Delivering manual cardiopulmonary resuscitation (CPR) in a diving bell: an analysis of head-to-chest and knee-to-chest compression techniques.

Introduction: Chest compression often cannot be administered using conventional techniques in a diving bell. Multiple alternative techniques are taught, including head-to-chest and both prone and seated knee-to-chest compressions, but there are no supporting efficacy data. This study evaluated the efficacy, safety and sustainability of these techniques. Methods: Chest compressions were delivered by a team of expert cardiopulmonary resuscitation (CPR) providers. The primary outcome was proportion of chest compressions delivered to target depth compared to conventional CPR. Techniques found to be safe and potentially effective by the study team were further trialled by 20 emergency department staff members. Results: Expert providers delivered a median of 98% (interquartile range [IQR] 1.5%) of chest compressions to the target depth using conventional CPR. Only 32% (IQR 60.8%) of head-to-chest compressions were delivered to depth; evaluation of the technique was abandoned due to adverse effects. No study team member could register sustained compression outputs using prone knee-to-chest compressions. Seated knee-to-chest were delivered to depth 12% (IQR 49%) of the time; some compression providers delivered > 90% of compressions to depth. Conclusions: Head-to-chest compressions have limited efficacy and cause harm to providers; they should not be taught or used. Prone knee-to-chest compressions are ineffective. Seated knee-to-chest compressions have poor overall efficacy but some providers deliver them well. Further research is required to establish whether this technique is feasible, effective and sustainable in a diving bell setting, and whether it can be taught and improved with practise.

An evaluation of the NUI Compact Chest Compression Device (NCCD), a mechanical CPR device suitable for use in the saturation diving environment.

Introduction: Provision of manual chest compressions in a diving bell using a conventional technique is often impossible, and alternative techniques are poorly evidenced in terms of efficacy and sustainability. The first mechanical cardiopulmonary resuscitation (CPR) device suitable for use in this environment, the NUI Compact Chest Compression Device (NCCD), has recently been designed and manufactured. This study assessed both the efficacy of the device in delivering chest compressions to both prone and seated manikins, and the ability of novice users to apply and operate it. Methods: Compression efficacy was assessed using a Resusi Anne QCPR intelligent manikin, and the primary outcome was the proportion of compressions delivered to target depth (50-60 mm). The gold standard was that achieved by expert CPR providers delivering manual CPR; the LUCAS 3 mCPR device was a further comparator. Results: The NCCD delivered 100% of compressions to target depth compared to 98% for the gold standard (interquartile range 1.5%) and 98% for the LUCAS 3 when applied to both supine and seated manikins. The NCCD sometimes became dislodged and had to be reapplied when used with a seated manikin. Conclusions: The NCCD can deliver chest compressions at target rate and depth to both supine and seated manikins with efficacy equivalent to manual CPR and the LUCAS 3. It can become dislodged when applied to a seated manikin; its design has now been altered to prevent this. New users can be trained in use of the NCCD quickly, but practise is required to ensure effective use.

ATM kinase inhibitor AZD0156 in combination with irinotecan and 5-fluorouracil in preclinical models of colorectal cancer.

BACKGROUND: AZD0156 is an oral inhibitor of ATM, a serine threonine kinase that plays a key role in DNA damage response (DDR) associated with double-strand breaks. Topoisomerase-I inhibitor irinotecan is used clinically to treat colorectal cancer (CRC), often in combination with 5-fluorouracil (5FU). AZD0156 in combination with irinotecan and 5FU was evaluated in preclinical models of CRC to determine whether low doses of AZD0156 enhance the cytotoxicity of irinotecan in chemotherapy regimens used in the clinic. METHODS: Anti-proliferative effects of single-agent AZD0156, the active metabolite of irinotecan (SN38), and combination therapy were evaluated in 12 CRC cell lines. Additional assessment with clonogenic assay, cell cycle analysis, and immunoblotting were performed in 4 selected cell lines. Four colorectal cancer patient derived xenograft (PDX) models were treated with AZD0156, irinotecan, or 5FU alone and in combination for assessment of tumor growth inhibition (TGI). Immunofluorescence was performed on tumor tissues. The DDR mutation profile was compared across in vitro and in vivo models. RESULTS: Enhanced effects on cellular proliferation and regrowth were observed with the combination of AZD0156 and SN38 in select models. In cell cycle analysis of these models, increased G2/M arrest was observed with combination treatment over either single agent. Immunoblotting results suggest an increase in DDR associated with irinotecan therapy, with a reduced effect noted when combined with AZD0156, which is more pronounced in some models. Increased TGI was observed with the combination of AZD0156 and irinotecan as compared to single-agent therapy in some PDX models. The DDR mutation profile was variable across models. CONCLUSIONS: AZD0156 and irinotecan provide a rational and active combination in preclinical colorectal cancer models. Variability across in vivo and in vitro results may be related to the variable DDR mutation profiles of the models evaluated. Further understanding of the implications of individual DDR mutation profiles may help better identify patients more likely to benefit from treatment with the combination of AZD0156 and irinotecan in the clinical setting.

Inhibition of DNA-PK with AZD7648 Sensitizes Tumor Cells to Radiotherapy and Induces Type I IFN-Dependent Durable Tumor Control.

PURPOSE: Combining radiotherapy (RT) with DNA damage response inhibitors may lead to increased tumor cell death through radiosensitization. DNA-dependent protein kinase (DNA-PK) plays an important role in DNA double-strand break repair via the nonhomologous end joining (NHEJ) pathway. We hypothesized that in addition to a radiosensitizing effect from the combination of RT with AZD7648, a potent and specific inhibitor of DNA-PK, combination therapy may also lead to modulation of an anticancer immune response. EXPERIMENTAL DESIGN: AZD7648 and RT efficacy, as monotherapy and in combination, was investigated in fully immunocompetent mice in MC38, CT26, and B16-F10 models. Immunologic consequences were analyzed by gene expression and flow-cytometric analysis. RESULTS: AZD7648, when delivered in combination with RT, induced complete tumor regressions in a significant proportion of mice. The antitumor efficacy was dependent on the presence of CD8+ T cells but independent of NK cells. Analysis of the tumor microenvironment revealed a reduction in T-cell PD-1 expression, increased NK-cell granzyme B expression, and elevated type I IFN signaling in mice treated with the combination when compared with RT treatment alone. Blocking of the type I IFN receptor in vivo also demonstrated a critical role for type I IFN in tumor growth control following combined therapy. Finally, this combination was able to generate tumor antigen-specific immunologic memory capable of suppressing tumor growth following rechallenge. CONCLUSIONS: Blocking the NHEJ DNA repair pathway with AZD7648 in combination with RT leads to durable immune-mediated tumor control.

Preclinical Evaluation of AZ12601011 and AZ12799734, Inhibitors of Transforming Growth Factor ß Superfamily Type 1 Receptors.

The transforming growth factor ß (TGFß) superfamily includes TGFß, activins, inhibins, and bone morphogenetic proteins (BMPs). These extracellular ligands have essential roles in normal tissue homeostasis by coordinately regulating cell proliferation, differentiation, and migration. Aberrant signaling of superfamily members, however, is associated with fibrosis as well as tumorigenesis, cancer progression, metastasis, and drug-resistance mechanisms in a variety of cancer subtypes. Given their involvement in human disease, the identification of novel selective inhibitors of TGFß superfamily receptors is an attractive therapeutic approach. Seven mammalian type 1 receptors have been identified that have context-specific roles depending on the ligand and the complex formation with the type 2 receptor. Here, we characterize the biologic effects of two transforming growth factor ß receptor 1 (TGFBR1) kinase inhibitors designed to target TGFß signaling. AZ12601011 [2-(2-pyridinyl)-4-(1H-pyrrolo[3,2-c]pyridin-1-yl)-6,7-dihydro-5H-cyclopenta[d]pyrimidine]; structure previously undisclosed] and AZ12799734 [4-({4-[(2,6-dimethyl-3-pyridinyl)oxy]-2-pyridinyl}amino)benzenesulfonamide] (IC50 = 18 and 47 nM, respectively) were more effective inhibitors of TGFß-induced reporter activity than SB-431542 [4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide] (IC50 = 84 nM) and LY2157299 [4-[2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]quinoline-6-carboxamide monohydrate]] (galunisertib) (IC50 = 380 nM). AZ12601011 inhibited phosphorylation of SMAD2 via the type 1 receptors activin A receptor type 1B (ALK4), TGFBR1, and activin A receptor type 1C (ALK7). AZ12799734, however, is a pan TGF/BMP inhibitor, inhibiting receptor-mediated phosphorylation of SMAD1 by activin A receptor type 1L, bone morphogenetic protein receptor type 1A, and bone morphogenetic protein receptor type 1B and phosphorylation of SMAD2 by ALK4, TGFBR1, and ALK7. AZ12601011 was highly effective at inhibiting basal and TGFß-induced migration of HaCaT keratinocytes and, furthermore, inhibited tumor growth and metastasis to the lungs in a 4T1 syngeneic orthotopic mammary tumor model. These inhibitors provide new reagents for investigating in vitro and in vivo pathogenic processes and the contribution of TGFß- and BMP-regulated signaling pathways to disease states.

pRAD50: a novel and clinically applicable pharmacodynamic biomarker of both ATM and ATR inhibition identified using mass spectrometry and immunohistochemistry.

BACKGROUND: AZD0156 and AZD6738 are potent and selective inhibitors of ataxia-telangiectasia-kinase (ATM) and ataxia-telangiectasia-mutated and Rad3-related (ATR), respectively, important sensors/signallers of DNA damage. METHODS: We used multiplexed targeted-mass-spectrometry to select pRAD50(Ser635) as a pharmacodynamic biomarker for AZD0156-mediated ATM inhibition from a panel of 45 peptides, then developed and tested a clinically applicable immunohistochemistry assay for pRAD50(Ser635) detection in FFPE tissue. RESULTS: We found moderate pRAD50 baseline levels across cancer indications. pRAD50 was detectable in 100% gastric cancers (n = 23), 99% colorectal cancers (n = 102), 95% triple-negative-breast cancers (TNBC) (n = 40) and 87.5% glioblastoma-multiformes (n = 16). We demonstrated AZD0156 target inhibition in TNBC patient-derived xenograft models; where AZD0156 monotherapy or post olaparib treatment, resulted in a 34-72% reduction in pRAD50. Similar inhibition of pRAD50 (68%) was observed following ATM inhibitor treatment post irinotecan in a colorectal cancer xenograft model. ATR inhibition, using AZD6738, increased pRAD50 in the ATM-proficient models whilst in ATM-deficient models the opposite was observed, suggesting pRAD50 pharmacodynamics post ATR inhibition may be ATM-dependent and could be useful to determine ATM functionality in patients treated with ATR inhibitors. CONCLUSION: Together these data support clinical utilisation of pRAD50 as a biomarker of AZD0156 and AZD6738 pharmacology to elucidate clinical pharmacokinetic/pharmacodynamic relationships, thereby informing recommended Phase 2 dose/schedule.

Discovery of a Series of 3-Cinnoline Carboxamides as Orally Bioavailable, Highly Potent, and Selective ATM Inhibitors.

We report the discovery of a novel series of 3-cinnoline carboxamides as highly potent and selective ataxia telangiectasia mutated (ATM) kinase inhibitors. Optimization of this series focusing on potency and physicochemical properties (especially permeability) led to the identification of compound 21, a highly potent ATM inhibitor (ATM cell IC50 0.0028 µM) with excellent kinase selectivity and favorable physicochemical and pharmacokinetics properties. In vivo, 21 in combination with irinotecan showed tumor regression in the SW620 colorectal tumor xenograft model, superior inhibition to irinotecan alone. Compound 21 was selected for preclinical evaluation alongside AZD0156.

The Identification of Potent, Selective, and Orally Available Inhibitors of Ataxia Telangiectasia Mutated (ATM) Kinase: The Discovery of AZD0156 (8-{6-[3-(Dimethylamino)propoxy]pyridin-3-yl}-3-methyl-1-(tetrahydro-2 H-pyran-4-yl)-1,3-dihydro-2 H-imidazo[4,5- c]quinolin-2-one).

ATM inhibitors, such as 7, have demonstrated the antitumor potential of ATM inhibition when combined with DNA double-strand break-inducing agents in mouse xenograft models. However, the properties of 7 result in a relatively high predicted clinically efficacious dose. In an attempt to minimize attrition during clinical development, we sought to identify ATM inhibitors with a low predicted clinical dose (<50 mg) and focused on strategies to increase both ATM potency and predicted human pharmacokinetic half-life (predominantly through the increase of volume of distribution). These efforts resulted in the discovery of 64 (AZD0156), an exceptionally potent and selective inhibitor of ATM based on an imidazo[4,5- c]quinolin-2-one core. 64 has good preclinical phamacokinetics, a low predicted clinical dose, and a high maximum absorbable dose. 64 has been shown to potentiate the efficacy of the approved drugs irinotecan and olaparib in disease relevant mouse models and is currently undergoing clinical evaluation with these agents.

The novel ATM inhibitor (AZ31) enhances antitumor activity in patient derived xenografts that are resistant to irinotecan monotherapy.

Irinotecan, a standard of care therapy for CRC, elicits cytotoxic effects by generating double strand breaks resulting in DNA damage. The activation of the ATM pathway plays a fundamental role in regulating the cellular response and repair to DNA damage. The objective of this preclinical study was to determine whether ATM inhibition would enhance sensitivity to irinotecan treatment. Treatment effects of AZ31, irinotecan or AZ31 + irinotecan were investigated in CRC cell lines and CRC patient derived xenografts. Activation of ATM and downstream targets p-RAD50 and p-H2AX were evaluated by immunohistochemistry. Combinational effects were demonstrated in 4 out of 8 CRC explants. Interestingly, each of the combinational sensitive CRC PDX models were shown to be more resistant to irinotecan single agent therapy. Treatment with irinotecan significantly elevated the ATM pathway evident by an increase in the activation of H2AX and RAD50. Combinational therapy reduced the activation of H2AX and RAD50 when compared to irinotecan alone in the combination sensitive CRC098. AZ31 + irinotecan was effective at reducing tumor growth in tumors that exhibited resistance to irinotecan in our CRC PDX model. These findings support further investigation of this combinational therapy for the treatment of CRC patients.

ZD6474 inhibits vascular endothelial growth factor signaling, angiogenesis, and tumor growth following oral administration.

ZD6474 [N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy]quinazolin-4-amine]is a potent, p.o. active, low molecular weight inhibitor of kinase insert domain-containing receptor [KDR/vascular endothelial growth factor receptor (VEGFR) 2] tyrosine kinase activity (IC(50) = 40 nM). This compound has some additional activity versus the tyrosine kinase activity of fms-like tyrosine kinase 4 (VEGFR3;IC(50) = 110 nM) and epidermal growth factor receptor (EGFR/HER1; IC(50) = 500 nM) and yet demonstrates selectivity against a range of other tyrosine and serine-threonine kinases. The activity of ZD6474 versus KDR tyrosine kinase translates into potent inhibition of vascular endothelial growth factor-A (VEGF)-stimulated endothelial cell (human umbilical vein endothelial cell) proliferation in vitro (IC(50) = 60 nM). Selective inhibition of VEGF signaling has been demonstrated in vivo in a growth factor-induced hypotension model in anesthetized rat: administration of ZD6474 (2.5 mg/kg, i.v.) reversed a hypotensive change induced by VEGF (by 63%) but did not significantly affect that induced by basic fibroblast growth factor. Once-daily oral administration of ZD6474 to growing rats for 14 days produced a dose-dependent increase in the femoro-tibial epiphyseal growth plate zone of hypertrophy, which is consistent with inhibition of VEGF signaling and angiogenesis in vivo. Administration of 50 mg/kg/day ZD6474 (once-daily, p.o.) to athymic mice with intradermally implanted A549 tumor cells also inhibited tumor-induced neovascularization significantly (63% inhibition after 5 days; P < 0.001). Oral administration of ZD6474 to athymic mice bearing established (0.15-0.47 cm(3)), histologically distinct (lung, prostate, breast, ovarian, colon, or vulval) human tumor xenografts or after implantation of aggressive syngeneic rodent tumors (lung, melanoma) in immunocompetent mice, produced a dose-dependent inhibition of tumor growth in all cases. Statistically significant antitumor activity was evident in each model with at least 25 mg/kg ZD6474 once daily (P < 0.05, one-tailed t test). Histological analysis of Calu-6 tumors treated with 50 mg/kg/day ZD6474 for 24 days showed a significant reduction (>70%) in CD31 (endothelial cell) staining in nonnecrotic regions. ZD6474 also restrained growth of much larger (0.9 cm(3) volume) Calu-6 lung tumor xenografts and induced profound regression in established PC-3 prostate tumors of 1.4 cm(3) volume. ZD6474 is currently in Phase I clinical development as a once-daily oral therapy in patients with advanced cancer.

Antitumor activity of the novel vascular targeting agent ZD6126 in a panel of tumor models.

PURPOSE: The purpose of this study was to examine the antitumor effects of the novel vascular targeting agent ZD6126 and to use histology, CD31 immunohistochemistry, and electron microscopy to gain an insight into the mechanism of action of this novel agent. EXPERIMENTAL DESIGN: The antitumor effects of ZD6126 were examined using a range of solid tumor models: (a) ras-transformed mouse 3T3 fibroblasts (Hras5); and (b) human lung (Calu-6), colorectal (LoVo and HT-29), prostate (PC-3), ovarian (SKOV-3), and breast (MDA-MB-231) tumors, grown as xenografts in nude mice. RESULTS: In vivo, a well-tolerated dose of ZD6126 was shown to cause rapid effects on tumor endothelium leading to exposure of the basal lamina after cell retraction and subsequent loss of endothelial cells. This led to thrombosis and vessel occlusion, resulting in extensive tumor necrosis 24 h after ZD6126 administration. Dose-response studies showed that these effects were seen at a dose 8- to 16-fold lower than the maximum tolerated dose, demonstrating that ZD6126 has a wide therapeutic margin in these mouse models. A single dose of ZD6126 (200 mg/kg) led to a significant growth delay in Calu-6 and LoVo tumors. Growth delay was increased when 100 mg/kg ZD6126 was given as a well-tolerated regime in five daily doses. Finally, combining ZD6126 with cisplatin resulted in greater than additive enhancement in growth delay in the Calu-6 model. CONCLUSIONS: These findings provide direct support that ZD6126 selectively disrupts tumor vasculature, demonstrate that it has activity in a range of tumor xenograft models, and show that it can significantly enhance the antitumor efficacy of cisplatin.