The SYDNEY Device Study: A Multicenter, Randomized, Open-label Usability Study of a 2-mL Alirocumab Autoinjector Device.

PURPOSE: The proprotein convertase subtilisin/kexin type 9 inhibitor alirocumab has produced significant reductions in LDL-C at a dose of 300 mg q4w administered as 2 separate 150-mg injections via a 1-mL autoinjector (AI). A recently developed 2-mL device (SYDNEY) permits the administration of a single 300mg dose of alirocumab. METHODS: We assessed the usability and product technical complaints (PTCs) reported by patients using the 2-mL SYDNEY device in unsupervised settings, adverse events, and effects on LDL-C, in a multicenter, randomized, open-label, 16-week study conducted in the United States. For their first dose, 69 patients with hypercholesterolemia despite receiving statin with or without other lipid-lowering therapy randomly received supervised, self-administered alirocumab 300 mg via 1 × 300 mg injection with the SYDNEY device (n = 35) or 2 × 150-mg injections with the currently approved AI (n = 34). All continuing patients subsequently received unsupervised, self-administered alirocumab 300 mg q4w using the SYDNEY device at weeks 4, 8, and 12. The primary end point was the proportion of SYDNEY device-associated PTCs related to the use of the unsupervised injections. FINDINGS: Baseline characteristics between the study arms varied only in a higher percentage of males being randomized to the study arm using the SYDNEY device (74.3%) compared with the AI arm (44.1%). A single PTC was reported during the unsupervised injections (0.5%; 1 of 196 injections; 95% CI, 0.0%-3.2%). This event was classified as patient related as opposed to device related. No PTCs occurred during supervised injections. Mean LDL-C reductions from baseline at week 4 were 66.2% with SYDNEY and 51.2% with the AI; after adjustment for sex differences between groups, mean LDL-C reductions were 63.5% and 53.9%, respectively. LDL-C reductions persisted for 16 weeks. The most common adverse event was upper respiratory tract infection (3 with SYDNEY and 0 with the AI during weeks 0-4). IMPLICATIONS: The SYDNEY device allowed for a single 2-mL injection of alirocumab 300 mg, providing substantial LDL-C reductions with no new product technical issues or no new safety concerns compared with the currently marketed 1-mL AI device. In conclusion, the 2-mL SYDNEY device provides patients with the possibility of injecting the 300-mg alirocumab dose as a single injection. ClinicalTrials.gov identifier: NCT03415178.

Association of Patient Profile with Glycemic Control and Hypoglycemia with Insulin Glargine 300 U/mL in Type 2 Diabetes: A Post Hoc Patient-Level Meta-Analysis.

AIMS: To examine the association of baseline patient characteristics with study outcomes in people with type 2 diabetes receiving insulin glargine 300 U/mL (Gla-300) versus glargine 100 U/mL (Gla-100), over a 6-month period. METHODS: A post hoc patient-level meta-analysis using data from three multicenter, randomized, open-label, parallel-group, phase 3a studies of similar design, in people previously receiving either basal and prandial insulin, basal insulin + oral antihyperglycemic drugs, or no prior insulin (EDITION 1, 2 and 3, respectively). The endpoints, glycated hemoglobin (HbA1c), hypoglycemia, body weight change, and insulin dose were investigated by subgroups: age (< 65 and ≥ 65 years), body mass index (BMI; < 30 and ≥ 30 kg/m2), age at onset (< 40, 40-50, and > 50 years), and diabetes duration (< 10 and ≥ 10 years). RESULTS: Reduction in HbA1c was comparable between insulins, regardless of subgroup. The lower risk of ≥ 1 nocturnal (00:00-05:59 h) confirmed (≤ 3.9 mmol/L [≤ 70 mg/dL]) or severe hypoglycemic event with Gla-300 versus Gla-100 was also unaffected by participant characteristics. While heterogeneity of treatment effect between diabetes duration subgroups was seen for the risk of ≥ 1 confirmed (≤ 3.9 mmol/L [≤ 70 mg/dL]) or severe hypoglycemic event at any time (24 h), treatment effect consistently favored Gla-300; no evidence of heterogeneity was observed for the other subgroups. Annualized rates of confirmed (≤ 3.9 mmol/L [≤ 70 mg/dL]) or severe hypoglycemia and body weight change were not influenced by participant characteristics; a similar pattern was observed with insulin dose. CONCLUSIONS: Comparable glycemic control was observed with Gla-300 versus Gla-100, with less hypoglycemia, regardless of age, BMI, age at onset or diabetes duration. FUNDING: Sanofi. Plain language summary available for this article.

The effect of concomitant DPPIVi use on glycaemic control and hypoglycaemia with insulin glargine 300 U/mL (Gla-300) versus insulin glargine 100 U/mL (Gla-100) in people with type 2 diabetes: A patient-level meta-analysis of EDITION 2 and 3.

AIMS: To evaluate the effect of concomitant dipeptidyl peptidase IV inhibitor (DPPIVi) use on efficacy and safety of insulin glargine 300 U/mL (Gla-300) versus glargine 100 U/mL (Gla-100) in people with type 2 diabetes on oral antihyperglycaemic drugs. METHODS: A post hoc patient-level meta-analysis was performed using data from EDITION 2 (basal insulin [N = 811]) and EDITION 3 (insulin-naïve [N = 878]), multicentre, randomised, open-label, parallel-group, phase 3a trials of similar design. Endpoints analysed included HbA1c, hypoglycaemia and adverse events, investigated in subgroups of participants with and without concomitant DPPIVi use. RESULTS: Of 1689 participants randomised, 107 (13%, Gla-300) and 133 (16%, Gla-100) received DPPIVi therapy. The least squares mean change in HbA1c (baseline to month 6) was comparable between treatment groups, irrespective of DPPIVi use (no evidence of heterogeneity of treatment effect across subgroups, p = 0.753), although group sizes were unbalanced. The cumulative mean number of confirmed (≤3.9 mmol/L [≤70 mg/dL]) or severe hypoglycaemic events, and the risk and annualised rate of such events, were consistently lower for Gla-300 than Gla-100 during the night (between 00:00 and 05:59 h) or at any time of day (24 h period), irrespective of DPPIVi use. Severe hypoglycaemia occurred in 8/838 and 10/844 participants in the Gla-300 and Gla-100 groups, respectively, and was not affected by DPPIVi use. The adverse event profile was similar between treatment groups and DPPIVi subgroups. CONCLUSIONS: Glycaemic control with Gla-300 was comparable to Gla-100, with less hypoglycaemia during the night and at any time of day (24 h), irrespective of concomitant DPPIVi use. TRIAL REGISTRATION: ClinicalTrials.gov NCT01499095; NCT01676220.

Setting up a wide panel of patient-derived tumor xenografts of non-small cell lung cancer by improving the preanalytical steps.

With the ongoing need to improve therapy for non-small cell lung cancer (NSCLC) there has been increasing interest in developing reliable preclinical models to test novel therapeutics. Patient-derived tumor xenografts (PDX) are considered to be interesting candidates. However, the establishment of such model systems requires highly specialized research facilities and introduces logistic challenges. We aimed to establish an extensive well-characterized panel of NSCLC xenograft models in the context of a long-distance research network after careful control of the preanalytical steps. One hundred fresh surgically resected NSCLC specimens were shipped in survival medium at room temperature from a hospital-integrated biobank to animal facilities. Within 24 h post-surgery, tumor fragments were subcutaneously xenografted into immunodeficient mice. PDX characterization was performed by histopathological, immunohistochemical, aCGH and next-generation sequencing approaches. For this model system, the tumor take rate was 35%, with higher rates for squamous carcinoma (60%) than for adenocarcinoma (13%). Patients for whom PDX tumors were obtained had a significantly shorter disease-free survival (DFS) compared to patients for whom no PDX tumors (P = 0.039) were obtained. We established a large panel of PDX NSCLC models with a high frequency of mutations (29%) in EGFR, KRAS, NRAS, MEK1, BRAF, PTEN, and PI3KCA genes and with gene amplification (20%) of c-MET and FGFR1. This new patient-derived NSCLC xenograft collection, established regardless of the considerable time required and the distance between the clinic and the animal facilities, recapitulated the histopathology and molecular diversity of NSCLC and provides stable and reliable preclinical models for human lung cancer research.

The vascular disrupting agent ombrabulin (AVE8062) enhances the efficacy of standard therapies in head and neck squamous cell carcinoma xenograft models.

Targeting tumor vasculature is an emerging strategy in cancer treatment. Promising results have been shown in preclinical studies when vascular disrupting agents (VDAs) are used in combination with other anticancer therapies. Because radiation therapy with concurrent cisplatin or cetuximab has become standard treatment for patients with locally advanced head and neck squamous cell carcinoma (HNSCC), we investigated whether the VDA ombrabulin (AVE8062) could improve the antitumor activity of radiation plus cisplatin and radiation plus cetuximab combinations. HNSCC HEP2 or FaDu tumor bearing mice were treated with ombrabulin, cisplatin, cetuximab, local radiation therapy or combinations of these treatments. Ombrabulin attenuated tumor growth of HEP2 and FaDu xenografts compared to control tumors. A more pronounced tumor growth delay and tumor regression were induced when ombrabulin was added to local irradiation, cisplatin or cetuximab in FaDu tumors compared to single agent treatments. Finally, triple agent therapies combining ombrabulin, irradiation, and either cisplatin or cetuximab were more effective than double combination treatment regimens and increased tumor growth delay in both HEP2 and FaDu tumor models. Of note, complete tumor regression was achieved in FaDu tumor model for the triple combination including platinum. Immunohistochemistry on FaDu tumors demonstrated a specificity of ombrabulin towards intratumoral vessels, in contrast to peritumoral vasculature. Our results provide a rationale for the use of ombrabulin in combination with two standard treatment regimens that are concurrent cisplatin-based chemoradiation and cetuximab plus ionizing radiation therapies, for the treatment of HNSCC.

Characterization of a large panel of patient-derived tumor xenografts representing the clinical heterogeneity of human colorectal cancer.

PURPOSE: Patient-derived xenograft models are considered to represent the heterogeneity of human cancers and advanced preclinical models. Our consortium joins efforts to extensively develop and characterize a new collection of patient-derived colorectal cancer (CRC) models. EXPERIMENTAL DESIGN: From the 85 unsupervised surgical colorectal samples collection, 54 tumors were successfully xenografted in immunodeficient mice and rats, representing 35 primary tumors, 5 peritoneal carcinoses and 14 metastases. Histologic and molecular characterization of patient tumors, first and late passages on mice includes the sequence of key genes involved in CRC (i.e., APC, KRAS, TP53), aCGH, and transcriptomic analysis. RESULTS: This comprehensive characterization shows that our collection recapitulates the clinical situation about the histopathology and molecular diversity of CRC. Moreover, patient tumors and corresponding models are clustering together allowing comparison studies between clinical and preclinical data. Hence, we conducted pharmacologic monotherapy studies with standard of care for CRC (5-fluorouracil, oxaliplatin, irinotecan, and cetuximab). Through this extensive in vivo analysis, we have shown the loss of human stroma cells after engraftment, observed a metastatic phenotype in some models, and finally compared the molecular profile with the drug sensitivity of each tumor model. Through an experimental cetuximab phase II trial, we confirmed the key role of KRAS mutation in cetuximab resistance. CONCLUSIONS: This new collection could bring benefit to evaluate novel targeted therapeutic strategies and to better understand the basis for sensitivity or resistance of tumors from individual patients.

Sorting nexin 5 is localized to a subdomain of the early endosomes and is recruited to the plasma membrane following EGF stimulation.

Sorting nexins are a large family of proteins that contain the phosphoinositide-binding Phox homology (PX) domain. A number of sorting nexins are known to bind to PtdIns3P, which mediates their localization to membranes of the endocytic pathway. We show here that sorting nexin 5 (SNX5) can be recruited to two distinct membrane compartments. In non-stimulated cells, the PX domain was independently targeted to endosomal structures and colocalized with full-length SNX5. The membrane binding of the PX domain was inhibited by the PI 3-kinase inhibitor, wortmannin. Although SNX5 colocalized with a fluid-phase marker and was found predominantly within a PtdIns3P-rich endosomal domain, very little colocalization was observed between SNX5 and the PtdIns3P-binding protein, EEA1. Using liposome-based binding assays, we have shown that the PX domain of SNX5 interacts not only with PtdIns3P but also with PtdIns3,4P2. In response to EGF stimulation, either the SNX5-PX domain or full-length SNX5 was rapidly recruited to the plasma membrane. The localization of SNX1, which does not bind PtdIns3,4P2, was unaffected by EGF signalling. Therefore, SNX5 is localized to a subdomain of the early endosome distinct from EEA1 and, following EGF stimulation and elevation of PtdIns3,4P2, is also transiently recruited to the plasma membrane. These results indicate that SNX5 may have functions not only associated with endosomal sorting but also with the phosphoinositide-signalling pathway.

Intracellular sorting and transport of proteins.

The secretory and endocytic pathways of eukaryotic organelles consist of multiple compartments, each with a unique set of proteins and lipids. Specific transport mechanisms are required to direct molecules to defined locations and to ensure that the identity, and hence function, of individual compartments are maintained. The localisation of proteins to specific membranes is complex and involves multiple interactions. The recent dramatic advances in understanding the molecular mechanisms of membrane transport has been due to the application of a multi-disciplinary approach, integrating membrane biology, genetics, imaging, protein and lipid biochemistry and structural biology. The aim of this review is to summarise the general principles of protein sorting in the secretory and endocytic pathways and to highlight the dynamic nature of these processes. The molecular mechanisms involved in this transport along the secretory and endocytic pathways are discussed along with the signals responsible for targeting proteins to different intracellular locations.

Activity and properties of alpha-L-fucosidase are dependent on the state of enterocytic differentiation of HT-29 colon cancer cells.

Previously we have demonstrated an impairment in the activity of alpha-L-fucosidase in colon tumours. In order to establish an in vitro model to study this enzyme in colon cancer, we have determined the activity and properties of the enzyme during the differentiation of HT-29 colon cancer cells. Cultures were committed to differentiate into enterocyte-like cells by placing them in a culture medium without glucose for 18-21 days. The state of differentiation was evaluated by assaying the activity of enterocytic marker enzymes, and the acquisition of enterocyte morphology was assessed by electron microscopy. The alpha-L-fucosidase activity was determined using a fluorometric method. Intracellular levels of alpha-L-fucosidase activity are lower in non-differentiated cells (3.0 +/- 1.01 U/mg) than in differentiated ones (9.2 +/- 4.09 U/mg) (P < 0.001). This variation is not due to a greater secretion of the enzyme to the culture medium, and properties such as pH optimum or the affinity towards substrate are not dependent on differentiation. The enzyme however, is more stable at acidic pH and at high temperatures, and V(max) is higher in differentiated cells. Moreover, in undifferentiated cells the enzyme is mainly in a monomeric form whereas multimeric forms of the enzyme appear only upon differentiation. Most of these changes are very similar to those previously observed between normal colon tissue and colon tumours. Thus, we suggest that differentiation of HT-29 colon cancer cells could be used as a model to study the alterations of the enzyme alpha-L-fucosidase during the progression of the tumoural process.