Process Mass Intensity (PMI): A Holistic Analysis of Current Peptide Manufacturing Processes Informs Sustainability in Peptide Synthesis.

Small molecule therapeutics represent the majority of the FDA-approved drugs. Yet, many attractive targets are poorly tractable by small molecules, generating a need for new therapeutic modalities. Due to their biocompatibility profile and structural versatility, peptide-based therapeutics are a possible solution. Additionally, in the past two decades, advances in peptide design, delivery, formulation, and devices have occurred, making therapeutic peptides an attractive modality. However, peptide manufacturing is often limited to solid-phase peptide synthesis (SPPS), liquid phase peptide synthesis (LPPS), and to a lesser extent hybrid SPPS/LPPS, with SPPS emerging as a predominant platform technology for peptide synthesis. SPPS involves the use of excess solvents and reagents which negatively impact the environment, thus highlighting the need for newer technologies to reduce the environmental footprint. Herein, fourteen American Chemical Society Green Chemistry Institute Pharmaceutical Roundtable (ACS GCIPR) member companies with peptide-based therapeutics in their portfolio have compiled Process Mass Intensity (PMI) metrics to help inform the sustainability efforts in peptide synthesis. This includes PMI assessment on 40 synthetic peptide processes at various development stages in pharma, classified according to the development phase. This is the most comprehensive assessment of synthetic peptide environmental metrics to date. The synthetic peptide manufacturing process was divided into stages (synthesis, purification, isolation) to determine their respective PMI. On average, solid-phase peptide synthesis (SPPS) (PMI ≈ 13,000) does not compare favorably with other modalities such as small molecules (PMI median 168-308) and biopharmaceuticals (PMI ≈ 8300). Thus, the high PMI for peptide synthesis warrants more environmentally friendly processes in peptide manufacturing.

Syndrome Valentino From a De Novo Aetiology - Acute Pancreatitis.

Valentino syndrome is one of the rare classical presentations of duodenal perforation, wherein the leaked contents collect at the right lower quadrant of the abdomen causing local peritonitis and mimicking appendicitis. Here we present a case profile of a 28-year-old gentleman, who presented with right lower quadrant abdominal pain and mass, which was clinically diagnosed as acute appendicular inflammatory mass. Later with laboratory reports and radiological imaging, he was confirmed to have acute pancreatitis, and actually the peripancreatic fluid collection has tracked down into the right iliac fossa and pelvis to present similar to Valentino syndrome. This article is reported to highlight acute pancreatitis as a cause of Valentino syndrome.

MicroRNA-432-5p regulates sprouting and intussusceptive angiogenesis in osteosarcoma microenvironment by targeting PDGFB.

Osteosarcoma (OS) is a type of bone tumor conferred with high metastatic potential. Attainable growth of tumors necessitates functional vasculature mediated by sprouting angiogenesis (SA) and intussusceptive angiogenesis (IA). However, the regulation of IA and SA is still unclear in OS. To understand the mechanisms adopted by OS to induce angiogenesis, initially, we assessed the expression profile of a set of miRNAs' in both OS cells (SaOS2 and MG63) and normal bone cells. Amongst them, miR-432-5p was found to be highly downregulated in OS. The functional role of miR-432-5p in OS was further analyzed using miR-432-5p mimic/inhibitor. Platelet-derived growth factor-B (PDGFB) was found to be a putative target of miR-432-5p and it was further confirmed that the PDGFB 3'UTR is directly targeted by miR-432-5p using the luciferase reporter gene system. PDGFB was found to be secreted by OS to regulate angiogenesis by targeting the cells in its microenvironment. The conditioned medium obtained from miR-432-5p mimic transfected MG63 and SaOS2 cells decreased cell viability, proliferation, migration, and aorta ring formation in endothelial cells. The miRNA mimic/inhibitor transfected MG63 and SaOS2 cells were placed on SA (day 6) and IA (day 9) phase of CAM development to analyze SA and IA mechanisms. It was found that miR-432-5p mimic transfection in OS promotes the transition of SA to IA which was documented by the angiogenic parameters and SA and IA-associated gene expression. Interestingly, this outcome was also supported by the zebrafish tumor xenograft model. Corroborating these results, it is clear that miR-432-5p expression in OS cells regulates SA and IA by targeting PDGFB genes. We conclude that targeting miR-432-5p/PDGFB signaling can be a potential therapeutic strategy to treat OS along with other existing strategies.

Characterization of Runx2 phosphorylation sites required for TGF-ß1-mediated stimulation of matrix metalloproteinase-13 expression in osteoblastic cells.

Transforming growth factor-beta1 (TGF-ß1), a highly abundant growth factor in skeletal tissues, stimulates matrix metalloproteinase-13 (MMP-13) expression in osteoblastic cells. MMP-13 plays a critical role in bone remodeling. Runx2, a bone transcription factor, is required for TGF-ß1-mediated stimulation of MMP-13 expression in osteoblastic cells. In this study, the molecular mechanism responsible for TGF-ß1-stimulation of MMP-13 expression via Runx2 in osteoblastic cells was elucidated. TGF-ß1 stimulated the phosphorylation of Runx2 at serine amino acids, and ERK inhibition blocked this effect in rat (UMR106-01) and human (MG-63) osteoblastic cells. Pretreatment with okadaic acid, a serine-threonine phosphatase inhibitor, increased Runx2 serine phosphorylation in osteoblastic cells. When cells were pretreated with an ERK inhibitor, TGF-ß1-mediated stimulation of MMP-13 mRNA expression decreased. Nano-ESI/LC/MS analysis identified that TGF-ß1 stimulates Runx2 phosphorylation at three serine amino acids. Transient transfection of mouse mesenchymal stem cells (C3H10T1/2) with Runx2 serine mutant constructs decreased TGF-ß1-mediated Runx2 serine phosphorylation. A luciferase reporter assay identified that TGF-ß1 stimulated MMP-13 promoter activity in these cells only in the presence of the wild Runx2 construct, and not with mutant Runx2. Thus, TGF-ß1 stimulates the phosphorylation of Runx2 at three serine amino acids, and this event is required for MMP-13 expression in osteoblastic cells. Hence, this study contributes to the knowledge of events governing bone remodeling and bone-related diseases.

Evaluating the efficacy of memantine on improving cognitive functions in epileptic patients receiving anti-epileptic drugs: A double-blind placebo-controlled clinical trial (Phase IIIb pilot study).

OBJECTIVES: People with epilepsy have greater cognitive and behavioral dysfunction than the general population. There is no specific treatment available for cognitive impairment of these patients. We aimed to evaluate the effects of memantine, an N-methyl-D-aspartate-type glutamate receptor noncompetitive antagonist, on improving cognition and memory functions in epileptic patients with cognitive and memory impairment, who received anti-epileptic drugs (AEDs). METHODS: We did a randomized, double-blind, placebo-controlled parallel group trial, in SRM Medical College Hospital and Research Centre, Kattankulathur, Kancheepuram, Tamil Nadu, India between April 2013 and September 2013. Fifty-nine epileptic patients taking AEDs with subjective memory complaints were recruited and randomized to either Group 1 to receive 16 weeks of once-daily memantine, (5 mg for first 8 weeks, followed by memantine 10 mg for next 8 weeks) or Group 2 to receive once daily placebo. This trial is registered with Clinical Trial Registry of India CTRI/2013/04/003573. RESULTS: Of 59 randomized patients, 55 patients completed the study (26 memantine and 29 placebo). Memantine group showed statistically significant improvement in total mini mental state examination score from baseline (P = 0.765) to 16(th) week (P < 0.001) in comparison with the placebo. The Weshler's Memory Scale total score in memantine group improved significantly after 8 weeks (P = 0.002) compared with baseline (P = 0.873) and highly significant at the end of 16(th) week (P < 0.001). The self-rated quality of life and memory in memantine group also significantly improved at the study end. CONCLUSION: We conclude that once-daily memantine (10 mg) treatment significantly improved cognition, memory and quality of life in epileptic patients with mild to moderate cognitive impairment and was found to have a favorable safety profile.

Recurrence patterns and management options in aggressive fibromatosis.

Aggressive fibromatosis is a rare neoplasm arising from musculoaponeurotic structures. Our aim is to share our experience with this rare tumor in our institute and to discuss the more perplexing recurrence patterns and the management options. This is a retrospective study of the disease, treated in our institute for the past fourteen years. A total of 36 patients were analyzed. The demographic pattern of the disease, various treatment modalities offered and their outcome along with patterns of recurrence were studied. Our study showed a demographic pattern mostly similar to the rest of the world. But the pattern of recurrence and the multicentric and the non-random pattern of presentation observed in our study showed some difference from the other studies. We suggest surgery as the primary modality with radiation reserved for select patients with margin positivity, inoperable tumors, and multiple tumors. Since the disease has a long natural history a wait and watch policy can be observed for giving adjuvant RT. There is need for prospective multi-institutional RCTs to shed light on the unknown facts about this disease.

Limb conservation in extremity soft tissue sarcomas with vascular involvement.

BACKGROUND: The major neurovascular involvement and large primary tumors are indication of amputation. The present study is an attempt to explore the feasibility of a limb salvage surgery in extremity sarcoma cases with major vessel involvement. Oncological outcomes and surgery-related morbidities are compared with those reported in literature. MATERIALS AND METHODS: A retrospective review of all limb salvage surgeries done in our department between 2005 and 2008 was done and four cases of extremity sarcoma of lower limb involving femoral vessels analyzed. Interpretation of data from these cases, along with review of literature, is done. RESULTS: In all these cases a wide monobloc excision was done adhering to oncological principles. This required resection of superficial femoral artery alone in two cases, resection of superficial femoral artery along with common femoral vein and femoral nerve in another, and of common femoral vein alone in yet another. Reconstruction was done in all these cases with reversed long saphenous vein graft. Histopathology of resected margins was free of tumor in all the four patients. One patient developed local recurrence and one developed distant metastsis. Two were disease free for one year with good functional limb, one has been disease-free for three years and another was disease-free at two years, after which he defaulted further follow-up. One patient developed arterial blowout which required ligation of common femoral artery which resulted in gangrene of the limb. He underwent amputation. CONCLUSION: Major neurovascular involvement in extremity sarcoma is not considered a contraindication for limb salvage surgery. Review of literature also supports our view. Post-operative wound related complications are more in this group of patients. However, long term functional outcome is good. Literature suggests a good long term local control after vascular resection and reconstruction.