Ongoing Clinical Program Research Articles

P098 Receptor-interacting protein 1 kinase inhibition therapeutically ameliorates experimental T-cell-dependent colitis in mice

Abstract Background Inflammatory bowel disease (IBD) is a multifactorial disorder characterised by chronic and relapsing intestinal inflammation. Receptor-interacting protein 1 (RIP1) kinase activity is emerging as a driver of pro-inflammatory cytokine production and cell death and has been implicated in multiple inflammatory diseases including IBD. To this end, recent work has shown that RIP1 kinase inhibition reduces the spontaneous production of cytokines from human ulcerative colitis (UC) and Crohn’s disease explants. Methods The aim of this study was to investigate the anti-inflammatory effect of a highly selective inhibitor of RIP1 kinase activity (GSK547A) in the mouse T-cell transfer model of colitis; a model that shares many features with human IBD. All animal studies were ethically reviewed and carried out in accordance with Animals (Scientific Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Animals. Chronic colitis was achieved by transferring CD4+CD45RBhigh T cells into immunodeficient female SCID mice (6/8 weeks old). After confirming the development of pathology using endoscopy and MRI, animals were treated therapeutically with the RIP1 inhibitor GSK547A (50 mg/kg twice a day PO) or vehicle (0.5% hydroxypropyl methylcellulose in water). The severity of colitis was measured 5 weeks after cell transfer both macro- and microscopically. Mucosal damage by endoscopy was also assessed. RT-PCR and MSD analysis were performed to detect colon cytokine/chemokine and calprotectin levels. SAA in the plasma was measured using ELISA. Results We found that treatment with GSK547 significantly ameliorated experimental T-cell-dependent colitis in mice. GSK547A-treated mice displayed decreased weight loss, colon density (ratio weight/length), macroscopic disease activity index, colon thickness compared with vehicle-treated animals. Mucosal damage, assessed by endoscopy and histopathology, was also reduced following treatment with RIP1 kinase inhibitor. In addition, GSK547A reduced the expression of cytokines in the colon (TNF-α, IL-17A, INF-γ, IL-6 and MCP-1), both at a protein and RNA level. Relevant translational biomarkers such as SAA in plasma and RNA calprotectin in the colon were also decreased in the GSK547A treated group when compared with vehicle. Conclusion Our results suggest that RIP1K inhibition is a strong protective factor with anti-inflammatory potential in the progression of chronic colitis, when applied to the translationally relevant T-cell transfer model of colitis. These findings suggest the potential application in the management of inflammatory bowel disease and support the ongoing clinical program evaluating the effect of RIP1 kinase inhibition in UC patients.

392P - Generation of a novel preclinical PK/PD model provides insights into PARP inhibitor clinical monotherapy activity

Background: In Phase II trials, an olaparib capsule dose that inhibits PARP (100 mg bd) was clinically inferior to the MTD (400 mg bd). Using an in vivo model, we investigated this discrepancy and the effect of dose and schedule on tumour progression. Methods: PK, PD and antitumour activity were assessed using olaparib (100, 50, 25, 10, 2.5 mg/kg qd) in a breast cancer patient-derived xenograft (PDX) with mutated BRCA2 and TP53. We studied the relationship between PARP1 inhibition and DNA single-strand breaks (SSBs) by creating a mathematical model. We also compared PK/PD model parameters with free minimum olaparib steady-state plasma concentration (Cmin,ss) in seven Phase I–III clinical studies. Results: Only 100 and 50 mg/kg olaparib doses caused tumour regression, yet all but the lowest led to acute PAR inhibition. The differentiating factor was time over PAR IC95; simulations using the mathematical model at steady state predicted a threshold where >95% reduction in PAR caused >20-fold increase in DNA SSBs. Overlaying clinical data, we saw that 400 mg bd capsule and 300 mg bd tablet doses provide continuous PAR inhibition (Cmin,ss>IC95 upper confidence level); 100 mg bd capsule does not. Dosing schedule assessment showed PDX regression with continuous 100 mg/kg olaparib (0/10 tumours progressed by day 100). By contrast, continuous dosing at 50 mg/kg or intermittent dosing (1 wk on/1 wk off) at 100 mg/kg resulted in 2/10 and 10/10 progressions, respectively. Treatment withdrawal upon regression led to regrowth in all cases; re-challenge at continuous 100 mg/kg was effective (9/10 regressions), 50 mg/kg was not (0/10). In a maintenance setting, switching to lower or intermittent dosing resulted in tumour response more similar to that of continuous 100 mg/kg (1/10, 2/10 and 0/10 progressions, respectively). Conclusions: Discrepancy between biologically and clinically effective olaparib doses is explained by a need for continuous >95% PAR inhibition. Both the 400 mg bd capsule and 300 mg bd tablet dose, used in the ongoing clinical programme, provide this. Dosing schedule data suggest continuous PAR inhibition is also needed for long-term responses. These data highlight important principles for treatment of patients with PARP inhibitor monotherapy. Legal entity responsible for the study: AstraZeneca Funding: AstraZeneca Disclosure: M. O'Connor, E. Cadogan, A. Hughes, M. Learoyd, H. Xu, J. Li, J. Yates: Employee of AstraZeneca and owns stock with AstraZeneca. E. Leo: Employee of AstraZeneca.

Observational Study on a Mindfulness Training for Smokers within a Smoking Cessation Program

Mindfulness is a skill of intentionally directing one’s attention to emotions, thoughts, or physical sensations with an open-minded, non-judgmental attitude. Mindfulness can enable a person to experience challenging feelings or situations without reacting impulsively or automatically. Mindfulness training may be provided to smokers to help them manage smoking triggers, urges, stress, or negative emotions without smoking. Randomized controlled trials have now shown that mindfulness training for smokers (MTS) yields smoking abstinence rates that are equal to or higher than matched behavioral treatments. We describe a comparative observational study on MTS within an ongoing clinical program. The study was conducted with a naturalistic design and utilized no procedures or evaluations beyond those already employed in clinical practice. All participants were advised to use FDA-approved smoking cessation medications and were provided with a choice of two behavioral treatments—MTS (selfpay) or the Wisconsin Tobacco Quit Line (WTQL) (free). Over a 12-month study period, 181 clinic patients enrolled; 65.75% (119) choose MTS and 34.25% (62) choose WTQL. MTS compared to WTQL treatment initiators showed significantly higher biochemically confirmed 7-day point prevalence smoking abstinence at 2 weeks post-quit attempt (MTS = 47.00%, QL = 24.52%, p = .011, OR = 2.71). Intent-to-treat analysis showed similar outcomes (p = .019, OR = 2.45). The study provides evidence that MTS provided within clinical program leads to similar outcomes as found in randomized controlled trials. The strategic use of MTS within a smoking cessation program is discussed.

Hypoxia-activated cytotoxic agent tirapazamine enhances hepatic artery ligation-induced killing of liver tumor in HBx transgenic mice

Transarterial chemoembolization (TACE) is the main treatment for intermediate stage hepatocellular carcinoma (HCC) with Barcelona Clinic Liver Cancer classification because of its exclusive arterial blood supply. Although TACE achieves substantial necrosis of the tumor, complete tumor necrosis is uncommon, and the residual tumor generally rapidly recurs. We combined tirapazamine (TPZ), a hypoxia-activated cytotoxic agent, with hepatic artery ligation (HAL), which recapitulates transarterial embolization in mouse models, to enhance the efficacy of TACE. The effectiveness of this combination treatment was examined in HCC that spontaneously developed in hepatitis B virus X protein (HBx) transgenic mice. We proved that the tumor blood flow in this model was exclusively supplied by the hepatic artery, in contrast to conventional orthotopic HCC xenografts that receive both arterial and venous blood supplies. At levels below the threshold oxygen levels created by HAL, TPZ was activated and killed the hypoxic cells, but spared the normoxic cells. This combination treatment clearly limited the toxicity of TPZ to HCC, which caused the rapid and near-complete necrosis of HCC. In conclusion, the combination of TPZ and HAL showed a synergistic tumor killing activity that was specific for HCC in HBx transgenic mice. This preclinical study forms the basis for the ongoing clinical program for the TPZ-TACE regimen in HCC treatment.

Identifying Impairments after Concussion

This study aimed to determine whether agreement exists between baseline comparison (comparison of postconcussion scores to individualized baseline scores) and normative comparison (comparison of postconcussion scores to a normative mean) in identifying impairments after concussion. A total of 1060 collegiate student-athletes completed baseline testing as part of an ongoing clinical program. Gender-specific normative means were obtained from a subset of 673 athletes with no history of self-reported concussion, learning disabilities, or attention-deficit disorders. Concussions were later diagnosed in 258 athletes who had completed baseline testing. The athletes completed their first assessment within 10 d after injury. Athletes completed a computerized neurocognitive test (Automated Neuropsychological Assessment Metrics), postural control assessment (Sensory Organization Test), and a 15-item graded symptom checklist at baseline and again after injury. We computed two postconcussion difference scores for each outcome measure: 1) baseline difference = postconcussion score--individualized baseline score and 2) normative difference = postconcussion score--normative mean. Athletes were considered impaired if postconcussion difference exceeded the reliable change parameters. McNemar tests were used to assess agreement on impairment status (impaired and unimpaired) between comparison methods for each outcome measure. The baseline comparison method identified 2.6 times more impairments than the normative comparison method for the Simple Reaction Time Test 1 (P = 0.043). The normative comparison method identified 7.6 times more impairments than the baseline comparison method for Mathematical Processing (P < 0.001). No other disagreements were observed for postural control or symptom severity. Our findings suggest that, when using these concussion assessment tools, clinicians may consider using normative data in lieu of individualized baseline measures. This may be especially useful to clinicians with limited resources and an inability to capture valid baselines on all athletes.

P234 The importance of appropriate reference equations for spirometry: lessons learned from the Cystic Fibrosis Gene Therapy “Run-In” study

Introduction and ObjectivesThe “Run-In” study is an on-going longitudinal, observational study of CF patients assessing outcome measures for a future gene therapy trial. Spirometry is performed at each visit and...

Outcome of Aurora Kinase Inhibition of Acute Myeloid Leukemia by AT9283 Is Dependent upon the Presence or Absence of Mutations in Type 1 Oncogenic Kinase Signalling Pathways.

Recent reports suggest that Aurora kinases (AK) A and B are overexpressed in a proportion of patients with AML and that the level of overexpression correlates with their sensitivity to AK inhibition in vitro. Inhibition of AKB results in mitotic exit in the absence of cell division resulting in polyploidy whilst AKA is responsible for the fidelity of mitotic spindle assembly and phosphorylates p53 at Ser315 leading to its ubiquitination by Mdm2 and subsequent proteolysis. Inhibition of this process increases p53 stability inducing cell-cycle arrest with 4N DNA. Clinical studies of the dual AK inhibitor AT9283 indicate that it is active in the treatment of a proportion of patients with relapsed/refractory AML. In order to explore the molecular basis of this varied sensitivity we investigated the effect of AT9283 in a panel of 10 AML cell lines from a variety of genetic backgrounds including a proportion driven by ras, Flt3 or c-kit mutations. Two phenotypes were observed;Accumulation of cells in the G2/M phase (4N) of the cell cycle followed by apoptosis, orAccumulation of cells with >4N DNA (polyploid) followed by apoptosis.Cell lines that exhibit the former phenotype were those driven by mutation in an oncogenic kinase such as ras, c-kit or Flt3. Further analysis of Cyclin B levels suggest that profile 1) results from a G2 block occurring as a consequence of a dominant AKA inhibitory effect in these cell lines. Profile 2) results from AKB inhibition; where cells continue to undergo rounds of DNA replication in the absence of cell division. Inhibition of both of these signaling pathways has been confirmed in AML blasts taken from patients treated with AT9283 in the ongoing clinical program. These results suggest that AT9283 triggers the mitotic checkpoint and induces apoptosis in patients harboring mutations in FLT3 or c-kit via AKA inhibition. These AML cases have been shown to be more likely to exhibit normal cytogenetics and this profile may be important in sustaining rapid peripheral blast proliferation typical of this subtype of AML. Cell lines that respond to treatment with AT9283 by becoming polyploid may be manifesting the effect of predominant AKB inhibition. The balance between these outcomes may reflect the corresponding levels of expression of AKA and AKB in individual subtypes of AML along with factors such as TP53 mutation being associated with genomic instability supporting the development of complex karyotypic abnormalities. Such preliminary findings indicate that patients with mutations in oncogenic signaling pathways may be particularly sensitive to treatment with AT9283 due to the presence of an intact mitotic checkpoint and a dominant Aurora A inhibitory phenotype. This hypothesis is currently being explored through the analysis of biological studies obtained from the ongoing clinical program.

Traitement des infections fongiques invasives en pédiatrie

Among children, an epidemiologic shift has caused an increase in non-albicans Candida infections and invasive aspergillosis, as well as infections by pathogens that were previously uncommon. These infections are frequent causes of infectious morbidity in immunocompromised children. Treatment options are limited for children. When choosing a therapy, it is important to consider pivotal clinical trial data, available pediatric pharmacokinetic profiles, safety and drug interactions profiles, and species-dependent susceptibilities. For most infections, the best initial treatment is still unassessed. Caspofungin is not yet approved for pediatric use, but an ongoing clinical program on this field gives promising results.

Effectiveness of mouthguards in reducing neurocognitive deficits following sports‐related cerebral concussion

Although it is widely accepted that mouthguards decrease the incidence of dental injuries, there is a controversy among sports medicine professionals as to the effectiveness of mouthguards in decreasing the incidence or severity of sports-related cerebral concussion (SRCC). While some experimental data suggest that this may be the case, there exist a number of reports suggesting that mouthguards do not serve this purpose. These conclusions have been drawn, however, without actually measuring the extent of neurocognitive dysfunction in athletes following sports-related concussion. The purpose of this study was to determine whether mouthguard use reduces the neurocognitive and symptomatic impairments that follow an injurious episode of SRCC. Preseason baseline data were collected as part of an ongoing clinical program that uses a computerized neurocognitive test to assess various faculties of brain function and symptoms reported at the time of testing. Follow-up testing from 180 student-athletes who had sustained an SRCC was analyzed for the purpose of this study. These athletes were separated into one of two groups: those who reported using mouthguards and those who did not. Neurocognitive testing was accomplished using the Immediate Post-Concussion and Assessment Test (ImPACT). Results suggest that neurocognitive deficits at the time of the athletes' first follow-up assessment did not differ between mouthguard users and non-users, suggesting that mouthguard use does little to reduce the severity of neurocognitive dysfunction and onset of symptoms following sports-related head trauma. However, an interesting finding in this study was that athletes experienced significantly lower neurocognitive test scores and reported higher symptom scores following SRCC regardless of mouthguard use. This emphasizes a thorough clinical evaluation of athletes that have sustained an SRCC. Although it was found in this study that mouthguard use does not decrease the severity of concussion, it is important to note that the use of mouthguards is paramount in reducing maxillofacial and dental trauma and their use should continue to be mandated by athletic associations and supported by all dental and sports medicine professionals.

Clinical application of two phonologically based treatment procedures.

Two phonological process-based treatment procedures were applied in an ongoing clinical program. Subjects were 4 children aged 3:1, 3:8, 4:1, and 5:1. Two subjects were assigned to a minimal pairs contrasting procedure, and 2 were assigned to a modified cycles procedure based on results of a detailed phonological analysis. All children demonstrated marked changes in their phonological systems as shown by the results of pretreatment and follow-up generalization probes. Correct production generalized to sounds affected by the treatment process that were not a focus of training. Correct production of untrained sounds lagged behind that of trained sounds for all subjects. Results support the hypothesis that articulation remediation is enhanced by treating phonological processes as well as the notion that the acquisition of phonology is a gradual process. Both treatment procedures used in this study were found to be effective and efficient, as evidenced by the elimination of up to three phonological processes within 2 1/2 months for each subject.

Application of Visual Reinforcement Audiometry (VRA) to Low-Functioning Children

Visual reinforcement audiometry (VRA) was studied on a group of low-functioning children in an ongoing clinical program. After establishing the specific reinforcing value of the visual stimulus, threshold assessments were made. VRA was an effective test procedure for most of the children tested, providing their developmental ages reached approximately the 10-month level or higher. On the basis of VRA thresholds and tympanometry, 52% of the children seen were referred for medical evaluation. A variation of VRA was used to assess syllable-discrimination performance on a pilot basis. Results are discussed in terms of the potential use of VRA to assess infant spedch-sound discrimination in addition to its current use as an indicator of hearing sensitivity.