The development and optimisation of an HPLC-based in vitro serum stability assay for a calcitonin gene-related peptide receptor antagonist peptide.

Evaluation of the stability of peptide drug candidates in biological fluids, such as blood serum, is of high importance during the lead optimisation phase. Here, we describe the optimisation and validation of a method for the evaluation of the stability of a lead calcitonin gene-related peptide antagonist peptide (P006) in blood serum. After initially determining appropriate peptide and human serum concentrations and selection of the quenching reagent, the HPLC method optimisation used two experimental designs, Plackett-Burman design and Taguchi design. The analytical method was validated as complying with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. The optimised method allowed the successful resolution of the parent peptide from its metabolites using RP-HPLC and identification of the major metabolites of P006 by mass spectrometry. This paradigm may be widely adopted as a robust early-stage platform for screening peptide stability to rule out candidates with low in vitro stability, which would likely translate into poor in vivo pharmacokinetics.

Novel peptide calcitonin gene-related peptide antagonists for migraine therapy.

OBJECTIVES: It has previously been shown that the peptide (34Pro,35Phe)CGRP27-37 is a potent calcitonin gene-related peptide, CGRP receptor antagonist, and in this project we aimed to improve the antagonist potency through the structural modification of truncated C-terminal CGRP peptides. METHODS: Six peptide analogues were synthesized and the anti-CGRP activity confirmed using both in vitro and in vivo studies. KEY FINDINGS: A 10 amino acid-containing peptide VPTDVGPFAF-NH2 (P006) was identified as a key candidate to take forward for in vivo evaluation, where it was shown to be an effective antagonist after intraperitoneal injection into mice. P006 was formulated as a preparation suitable for nasal administration by spray drying with chitosan to form mucoadhesive microcarriers (9.55 ± 0.91 mm diameter) and a loading of 0.2 mg peptide per 20 mg dose. CONCLUSIONS: The project has demonstrated the potential of these novel small peptide CGRP antagonists, to undergo future preclinical evaluation as anti-migraine therapeutics.

PTEN Protein Phosphatase Activity Is Not Required for Tumour Suppression in the Mouse Prostate.

Loss PTEN function is one of the most common events driving aggressive prostate cancers and biochemically, PTEN is a lipid phosphatase which opposes the activation of the oncogenic PI3K-AKT signalling network. However, PTEN also has additional potential mechanisms of action, including protein phosphatase activity. Using a mutant enzyme, PTEN Y138L, which selectively lacks protein phosphatase activity, we characterised genetically modified mice lacking either the full function of PTEN in the prostate gland or only lacking protein phosphatase activity. The phenotypes of mice carrying a single allele of either wild-type Pten or PtenY138L in the prostate were similar, with common prostatic intraepithelial neoplasia (PIN) and similar gene expression profiles. However, the latter group, lacking PTEN protein phosphatase activity additionally showed lymphocyte infiltration around PIN and an increased immune cell gene expression signature. Prostate adenocarcinoma, elevated proliferation and AKT activation were only frequently observed when PTEN was fully deleted. We also identify a common gene expression signature of PTEN loss conserved in other studies (including Nkx3.1, Tnf and Cd44). We provide further insight into tumour development in the prostate driven by loss of PTEN function and show that PTEN protein phosphatase activity is not required for tumour suppression.

How to do a thoracoscopic-assisted oesophagectomy with oesophagogastric anastomosis using a circular stapler.

Thoracoscopic mobilization of the oesophagus during oesophagectomy has many advantages over the traditional open approach including less blood loss, reduced pulmonary complications and shorter hospital stay. Minimally invasive intrathoracic oesophagogastric anastomosis can be technically challenging, with several different techniques described in the literature. Here, we describe a nuanced technique to perform an intracorporeal anastomosis using a circular stapler.

Identifying areas of weakness in thoracic surgery residency training: a comparison of the perceptions of residents and program directors.

OBJECTIVE: To identify core thoracic surgery procedures that require increased emphasis during thoracic surgery residency for residents to achieve operative independence and to compare the perspectives of residents and program directors in this regard. METHODS: A modified Delphi process was used to create a survey that was distributed electronically to all Canadian thoracic surgery residents (12) and program directors (8) addressing the residents' ability to perform 19 core thoracic surgery procedures independently after the completion of residency. Residents were also questioned about the adequacy of their operative exposure to these 19 procedures during their residency training. A descriptive summary including calculations of frequencies and proportions was conducted. The perceptions of the 2 groups were then compared using the Fisher exact test employing a Bonferroni correction. The relationship between residents' operative exposure and their perceived operative ability was explored in the same fashion. RESULTS: The response rate was 100% for residents and program directors. No statistical differences were found between residents' and program directors' perceptions of residents' ability to perform the 19 core procedures independently. Both groups identified lung transplantation, first rib resection, and extrapleural pneumonectomy as procedures for which residents were not adequately prepared to perform independently. Residents' subjective ratings of operative exposure were in good agreement with their reported operative ability for 13 of 19 procedures. CONCLUSION: This study provides new insight into the perceptions of thoracic surgery residents and their program directors regarding operative ability. This study points to good agreement between residents and program directors regarding residents' surgical capabilities. This study provides information regarding potential weaknesses in thoracic surgery training, which may warrant an examination of the curricula of existing programs as well as a reconsideration of what the scope of practice of a general thoracic surgeon should entail.

Caspofungin Etest endpoint for Aspergillus isolates shows poor agreement with the reference minimum effective concentration.

The Clinical and Laboratory Standards Institute (CLSI) M38-A2 reference broth microdilution (BMD) method for the antifungal susceptibility testing of filamentous fungi now includes guidelines for testing echinocandin activity using the minimum effective concentration (MEC) as the endpoint measurement. In this study, we compared the caspofungin Etest MIC on RPMI agar and Mueller-Hinton agar (supplemented with glucose and methylene blue [MGM]) to the BMD MEC for 345 clinical Aspergillus isolates, including A. flavus, A. fumigatus, A. nidulans, A. niger, and A. terreus. The essential agreement (+/-1 log(2) dilution) of the Etest on MGM and RPMI agar with the reference BMD MEC was 18 and 26%, respectively. The geometric mean values for BMD MEC and MGM Etest were 0.137 and 0.024 microg/ml, respectively, and the geometric mean values for BMD and RPMI agar were 0.128 and 0.031 microg/ml, respectively. Comparatively, 91% of paired MGM and RPMI Etest results were within 2 log(2) dilutions of each other and consistently produced clearly defined endpoints. In conclusion, the caspofungin Etest MIC, like the BMD MEC, is a reproducible endpoint but is markedly lower than the reference BMD. In anticipation of susceptibility breakpoint assignments, optimization studies will be required to improve the concordance of these two assays so that the potential for underreporting echinocandin resistance in Aspergillus is mitigated.