Phase II activity of belinostat (PXD-101), carboplatin, and paclitaxel in women with previously treated ovarian cancer.

BACKGROUND: Preclinical data show that belinostat (Bel) is synergistic with carboplatin and paclitaxel in ovarian cancer. To further evaluate the clinical activity of belinostat, carboplatin, and paclitaxel (BelCaP), a phase 1b/2 study was performed, with an exploratory phase 2 expansion planned specifically for women with recurrent epithelial ovarian cancer (EOC). METHODS: Thirty-five women were treated on the phase 2 expansion cohort. BelCap was given as follows: belinostat, 1000 mg/m² daily for 5 days with carboplatin, AUC 5; and paclitaxel, 175 mg/m² given on day 3 of a 21-day cycle. The primary end point was overall response rate (ORR), using a Simon 2 stage design. RESULTS: The median age was 60 years (range, 39-80 years), and patients had received a median of 3 prior regimens (range, 1-4). Fifty-four percent had received more than two prior platinum-based combinations, sixteen patients (46%) had primary platinum-resistant disease, whereas 19 patients (54%) recurred within 6 months of their most recent platinum treatment. The median number of cycles of BelCaP administered was 6 (range, 1-23). Three patients had a complete response, and 12 had a partial response, for an ORR of 43% (95% confidence interval, 26%-61%). When stratified by primary platinum status, the ORR was 44% among resistant patients and 63% among sensitive patients. The most common drug-related adverse events related to BelCaP were nausea (83%), fatigue (74%), vomiting (63%), alopecia (57%), and diarrhea (37%). With a median follow-up of 4 months (range, 0-23.3 months), 6-month progression-free survival is 48% (95% confidence interval, 31%-66%). Median overall survival was not reached during study follow-up. CONCLUSIONS: Belinostat, carboplatin, and paclitaxel combined was reasonably well tolerated and demonstrated clinical benefit in heavily-pretreated patients with EOC. The addition of belinostat to this platinum-based regimen represents a novel approach to EOC therapy and warrants further exploration.

Plasma and cerebrospinal fluid pharmacokinetics of the histone deacetylase inhibitor, belinostat (PXD101), in non-human primates.

PURPOSE: Histone deacetylases (HDAC) are involved in the regulation of gene transcription. Aberrant HDAC activity has been associated with tumorigenesis, and, therefore, HDACs are potential targets for the treatment of cancers, including tumors of the central nervous system (CNS). Belinostat is a novel, potent, pan-HDAC inhibitor with antiproliferative activity on a wide variety of tumor cell lines. We studied the cerebrospinal fluid (CSF) penetration of intravenous (IV) belinostat in a non-human primate model as a surrogate for blood:brain barrier penetration. DESIGN: Five adult rhesus monkeys received increasing doses of belinostat (10-60 mg/kg) as a 30-min IV infusion. Serial blood and CSF samples were collected over 48 h. Plasma and CSF concentrations of belinostat were quantified with an LC/MS/MS assay. Pharmacokinetic parameters were calculated using non-compartmental methods, and CSF penetration is expressed as the ratio of the area under the concentration-time curve (AUC) in CSF to the AUC in plasma. RESULTS: Belinostat was cleared rapidly from plasma with a half-life of 1.0 h, a mean residence time of 0.47 h, and a clearance of 425 ml/min/m(2). CSF penetration of belinostat was limited. CSF drug exposure was <1% of plasma drug exposure and <10% of free (non-protein bound) plasma drug exposure. CONCLUSION: IV belinostat is rapidly cleared from plasma and has limited penetration into the CSF.

Activity of the histone deacetylase inhibitor belinostat (PXD101) in preclinical models of prostate cancer.

Histone deacetylase inhibitors (HDACi) represent a promising new class of anticancer agents. In the current investigation, we examined the activity of the HDACi belinostat in preclinical models of prostate cancer. In vitro proliferation assays demonstrated that belinostat potently inhibited the growth of prostate cancer cell lines (IC(50) < 1.0 microM) and was cytotoxic to these cells. Washout experiments indicated that exposure to belinostat for relatively short periods of time (<12 hr) induced suboptimal growth-inhibition and that cells exposed to 1.0 microM belinostat for 48 hr retained the capacity for regrowth following drug withdrawal, while cells exposed to 4.0 microM belinostat were irreversibly growth-inhibited. Cell cycle analyses demonstrated that belinostat induced G2/M arrest and increased the percentage of cells with subG1 DNA content, thus confirming the growth-inhibitory and cytotoxic effects of this compound. Normal prostate epithelial cells were generally less susceptible to the effects of belinostat than were prostate cancer cells. In an orthotopic prostate cancer tumor model, belinostat inhibited tumor growth by up to 43%. Moreover, metastatic lung lesions were present in 47% of vehicle-treated animals but in none of the animals administered belinostat. Consistent with its observed antimetastatic activity, belinostat inhibited the migration of prostate tumor cells and increased the production of tissue inhibitor of metalloproteinase-1 (TIMP-1) by these cells, the latter effect being replicated by siRNA knockdown of HDAC3. Belinostat also increased the expression of p21 and decreased the expression of potentially oncogenic proteins (mutant p53 and ERG). These results support the clinical evaluation of belinostat for the treatment of prostate cancer.

Determination of the class and isoform selectivity of small-molecule histone deacetylase inhibitors.

The human HDAC (histone deacetylase) family, a well-validated anticancer target, plays a key role in the control of gene expression through regulation of transcription. While HDACs can be subdivided into three main classes, the class I, class II and class III HDACs (sirtuins), it is presently unclear whether inhibiting multiple HDACs using pan-HDAC inhibitors, or targeting specific isoforms that show aberrant levels in tumours, will prove more effective as an anticancer strategy in the clinic. To address the above issues, we have tested a number of clinically relevant HDACis (HDAC inhibitors) against a panel of rhHDAC (recombinant human HDAC) isoforms. Eight rhHDACs were expressed using a baculoviral system, and a Fluor de Lystrade mark (Biomol International) HDAC assay was optimized for each purified isoform. The potency and selectivity of ten HDACs on class I isoforms (rhHDAC1, rhHDAC2, rhHDAC3 and rhHDAC8) and class II HDAC isoforms (rhHDAC4, rhHDAC6, rhHDAC7 and rhHDAC9) was determined. MS-275 was HDAC1-selective, MGCD0103 was HDAC1- and HDAC2-selective, apicidin was HDAC2- and HDAC3-selective and valproic acid was a specific inhibitor of class I HDACs. The hydroxamic acid-derived compounds (trichostatin A, NVP-LAQ824, panobinostat, ITF2357, vorinostat and belinostat) were potent pan-HDAC inhibitors. The growth-inhibitory effect of the HDACis on HeLa cells showed that both pan-HDAC and class-I-specific inhibitors inhibited cell growth. The results also showed that both pan-HDAC and class-I-specific inhibitor treatment resulted in increased acetylation of histones, but only pan-HDAC inhibitor treatment resulted in increased tubulin acetylation, which is in agreement with their activity towards the HDAC6 isoform.

Treatment parameters modulating regression of human melanoma xenografts by an antibody-drug conjugate (CR011-vcMMAE) targeting GPNMB.

PURPOSE: To investigate the pharmacological properties of the CR011-vcMMAE fully human antibody-drug conjugate (ADC), such as dose titrations, quantitation of the time (days) to complete regression, pharmacokinetics, and schedule dependency. Our prior study characterized a fully human antibody to GPNMB covalently linked to monomethylauristatin E, CR011-vcMMAE, and further demonstrated cell surface staining of melanoma lines susceptible to the immunoconjugate's cytotoxicity (Clin Cancer Res 2005; 12(4): 1373-1382). METHODS: The human SK-MEL-2 and SK-MEL-5 melanoma xenografts were used in athymic mice to assess anti-tumor efficacy. After s.c. implantation, tumors became established (60-100 mg), and treatment commenced by i.v. injection of the immunoconjugate or vinblastine or paclitaxel. Short-term anti-tumor effects (inhibition of tumor growth) and long-term effects (complete regression) were observed. RESULTS: CR011-vcMMAE induced regression of established human SK-MEL-2 and SK-MEL-5 xenografts at doses from 1.25 to 80 mg/kg treatment when administered intravenously every 4 days (4 treatments); strikingly, regressions were not associated with re-growth during the observation period (200 days). The disappearance rate of implants was dose dependent (minimum time, 18.5 days). Detectable serum CR011-vcMMAE >or=1 microg/mL (approximately 0.01 microM) was observed for >30 days post-dose; CR011-vcMMAE showed an elimination half-life of 10.3 days. A low volume of distribution suggested that CR011-vcMMAE was confined to blood and interstitial fluid. CR011-vcMMAE could be delivered by either a single bolus dose or by intermittent dosing (i.e., every 1, 2, 4, 8, or 16 days) with no discernible differences in the proportion of tumor-free survivors, indicating a lack of schedule dependency. The antibody-drug conjugate produced complete regressions, but the equivalent doses of free monomethylauristatin E or unconjugated antibody did not show anti-tumor effects. In addition, decreases in plasma tumor-derived human interleukin-8 coincided with tumor nodule disappearance. CONCLUSIONS: Short-term anti-tumor effects and long-term effects (complete regression) were observed with CR011-vcMMAE, but not with the reference agents. These results suggest that CR011-vcMMAE may provide therapeutic benefit in malignant melanoma.

Activity of PXD101, a histone deacetylase inhibitor, in preclinical ovarian cancer studies.

Histone deacetylase inhibitors represent a promising new class of anticancer agents. In the current investigation, we examined the activity of PXD101, a potent histone deacetylase inhibitor, used alone or in combination with clinically relevant chemotherapeutics (docetaxel, paclitaxel, and carboplatin), in preclinical in vitro and in vivo models of ovarian cancer. In vitro activity was examined in ovarian cancer and multidrug-resistant cell lines grown in monolayer culture, and in primary clinical ovarian cancer specimens grown in three-dimensional organoid culture. PXD101 was found to inhibit in vitro cancer cell growth at sub- to low micromolar IC(50) potency, exhibited synergistic activity when used in combination with relevant chemotherapeutics, and effectively inhibited the growth of multidrug-resistant cells. In vivo, PXD101 displayed single-agent antitumor activity on human A2780 ovarian cancer s.c. xenografts which was enhanced via combination therapy with carboplatin. In support of these findings, PXD101 was shown to increase the acetylation of alpha-tubulin induced by docetaxel and the phosphorylation of H2AX induced by carboplatin. Taken together, these results support the clinical evaluation of PXD101 used alone or in combination therapy for the treatment of ovarian cancer.

CR011, a fully human monoclonal antibody-auristatin E conjugate, for the treatment of melanoma.

PURPOSE: Advanced melanoma is a highly drug-refractory neoplasm representing a significant unmet medical need. We sought to identify melanoma-associated cell surface molecules and to develop as well as preclinically test immunotherapeutic reagents designed to exploit such targets. EXPERIMENTAL DESIGN AND RESULTS: By transcript profiling, we identified glycoprotein NMB (GPNMB) as a gene that is expressed by most metastatic melanoma samples examined. GPNMB is predicted to be a transmembrane protein, thus making it a potential immunotherapeutic target in the treatment of this disease. A fully human monoclonal antibody, designated CR011, was generated to the extracellular domain of GPNMB and characterized for growth-inhibitory activity against melanoma. The CR011 monoclonal antibody showed surface staining of most melanoma cell lines by flow cytometry and reacted with a majority of metastatic melanoma specimens by immunohistochemistry. CR011 alone did not inhibit the growth of melanoma cells. However, when linked to the cytotoxic agent monomethylauristatin E (MMAE) to generate the CR011-vcMMAE antibody-drug conjugate, this reagent now potently and specifically inhibited the growth of GPNMB-positive melanoma cells in vitro. Ectopic overexpression and small interfering RNA transfection studies showed that GPNMB expression is both necessary and sufficient for sensitivity to low concentrations of CR011-vcMMAE. In a melanoma xenograft model, CR011-vcMMAE induced significant dose-proportional antitumor effects, including complete regressions, at doses as low as 1.25 mg/kg. CONCLUSION: These preclinical results support the continued evaluation of CR011-vcMMAE for the treatment of melanoma.

Angioarrestin: a unique angiopoietin-related protein with anti-angiogenic properties.

The process of angiogenesis plays a pivotal role in embryogenesis, wound healing, and tumorigenesis through the growth of new blood vessels from pre-existing vasculature. Among the angiogenic factors recently identified as specific for vascular endothelium are the angiopoietins. In depth characterization of the angiopoietins has allowed investigators to better understand the molecular basis of blood vessel formation and vascular endothelial cell function. In this review, we describe angiopoietins and related family members, with particular emphasis on a recently identified protein known as angioarrestin. Our investigations clearly demonstrate that angioarrestin is an anti-angiogenic molecule. The effects of angioarrestin on tumor cell progression and specific aspects of the angiogenic cascade in in vitro models are further discussed.

Recombinant semaphorin 6A-1 ectodomain inhibits in vivo growth factor and tumor cell line-induced angiogenesis.

The Semaphorins are a large family of transmembrane, GPI-anchored and secreted proteins that play an important role in neuronal and endothelial cell guidance. A human gene related to the class 6 Semaphorin family, Semaphorin 6A-1 (Sema 6A-1) was identified by homology-based genomic mining. Recent implication of Sema 3 family members in tumor angiogenesis and our expression analysis of Sema 6A-1 suggested that class 6 Semaphorin might effect tumor neovascularization. The mRNA expression of Sema 6A-1 was elevated in several renal tumor tissue samples relative to adjacent nontumor tissue samples from the same patient. Sema 6A-1 transcript was also expressed in the majority of renal clear cell carcinoma (RCC) cell lines and to a lesser extent in endothelial cells. To test the role of Sema 6A-1 in tumor angiogenesis, we engineered, expressed and purified the Sema 6A-1 soluble extracellular domain (Sema-ECD). The purified Sema-ECD was screened in a variety of endothelial cell-based assays both in vitro and in vivo. In vitro, Sema-ECD blocked VEGF-mediated endothelial cell migration. These effects were explained in part by our observation in endothelial cells that Sema-ECD inhibited VEGF-mediated Src, FAK and ERK phosphorylation. In vivo, mouse Matrigel assays demonstrated that the intraperitoneal administration of recombinant Sema-ECD inhibited both bFGF/VEGF and tumor cell line-induced neovascularization. These findings reveal a novel therapeutic utility for Sema 6A-1 (Sema-ECD) as an inhibitor of growth factor as well as tumor-induced angiogenesis.

Preclinical characterization of CG53135 (FGF-20) in radiation and concomitant chemotherapy/radiation-induced oral mucositis.

PURPOSE: The purpose of this study was to evaluate the activity of CG53135 (FGF-20), a protein with in vitro mitogenic activity on epithelial and mesenchymal cells, in two in vivo models of oral mucositis (OM). EXPERIMENTAL DESIGN: Radiation or concomitant chemotherapy/radiation-induced OM was elicited in hamsters. Activity of CG53135 was assessed at different doses and regimens in the models. Bromodeoxyuridine (BrdUrd) incorporation and pharmacokinetic studies were also performed to correlate in vivo activity of CG53135 with exposure. RESULTS: In the hamster radiation model, administration of CG53135 (600 or 1200 micro g/day, i.p.) on days 3-15 resulted in a statistically significant (P < 0.001) reduction in days spent with severe mucositis. CG53135 administered at 12 mg/kg, i.p. (days 1-2 or 1-8) in the concomitant chemotherapy/radiation model resulted in a statistically significant (P < 0.001) reduction in severe mucositis. Maximal BrdUrd incorporation was observed in cheek pouch and jejunal tissues at 8 h, and peak plasma levels of CG53135 were reached 1 h after administration. CONCLUSIONS: CG53135 demonstrates potent, regimen-dependent activity in hamster models of OM. The activity was regimen dependent. BrdUrd incorporation studies confirmed that CG53135 had proliferative activity in vivo with a favorable pharmacokinetic profile. Based in part on work described herein, CG53135 has received approval from the United States Food and Drug Administration to be evaluated in a Phase I clinical trial of cancer patients at risk for developing OM.

A fully human monoclonal antibody (CR002) identifies PDGF-D as a novel mediator of mesangioproliferative glomerulonephritis.

PDGF-B is of central importance in mesangioproliferative diseases. PDGF-D, a new PDGF isoform, like PDGF-B, signals through the PDGF betabeta-receptor. The present study first determined that PDGF-D is mitogenic for rat mesangial cells and is not inhibited by a PDGF-B antagonist. Low levels of PDGF-D mRNA were detected in normal rat glomeruli. After induction of mesangioproliferative nephritis in rats by anti-Thy 1.1 mAb, glomerular PDGF-D mRNA and protein expression increased significantly from days 4 to 9 in comparison with nonnephritic rats. Peak expression of PDGF-D mRNA occurred 2 d later than peak PDGF-B mRNA expression. In addition, PDGF-D serum levels increased significantly in the nephritic animals on day 7. For investigating the functional role of PDGF-D, neutralizing fully human mAb were generated using the XenoMouse technology. Rats with anti-Thy 1.1-induced nephritis were treated on days 3 and 5 with different amounts of a fully human PDGF-DD-specific neutralizing mAb (CR002), equal amounts of irrelevant control mAb, or PBS by intraperitoneal injection. Specific antagonism of PDGF-D led to a dose-dependent (up to 67%) reduction of glomerular cell proliferation. As judged by double immunostaining for 5-bromo-2'-deoxyuridine and alpha-smooth muscle actin, glomerular mesangial cell proliferation was reduced by up to 57%. Reduction of glomerular cell proliferation in the rats that received CR002 was not associated with reduced glomerular expression of PDGF-B mRNA. PDGF-D antagonism also led to reduced glomerular infiltration of monocytes/macrophages (day 5) and reduced accumulation of fibronectin (day 8). In contrast, no effect was noted in normal rats that received an injection of CR002. These data show that PDGF-D is overexpressed in mesangioproliferative states and can act as an auto-, para-, or even endocrine glomerular cell mitogen, indicating that antagonism of PDGF-D may represent a novel therapeutic approach to mesangioproliferative glomerulonephritides.

A novel human fibroblast growth factor treats experimental intestinal inflammation.

BACKGROUND & AIMS: We recently identified a novel member of the human fibroblast growth factor (FGF) family of signaling molecules, designated FGF-20. In the present study, we examined the activity of this protein in 2 animal models of acute intestinal inflammation and in mechanistic studies in vitro. METHODS: In vivo experiments consisted of a murine dextran sulfate sodium (DSS) model of colitis and a rat indomethacin model of small intestinal ulceration/inflammation. Cell growth, restitution, gene expression (cyclooxygenase-2 [COX-2] and intestinal trefoil factor [ITF]), and prostaglandin E2 (PGE2) levels were examined in vitro. RESULTS: In the DSS-colitis model, prophylactic administration of FGF-20 significantly reduced the severity and extent of mucosal damage as indicated by a 55%-93% reduction in luminal blood loss, distal colonic edema, histologic inflammation, and epithelial cell loss relative to animals administered vehicle control. No toxicity was noted during administration of FGF-20 to normal controls. In addition, therapeutic administration of FGF-20 enhanced survival in this model. In the indomethacin-small bowel ulceration/inflammation model, administration of FGF-20 reduced small intestinal weight gain, necrosis, inflammation, and weight loss (36%-53% relative to vehicle control). In vitro studies demonstrated that FGF-20 stimulates growth, restitution, mRNA expression of COX-2 and ITF, and PGE2 levels in human intestinal epithelial cells and enhances the growth of human intestinal fibroblasts. CONCLUSIONS: FGF-20, having demonstrated therapeutic activity in 2 experimental models of intestinal inflammation, represents a promising new candidate for the treatment of human inflammatory bowel disease.

Fibroblast growth factors in cancer: therapeutic possibilities.

The fibroblast growth factor (FGF) family of signalling molecules and its receptors (FGFRs) contribute to normal developmental and physiological processes. However, the subversion of this powerful growth stimulatory pathway has been implicated in the generation of a variety of pathological conditions. This review focuses on the role of FGF/FGFRs in cancer. The case will be made that this signalling pathway is associated with and functionally important for the growth of some human tumours. As such, FGF/FGFRs can be viewed as rational therapeutic oncology targets and strategies used to inhibit these molecules are discussed. The therapeutic exploitation of tumour-associated FGFR expression to deliver toxins or antiproliferative signals to tumour cells is also reviewed, as is the use of FGFs as protein therapeutics to alleviate the side effects of cancer therapy.

Angioarrestin: an antiangiogenic protein with tumor-inhibiting properties.

The angiopoietins comprise a family of proteins that have pro or antiangiogenic activities. Through a proprietary technology designed to identify transcripts of all expressed genes, we isolated a cDNA encoding an angiopoietin-related protein that we designate angioarrestin. The mRNA expression profile of angioarrestin was striking in that it was down-regulated in many tumor tissues when compared with adjacent nontumor tissue, suggesting a role for this protein in tumor inhibition. To test this hypothesis, we ectopically expressed angioarrestin in HT1080 tumor cells and measured pulmonary tumor nodule formation in nude mice. HT1080 cells expressing angioarrestin showed a marked reduction in the number and size of tumor nodules. In vitro, the recombinant protein was systematically tested in a number of endothelial cell assays and found to block critical processes involved in the angiogenic cascade, such as vascular endothelial growth factor/basic fibroblast growth factor-mediated endothelial cell proliferation, migration, tubular network formation, and adhesion to extracellular matrix proteins. These findings reveal a novel function for angioarrestin as an angiogenesis inhibitor and indicate that the molecule may be a potential cancer therapeutic.

Platelet-derived growth factor D: tumorigenicity in mice and dysregulated expression in human cancer.

Platelet-derived growth factor (PDGF) has been directly implicated in developmental and physiological processes, as well as in human cancer and other proliferative disorders. We have recently isolated and characterized a novel protease-activated member of the PDGF family, PDGF D. PDGF D has been shown to be proliferative for cells of mesenchymal origin, signaling through PDGF receptors. Comprehensive and systematic PDGF D transcript analysis revealed expression in many cell lines derived from ovarian, renal, and lung cancers, as well as from astrocytomas and medulloblastomas. beta PDGF receptor profiling further suggested autocrine signaling in several brain tumor cell lines. PDGF D transforming ability and tumor formation in SCID mice was further demonstrated. Exploiting a sensitive PDGF D sandwich ELISA using fully human monoclonal antibodies, PDGF D was detected at elevated levels in the sera of ovarian, renal, lung, and brain cancer patients. Immunohistochemical analysis confirmed PDGF D localization to ovarian and lung tumor tissues. Together, these data demonstrate that PDGF D plays a role in certain human cancers.