Arno Therapeutics (United States)

<div>Abstract<p><b>Purpose:</b> 7-<i>t</i>-Butyldimethylsilyl-10-hydroxycamptothecin (AR-67) is a novel third generation camptothecin selected for development based on the blood stability of its pharmacologically active lactone form and its high potency in preclinical models. Here, we report the initial phase I experience with i.v. AR-67 in adults with refractory solid tumors.</p><p><b>Experimental Design and Methods:</b> AR-67 was infused over 1 hour daily five times, every 21 days, using an accelerated titration trial design. Plasma was collected on the 1st and 4th day of cycle 1 to determine pharmacokinetic parameters.</p><p><b>Results:</b> Twenty-six patients were treated at nine dosage levels (1.2-12.4 mg/m<sup>2</sup>/d). Dose-limiting toxicities were observed in five patients and consisted of grade 4 febrile neutropenia, grade 3 fatigue, and grade 4 thrombocytopenia. Common toxicities included leukopenia (23%), thrombocytopenia (15.4%), fatigue (15.4%), neutropenia (11.5%), and anemia (11.5%). No diarrhea was observed. The maximum tolerated dosage was 7.5 mg/m<sup>2</sup>/d. The lactone form was the predominant species in plasma (>87% of area under the plasma concentration-time curve) at all dosages. No drug accumulation was observed on day 4. Clearance was constant with increasing dosage and hematologic toxicities correlated with exposure (<i>P</i> < 0.001). A prolonged partial response was observed in one subject with non–small cell lung cancer. Stable disease was noted in patients with small cell lung cancer, non–small cell lung cancer, and duodenal cancer.</p><p><b>Conclusions:</b> AR-67 is a novel, blood-stable camptothecin with a predictable toxicity profile and linear pharmacokinetics. The recommended phase II dosage is 7.5 mg/m<sup>2</sup>/d five times every 21 days. Clin Cancer Res; 16(2); 673–80</p></div>

IntroductionOnapristone is a type I progesterone receptor (PR) antagonist, which prevents PR- mediated DNA transcription. Onapristone is active in multiple preclinical models and two prior studies demonstrated promising activity in patients with breast cancer. We conducted a study of extended release (ER) Onapristone to determine a recommended dose and explore the role of transcriptionally-activated PR (APR), detected as an aggregated subnuclear distribution pattern, as a predictive biomarker.MethodsAn open-label, multicenter, randomized, parallel-group, phase 1 study (target n = 60; NCT02052128) included female patients ≥18 years with PRpos tumors. APR analysis was performed on archival tumor tissue. Patients were randomized to five cohorts of extended release (ER) onapristone tablets 10, 20, 30, 40 or 50 mg BID, or immediate release 100 mg QD until progressive disease or intolerability. Primary endpoint was to identify the recommended phase 2 dose. Secondary endpoints included safety, clinical benefit and pharmacokinetics.ResultsThe phase 1 dose escalation component of the study is complete (n = 52). Tumor diagnosis included: endometrial carcinoma 12; breast cancer 20; ovarian cancer 13; other 7. Median age was 64 (36–84). No dose limiting toxicity was observed with reported liver function test elevation related only to liver metastases. The RP2D was 50 mg ER BID. Median therapy duration was 8 weeks (range 2–44), and 9 patients had clinical benefit ≥24 weeks, including 2 patients with APRpos endometrial carcinoma.ConclusionClinical benefit with excellent tolerance was seen in heavily pretreated patients with endometrial, ovarian and breast cancer. The data support the development of Onapristone in endometrial endometrioid cancer. Onapristone should also be evaluated in ovarian and breast cancers along with APR immunohistochemistry validation.

590 Background: The Androgen Receptor is present in BC, and ongoing studies are testing the activity of antiandrogens in BC. AR has been associated with a better prognosis in ERα positive tumors. AR has a nuclear biology dynamics common to the steroid nuclear receptors. In the absence of ligand, it is evenly distributed in nuclei; when exposed to androgens, it migrates to form sub-nuclear aggregates that can be detected by fluorescence microscopy. Thus, two distinct nuclear patterns, diffuse (D) or aggregated (A), correspond to two receptor functional states. Similarly to previous work on ER and PR (ASCO 2013 abstr # 11535 & 593), we have devised an immunohistochemical (IHC) method to characterize the nuclei patterns that may indicate whether AR is transcriptionally active or not in PEFF tissues. The goal of this study is to explore the AR biological or clinical relevance. Methods: As of February 4th, 662 archived BC biopsies have been obtained along with associated clinical data. Biopsies were analyzed for standard HES, ER, PR, AR and Ki67. AR positivity was determined with clone AR27 antibody (Novocastra). The A-AR (aggregated AR) and D-AR (diffuse AR) pattern were analyzed at x1000 magnification. Results: 490 cases have been analyzed to date. Mean Age: 57 (17-89). Histology: ductal 82%, lobular 15%, and other 3%; ERapos , 78%, PRpos 78%. Adj Chemotherapy 46% Hormonotherapy 85%. Stage I 44%, II 48%, III 8%. Grade I 25%, II 50%, III 25%. AR was positive (ARpos) in 51%. AR status was diffuse (D-AR) in 66% and aggregated (activated, A-AR) in 34% of the ARpos biopsies. ARpos and D-AR were associated with lower Grade (p < 0.04, p = 0.1), ERα (p <0.000,= 0.03), PR (p <0.000, 0.1), but not HER2, Ki67, or Staging. Median follow up was 36 months (mo). Progressive disease (PD), local or distant, was 19%, and PD was not associated with A-AR (p=0.25). With DFS defined as time to PD or death (5 year cut-off), ARposwas associated with a HR of 0.61 (p = 0.08) but not in a Cox model including both AR and ER positivity (HR for ER = 0.41, for AR 0.92, with p = 0.006 and 0.8 respectively). When added, grade was an independent predictor (HR= 0.44, p = 0.004). Conclusions: A-AR is associated with grade and other steroid receptors suggesting biological relevance.

e11535 Background: : About 50% of ER positive (ERpos) BC are resistant to hormone treatment. In absence of ligand, ERs are evenly distributed in nuclei in normal tissue. Upon ligand binding, ERs dimerizes and form a discrete focal subnuclear distribution pattern (FDP), which are associated with transcriptional activation of ER. This ER FDP is observed in BC. We hypothesized that in BC that the presence/absence of ER in the FDP could predict antiestrogen activity. This study describes an immunohistochemistry (IHC) method, by which a biomarker could be developed to investigate this hypothesis. Methods: 37 paraffin embedded, formalin fixed archived BC samples were processed using 4 ER-alpha antibodies (Ab) under different IHC conditions to develop and refine this biomarker, the ER nuclear morphology was analyzed with standard microscopy at x1000. Interpretation of the IHC slides was done by an experienced pathologist. Standard ER, progesterone receptor (PR), and Ki67 testing were done. Tumor grade was obtained from the patients’ records. Analysis of the different Ab was with the kappa statistics. Comparison of the IHC technique to immunofluorescence (IHF) was done on 8 samples. Results: Consistent with prior research observations, tumors had two ER nuclear morphologies: 1. Diffuse pattern (D) where the ER was distributed evenly in a fine granular pattern, 2. Aggregate pattern (A) where the ER is distributed in distinct clumps or aggregates. This defined 3 tumor phenotypes: A cells only (A) 1% (4/37), D cells only (D) 65% (24/37), and a heterogenous mix of A + D cells (AD) 18% (7/37), ER Neg 14% (5/37). IHC and IHF analysis for ER nuclear morphology was concordant. The ER activation status (A/AD or D) was not correlated with staining ER intensity, ERpos %, PRpos%, but was correlated with % Ki67 staining (p=0.09) and tumor grade (0.035). Conclusions: ERpos early BC biopsies can be grouped in two categories based on ER nuclear morphology: A group with diffuse and homogenous nuclear staining, and a different group with heterogeneous area of cells having a nuclear pattern consistent with a functional or activated ER. These observations warrant clinical pathological studies to determine if this biomarker is potentially predictive of clinical outcomes.

Abstract Background: Antihormonal agents (AH) are some of the most clinically useful anticancer drugs. AH resistance and the availability of alternative treatments have made patient selection increasingly important for AH drug development. We hypothesized that the progesterone receptor (PR) may be constitutively active because of the aberrant biological pathways and that selecting patients with baseline tumor PR activation would predict for antiprogestin activity. Activated PR (APR) can be visualized in nuclei as fluorescent aggregates via fluorescent green protein engineered PR cell lines in vitro and by immunofluorescence (IF) in tissues. The detection of PR foci has been associated with transcription and gene activation in vitro. PR phosphorylation (phos) is induced by a variety of pathways. We have developed a IHC method that allows APR determination in tumor biopsies on a routine basis (SABCS 2012). The predictive nature of the observation of APR by IHC in vitro has been tested with onapristone (ONA) for cell survival and proliferation. Methods: 8 human breast cancer (BT-474, CAMA-1, EVSA-T, HCC-1954, MCF-7, MDA-MB-231, T-47D, ZR-75-1) and 2 human endometrial cancer (Ishikawa, HEC-1-A) cell lines were studied. Cytoblocks for APR analysis were made for each time point and experimental condition, baseline ER/PR expression was determined by IHC and PR phos was analyzed with specific antibodies (pSER162, 190, 294, 400, 554) by Western Blotting and IHC. Culture conditions: normal and stripped FBS, +/- ONA and growth factor/hormone exposure (EGF, FGF2, E2 and P4) in triplicate. Analysis was performed at baseline (BL), 6h, 96h and 7 days. Cell viability was assessed by MTS assay, proliferation was studied by Ki67 analysis. APR foci were determined by IHC and read by a pathologist blinded to the experimental conditions. Results: At BL only two cell lines were PR pos/APR pos (T47D, CAMA-1) and all other cell lines where PR pos/APR neg (BT-474, EVSA-T, HCC-1954, MCF-7) or PR neg/APR neg (Ishikawa, HEC-1-A, MDA-MB-231, ZR-75-1). ONA exerted inhibitory effect only in APR pos cell lines. In stripped FBS at 6h, T47D APR pos status was converted to APR neg by ONA except with E2 stimulation which required longer treatment. The CAMA-1 data are not yet available. No APR neg cell lines were converted into APR pos except in one experiment after protracted exposure to EGF. ONA generally decreased phos; phos decrease measured by WB did not correlate with growth inhibition. Phos status determined by IHC and Ki67 proliferation assessment are currently in process and will be presented with the confirmatory data. Conclusions: Baseline APR positivity is predictive of ONA activity in vitro. ONA converts APR pos cells to APR neg within 6h in most cases, a time frame consistent with PR biology. The growth inhibitory effects of ONA were not correlated to PR phos in the cell lines and conditions tested. The APR IHC methodology may be applicable in the clinical setting. Citation Format: Erard Gilles, Laura Caplier, Alexander Valent, Guillaume Serin, Anne Gompel, Jacques Bosq, Alexander Zukiwski. Antiprogestin drug development: in vitro validation of a potential clinical biomarker. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1317. doi:10.1158/1538-7445.AM2013-1317

2534 Background: AR-67 is a 3rd generation camptothecin analog selected for development based on the high in vitro stability of its pharmacologically active lactone form and high potency in preclinical models. This report describes the initial phase I study of intravenous AR-67 in adults with refractory solid tumors. Methods: AR-67 was infused over 1 hour for 5 days of a 21-day cycle using an accelerated titration phase I trial design. PK was performed on the 1st and 4th day of cycle 1. AR-67 was assayed with a validated chromatography method. Toxicity and response were assessed using NCI CTC (v3) grading scale and RECIST. Results: In total, 26 patients were treated at 9 dose levels (mg/m2/day): 1.2 (n=2), 1.67 (n=3), 2.34 (n=3), 3.2 (n=3); 4.5 (n=1), 6.3 (n=1), 7.5 (n=7), 8.9 (n=4) and 12.4 (n=2). Median age 62 (range 31–79), 15M/11F, median prior therapies 3 (range 1 to 6). Tumor types included: colorectal (8), non-small cell lung (NSCLC) (4), small cell lung (3), soft tissue sarcoma, (3), head and neck (2), prostate (2), and other (4). 21 subjects completed 2 or more cycles of therapy, 5 subjects received 1 cycle of therapy and had rapid disease progression (1 received 2d of drug prior to PD), 1 subject is still under treatment after 9 cycles. DLTs were observed in 5 patients: 2 of 2 at 12.4 mg/m2/day (Gr 4 febrile neutropenia, Gr 3 fatigue); 2 of 4 at 8.9 mg/m2/day (Gr 4 thrombocytopenia), 1 of 7 at 7.5 mg/m2/day (Gr 4 thrombocytopenia). Common C1 worst-grade drug related toxicities (CTC I/II % vs III/IV %): Hg (27/8), WBC (11/19), ANC (19/8), platelets (19/12), fatigue (15/8) insomnia (8/0), flushing (15//0), constipation (8/0), nausea (23/0), ALT elevation (12/0), hiccups (8/0). Antitumor activity, assessed by development of PR and SD, was observed in NSCLC, SCLC, colon and bladder cancer. The lactone form was predominant in plasma (&gt;85% of AUC) at all time points. Clearance was constant with increasing dose and exposure (AUC) correlated with toxicity. Conclusions: AR-67 has superior lactone stability compared to approved analogs, has a predictable toxicity profile that did not include diarrhea and has activity in NSCLC. The RP2D is 7.5 mg/m2/day for 5 days of a 21-day cycle. This work was supported by R21-CA-123867 and Arno Therapeutics. [Table: see text]

To assess the quality of antibiotic prescribing by Victorian general practitioners, and the effectiveness of educational intervention techniques in improving prescribing. A randomised, controlled, parallel group trial. In rural and metropolitan Victoria, 182 general practitioners (78 control, 104 intervention) began and 103 (41 control, 62 intervention) completed the study. Participants recorded their antibiotic prescribing for tonsillitis. The intervention group received an educational mailing campaign. A project pharmacist visited each doctor to discuss campaign messages. The percentages of prescriptions of antibiotics for tonsillitis complying with those recommended in Antibiotic guidelines. In the intervention group, prescriptions consistent with recommendations in the guidelines increased from 60.5% before the campaign to 87.7% afterwards. Improvement also occurred in the control group, from 52.9% to 71.7% of prescriptions. The improvement within the intervention group was significantly greater than that within the control group. The educational campaign significantly improved the prescribing of appropriate antibiotics for tonsillitis by general practitioners.