Targeting EGFR Induced Oxidative Stress by PARP1 Inhibition in Glioblastoma Therapy

Despite the critical role of Epidermal Growth Factor Receptor (EGFR) in glioblastoma pathogenesis [1,2], EGFR targeted therapies have achieved limited clinical efficacy [3]. Here we propose an alternate therapeutic strategy based on the conceptual framework of non-oncogene addiction [4,5]. A directed RNAi screen revealed that glioblastoma cells overexpressing EGFRvIII [6], an oncogenic variant of EGFR, become hyper-dependent on a variety of DNA repair genes. Among these, there was an enrichment of Base Excision Repair (BER) genes required for the repair of Reactive Oxygen Species (ROS)-induced DNA damage, including poly-ADP ribose polymerase 1 (PARP1). Subsequent studies revealed that EGFRvIII overexpression in glioblastoma cells caused increased levels of ROS, DNA strand break accumulation, and genome instability. In a panel of primary glioblastoma lines, sensitivity to PARP1 inhibition correlated with the levels of EGFR activation and oxidative stress. Gene expression analysis indicated that reduced expression of BER genes in glioblastomas with high EGFR expression correlated with improved patient survival. These observations suggest that oxidative stress secondary to EGFR hyperactivation necessitates increased cellular reliance on PARP1 mediated BER, and offer critical insights into clinical trial design.

Graphene Nanoribbons as a Drug Delivery Agent for Lucanthone Mediated Therapy of Glioblastoma Multiforme

We report use of PEG-DSPE coated oxidized graphene nanoribbons (O-GNR-PEG-DSPE) as agent for delivery of anti-tumor drug Lucanthone (Luc) into Glioblastoma Multiformae (GBM) cells targeting base excision repair enzyme APE-1 (Apurinic endonuclease-1). Lucanthone, an endonuclease inhibitor of APE-1, was loaded onto O-GNR-PEG-DSPEs using a simple non-covalent method. We found its uptake by GBM cell line U251 exceeding 67% and 60% in APE-1-overexpressing U251, post 24 h. However, their uptake was ~ 38% and 29% by MCF-7 and rat glial progenitor cells (CG-4), respectively. TEM analysis of U251 showed large aggregates of O-GNR-PEG-DSPE in vesicles. Luc-O-GNR-PEG-DSPE was significantly toxic to U251 but showed little/no toxicity when exposed to MCF-7/CG-4 cells. This differential uptake effect can be exploited to use O-GNR-PEG-DSPEs as a vehicle for Luc delivery to GBM, while reducing nonspecific cytotoxicity to the surrounding healthy tissue. Cell death in U251 was necrotic, probably due to oxidative degradation of APE-1.

Graphical abstract

We used O-GNR-PEG-DSPE as a reliable, non-toxic vehicle for delivery of APE-1 inhibiting Lucanthone into GBM tumor cell lines. LUC-O-GNR-PEG-DSPE particles showed 60% or more uptake by CMV/U251 and A1-5/CMV/U251 where as the uptake by MCF7 and normal CG4 glial cells was much lower (38% and 29% respectively). Different concentrations of Luc (5–80 μM) loaded onto O-GNR-PEG-DSPE showed lower toxicity in the exposed cells compared to the free drug, due to possible slow release of the drug from this particle, which ensures minimum non-specific release of the drug from the particle once it is injected in vivo.
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Cathepsin S expression: An independent prognostic factor in glioblastoma tumours - A pilot Study

Cysteine proteinases have been implicated in astrocytoma invasion. We recently demonstrated that cathepsin S (CatS) expression is up-regulated in astrocytomas and provided evidence for a potential role in astrocytoma invasion (Flannery et al., Am J Path 2003;163(1):175–82). We aimed to evaluate the significance of CatS in human astrocytoma progression and as a prognostic marker. Frozen tissue homogenates from 71 patients with astrocytomas and 3 normal brain specimens were subjected to ELISA analyses. Immunohistochemical analysis of CatS expression was performed on 126 paraffin-embedded tumour samples. Fifty-one astrocytoma cases were suitable for both frozen tissue and paraffin tissue analysis. ELISA revealed minimal expression of CatS in normal brain homogenates. CatS expression was increased in grade IV tumours whereas astrocytoma grades I–III exhibited lower values. Immunohistochemical analysis revealed a similar pattern of expression. Moreover, high-CatS immunohistochemical scores in glioblastomas were associated with significantly shorter survival (10 vs. 5 months, p = 0.014). With forced inclusion of patient age, radiation dose and Karnofsky score in the Cox multivariate model, CatS score was found to be an independent predictor of survival. CatS expression in astrocytomas is associated with tumour progression and poor outcome in glioblastomas. CatS may serve as a useful prognostic indicator and potential target for anti-invasive therapy.

Evaluation of a Water-soluble Bioadhesive Patch for Photodynamic Therapy of Vulval Lesions

An innovative bioadhesive patch intended primarily as a vulval drug delivery system and, specifically, as a means to deliver photosensitisers, or their prodrugs, for photodynamic purposes is described. The patch was formulated with a copolymer of methyl vinyl ether and maleic anhydride (PMVE/MA) as a bioadhesive matrix and poly(vinyl chloride) as a drug-impervious backing layer. Adhesive strength to neonate porcine skin, as a model substrate, was evaluated using peel and tensile testing measurements. Acceptabilities of non-drug loaded patches were appraised using human volunteers and visual-analogue scoring devices. An optimal formulation, with water uptake and peel strengths appropriate for vulval drug delivery, was cast from a 20% (w/w) PMVE/MA solution and adhered with a strength of approximately 1.7 N cm-2. Patient evaluation demonstrated comfort and firm attachment for up to 4 h in mobile patients. Aminolevulinic acid, a commonly used photosensitiser, was formulated into the candidate formulation and applied to vulval intraepithelial neoplastic lesions. Fluorescence under ultraviolet illumination revealed protoporphyrin synthesis. The patch achieves the extended application times obligatory in topical photodynamic therapy of vulval lesions, thereby contributing to potential methods for the eradication of neoplastic lesions in the lower female reproductive tract.

Rheumatologists' judgements about the efficacy of anti-TNF therapy in two neighbouring regions

Objectives: The requirement in Northern Ireland to prescribe biologic agents according to NICE/BSR guidelines and within a fixed budget has created a waiting list for treatment that has no parallel in the Republic of Ireland. The study investigated the bearing this situation may have on had on the consultants’ judgements in the respective areas.

Methods: 78 case vignettes created from the data on real patients with RA treated with biologics in the north and south of Ireland were appraised by 9 southern and 8 northern consultants who judged the clinical benefit and significance of the patients’ condition after a trial of therapy. Quantitative (Clinical Judgement Analysis) and Qualitative (Focus groups) techniques were used.

Results: Northern consultants perceived a slightly greater degree of clinical benefit after a trial of therapy than southern consultants. Judgment models of northern and southern consultants were broadly comparable. The latter tended to be more uniform in their judgments than the southern group. Focus group discussions with consultants largely validated the findings of the quantitative analysis but revealed how clinical judgment analysis might be misled by gaming strategies.

Conclusions: Despite the absence of overt rationing in the south of Ireland, as far as the judgment of therapeutic benefit from biologics was concerned, the clinical judgment policies of practitioners were very similar to those in the north. The adoption of NICE/BSR guidelines in the north may have improved the uniformity of clinical practice in Northern Ireland.

Predictors of therapy resistant asthma: outcome of a systematic evaluation protocol

Background: It has been suggested that asthmatic subjects with persisting symptoms despite adequate maintenance therapy should be systematically evaluated to identify factors contributing to poor control. The aims of this study were to examine the prevalence of these factors in a cohort of sequentially referred poorly controlled asthmatics, and to determine if any factor or combination of factors predicted true therapy resistant asthma (TRA).

Methods: Patients were evaluated using a systematic evaluation protocol including induced sputum analysis, psychiatric assessment, ear, nose and throat examination, pulmonary function testing, high resolution CT scan of the thorax, and 24 hour dual probe ambulatory oesophageal pH monitoring; any identified provoking factor was treated. Asthma was managed according to BTS guidelines.

Results: Of 73 subjects who completed the assessment, 39 responded to intervention and 34 had TRA. Subjects with TRA had a greater period of instability, a higher dose of inhaled steroids at referral, more rescue steroid use, and a lower best percentage forced expiratory volume in 1 second (FEV1%). Oesophageal reflux, upper airway disease, and psychiatric morbidity were common (57%, 95%, 49%, respectively) but were not more prevalent in either group. Using multivariate logistic regression analysis, inhaled steroid dose >2000 µg BDP, previous assessment by a respiratory specialist, and initial FEV1% of <70% at referral predicted a final diagnosis of TRA.

Conclusions: In poorly controlled asthmatics there is a high prevalence of co-morbidity, identified by detailed systematic assessment, but no difference in prevalence between those who respond to intervention and those with TRA. Targeted treatment of identified co-morbidities has minimal impact on asthma related quality of life in those with therapy resistant disease.

Randomized controlled trial of interferential therapy and manipulative therapy for acute low back pain.

Study Design. A multi-center assessor-blinded randomized clinical trial was conducted. Objectives. To investigate the relative effectiveness of interferential therapy and manipulative therapy for patients with acute low back pain when used as sole treatments and in combination. Summary of Background Data. Both manipulative therapy and interferential therapy are commonly used treatments for low back pain. Evidence for the effectiveness of manipulative therapy is available only for the short term. There is no evidence for interferential therapy and no study has investigated the effectiveness of interferential therapy combined with manipulative therapy. Methods. Consenting subjects (n=240) were randomly assigned to receive a copy of the Back Book and either manipulative therapy (MT; n=80), interferential therapy (IFT; n=80) or combined manipulative therapy and interferential therapy (CT; n=80). Follow-up outcome questionnaires were posted at discharge, 6 and 12 months. Results. The groups were balanced at baseline for low back pain and demographic characteristics. All interventions were found to significantly reduce functional disability and pain and increase quality of life at discharge and to maintain these improvements at 6 and 12 months. No significant differences were found between groups for reported LBP recurrence, work absenteeism, medication consumption, exercise participation and healthcare use at 12 months. Conclusions. For acute low back pain, interferential therapy whether used in isolation or in combination with manipulative therapy was as effective as manipulative therapy alone (in addition to the Back Book).

A descriptive study of the usage of spinal manipulative therapy techniques within a randomised clinical trial in acute low back pain

The majority of randomized clinical trials (RCTs) of spinal manipulative therapy have not adequately de?ned the terms ‘mobilization’ and ‘manipulation’, nor distinguished between these terms in reporting the trial interventions. The purpose of this study was to describe the spinal manipulative therapy techniques utilized within a RCT of manipulative therapy (MT; n=80), interferential therapy (IFT; n=80), and a combination of both (CT; n=80) for people with acute low back pain (LBP). Spinal manipulative therapy was de?ned as any ‘mobilization’ (low velocity manual force without a thrust) or ‘manipulation’ (high velocity
thrust) techniques of the spine described by Maitland and Cyriax.
The 16 physiotherapists, all members of the Society of Orthopaedic Medicine, utilized three spinal manipulative therapy patterns in the RCT: Maitland Mobilization (40.4%, n=59), Maitland Mobilization/Cyriax Manipulation (40.4%, n=59) and Cyriax Manipulation (19.1%, n=28). There was a signi?cant difference between the MT and CT groups in their usage of spinal manipulative therapy techniques (w2=9.178; df=2;P=0.01); subjects randomized to the CT group received three times more Cyriax Manipulation (29.2%, n=21/72) than those randomized to the MT group (9.5%, n=7/74; df=1; P=0.003).
The use of mobilization techniques within the trial was comparable with their usage by the general population of physiotherapists in Britain and Ireland for LBP management. However, the usage of manipulation techniques was considerably higher than reported in physiotherapy surveys and may re?ect the postgraduate training of trial therapists.