arms Adverse events were in line with the expected pharmacologic effects of an ETAR antagonist Schelman I Metastatic CRPC Zibotentan Z 10 – 200 mg/day The MWTD for zibotentan 2009 [82] p.o. was 15 mg Dose-limiting toxicity was similar to that reported for other drugs of this class Some patients experienced prolonged stable disease Bosentan Kefford II Metastatic Bosentan B 500 mg/day p.o. Beneﬁt in disease 2007 [83] melanoma b.i.d. stabilization The most frequent adverse events were headache,

For personal use only. fatigue, nausea, back pain and abnormal hepatic function

metastatic CRPC patients. The maximum well-tolerated dose 7. Conclusions for zibotentan was 15 mg/day p.o. Dose-limiting toxicity was similar to that reported for other drugs of this class. Some ETs belong to a family of proteins with regulatory activity in

Expert Opin. Emerging Drugs Downloaded from informahealthcare.com by Eli Lilly and Company on 02/18/15 patients experienced prolonged stable disease, suggesting that different cellular pathways, including cell growth, survival, new methods are needed to test antitumor activity [82]. invasion and angiogenesis. ET-1, which mainly exert their Phase III studies with zibotentan in the same setting of function on the ETAR, has shown a role in the various phases patients are ongoing. of carcinogenesis and cancer progression of different tumor types. Preclinical studies have demonstrated the capability of ET antagonists in inhibiting tumor cell proliferation, invasive- 6.3 Bosentan ness and new vessel formation, as well as their effects on Bosentan, the dual ETAR/ETBR antagonist, was also studied osteogenesis and cancer-related pain. The clinical investi- in a Phase II trial with metastatic melanoma patients. No gation of an orally bioavailable ET antagonist, atrasentan, tumor responses were obtained, but disease stabilization was has suggested its benefit in PSA progression, markers of bone found in about 20% of patients suggesting the need for further turnover, and pain in men with prostate cancer, but improve- investigation in combination with other anticancer ment in time to progression and overall survival has not yet drugs [83]. Table 2 summarizes all the clinical trials with ET been demonstrated with sufficient statistical significance, receptor antagonists studied for cancer treatment. perhaps because of the small number of patients included in

36 Expert Opin. Emerging Drugs (2010) 15(1) Russo, Bronte, Rizzo, Fanale, Di Gaudio, Gebbia & Bazan

the clinical trials. This class of drugs seems quite promising, confirmed a non-positive trend in favor of patients treated above all for CRPC patients. Nevertheless, we should define with atrasentan independently of its dose. All these results their exact role in different cancer treatments by means of suggest that more effective clinical trials are required in order future clinical trials. to evaluate ETAR antagonists. To date, the treatment of prostate cancer patients is the only clinical setting to be extensively studied for therapy with 8. Expert opinion atrasentan. Unsatisfactory results have been obtained from The ET axis, which includes the biological functions of the a Phase II trial with bosentan in metastatic melanoma patients. ETs and their receptors, has played a physiological role in The improved characterization of the ET-1 axis will normal tissue, acting as a modulator of vasomotor tone, tissue provide a valid target for more promising molecular treat- differentiation and development, cell proliferation and hor- ment strategies to combine with known anticancer drugs. mone production. Interestingly, it also functions in the growth The synergistic action of ZD-4054 and gefitinib on tumor and progression of various tumors. In vitro and in vivo studies burden reduction is the best demonstration of this have suggested that it can trigger signaling pathways impli- therapeutic potential. cated in various hallmarks of cancer, including aberrant cell The choice of the most adequate end point to define the proliferation, adhesion, migration, invasion, angiogenesis and clinical efficacy of these drugs remains the most controversial antiapoptotic activity. feature. Statistical power, baseline characteristics of patients Several researchers have identified the blockade of the ET-1 and pharmaceutical doses are the parameters to emphasize in receptor as a promising therapeutic approach. order to reach a definite understanding of the role of these new Preclinical findings have highlighted the fact that ET-1 agents in cancer treatment. receptor antagonists enhance the efficacy of cytotoxic drugs. Combination clinical trials with chemotherapy or other anticancer agents in various malignancies are needed. These When ETAR is blocked by its antagonists, the cancer cells are sensitized to the apoptotic potential of chemotherapeutics studies will found a proper rationale in the results of the drugs. The finding of sensitization to paclitaxel is an combination Phase I/II trials with atrasentan and standard example of this phenomenon obtained by ovarian and cervical chemotherapeutic drugs in prostate and lung cancers. carcinoma preclinical models. Nevertheless, different end points, not only clinical but also molecular, should be identified in order to reach a better Atrasentan – one of the most potent and selective ETAR antagonists – has been the most effectively studied, because it understanding of the most appropriate application of these has been included in two Phase III clinical trials as mono- agents in the treatment of solid tumors.

For personal use only. therapy and Phase I/II combination trials with chemotherapy. It has shown a quite amazing tolerability profile and even the Acknowledgements means of administration – oral – would allow a wide applicability if its preliminary efficacy results are subsequently A Russo and G Bronte have contributed equally to this work. confirmed. Time to progression, which is the primary end point of the Phase III trials with atrasentan, has been achieved Declaration of interest only in the evaluable set of metastatic patients. No statistically significant efficacy has been observed in the intent-to-treat The authors state no conflict of interest and have received no analysis. Overall survival, one of the secondary end points, has payment in preparation of this manuscript. Expert Opin. Emerging Drugs Downloaded from informahealthcare.com by Eli Lilly and Company on 02/18/15

Expert Opin. Emerging Drugs (2010) 15(1) 37 Anti-endothelin drugs in solid tumors

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72. Khodorova A, Navarro B, Jouaville LS, 78. Michaelson MD, Kaufman DS, Kantoff P, prostate cancer. Invest New Drugs 2009, et al. Endothelin-B receptor activation et al. Randomized phase II study of doi: 10.1007/s10637-009-9318-5 triggers an endogenous analgesic cascade atrasentan alone or in combination with 83. Kefford R, Beith JM, Van Hazel GA, et al. at sites of peripheral injury. Nat Med zoledronic acid in men with metastatic A phase II study of bosentan, a dual 2003;9(8):1055-61 prostate cancer. Cancer endothelin receptor antagonist, as 73. Nelson JB. Endothelin receptor 2006;107(3):530-5 monotherapy in patients with stage IV antagonists. World J Urol 79. Armstrong AJ, Creel P, Turnbull J, et al. A metastatic melanoma. Invest New Drugs 2005;23(1):19-27 phase I-II study of docetaxel and atrasentan 2007;25(3):247-52 74. Nelson JB, Nabulsi AA, Vogelzang NJ, in men with castration-resistant metastatic et al. Suppression of prostate cancer prostate cancer. Clin Cancer Res Affiliation † induced bone remodeling by the 2008;14(19):6270-6 Antonio Russo 1 MD, Giuseppe Bronte1, endothelin receptor A antagonist 80. Chiappori AA, Haura E, Rodriguez FA, Sergio Rizzo1, Daniele Fanale1, atrasentan. J Urol 2003;169(3):1143-9 et al. Phase I/II study of atrasentan, an Francesca Di Gaudio2, Nicola Gebbia1 & 1 75. Takuwa Y, Ohue Y, Takuwa N, endothelin A receptor antagonist, in Viviana Bazan † Yamashita K. Endothelin-1 activates combination with paclitaxel and Author for correspondence 1Universita` di Palermo, phospholipase C and mobilizes Ca2+ from carboplatin as first-line therapy in extra- and intracellular pools in osteoblastic advanced non-small cell lung cancer. Section of Medical Oncology, cells. Am J Physiol Clin Cancer Res 2008;14(5):1464-9 Department of Surgical and Oncological Sciences, 1989;257(6 Pt 1):E797-803 81. James ND, Caty A, Borre M, et al. Safety Via del Vespro 129, 76. Carducci MA, Saad F, Abrahamsson PA, and efficacy of the specific endothelin-A receptor antagonist ZD4054 in patients 90127 Palermo, Italy et al. A phase 3 randomized controlled trial Tel: +39 091 6552500; Fax: +39 091 6554529; of the efficacy and safety of atrasentan in with hormone-resistant prostate cancer and bone metastases who were pain free or E-mail: [email protected] men with metastatic hormone-refractory 2 mildly symptomatic: a double-blind, Professor, prostate cancer. Cancer Universita` di Palermo, 2007;110(9):1959-66 placebo-controlled, randomised, phase 2 trial. Eur Urol 2009;55(5):1112-23 Department of Medical Biotechnology 77. Nelson JB, Love W, Chin JL, et al. Phase 3, and Legal Medicine, randomized, controlled trial of atrasentan 82. Schelman WR, Liu G, Wilding G, et al. A Palermo, Italy in patients with nonmetastatic, phase I study of zibotentan (ZD4054) in hormone-refractory prostate cancer. patients with metastatic, castrate-resistant Cancer 2008;113(9):2478-87 For personal use only. Expert Opin. Emerging Drugs Downloaded from informahealthcare.com by Eli Lilly and Company on 02/18/15

40 Expert Opin. Emerging Drugs (2010) 15(1)