Enhanced physical stability of human calcitonin after methionine oxidation

Calcitonin is a blood-calcium-lowering peptide, present in different species, which inhibits the resorption of bone by osteoclasts. Human calcitonin (hCT) is one of the few calcitonin peptides, which contains a methionine residue; this residue is in position 8. Methionines are known to be readily oxidized to sulfoxides both in vivo and in vitro. The current work describes the effect of methionine oxidation on the physical stability of hCT. Aggregation kinetics of human calcitonin were studied at different pH values by intrinsic fluorescence spectroscopy, turbidity at 350 nm, microscopy analyses, Nile Red, and 1,8-ANS fluorescence emission. In all the experiments, methionine oxidation reduced the aggregation rate of human calcitonin. The effect of methionine oxidation was independent of pH. Fluorescence lifetime data also showed that the conformation of hCT in the aggregated state can be influenced by methionine oxidation. A hypothesis for the enhanced physical stability of oxidized hCT is presented and discussed.     

In vitro and in vivo characterization of the novel GABAB receptor positive allosteric modulator, 2-{1-[2-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-yl]-2-piperidinyl}ethanol (CMPPE)

There is preclinical evidence supporting the finding that the GABA(B) receptor orthosteric agonist, baclofen, has significant effects on eating behavior suggesting the potential therapeutic application of this compound for the treatment of eating related disorders. However, the wide clinical use of baclofen might be limited by the appearance of sedative and motor impairment effects. The identification of positive allosteric modulators (PAMs) of GABA(B) receptors represents a novel therapeutic approach to reduce the centrally-mediated adverse effects typical of the GABA(B) receptor orthosteric agonist. In the present work, we report the in vitro profile of a novel chemical structure, 2-{1-[2-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-yl]-2-piperidinyl}ethanol (CMPPE) identified by screening the GSK compound collection. CMPPE potentiates GABA-stimulated [(35)S]GTPγS binding to membranes of human recombinant cell line and of rat brain cortex. GABA concentration-response curves (CRC) in the presence of fixed concentrations of CMPPE, in rat native tissue, revealed an increase of both the potency and maximal efficacy of GABA. A similar modulatory effect was observed in GABA(B) receptor-mediated activation of inwardly rectifying potassium channels in hippocampal neurons. CMPPE (30-100 mg/kg) and GS39783 (100 mg/kg) significantly decreased food consumption in rat without impairment on the animal locomotor activity. On the contrary, baclofen (2.5 mg/kg) decreased both food intake and motor performance. All together these findings confirm the role of GABA(B) system in controlling animal food intake and for the first time demonstrate that GABA(B) receptor PAMs may represent a novel pharmacological approach to treat eating disorders without unwanted sedative effects.     

Radiotherapy and concomitant temozolomide during the first and last weeks in high grade gliomas: long-term analysis of a phase II study

We tested the efficacy and safety of temozolomide (TMZ) when given concomitantly to radiotherapy only in the first and last weeks of treatment to patients affected by high grade gliomas. Conformal radiotherapy (CTV1: tumor bed + residual tumor if present + 1.5 cm, 5,940 cGy, 180 cGy/day; CTV2: oedema, 3,960 cGy, 180 cGy/day) was associated with TMZ, 75 mg/m² × 5 days, the first and last weeks of radiotherapy. Adjuvant chemotherapy with TMZ (150 mg/mq daily × 5 days, q28 on the first cycle, 200 mg/mq daily × 5 days, q28 for the following cycles) was given, after chemoradiation, until disease progression or up to 6 cycles. From October 2000 to December 2003, 29 patients (25 GBL, 86.2%; 4 AA, 13.8%) were enrolled in this study. Twenty-two patients (75.8%) received a median 6 cycles of adjuvant chemotherapy with TMZ (range 1–20). Hematological toxicity was absent during concomitant chemoradiation and mild in adjuvant therapy, while neurological toxicity (seizures) was observed only in one case. At a median follow-up of 66 months (range 3–96), median progression-free survival (PFS) was 8 months, with a 1- and 2-year PFS of 46.7 and 28.7%, respectively; median overall survival (OS) time was 21 months, with a 1- and 2-year OS of 69.2 and 42.3%, respectively. In our experience, TMZ proved to be effective even when given only during the first and the last week of radiotherapy, with lower hematological toxicity.     

The Italian Research Group for Gastric Cancer (GIRCG) guidelines for gastric cancer staging and treatment: 2015

Methodology for the diagnosis and staging of early gastric cancer (EGC) has improved in Japan since the development of the gastro-camera and determination of a definition of EGC. Imaging technology has been steadily evolving in the endoscopy field. Improvements in the resolution of standard endoscopy images used in screening and surveillance provide greater opportunities to find gastric cancer earlier. Image enhancement endoscopy (IEE), such as narrow band imaging (NBI), highlights mucosal structures and vascularity. In particular, when NBI is used with magnifying endoscopy, it reveals fine details of subtle superficial abnormalities of EGC that are difficult to recognize using standard white light endoscopy. IEE-assisted magnifying endoscopy has improved the accuracy of the differentiation of superficial gastric cancer as well as delineation of the diseased mucosa. The advanced imaging technology enables precise assessment of the risk of lymph node metastasis of EGC and is widely used to determine indications for endoscopic treatment. It is not an overstatement to say that this has become the basis for the current development and dissemination of endoscopic treatments. Moreover, the resolution of endoscopic imaging has been upgraded to the microscopy level by the development of endomicroscopy, including endocytoscopy and confocal laser endomicroscopy. Endomicroscopy allows real-time histological analysis of living tissue during routine endoscopy and may reduce the number of biopsies needed to reach the correct diagnosis, minimizing the risk of sampling errors.     

Phospho-TCTP as a therapeutic target of dihydroartemisinin for aggressive breast cancer cells

Upregulation of Translationally Controlled Tumor Protein (TCTP) is associated with poorly differentiated aggressive tumors, including breast cancer, but the underlying mechanism(s) are still debated. Here, we show that in breast cancer cell lines TCTP is primarily localized in the nucleus, mostly in the phosphorylated form.The effects of Dihydroartemisinin (DHA), an anti-malaria agent that binds TCTP, were tested on breast cancer cells. DHA decreases cell proliferation and induces apoptotic cell death by targeting the phosphorylated form of TCTP. Remarkably, DHA enhances the anti-tumor effects of Doxorubicin in triple negative breast cancer cells resulting in an increased level of apoptosis. DHA also synergizes with Trastuzumab, used to treat HER2/neu positive breast cancers, to induce apoptosis of tumor cells.Finally, we present new clinical data that nuclear phospho-TCTP overexpression in primary breast cancer tissue is associated with high histological grade, increase expression of Ki-67 and with ER-negative breast cancer subtypes. Notably, phospho-TCTP expression levels increase in trastuzumab-resistant breast tumors, suggesting a possible role of phospho-TCTP as a new prognostic marker.In conclusion, the anti-tumor effect of DHA in vitro with conventional chemotherapeutics suggests a novel therapeutic strategy and identifies phospho-TCTP as a new promising target for advanced breast cancer.     

Dynamic mechanical thermal analysis of hypromellose 2910 free films

It is common practice to coat oral solid dosage forms with polymeric materials for controlled release purposes or for practical and aesthetic reasons. Good knowledge of thermo-mechanical film properties or their variation as a function of polymer grade, type and amount of additives or preparation method is of prime importance in developing solid dosage forms. This work focused on the dynamic mechanical thermal characteristics of free films of hypromellose 2910 (also known as HPMC), prepared using three grades of this polymer from two different manufacturers, in order to assess whether polymer chain length or origin affects the mechanical or thermo-mechanical properties of the final films. Hypromellose free films were obtained by casting their aqueous solutions prepared at a specific concentrations in order to obtain the same viscosity for each. The films were stored at room temperature until dried and then examined using a dynamic mechanical analyser. The results of the frequency scans showed no significant differences in the mechanical moduli E′ and E″ of the different samples when analysed at room temperature; however, the grade of the polymer affected material transitions during the heating process. Glass transition temperature, apparent activation energy and fragility parameters depended on polymer chain length, while the material brand showed little impact on film performance.     

Effects of intravenous zoledronic acid and oral ibandronate on early changes in markers of bone turnover in patients with bone metastases from non-small cell lung cancer

Background

The aim of this study was to assess the early effects of zoledronic acid (ZOL) and oral ibandronate (IBA) on the bone resorption marker s-CTX (serum C-telopeptide of collagen type I) and the bone formation marker B-ALP (bone-alkaline phosphatase) in patients with bone metastases from non-small cell lung cancer (NSCLC).

Methods

Fifty-five patients with at least one site of bone metastasis secondary to NSCLC were randomly assigned to receive intravenous ZOL 4?mg every 4?weeks, or oral IBA 50?mg/day.

Results

At 1?month of treatment, s-CTX was reduced by 54.8% (95% CI 40.4?C59.8%) in the ZOL group (26 evaluable patients) compared with 38.2% (95% CI 29.8?C48.7%) in the oral IBA group (27 evaluable patients) (p?=?0.03). At 3?months, s-CTX was reduced by 72.6% (95% CI 58.6?C71.3%) in the ZOL group, compared with 66.4% (95% CI 54.3?C79.5%) in the oral IBA group (p?=?0.22). Both bisphosphonates similarly decreased the bone marker B-ALP at 1?month (ZOL 24.7%, 95% CI 3.6?C39.5%, and IBA 24.2%, 95% CI 2.8?C43.4%) and 3?months (ZOL 28.6%, 95% CI +2.8?C43.3%, and IBA 24.2%, 95% CI 3.2?C47.4%). Both bisphosphonates were well tolerated.

Conclusion

Considering the changes in bone markers, ZOL and oral IBA show comparable efficacy in patients with NSCLC and bone metastases.     

Oxidized Recombinant Human Growth Hormone That Maintains Conformational Integrity

Chemical degradations often induce changes in protein conformation and thus influence protein activity and protein stability in solutions. One difficulty in studying of chemical degradations on protein aqueous properties is to obtain sufficient amount of chemically degraded protein which is well characterized. Chemical degradation protocols that are often used may induce also conformation changes and aggregation of the protein. In this article we studied the effect of methionine oxidation on the conformation of recombinant human growth hormone (r-hGH). In literature it is reported that oxidation of methionine residues induces conformation changes on r-hGH. In our study, oxidation of r-hGH was performed by incubation with hydrogen peroxide under mild conditions. Mass spectrometry and chromatographic analysis revealed that oxidation with hydrogen peroxide resulted in more than 90% of oxidized r-hGH. By extensive spectroscopic characterizations no detectable change in conformation and aggregation of r-hGH after oxidation was found. In conclusion, mild oxidation conditions led to selective oxidation of the two more accessible methionine residues of r-hGH (Met¹⁴ and Met¹²⁵) and did not results in any conformation change of the protein. These findings prove that oxidation of human growth hormone does not influence protein conformation and demonstrate the importance of employing mild conditions during production of oxidized protein.