In Vitro Immunomodulation of the Polysaccharides from Yam (Dioscorea opposita Thunb.) in Response to a Selenylation of Lower Extent

The immunomodulation of chemically selenylated polysaccharides has been attracting more attention recently, but the corresponding performance of the yam polysaccharides (YPS) with lower selenylation extent remains, thus far, unsolved. In this study, the YPS was selenylated with Na2SeO3 under acidic conditions generated by HNO3 to reach two lower selenylation extents, yielding two selenylated YPSs, namely SeYPS-1 and SeYPS-2 with selenium contents of 715 and 1545 mg/kg, respectively. The results indicated that YPS, SeYPS-1, and SeYPS-2 all had in vitro immuno-modulation when using RAW 264.7 macrophages and murine splenocytes as cell models. In detail, the three polysaccharide samples at dose levels of 5–160 μg/mL showed insignificant cytotoxicity to the macrophages and splenocytes with cell exposure times of 12–24 h, because of the measured values of cell viability larger than 100%. However, Na2SeO3 at dose levels of 1.3–3.25 μg/mL mostly caused obvious cytotoxic effects on the cells, resulting in reduced cell viability values or cell death, efficiently. The results demonstrated that, compared with YPS, both SeYPS-1 and SeYPS-2 at a lower dose level (5 μg/mL) were more active at promoting phagocytosis activity, increasing the CD4+/CD8+ ratio of the T-lymphocyte sub-population in the murine splenocyte, improving cytokine secretion, including interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α in the macrophages, or increasing interferon-γ secretion, but suppressing IL-4 production in the splenocytes. Consistently, SeYPS-2 has more potential than SeYPS-1 at exerting these assessed bioactivities in the cells. Thus, we conclude that a chemical modification of YPS using trace element Se at a lower selenylation extent could bring about higher immunomodulatory activity towards macrophages and splenocytes, while selenylation extent of YPS is a critical factor used to govern the assessed activity changes of YPS.

Randomized, Phase II Dose Optimization Study Of Chimeric Antigen Receptor Modified T Cells Directed Against CD19 (CTL019) In Patients With Relapsed, Refractory CLL

Background Patients (pts) with relapsed, and/or refractory (R/R) CLL have a poor prognosis with few effective treatment options. We have shown that infusion of autologous T cells genetically modified to express a chimeric antigen receptor (CAR) consisting of an external anti-CD19 domain, with the CD3ζ and 4-1BB signaling domains (CTL019 cells), can mediate potent anti-tumor effects in pts with advanced, relapsed refractory CLL. In our initial pilot study, doses of 1.7-50, x 108 mononuclear cells, corresponding to 0.14-5.9 x 108genetically modified cells, were given as a split dose infusion on days 0, 1 and 2 to 14 pts with R/R CLL and overall response rate (PR plus CR) was 57%. The majority of responses were sustained, and associated with marked expansion and long-term persistence of transduced cells. Notably, there was no obvious dose:reponse or dose:toxicity effect noted over a wide range of cell doses. To better define an optimal CTL019 cell dose, we are performing a randomized phase II study of 2 doses of CTL019 cells in pts with R/R CLL. Methods Pts with R/R CLL are randomly assigned to receive either 5x108 vs. 5x107transduced CTL019 cells, with the rationale that both doses induced CRs in pts on our initial pilot trial. In the initial stage, 12 evaluable pts will be treated in each arm and in stage 2, an additional 8 pts will be treated with the selected dose level. Pts have to have relapsed or persistent disease after at least 2 previous treatments and progress within 2 years of their last therapy. All pts receive lymphodepleting chemotherapy ending 3-5 days before T cell infusion. Cell infusions are given as a single dose. Results As of 7/15/2013, 27 pts have been enrolled; T cells did not adequately expand in 3, 1 patient was not eligible after screening, and 10 pts have been treated including 7 men and 3 women with a median age of 63 yrs (range 59-76). 5 pts had a mutation of p53. All pts had active disease at the time of CTL019 cell infusion. Lymphodepleting chemotherapy was Fludarabine/cyclophosphamide (8), pentostatin/cyclophosphamide (1), or bendamustine (1). 4 pts have been randomized to the higher dose level (5 x 108 CTL019 cells) and 6 pts have been randomized to the lower dose level (5 x 107CTL019 cells). There were no significant infusional toxicities. Median follow-up as of July 15, 2013 was 3 mo (1.3-5) for all pts and 3.3 mo (1.3-4) for responding pts. 2 pts have achieved a CR and 2 pts achieved PR, both with clearance of CLL from the blood and marrow and >50 reduction in adenopathy, for an overall response rate of 40%. In other recipients of CTL019 cells, we have observed ongoing improvement in adenopathy over time implying there can be a continued anti-tumor response. No responding patient has progressed. Seven of 10 pts experienced a delayed cytokine release syndrome (CRS) manifested by symptoms that included high fevers, nausea, myalgias and in some cases, capillary leak, hypoxia, and hypotension, typically correlated with peak CTL019 cell expansion. We have noted that the CRS accompanying CTL019 therapy has been associated with marked increases of serum IL6 and can be rapidly reversed with the IL6-receptor antagonist tocilizumab. The CRS required intervention in 2 pts, one who responded and one who did not respond to CTL019. Treatment was initiated for hemodynamic or respiratory instability and was effective in reversing signs and symptoms of CRS in both pts. A preliminary analysis through July 15, 2013 does not yet suggest a dose:response or dose:toxicity relationship. 2 of 4 recipients of the higher dose CTL019 responded, and 2 of 6 recipients at the lower dose level responded. The 7 pts who experienced a CRS included all 4 responding pts and 3 pts who did not respond. The CRS occurred in 3/4 recipients of higher dose CTL019 cells and 4/6 of recipients of lower dose CTL019 cells. CTL019 expansion in-vivo and persistence over the follow up period was noted in all responding pts. Conclusions In this ongoing dose optimization study of CTL019 cells, 4 of the first 10 pts treated have responded within 3 months. With short follow-up, as yet there is no suggestion that there is a dose:response or dose:toxicity relationship at the dose ranges being studied. These cells can undergo robust in-vivo expansion and from other studies (ASH 2013) can persist for at least 3 yrs. This trial confirms that CTL019 cells can induce potent responses for pts with advanced, relapsed and refractory CLL. Disclosures: Porter: Novatis: IP and potential royalties with COI managed according to policies of the University of Pennsylvania, IP and potential royalties with COI managed according to policies of the University of Pennsylvania Patents & Royalties, Research Funding; Genentech: Spouse employment, Spouse employment Other. Off Label Use: CTL019 cells to treat CLL. Kalos:Novartis corporation: CART19 technology, CART19 technology Patents & Royalties; Adaptive biotechnologies: Member scientific advisory board , Member scientific advisory board Other. Grupp:Novartis: Research Funding. Chew:Novartis: Patents & Royalties. Shen:Novartis Pharmaceuticals: Employment, Equity Ownership. Wood:Novartis Pharmaceuticals: Employment, Equity Ownership. Litchman:Novartis Pharmaceuticals Corporation: Employment, Equity Ownership. Zheng:Novartis: Patents & Royalties. Levine:Novartis: cell and gene therapy IP, cell and gene therapy IP Patents & Royalties. June:Novartis: Patents & Royalties, Research Funding.

Phase I trial of pemetrexed (P) in combination with cetuximab (C) and concurrent radiotherapy (RT) in patients (pts) with head and neck cancer (HNC)

16516 Background: P is a multitargeted antifolate that has demonstrated activity in HNC and radiosensitizing properties in animal models. C is a chimeric IgG1 monoclonal antibody directed against the epidermal growth factor receptor that has increased the survival of pts with stage III/IV HNC when added to RT. We conducted a phase I study to evaluate the dose-limiting toxicities (DLT) and maximum tolerated dose of P when combined with standard RT and C. Methods: Pts with high-risk HNC were enrolled and evaluated separately in 2 cohorts: pts with no prior head and neck RT (cohort A) and previously irradiated pts (cohort B). A loading dose of C 400 mg/m2 was given intravenously 7 days prior to starting RT followed by 250 mg/m2 weekly throughout RT. RT was given 2 Gy/day to a total dose of 70 Gy (cohort A) or 60–66 Gy (cohort B). P was administered intravenously on days 1 and 22 (and day 43 if >60 Gy was delivered) according to the following design: Starting dose level (0)-350 mg/m2, dose level (-1)-200 mg/m2, dose level (+1)-500 mg/m2. Additional pts could be treated at a lower dose level while other pts were being evaluated at a higher dose level. Results: 18 pts have been enrolled (A: 14; B:4). 2 pts in cohort A were not evaluable and replaced (1 pt was noncompliant due to transportation issues and 1 pt had sudden death attributable to aspiration). Histological types: squamous cell (13), undifferentiated (1), medullary thyroid (1), adenoid cystic (1), and adenosquamous carcinomas (2). In cohort A, 2 DLT (both neutropenic fever with ANC <500) occurred: 1/6 at dose level 0 and 1/6 at dose level -1. In cohort B, no DLT has occurred: 0/3 pts at dose level 0 and 0/1 pts at dose level +1. There were no grade (G) 4 non- hematologic toxicities. Other serious toxicities included: neutropenic fever (NF) with ANC <1000 (n=1), G 3/4 neutropenia (n=3/n=4), G 3 dysphagia (n=5), G 3 dermatitis (n=3), G 3 mucositis (n=4), G 3 fatigue (n=1), and G 3 anemia (n=1). Due to the development of 3 cases of NF in cohort A the protocol was amended and prophylactic antibiotics were added. Conclusions: The addition of P to RT and C was feasible in pts with or without a history prior RT. Neutropenia and in-field toxicities were predominant but manageable. Pts are currently being treated at dose level +1 (P 500 mg/m2). No significant financial relationships to disclose.

Validating the alkane pair technique to estimate dry matter intake in equids

The estimation of dry matter intake (DMI) using the alkane pair technique has been validated in ruminants, but not in equids. The current paper reports the finding of three comparative validation studies carried out using a total of 12 cattle, 29 donkeys and 10 horses during which directly measured intake was compared to estimated intake using the alkane pair technique. Two methods were developed to dose the even chain alkanes that were used as external markers. Study I, carried out in Zimbabwe, compared the accuracy of estimated intake with measured intake in cattle and donkeys using hexatriacontane (C36) as the external marker. Studies II and III were carried out in the UK with horses and donkeys and compared the accuracy of estimated intake with measured intake using dotriacontane (C32) as the external marker. Study III also tested the effect on the accuracy of intake estimates of two marker dosing levels (mean daily dose of 224 mg per animal and 448 mg per animal) and two dosing frequencies (2× and 3× daily). Twice daily dosing of even-chain alkane at the lower dose level provided an estimate of DMI similar to that obtained by thrice daily dosing at this low level. The higher dose level given twice daily tended to produce large variation in faecal concentrations of dosed even-chain alkanes, this variation was reduced when dosing frequency was increased to thrice daily. The accuracy of estimated intake improved progressively as the number of faecal sampling days was increased from one to six with no significant difference between estimated intake based on day 5 or 6 of faecal sampling.The results of all three studies indicate that the alkane pair technique provides a robust method of estimating intake in equids with no significant difference between measured and estimated values in all but one case. Using C31 as an internal marker provided a more accurate estimated intake than using C33 as the internal marker in all cases. Faecal recoveries of alkanes in equids do not appear to show the same influence of carbon chain length that has been observed in ruminant studies.

Clofarabine Plus Anthracycline Combinations in Acute Myeloid Leukemia (AML) Salvage.

Clofarabine (CLO) is a second-generation nucleoside analog with activity in acute leukemias. Whereas it has received FDA approval in children with relapsed acute lymphoblastic leukemia, the focus of clinical research in adults has shifted to AML. To improve single agent activity, various CLO combinations are being studied. Here we present the results of two dose-finding phase I studies, exploring combinations of CLO with idarubicin (IDA) [CI] and CLO+IDA+Ara−C [CIA] in patients (pts) with relapsed or refractory AML and high-risk MDS. Eligibility for CI required previous Ara-C and primary refractory disease, or relapsed AML with first remission duration (CRD1) < 12 mos. Eligibility for CIA required either no previous Ara-C or Ara-c with CRD1 ≥ 12 mos. Maximum tolerated dose (MTD) was determined by “3+3” method. Forty-four pts (CI 23; CIA 21) have been accrued and are evaluable. The median age of pts on CI was 56 yrs (range 24–71), 9 were primary refractory and 14 in first relapse (median CRD1 2 mos. [0–9]). Fifteen pts had abnormal cytogenetics (including 6 with -5, -7, or 11q23). Sixteen pts had intermediate- or high-dose Ara-C-based prior therapy, 2 relapsed from allogeneic transplant (SCT). The median age of pts on CIA was 56 yrs (23–78), 8 were primary refractory and 13 in first relapse (median CRD1 9 mos [0–61]). Twelve pts had an abnormal karyotype (-5, -7, or 11q23 in 6 pts). Twelve pts received intermediate dose Ara-C-based prior therapy and 2 failed unrelated donor SCT. The starting dose level of the CI group was CLO 22.5 mg/m2 iv daily x 5d and IDA 12 mg/m2 iv daily x 3d. Three DLTs occurred (diarrhea, rash, ↑ bilirubin) requiring dose de-escalation to CLO 15 mg/m2/d x 5d and IDA 8 mg/m2/d x 3d. Gradual re-escalation led to CLO 30 mg/m2/d x 5d and IDA 10 mg/m2/d x 3d where 2/5 pts experienced DLTs (↑ SGPT, ↑ bilirubin, headache). MTD was defined by the next lower dose level: CLO 22.5 mg/m2/d x 5d and IDA 10 mg/m2/d x 3d. Three (13%) CR occurred. The CIA group started at CLO 22.5 mg/m2/d x 5d, IDA 8 mg/m2/d x 3d, and Ara-C 1g/m2/d x 5d. Two pts developed DLTs (diarrhea, ↑ bilirubin, renal failure) requiring de-escalation to CLO 15 mg/m2/d x 5d, IDA 6 mg/m2/d x 3d, and Ara-C 0.75 g/m2/d x 5d. Re-escalation to CLO 30mg/m2/d x 5d, IDA 6mg/m2/d x 3d, and Ara-C 0.75 g/m2/d x 5d revealed DLTs in 2/6 pts (diarrhea, mucositis, ↑ bilirubin). MTD was thus defined at next lower dose level of CLO 22.5 mg/m2/d x 5d, IDA 6 mg/m2/d x 3d, and Ara-C 0.75 g/m2/d x 5d. Ten (48%) responses (9 CR, 1 CRp) occurred. The higher response rate of CIA may be due to the difference in pt characteristics between the two trials. A phase II study is now in progress adaptively randomizing patients with AML relapse to CI versus CIA versus CLO (40 mg/m2/d x 5d) plus Ara-C (1 g/m2/d x 5d) (as established in an earlier study) to assess activity of the combinations.

Phase I dose escalation study to determine the safety, pharmacokinetics and pharmacodynamics of BMS-582664, a VEGFR/FGFR inhibitor in patients with advanced/metastatic solid tumors

3051 Background: This is the first multiple-dose study with BMS-582664, an oral VEGFR/FGFR tyrosine kinase inhibitor (IC50 34, 10, 145, 125 nM for VEGFR2, VEGFR3, FGFR1 and FGFR2 respectively). Patients (pts) with metastatic solid tumors refractory to standard therapy were enrolled. Part A of this study has completed. Part B is ongoing to evaluate the effect of BMS-582664 on dynamic contrast enhanced MRI (DCE-MRI) parameters. Methods: Part A: Open label dose escalation, continuous qd schedule, 3 patients per cohort. The starting dose was 180 mg. Dose escalation continued until dose limiting toxicities (DLTs) at 1000 mg in 2/3 pts were observed. The next lower dose level (800 mg) was expanded to 6 pts. Blood sampling for pharmacodynamic [circulating endothelial cells (CECs) and Collagen IV ELISA] and PK analysis was performed on Days 1, 8 and 26. BMS-582664 (prodrug) and its active metabolite, BMS-540215 were analyzed using an LC/MS/MS assay. DCE-MRI was performed at 320 mg, 600 mg and 800 mg at baseline ×2, Days 2, 8 and 26. Results: Negligible BMS-582664 was detected. BMS-540215 AUC increased proportionally to dose increase across 180 mg to 1000 mg. The accumulation ratio (Day 26:Day 1) of C24hr, Cmax and AUC was 4.8, 2.0 and 2.1 respectively at 1000 mg, but close to 1 at lower doses. The median time on study for all patients in Part A was 84 days. Conclusions: DLT was dizziness/fatigue. BMS-582664 has pharmacodynamic and anti-tumor activity and was well tolerated at ≤ 800 mg qd. [Table: see text] [Table: see text]

A phase 1, dose-escalation trial of STA-5312, a microtubule inhibitor with a novel binding site, in advanced or metastatic solid malignancies

13040 Background: STA-5312 is a novel microtubule inhibitor with a distinct binding site from other agents such as vincristine, colchicine, or paclitaxel, which has demonstrated antitumor activity against a range of solid tumors in chemotherapy-resistant cancer. Methods: The trial assessed the safety, toxicity, pharmacokinetics (PK) and maximum tolerated dose (MTD) level of STA-5312 when administered as an IV infusion weekly for the first 3 weeks of a 4 week cycle. Assessment of activity is a secondary objective. The starting dose level was 6mg/m2. Dose escalation was based on evaluation of toxicity through the first cycle, with dose doubling in cohorts of 1–2 patients (pts) until a grade (gr) 2 or greater toxicity occurred, followed by at least 3 pts/cohort and 50% dose escalations. After a dose limiting toxicity (DLT), cohorts were expanded to at least 6 pts and subsequent escalations limited to 33%. The dose below the lowest dose at which 1/3 of 6 or more patients show DLT will be declared the MTD. PK sampling was performed during cycle 1. Results: To date, 25 pts have been treated: median age 58 years (35–77), ECOG performance status of 0–2, and median treatment duration 43 days. No serious drug-related toxicity was observed until a DLT (ataxia) was reported in the only patient treated at 64mg/m2, leading to de-escalation to 48 mg/m2, where the only patient experienced aphasia and ataxia. Enrollment is being expanded at a lower dose level of 32 mg/m2. Other adverse events attributable to STA-5312 include (>10%) anemia, fatigue, constipation, nausea, and anorexia. Exposure (AUC) was linear with respect to dose. Five patients have had disease stabilization. Conclusions: STA-5312 appears to be well tolerated when given weekly in doses up to 32mg/m2. The MTD has not been determined and enrollment is ongoing. [Table: see text]

The effect of implantation of lowland ewes with melatonin on the time of mating and reproductive performance

Two trials, involving a total of 1006 Mule ewes and 1862 Suffolk-× ewes, were conducted to investigate the ability of implantation with melatonin to induce early breeding in lowland sheep flocks. Dates of implantation ranged from mid May to late June for Suffolk-× flocks and mid May to early July for Mule flocks.From the mating patterns obtained it is suggested that the optimum implantation date is mid May to mid June for Suffolk-× ewes and mid June to mid July for Mule-type ewes.Melatonin treatment also significantly increased the mean litter size per ewe exposed to the ram (resulting from a 6-week mating period) when the data were pooled across flocks, but the magnitude of this effect varied across flocks. Part of this overall mean increase in Mule ewes (+0·84 and +0·50 extra lambs per ewe in trials 1 and 2 respectively) was due to an increase in the proportion of ewes mating and lambing and part of it (+0·13 and +0·17 extra lambs per ewe, respectively) was due to an increase in litter size per ewe pregnant. In the Suffolk-× ewes there was evidence that the overall mean benefit following implantation with two implants (+0·31 extra lambs per ewe) was greater than that following implantation with one implant (+0·14 extra lambs per ewe), with all of the increase at the lower dose level and +0·21 extra lambs per ewe at the higher dose being due to an increase in litter size per ewe pregnant.

An evaluation of continuation therapy with tricyclic antidepressants in depressive illness

SynopsisA double-blind clinical trial has been carried out to ascertain whether patients making a good recovery from depressive illness with tricyclic antidepressant medication derive any benefit from continuation of therapy with the same drug at a lower dose level. Of the 92 patients who entered the trial significantly fewer on active treatment relapsed during the six-month trial period: 22% as compared with 50% of patients receiving placebo. Patients with residual symptoms on entry to the trial derived more benefit from continuation therapy than patients who had made a complete recovery. The findings relate to a six-month trial period only, and any possible advantage of continuation therapy over a longer period remains uncertain.