Feasible Dose Research Articles

Determination of the minimum protective dose of a glycoprotein-G-deficient infectious laryngotracheitis virus vaccine delivered via eye-drop to week-old chickens.

Infectious laryngotracheitis (ILT) is an upper respiratory tract disease of chickens that is caused by infectious laryngotracheitis virus (ILTV), an alphaherpesvirus. This disease causes significant economic loses in poultry industries worldwide. Despite widespread use of commercial live attenuated vaccines, many poultry industries continue to experience outbreaks of disease caused by ILTV. Efforts to improve the control of this disease have resulted in the generation of new vaccine candidates, including ILTV mutants deficient in virulence factors. A glycoprotein G deletion mutant vaccine strain of ILTV (ΔgG ILTV), recently licenced as Vaxsafe ILT (Bioproperties Pty Ltd), has been extensively characterised in vitro and in vivo, but the minimum effective dose required to protect inoculated animals has not been determined. This study performed a vaccination and challenge experiment to determine the minimum dose of ΔgG ILTV that, when delivered by eye-drop to seven-day-old specific pathogen-free chickens, would protect the birds from a robust challenge with a virulent field strain of virus (class 9 ILTV). A dose of 103.8 plaque forming units was the lowest dose capable of providing a high level of protection against challenge, as measured by clinical signs of disease, tracheal pathology and virus replication after challenge. This study has shown that the ΔgG ILTV vaccine strain is capable of inducing a high level of protection against a virulent field virus at a commercially feasible dose. These results lay the foundations upon which a commercial vaccine can be developed, thereby offering the potential to provide producers with another important tool to help control ILTV.

Breaking up sitting time after stroke - How much less sitting is needed to improve blood pressure after stroke (BUST-BP-Dose): Protocol for a dose-finding study.

Excessive sitting is detrimentally associated with cardiovascular disease and all-cause mortality. Frequent breaks in prolonged sitting can improve cardiometabolic responses in non-stroke populations. However, this has not been established in stroke survivors. This study will determine the most effective dose of activity breaks that (i) produce clinically meaningful improvements in mean systolic blood pressure (primary outcome), postprandial glucose, and insulin responses (secondary outcomes), and (ii) is safe and feasible. We hypothesis that systolic blood pressure, postprandial insulin, and glucose responses will improve with increasing doses of activity and be most effective at the maximum safe and feasible dose of activity. Thirty participants in the most effective dose will provide 80% power to detect a within-person, between-condition, difference of 3.5 mmHg in systolic blood pressure assuming a SD of 15 mmHg, within-person correlation of 0.9, and α = 0.05. Stroke survivors will complete 3 experimental conditions in a within-participant, dose escalation design including (i) uninterrupted sitting (8 h), (ii) Dose 1: uninterrupted sitting with bouts of light-intensity exercises while standing (initial dose involves two 5-min breaks), and (iii) Dose 2: two additional 5-min breaks above Dose 1. Ambulatory blood pressure will be collected every 30 min during experimental conditions and hourly for 24-h post-experimental conditions. Blood samples will be collected every 30 min during 2-h postprandial periods. This study will identify the most effective dose of light-intensity exercises while standing to improve cardiometabolic responses in stroke survivors.

P066 A Phase I Study of Apatinib Combined with Pemetrexed and Carboplatin in Untreated EGFR-Negative Stage IV Non-Squamous NSCLC

This phase I study aimed to establish the feasible dose of Apatinib in combination with pemetrexed plus carboplatin as first-line therapy for EGFR-negative stage IV non-squamous non-small-cell lung cancer (NSCLC). Using a 3+3 dose-reduction design, patients received oral Apatinib at four dose levels: 750 mg qd, 500 mg qd, 500 mg/day 2 weeks on/1 week off schedule (schedule 2/1) or 250mg qd. Pemetrexed (500 mg/m2) plus carboplatin (AUG=5) was administered every 3 weeks. The feasible dose was determined based on cycle 1 dose-limiting toxicities (DLT); other assessments included safety and antitumor activity according to response evaluation criteria in solid tumors. A total of 12 patients were enrolled and cycle 1 DLTs were observed in two patients at 750 mg qd treatment (both Grade 3 hypertension), two patients at 500 mg qd (Grade 3 hypertension and Grade 3 hand-foot syndrome), and only one of six patients at 500 mg/day schedule 2/1 (Grade 3 hypertension). The most frequently drug-related adverse events were hematological toxicity, hypertension, hand-foot syndrome and hepatic transaminases elevation. Partial response was observed in 4 patients of 11 evaluable patients (ORR 36.4%), and 6 patients exhibited stable disease (DCR 90.9%). In patients with advanced non-squamous NSCLC, the feasible dose of Apatinib given with standard-dose pemetrexed and carboplatin was 500 mg/day schedule 2/1. The schedule was generally well tolerated and demonstrated promising clinical benefit in NSCLC.

Low-dose CT imaging of the acute abdomen using model-based iterative reconstruction: a prospective study.

Performance of a modified abdominopelvic CT protocol reconstructed using full iterative reconstruction (IR) was assessed for imaging patients presenting with acute abdominal symptoms. Fifty-seven patients (17 male, 40 female; mean age of 56.5 ± 8years) were prospectively studied. Low-dose (LD) and conventional-dose (CD) CTs were contemporaneously acquired between November 2015 and March 2016. The LD and CD protocols imparted radiation exposures approximating 10-20% and 80-90% those of routine abdominopelvic CT, respectively. The LD images were reconstructed with model-based iterative reconstruction (MBIR), and CD images with hybrid IR (40% adaptive statistical iterative reconstruction (ASIR)). Image quality was assessed quantitatively and qualitatively. Independent clinical interpretations were performed with a 6-week delay between reviews. A 74.7% mean radiation dose reduction was achieved: LD effective dose (ED) 2.38 ± 1.78mSv (size-specific dose estimate (SSDE) 3.77 ± 1.97mGy); CD ED 7.04 ± 4.89mSv (SSDE 10.74 ± 5.5mGy). LD-MBIR images had significantly lower objective and subjective image noise compared with CD-ASIR (p < 0.0001). Noise reduction for LD-MBIR studies was greater for patients with BMI < 25kg/m2 than those with BMI ≥ 25kg/m2 (5.36 ± 3.2Hounsfield units (HU) vs. 4.05 ± 3.1HU, p < 0.0001). CD-ASIR studies had significantly better contrast resolution, and diagnostic acceptability (p < 0.0001 for all). LD-MBIR studies had significantly lower streak artifact (p < 0.0001). There was no difference in sensitivity for primary findings between the low-dose and conventional protocols with the exception of one case of enteritis. Low-dose abdominopelvic CT performed with MBIR is a feasible radiation dose reduction strategy for imaging patients presenting with acute abdominal pain.

Preclinical safety evaluation of the human-identical milk oligosaccharide lacto-N-tetraose

Lacto-N-tetraose (LNT) is one of the most abundant oligosaccharides that are endogenously present in human breast milk. To simulate the composition of human breast milk more closely, commercial infant formula can be supplemented with human-identical milk oligosaccharides, which are manufactured structurally identical versions of their naturally occurring counterparts. As part of the safety evaluation of LNT, in vitro genotoxicity tests and a subchronic oral gavage toxicity study (in neonatal Sprague-Dawley rats) were conducted. In the subchronic study, LNT was administered at dose levels of 0, 1,000, 2500 or 4000 mg/kg body weight (bw)/day, once daily for at least 90 days, followed by a 4-week treatment-free period. An identically comprised reference control group received fructooligosaccharides powder (a non-digestible oligosaccharide used in infant formula) at 4000 mg/kg bw/day, to allow for direct comparison against the high-dose LNT group. LNT was non-genotoxic in the in vitro tests. There were no compound-related adverse effects in the 90-day study; therefore, 4000 mg/kg bw/day (the highest feasible dose) was established as the no-observed-adverse-effect-level. These results support the safe use of LNT in infant formula and as a food ingredient, at levels not exceeding those found naturally in human breast milk.

Effect of pterostilbene complexed with cyclodextrin on rat liver: potential reduction of oxidative damage and modulation redox-sensitive proteins

The objectives of this study were to promote the aqueous solubility of pterostilbene (PTS) by complexation with hydroxypropyl-β-cyclodextrin (HPβCD), characterize the complex under physical aspects, to make its oral administration feasible in biological tests, and to investigate their pharmacological properties. For 14 days, rats received daily PTS:HPΒCD complex at doses of 25, 50, or 100 mg kg−1 per day orally. The results showed no kidney or liver damage, nor any induction of apoptosis by the administered doses. Also, the complex showed dose-dependent antioxidant effects in the rat liver, as evidenced by a reduction in lipid peroxidation and reactive oxygen species, as well as an increase in non-enzymatic antioxidant. PTS:HPΒCD complex also increased the expression of sensitive redox proteins such as AKT and GSK-3β related to the insulin signaling pathway in the liver. Thus, the complexation demonstrated to be able to increase the apparent solubility of PTS making feasible dose curve administration and could be a food alternative complementary to antioxidant therapeutic. Therefore, the PTS:HPβCD complex can be used for prevention of diseases related to oxidative damage and insulin signaling.

Increasing Levels of Sulfur on Lowland Rice in Different Soil Textures

A viable alternative to increase productivity in rice cultivation areas has been the investment in macronutrient fertilization. Sulfur (S) is an essential nutrient for plants, as it participates in the formation of several organic molecules, being essential to ensure high yields of rice. The aim of this paper is to analyze the rice crop performance in two types of irrigated floodplain soils under different doses of sulfur fertilization. The experiment was carried out on two lowland soils in Formoso do Araguaia-Tocantins, Brazil, under a randomized block design, in a 2 × 8 factorial arrangement, two soil types and eight sulfur doses, using ammonium sulfate as the source of S (0, 10, 20, 30, 40, 50, 60 and 70 kg ha-1 of S). The doses were applied when the plants were in the V3-V4 development stage in both soils. S provided plant a growth up to 40 kg ha-1, which is the economical feasible dose for plant height in soil 1. With the increase of the dose of S over 60 kg ha-1 in soil 2, there was a decrease in the number of panicles per m2. There was an increase in productivity in soil 2 and reduction in soil 1. The highest grain yield was obtained in soil with higher clay content. The dynamics of sulfur fertilization in floodplain soils requires further studies for a better understanding.

Microemulsion containing triamcinolone acetonide for buccal administration

The aim of the present work was to investigate the potential of microemulsions for the buccal administration of triamcinolone acetonide. Microemulsions were developed by the construction of pseudoternary phase diagrams, using the aqueous titration method. Among all microemulsions prepared and tested for stability, three were selected and submitted to characterization and in vitro permeation/retention experiments, using pig esophageal epithelium, an accepted model of the buccal mucosa. Furthermore, one microemulsion was added of excipients (stearylamine, CTAB and chitosan) able to alter the charge of droplets.The results obtained show that the permeation of triamcinolone acetonide across pig esophageal epithelium was not influenced by the droplet size nor by the composition, but only by the presence of chitosan, polysaccharide able to increase the transport across mono and stratified epithelia. The determination of the permeation parameters allowed us to show that chitosan acts on the diffusion parameter across the tissue and not on the partitioning parameter; for the same reason the tissue retention of triamcinolone acetonide was not modified. Triamcinolone flux (2.6 μg cm−2 h−1) was too low to make systemic administration feasible (dose required 2.5 to 60 mg/day).The amount of triamcinolone acetonide recovered in the mucosa after only 10 min. of microemulsion application was much higher than after overnight application of the commercial paste Omicilon® A. This suggests that triamcinolone acetonide microemulsions can be an interesting alternative to the commercial formulation to treat diseases of the buccal mucosa. Owing to the fast uptake by the tissue, the formulation can be used as a mouthwash.

Alfaxalone anaesthesia in Lemur catta following dexmedetomidine–butorphanol–midazolam sedation

ObjectiveTo evaluate the clinical effects and quality of sedation, induction, maintenance and recovery in Lemur catta after dexmedetomidine–butorphanol–midazolam sedation and alfaxalone anaesthesia. Study designProspective, observational study. AnimalsSix male L. catta weighing 3.0 ± 0.6 kg undergoing surgical castration. MethodsLemurs were sedated with intramuscular dexmedetomidine (0.015 mg kg−1), butorphanol (0.2 mg kg−1) and midazolam (0.2 mg kg−1). Anaesthesia was induced with intravenous alfaxalone 0.5 mg kg−1 over 60 seconds; further boluses were administered until tracheal intubation was feasible and final dose recorded. Alfaxalone continuous infusion was used to maintain anaesthesia. Atipamezole (0.15 mg kg−1) was administered during recovery. The quality of sedation, induction, intubation, maintenance and recovery was assessed using a scoring system. Physiological parameters were recorded during sedation, maintenance and recovery. ResultsSedation was achieved in 13.6 ± 5.6 minutes and no reactions were observed during handling or venepuncture. The mean dose of alfaxalone required for induction and maintenance was 2.09 ± 0.65 and 0.08 ± 0.02 mg kg−1 minute−1, respectively. Quality of induction, intubation and maintenance was good in almost all animals. Mild self-limiting muscle twitching was observed after alfaxalone administration in three animals. Cardiorespiratory function was stable in all animals but one. One lemur showed respiratory depression and required oxygen administration and manual ventilation. The mean maintenance time was 29.2 ± 7.4 minutes. The mean times from the end of alfaxalone administration to extubation, atipamezole administration and full recovery were: 15.3 ± 8.0, 22.2 ± 4.6 and 60.0 ± 8.4 minutes, respectively. Recovery was considered good in all animals. Conclusions and clinical relevanceDexmedetomidine–butorphanol–midazolam combination provided reliable sedation and adequate muscle relaxation in L. catta. Alfaxalone proved to be a useful drug for induction and maintenance of anaesthesia and might be considered an option for injectable anaesthesia in lemurs.

Editor's Highlight: Formulation and Toxicology Evaluation of the Intrathecal AYX1 DNA-Decoy in Sprague Dawley Rats.

The longevity of pain after surgery is debilitating and limits the recovery of patients. AYX1 is a double-stranded, unprotected, 23 base-pair oligonucleotide designed to reduce acute post-surgical pain and prevent its chronification with a single intrathecal perioperative dose. AYX1 mimics the DNA sequence normally bound by EGR1 on chromosomes, a transcription factor transiently induced in the dorsal root ganglia-spinal cord network following a noxious input. AYX1 binds to EGR1 and prevents it from launching waves of gene regulation that are necessary to maintain pain over time. A formulation suitable for an intrathecal injection of AYX1 was developed, including a specific ratio of AYX1 and calcium so the ionic homeostasis of the cerebrospinal fluid is maintained and no impact on neuromuscular control is produced upon injection. A GLP toxicology study in naïve Sprague Dawley rats was conducted using 3 dose levels up to the maximum feasible dose. Clinical observations, neurobehavioral observations, clinical pathology and histopathology of the nervous system and peripheral tissues were conducted. An additional nonGLP study was conducted in the spared nerve injury model of chronic neuropathic pain in which EGR1 is induced in the dorsal root ganglia and spinal cord. Similar testing was performed, including a modified Irwin test to assess a potential impact of AYX1 on autonomic nervous system responses, locomotion, activity, arousal, sensorimotor, and neuromuscular function. No AYX1-related adverse events were observed in any of the studies and the no-observed-adverse-effect-level was judged to be the maximum feasible dose.

Regrowth ability and coagulation behavior by second dose: Breakage during the initial flocculation phase

Breakage and regrowth of flocs has been studied for several years, however little attention was paid to the regrowth ability of broken micro-flocs which are mainly formed during the initial flocculation stage. In this paper, a continuous optical monitoring method was employed to investigate the kinetics of flocs formation, and especially more evident flocs properties (strength and recovery factor) were also explored. The results showed that breakage at the beginning of flocculation stage can improve flocs regrowth to greater sizes when the coagulation was initially dominated by charge neutralization at a predetermined dosage. Furthermore, second dose of different coagulants during this breakage process improved kaolin particles removal efficiency to varying degrees. Separately, the second dose of Al2(SO4)3 continuously enlarged the floc size with the increasing dosage, contrarily that of PACl reduced the floc size. Based on the analysis of flocs properties, this phenomenon can be well explained by the difference between coagulation mechanisms under the second dose of Al2(SO4)3 and PACl respectively: newly formed hydroxides of Al2(SO4)3 enhanced broken flocs regrowth to larger sizes and entrapped more particles (lower residual turbidity) by sweep flocculation; that of PACl formed compact flocs due to stronger electric density of Al hydroxides. It was worth noting that turbidity removal performance was even better when the overall dosage by second dose only counted for ∼70% of one-time dose. Importantly, this study aimed at providing a feasible economic dose strategy for the real coagulation-flocculation application.

Phase I Study of Clofarabine and 2-Gy Total Body Irradiation as a Nonmyeloablative Preparative Regimen for Hematopoietic Stem Cell Transplantation in Pediatric Patients with Hematologic Malignancies: A Therapeutic Advances in Childhood Leukemia Consortium Study

Clofarabine is a purine nucleoside analog with immunosuppressive and antileukemic activity and its inclusion in reduced-intensity regimens could potentially improve outcomes. We performed a prospective phase I study of clofarabine combined with 2 Gy total body irradiation (TBI) as a nonmyeloablative preparative regimen for allogeneic stem cell transplantation in pediatric patients who were considered at high risk of mortality from standard myeloablative regimens. The main goal of the study was to delineate the maximum feasible dose (MFD) of clofarabine in combination with 2 Gy TBI. Eighteen patients, 1 to 21 years of age and in complete remission, were enrolled in 2 strata (matched related donor and unrelated donor) and evaluated for day100 dose-limiting events (DLE) (nonengraftment, nonrelapse mortality [NRM], and severe renal insufficiency) after receiving clofarabine at the starting dose level of 40 mg/m2. All 6 patients (3 in each stratum) engrafted with no day 100 DLE seen in the first cohort. The dose was increased to 52 mg/m2 in the next and an expanded cohort (total of 12 patients) and no DLE were observed at day 100 and at the 1-year study endpoint. The regimen was well tolerated with transient transaminitis and gastrointestinal and skin reactions as the common reversible toxicities observed with clofarabine. The dose of 52 mg/m2 of clofarabine was deemed the MFD. Disease relapse led to mortality in 6 (33%) patients during follow-up with 1-year event-free survival and overall survival of 60% (95% confidence interval [CI], 34 to 79) and 71% (95% CI, 44 to 87), respectively. This regimen leads to successful engraftment using both related and unrelated donors with exceptionally low rates of NRM.

Combination of TLR9 agonists EnanDIM with checkpoint inhibitors for cancer immunotherapy.

134 Background: TLR9 agonists have shown anti-tumor effects by modulating the innate and adaptive immune system. Ongoing clinical studies support the use of TLR9 agonists for immunotherapeutic approaches. The new family of TLR9 agonists, EnanDIM, consists of linear single-stranded ODN synthesized using L-deoxyribonucleotides (natural enantiomers of D-deoxyribonucleotides) at their 3’-ends to prevent degradation. Therefore, EnanDIM does not own the off-target effects of PTO-modified CpG-ODN. Methods: EnanDIM with varying nucleotid sequences were compared for IFN-alpha response from human peripheral blood mononuclear cells and those molecules inducing the stronges response were selected. A maximum feasible dose (MFD) approach was employed to evaluate their acute toxicity and immunomodulatory properties. In addition, a combinatory approach with aPD-1 was evaluated in an syngeneic colon carcinoma CT26 mouse tumor model. Results: EnanDIM581 and EnanDIM532 were selected due to their pronounced activation of the IFN-alpha pathway in vitro. Safety assessments throughout and a gross necropsy at the end of the study revealed no signs of toxicity despite extremely high doses (300 - 1700 mg/kg). Dose-dependent increase of IP-10 levels in serum was observed between 6 and 24 hours after injection. In the colon carcinoma CT26 model subcutaneous injection of EnanDIM532 and intraperitoneal injection of aPD-1 had a moderate effect on the tumor growth when used in monotherapy (28.3% and 57.0% tumor growth inhibition, TGI). Notably, a combination of EnanDIM532 and aPD-1 further reduced tumor growth (74.7% TGI) and thus prolonged survival of the mice. Conclusions: In conclusion, EnanDIM, a new family of TLR9 agonists and immune surveillance reactivators (ISR), broadly activates the immune system, shows no toxicity in an MFD study and enhances the anti-tumor effects of the aPD-1 checkpoint inhibitor in a pilot study of a murine colon carcinoma tumor model. These data show the promising potential of EnanDIM not only for monotherapeutic but also combinatory approaches.

Abstract P6-12-03: A phase I study of AZD9496, a novel oral, selective estrogen receptor degrader (SERD) in women with estrogen receptor positive, HER-2 negative advanced breast cancer (ABC)

Abstract Background AZD9496 is a potent orally bioavailable ER antagonist and degrader that has shown antitumor efficacy in a range of preclinical xenograft models including ESR1wild-type tamoxifen-resistant and long term estrogen deprived models and an ESR1 mutant model. Methods This is a phase I, open label global multicenter study in women with ER+ HER2–ve BC either metastatic or locoregionally recurrent, not amenable to treatment with curative intent. Patients are post-menopausal, or pre-menopausal women receiving LHRH agonist therapy, with disease progression after ≥6 months endocrine therapy for ER+ BC (no limit on number of prior endocrine therapies; ≤2 prior chemotherapies in advanced setting). The primary objective is to determine the safety and tolerability of AZD9496. Cohorts of 3-6 patients received daily oral therapy and dose limiting toxicities (DLTs) occurring in cycle 1 (28 days) were assessed. Patients are dosed until MTD (defined as ≤1/6 patients with a DLT) or maximum feasible dose (MFD) is reached. Key secondary objectives include determination of single and multiple dose pharmacokinetics (PK), and preliminary antitumor efficacy. ER target modulation by protein and gene expression is evaluated in circulating tumor cells and paired tumor biopsies. In addition to the dose escalation phase, expansion cohort(s) in patients with or without ESR1 mutations can be enrolled to examine the safety, tolerability, PK and biological activity of AZD9496 further. Results Preliminary data as of 30th April 2016: 45 patients (median age 62 (range 41-83); 38 post-menopausal, 7 pre/perimenopausal; visceral metastases 76%, prior fulvestrant 25/45) received AZD9496 in 7 dose escalation cohorts: 20mg QD n=4, 40mg BID n=6, 80mg BID n=5, 150mg BID n=6, 250mg BID n=6, 400mg BID n=6, 600mg BID n=6 and also a 250mg BID expansion cohort n=6. The majority of adverse events (AEs) were grade 1 or 2; the most common treatment-related AEs (≥10%) have been diarrhoea (33%), fatigue (27%), nausea (22%), upper abdominal pain (13%) and increased liver function tests (13%). Six patients had treatment-related grade 3 AEs, 5 of which were manageable with dose interruption +/- dose reduction. Specifically, three had DLTs: grade 3 increased AST/ALT/GGT-150mg BID, serious adverse reaction (SAR) leading to withdrawal; grade 3 diarrhoea and grade 3 increased AST/ALT/GGT-400mg BID, SAR, manageable with dose reductions; grade 3 diarrhoea 600mg BID, manageable with dose reduction. The MTD/MFD has not been reached. Following the first dose up to 400mg the AZD9496 exposure increased in reasonable proportion to increasing dose. At 600mg a more than dose-proportional increase in exposure was observed. Evidence of reduced ER and Ki67 has been observed in on-study biopsies at 150mg BID and above. 10 subjects received treatment for &amp;gt;3-&amp;lt;6 months (5 ongoing, 5 discontinued),4 subjects &amp;gt;6-&amp;lt;12 months (3 ongoing, 1 discontinued), 3 subjects ≥1 year (2 ongoing, 1 discontinued). Conclusions AZD9496 has a tolerable safety profile, evidence of PD biomarker modulation and prolonged stabilisation of disease in women with heavily pre-treated ER+ve ABC. Citation Format: Hamilton E, Patel M, Armstrong A, Baird R, Jhaveri K, Hoch M, Morgan S, Dowdall T, Schiavon G, Klinowska T, Weir H, Bujac S, Nash T, Im S-A. A phase I study of AZD9496, a novel oral, selective estrogen receptor degrader (SERD) in women with estrogen receptor positive, HER-2 negative advanced breast cancer (ABC) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-12-03.

Pharmacology, pharmacokinetics, and metabolism of the DNA-decoy AYX1 for the prevention of acute and chronic post-surgical pain.

BackgroundAYX1 is an unmodified DNA-decoy designed to reduce acute post-surgical pain and its chronification with a single intrathecal dose at the time of surgery. AYX1 inhibits the transcription factor early growth response protein 1, which is transiently induced at the time of injury and triggers gene regulation in the dorsal root ganglia and spinal cord that leads to long-term sensitization and pain. This work characterizes the AYX1 dose-response profile in rats and the link to AYX1 pharmacokinetics and metabolism in the cerebrospinal fluid, dorsal root ganglia, and spinal cord.ResultsThe effects of ascending dose-levels of AYX1 on mechanical hypersensitivity were measured in the spared nerve injury model of chronic pain and in a plantar incision model of acute post-surgical pain. AYX1 dose-response profile shows that efficacy rapidly increases from a minimum effective dose of ∼ 0.5 mg to a peak maximum effective dose of ∼ 1 mg. With further dose escalation, the efficacy paradoxically appears to decrease by ∼ 30% and then returns to full efficacy at the maximum feasible dose of ∼ 4 mg. The reduction of efficacy is associated to doses triggering a near-saturation of AYX1 metabolism by nucleases in the cerebrospinal fluid and a paradoxical reduction of AYX1 exposure during the period of early growth response protein 1 induction. This effect is overcome at higher doses that compensate for the effect of metabolism.DiscussionAYX1 is a competitive antagonist of early growth response protein 1, which is consistent with the overall increased efficacy observed as dose-levels initially escalate. Chemically, AYX1 is unprotected against degradation by nucleases. The sensitivity to nucleases is reflected in a paradoxical reduction of efficacy in the dose-response curve.ConclusionsThese findings point to the importance of the nuclease environment of the cerebrospinal fluid to the research and development of AYX1 and other intrathecal nucleotide-based therapeutics.

Safety and Feasibility of Administration of High Doses of Ex Vivo Expanded NK Cells for Prevention of Disease Relapse after Transplantation for Patients with Myeloid Malignancies - Final Results of a Phase I Clinical Trial

Background: Disease relapse remains the most common cause of treatment failure after transplantation. To enhance anti-tumor effect of the graft we explored the use of high doses of donor-derived ex vivo expanded NK cells administered after haploidentical stem cell transplantation (HaploSCT) with the ultimate goal to decrease relapsed rate post-transplant.Methods: We aimed to study safety and determine the maximum tolerated dose (MTD) of high doses of mbIL-21 ex vivo expanded NK cells in a phase I clinical trial (clinicaltrials.gov NCT01904136) administered post-transplant in patients with myeloid malignancies (AML, CML, MDS). We hypothesized that infusion of mature NK cells would compensate for the lower NK-cell numbers and poor function previously observed by our group in the first month post-transplant. Patients received conditioning with melphalan 140mg/m2, fludarabine 160mg/m2 and 2GyTBI, and GVHD prophylaxis with post-transplant cyclophosphamide, tacrolimus and mycophenolate (Gaballa S, et al. Cancer. 2016). All patients had a bone marrow graft. NK cells were generated from peripheral blood mononuclear cells of the same donor with infusions on Days -2, +7 and on/after +28. The first infusion was with fresh and the other two were with cryopreserved NK cells. Dose escalation was planned in cohorts of 2 patients starting at 1x105 to 1x109 NK cells/Kg. Having an NK-cell alloreactive donor or certain KIR genotype was not a requirement to participate in this study, although these characteristics were evaluated in all patients.Results: Thirteen patients were enrolled. Eight patients had AML (7 in CR1, 4 with high-risk cytogenetics, 3 had primary induction failure AML and 3 had +minimal residual disease (MRD) at transplant, and one FLT3+ AML in CR2 with persistent MRD by flow cytometry), and 5 had CML (4 in second chronic phase, 2 with prior CNS disease, one had associated MDS with monosomy 7, and 2 who failed multiple TKIs). The median age was 41.5 years (range 18-60). Five patients were males and 8 females. The NK-cell dose escalation was as follows: 1x105/kg (N=2), 1x106/kg (N=3), 1x107/kg (N=3), 3x107/kg (N=2) and 1x108/kg (N=2). One patient was treated with 1x104/kg (Dose -1). All patients received the 3 planned infusions except one. Median purity of the NK-cell product was 98.98%. All patients achieved primary engraftment (100%) with 100% donor chimerism except one patient (who received 1x104/kg) who had secondary graft failure with concurrent parainfluenza pneumonia and died of treatment-related mortality (TRM). The median time to neutrophil and platelet engraftment was 18 and 26 days, respectively. Of 12 patients evaluable for aGVHD, the maximum grade was II in 7 patients. No grade III-IV aGVHD or cGVHD was observed. Only 5/12 patients had CMV reactivation (41.6% compared with 71% in retrospective data with the same treatment without NK cells), while none developed BK virus hemorrhagic cystitis. All patients (N=12) achieved remission with negative MRD after transplant. One patient treated NK cells at 1x105/kg/dose relapsed, received salvage treatment and was alive at last follow-up. All other patients are alive and in complete remission (N=11) after a median follow-up of 12.8 months (range 6-25.1). Compared with patients treated on a previous clinical trial with the same conditioning without NK cells (AML in CR1/2 and CML in CP), the patients who received NK cells had marked improvement in NK-cell function and cytotoxicity, as well as a significantly increase in INF gamma and TNF alpha production. A lower relapse rate and improved survival was observed compared with the retrospective cohort of patients treated without NK cells, although not statistically significant (p=0.21) (Figure 1).Conclusions: Doses up to 3x108/kg total dose of mbIL-21 ex vivo expanded NK cells obtained from the same donor can be safely administered in the early post-transplant period after HaploSCT. This was the maximum feasible dose to be manufactured in the current system, while no toxicity or increase in GVHD was observed. Three infusions of 1x108 NK cells per kg will be administered in the phase II study [Display omitted] DisclosuresCiurea:Spectrum Pharmaceuticals: Other: Advisory Board; Cyto-Sen Therapeutics: Equity Ownership. Lee:Sanofi: Consultancy; Cyto-Sen Therapeutics: Equity Ownership, Other: Board of Directors; Intellia Therapeutics: Other: Advisory Board; Courier Therapeutics: Other: Advisory Board; Intrexon: Consultancy, Patents & Royalties; Shire: Other: Advisory Board; Ziopharm: Consultancy, Patents & Royalties. Bashir:Spectrum: Consultancy; Celgene: Research Funding; Takeda: Research Funding; Takeda: Consultancy. Champlin:Intrexon: Equity Ownership, Patents & Royalties; Ziopharm Oncology: Equity Ownership, Patents & Royalties.

A Phase I Study of Donor Regulatory T Cells As Treatment for Steroid Dependent/Refractory Chronic Graft Versus Host Disease

Background: Chronic graft versus host disease (CGVHD) remains a notable cause of post transplant morbidity and mortality, limiting quality of life and survival in the allogeneic transplant survivor. CD4+CD25+CD127dimFoxP3+ regulatory T cells (Treg) are a naturally occurring cell population isolatable in the peripheral blood (PB) with key immune regulatory function. Treg have been shown to protect against GVHD in a number of murine models and in patients. Further, clinical correlations have been observed between the presence of CGVHD and a decreased frequency of Treg post allogeneic hematopoietic cell transplant (HCT) as well as the use of low dose IL-2 resulting in clinical responses with an associated increase in Treg. Based on these observations we embarked on a phase I safety and tolerability trial of the treatment of steroid-refractory or dependent CGVHD with infusion of donor Treg in matched related donor recipients.Methods: Eligible patients were ³ 18 years of age with steroid refractory or dependent CGVHD with a matched related HCT donor. All patients were on systemic immunosuppression at the time of enrollment. Donor derived Treg were enriched from a non-mobilized PB apheresis product over 2 days of apheresis. The fresh apheresis product underwent enrichment by CD25+ immunomagnetic selection on the CliniMACS¨ Cell Selection System (Miltenyi Biotec) and Treg were purified by high-speed flow cytometry and gated on the CD4+CD127dim population. The functional activity of the Treg product was assessed via a mixed lymphocyte reaction (MLR). The trial dosing was through a 3+3 dose escalation design with three Treg dose cohorts: 1 x 10E5 Treg/kg, 5 x 10E5 Treg cells/kg and 1.5 x 10E6 Treg cells/kg.Results: Ten patients were enrolled and received donor Treg cell infusions. Isolation of highly purified Treg was successful from all donors. A median of 1.9x10E8 (range 0.9-2.78x10E8) Treg were collected with a purity that ranged from 87.1-99.3% FoxP3+ (Table 1). Function of the Treg enriched apheresis product confirmed Treg suppressive activity with a median suppression of 69.1% (48.7-77.2%) at a ratio of responder cells:CD4+/CD8+:Treg cells of 1:2:1, with sustained suppression to a ratio of 1:2:0.1. All 10 pateints were treated with three patients treated on all three dose cohorts and an additional patient was treated on the third cohort due to the planned cell dose being not reached with the third patient-donor pair. There were no adverse toxicities with the Treg infusion and no incidence of acute GVHD or acute exacerbation of CGVHD. The ratio of Treg/CD4+ cells in the patients’ PB ranged from 2-4.4% at the time of the infusion and remained stable <3 months post infusion. Six of 10 patients have had stable to improved CGVHD with one of these patients having had a flare of CGVHD in the setting of a lapse in immunosuppressive medication and response with reinstitution of immunosuppression. Four patients have had unresponsive or progressive disease requiring the addition of new immunosuppressive agents <2 months post Treg cell infusion. All enrolled patients remain alive at a follow up of >150 days post Treg cell infusion.Summary: This study is the first clinical experience utilizing highly purified donor derived Treg isolated through clinical scale high speed cell sorting. Donor Treg cell therapy for the treatment of steroid refractory or dependent CGVHD at doses of 1.5x10E5-1.5x10E6 Treg/kg were administered without toxicity including absence of AGVHD or exacerbation of CGVHD. The Treg dose of 1.5 x 10E6/kg of recipient weight may be the maximum feasible dose attainable without ex vivo Treg cell expansion. Treg mediated suppression in the MLR documented the in vitro suppressive capability of the Treg cells with investigations ongoing to characterize the functionality of the Treg cells infused. Although this trial was designed to describe the feasibility, safety and tolerability of Treg cell infusions in patients with active CGVHD, the preliminary outcomes of clinical responses and survival are encouraging and comparable to other therapies for steroid refractory/dependent CGVHD with only a single infusion of cells. Further strategies such as multiple infusions or the addition of low dose IL-2 could be considered to attempt to augment the clinical efficacy of Treg based cellular immunotherapy. [Display omitted] DisclosuresMeyer:Stanford University: Patents & Royalties. Negrin:Stanford University: Patents & Royalties.

Abstract B067: Preclinical data of novel enantiomeric oligonucleotides for cancer immunotherapy: The TLR9 agonist EnanDIM

Abstract Introduction: The use of TLR9 agonists as immunomodulators is supported by preclinical and clinical studies, showing their anti-tumor effect by enhancing both cellular and humoral responses. Two different families of DNA molecules containing non-methylated CG-motifs for TLR9 activation have been established so far: Dumbbell-shaped dSLIM molecules are protected against exonucleolytic degradation by their covalently-closed, natural phosphodiester (PO) backbone. In contrast, single-stranded, oligodeoxynucleotides (CpG-ODN) are most commonly chemically-stabilized by phosphorothioates (PTO) in their phosphate moieties. However, PTO modifications produce off-target effects in immune cell populations and have resulted in an unfavorable risk-to-benefit ratio. Methods: Linear single-stranded ODN were synthesized using L-deoxyribonucleotides at their 3'-ends, which are the natural enantiomers of D-deoxyribonucleotides, to ensure protection against exonucleases and avoid the off-target effects of PTO-modified CpG-ODN. The vast majority of deoxyribose in present organisms are D-deoxyribose, thus co-evolved nucleases are blind for L-deoxyribose thereby leaving L-protected ODN intact. Using high secretion of IFN-alpha and IP-10 from human peripheral blood mononuclear cells as marker, we selected nucleotide sequences of such L-protected, CG-motif containing, single-stranded ODN, EnanDIM. First, we employed a maximum feasible dose (MFD) approach. Mice received subcutaneous injection of single doses of 10 to 50 mg EnanDIM to evaluate its acute toxicity. Immunomodulatory properties were evaluated as well. Finally, a pilot tumor model in mice was used to investigate the anti-tumor effect of EnanDIM. Results: EnanDIM581 and EnanDIM532 were selected due to their pronounced activation of immune cells (e.g. monocytes, NK cells and plasmacytoid dendritic cells) and their prominent induction of IFN-alpha and IP-10 secretion in vitro. EnanDIM744, comprising EnanDIM581 with additional 5'-end L-nucleotide protection and exhibiting an activation pattern similar to EnanDIM581, was also used for MFD studies. Safety assessments were performed throughout the study period and no mortality, clinical signs and body weight changes were observed, despite the extremely high doses of app. 300 to 1700 mg/kg. A gross necropsy consisting of a macroscopic organ evaluation at day 15 revealed also no toxicity. Dose-dependent increase of IP-10 levels in serum was observed between 6 and 24 hours after injection (maximum at 6h) but after 15 days IP-10 levels were undetectable, confirming that L-nucleotides in EnanDIM do not change the kinetic profile known from other DNA-based TLR9 agonists. Preliminary data from a murine tumor model suggest that multiple doses of EnanDIM can reduce tumor growth. Conclusions: EnanDIM, a new family of TLR9 agonists, broadly activates the immune system in vitro and in vivo. While maximal feasible doses of EnanDIM resulted in no signs of toxicity, an indication of reduced tumor growth was observed in a murine tumor model in vivo. Therefore EnanDIM has the potential for clinical development in the treatment of cancer. Citation Format: Kerstin Kapp, Barbara Volz, Detlef Oswald, Burghardt Wittig, Manuel Schmidt. Preclinical data of novel enantiomeric oligonucleotides for cancer immunotherapy: The TLR9 agonist EnanDIM [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B067.

TU‐AB‐BRA‐11: Indications for Online Adaptive Radiotherapy Based On Dosimetric Consequences of Interfractional Pancreas‐To‐Duodenum Motion in MRI‐Guided Pancreatic Radiotherapy

Purpose:Dose limiting structures, such as the duodenum, render the treatment of pancreatic cancer challenging. In this multi‐institutional study, we assess dosimetric differences caused by interfraction pancreas‐to‐duodenum motion using MR‐IGRT to determine the potential impact of adaptive replanning.Methods:Ten patients from two institutions undergoing MRI‐guided radiotherapy with conventional fractionation (n=5) or SBRT (n=5) for pancreatic cancer were included. Initial plans were limited by duodenal dose constraints of 50 Gy (0.5 cc)/31 Gy (0.1 cc) for conventional/SBRT with prescriptions of 30 Gy/5 fractions (SBRT) and 40–50 Gy/25 fractions (conventional). Daily volumetric MR images were acquired under treatment conditions on a clinical MR‐IGRT system. The correlation was assessed between interfractional GTV‐to‐duodenum positional variation and daily recalculations of duodenal dose metrics. Positional variation was quantified as the interfraction difference in Hausdorff distance from simulation baseline (ΔHD) between the GTV and proximal duodenal surface, or volume overlap between GTV and duodenum for cases with HD0=0 (GTV abutting duodenum). Adaptation was considered indicated when daily positional variations enabled dose escalation to the target while maintaining duodenal constraints.Results:For fractions with ΔHD&gt;0 (n=14, SBRT only), the mean interfraction duodenum dose decrease from simulation to treatment was 44±53 cGy (maximum 136 cGy). A correlation was found between ΔHD and dosimetric difference (R2=0.82). No correlation was found between volume of overlap and dosimetric difference (R2=0.31). For 89% of fractions, the duodenum remained overlapped with the target and the duodenal dose difference was negligible. The maximum observed indication for adaptation was for interfraction ΔHD=11.6 mm with potential for adaptive dose escalation of 136 cGy.Conclusion:This assessment showed that Hausdorff distance was a reasonable metric to use to determine the indication for adaptation. Adaptation was potentially indicated in 11% of the treatments (fractions where GTV‐to‐duodenum distance increased from simulation), with a feasible average dose escalation of 7.0%.MB, LH, JO, RK, PP: research and/or travel funding from ViewRay Inc. PP: research grant from Varian Medical Systems and Philips Healthcare

740. The TLR9 Agonist EnanDIM® - Evaluation of New Enantiomeric Oligonucleotides for Cancer Immunotherapy In Vitro and In Vivo

Introduction The use of TLR9 agonists as immunomodulators is supported by preclinical and clinical studies, showing their anti-tumor effect by enhancing both cellular and humoral immune responses. So far, two different families of DNA molecules containing non-methylated CG-motifs for TLR9 activation have been established: Dumbbell-shaped dSLIM® molecules are protected against exonucleolytic degradation by their covalently-closed, natural phosphodiester (PO) backbone. In contrast, single-stranded, oligodeoxynucleotides (CpG-ODN) are most commonly chemically-stabilized by phosphorothioates (PTO) in their phosphate moieties. PTO modification, however, produce off-target effects in immune cell populations and have resulted in an unfavorable risk-to-benefit ratio.Methods To avoid the off-target effects of PTO-modified CpG-ODN, linear single-stranded ODN were synthezised using L-deoxyribonucleotides at their 3’-ends, which are the natural enantiomers of Ddeoxyribonucleotides. The vast majority of deoxyribose in present organisms are D-deoxyribose, thus co-evolved nucleases are blind for L-deoxyribose thereby leaving L-protected ODN intact. We selected nucleotide sequences of such L-protected, CG-motif containing, single-stranded ODN, EnanDIM(R), for high secretion of IFN-alpha and IP-10 from human peripheral blood mononuclear cells (PBMC). In a maximum feasible dose approach in CD-1 mice EnanDIM(R) doses of 10 to 50 mg per mice were injected subcutaneously to evaluate the acute toxicity and immunomodulatory properties of EnanDIM(R) molecules in vivo.Results EnanDIM581 and EnanDIM532 were chosen since they caused high secretion of IFN-alpha and IP-10 from human PBMC and resulted in a strong activation of monocytes, NK cells and plasmacytoid dendritic cells (pDC) in vitro. Notably, both showed a distinct immune activation pattern, with the highest secretion of IFN-alpha by EnanDIM581 and the strongest maturation of TLR9-bearing pDC by EnanDIM532. EnanDIM744, comprising EnanDIM581 with additional 5’-end L-nucleotide protection and exhibiting an immune activation pattern similar to EnanDIM581, was selected as third EnanDIM(R) for in vivo studies. In the maximum feasible dose approach, safety assessments were performed throughout the study period and no mortality, clinical signs and body weight changes were observed, despite the fact that extremely high doses of app. 300 to 1700 mg/kg were used. A gross necropsy consisting of a macroscopic organ evaluation at day 15 revealed also no toxicity. Regarding immune activation, increased levels of IP-10 in serum were observed 24 hours after injection but not after 15 days confirming that L-nucleotides in EnanDIM(R) do not change the kinetic profile known from other DNA-based TLR9 agonists.Conclusions EnanDIM®, a new family of TLR9 agonists with conformation-mediated nuclease-resistance, broadly activates the immune system in vitro. Maximal feasible doses of EnanDIM(R) resulted in no signs of toxicity and confirmed immunomodulatory effects in vivo. Therefore EnanDIM(R) has the potential for clinical development in the treatment of cancer.