Characterization and regulation of MT1‐MMP cell surface‐associated activity

Quantitative assessment of MT1‐MMP cell surface‐associated proteolytic activity remains undefined. Presently, MT1‐MMP was stably expressed and a cell‐based FRET assay developed to quantify activity toward synthetic collagen‐model triple‐helices. To estimate the importance of cell surface localization and specific structural domains on MT1‐MMP proteolysis, activity measurements were performed using a series of membrane‐anchored MT1‐MMP mutants and compared directly with those of soluble MT1‐MMP. MT1‐MMP activity (k_cat/K_M) on the cell surface was 4.8‐fold lower compared with soluble MT1‐MMP, with the effect largely manifested in k_cat. Deletion of the MT1‐MMP cytoplasmic tail enhanced cell surface activity, with both k_cat and K_M values affected, while deletion of the hemopexin‐like domain negatively impacted K_M and increased k_cat. Overall, cell surface localization of MT1‐MMP restricts substrate binding and protein‐coupled motions (based on changes in both k_cat and K_M) for catalysis. Comparison of soluble and cell surface‐bound MT2‐MMP revealed 12.9‐fold lower activity on the cell surface. The cell‐based assay was utilized for small molecule and triple‐helical transition state analog MMP inhibitors, which were found to function similarly in solution and at the cell surface. These studies provide the first quantitative assessments of MT1‐MMP activity and inhibition in the native cellular environment of the enzyme.     

Medicine‐associated constipation in primary care

A pharmacoepidemiological study was undertaken to identify drugs associated with constipation Prescribing records of 141,056 individuals were searched of which 10,443 had constipation Antipsychotics, antidepressants, analgesics, drugs for genito‐urinary disorders and preparations used in anaemia were associated with constipation Antispasmodics or antihaemorrhoidal preparations were associated with constipation but this probably reflects management of the underlying disease NSAIDs were not found to be associated with drug‐induced constipation     

Murrayanine Attenuates Lipopolysaccharide‐induced Inflammation and Protects Mice from Sepsis‐associated Organ Failure

Murrayanine (MK) is the main compound isolated from Murraya koenigii, an aromatic plant belonging to the Rutaceae family, also known as curry leaf tree. Murrayanine was reported to possess potential antioxidant, antimycobacterial and antifungal effects. However, its effect in sepsis remains unclear. This study was designed to investigate the anti‐inflammatory effect of MK using both in vitro and in vivo assay. Results of this study indicated that MK decreased NO, TNF‐α and IL‐6 production in both lipopolysaccharide (LPS)‐stimulated RAW 264.7 cells and murine peritoneal macrophages. Moreover, iNOS and COX‐2 protein expression as well as their downstream product, PGE2, was also decreased effectively in RAW 264.7 cells. Furthermore, MK decreased the phosphorylation of IKB and repressed NF‐kB activity in LPS‐activated RAW 264.7 cells. Additionally, we evaluated MK efficacy in vivo using LPS‐induced sepsis, a systemic inflammation model in mice. Administration of MK inhibits pro‐inflammatory cytokines (TNF‐α and IL‐6) secretion; decreases AST, ALT, BUN and CRE level in mouse sera; mitigates lung, liver and kidney injuries; and also increases LPS‐challenged mice survival rate. Collectively, our results suggest that MK exerts potential as a new anti‐inflammatory and immunosuppressive drug in sepsis treatment.     

Ramipril and resveratrol co‐treatment attenuates RhoA/ROCK pathway‐regulated early‐stage diabetic nephropathy‐associated glomerulosclerosis in streptozotocin‐induced diabetic rats

Clinical studies have shown that hyperglycemia can induce early‐stage diabetic nephropathy (DN). Furthermore, oxidative stress, tubular epithelial‐mesenchymal transition and extracellular matrix accumulation promote the progression of DN to chronic kidney disease and tubulointerstitial fibrosis. It is necessary to initiate treatment at the early stages of DN or even during the early stages of diabetes. In this work, rats with streptozotocin (STZ)‐induced diabetes mellitus (DM) presented early DN symptoms within 45 days, and collagen accumulation in the glomerulus of the rats was primarily mediated through the RhoA/ROCK pathway instead of the TGF‐β signaling pathway. Resveratrol (15 mg/kg/day) and ramipril (10 mg/kg/day) co‐treatment of STZ‐induced DN rats showed that glomerulosclerosis in early‐stage DN was reversible (P < .05 compared with that in STZ‐induced DM rats). The results of this study support early intervention in diabetes or DN as a more efficient therapeutic strategy.     

Design and synthesis of pyrazole–pyrazoline hybrids as cancer‐associated selective COX‐2 inhibitors

In continuation of our previous work on cancer and inflammation, 15 novel pyrazole–pyrazoline hybrids ( WSPP1 – 15 ) were synthesized and fully characterized. The formation of the pyrazoline ring was confirmed by the appearance of three doublets of doublets in ¹H nuclear magnetic resonance spectra exhibiting an AMX pattern for three protons (H_A, H_M, and H_X) of the pyrazoline ring. All the synthesized compounds were screened for their in vitro anticancer activity against five cell lines, that is, MCF‐7, A549, SiHa, COLO205, and HepG2 cells, using the MTT growth inhibition assay. 5‐Fluorouracil was taken as the positive control in the study. It was observed that, among them, WSPP11 was found to be active against A549, SiHa, COLO205, and HepG2 cells, with IC₅₀ values of 4.94, 4.54, 4.86, and 2.09 µM. All the derivatives were also evaluated for their cytotoxicity against HaCaT cells. WSPP11 was also found to be nontoxic against normal cells (cell line HaCaT), with an IC₅₀ value of more than 50 µM. The derivatives were also evaluated for their in vitro anti‐inflammatory activity by the protein (egg albumin) denaturation assay and the red blood cell membrane stabilizing assay, using diclofenac sodium and celecoxib as standard. Compounds that showed significant anticancer and anti‐inflammatory activities were further studied for COX‐2 inhibition. The manifestation of a higher COX‐2 selectivity index of WSPP11 as compared with other derivatives and an in vitro anticancer activity against four cell lines further established that compounds that were more selective toward COX‐2 also exhibited a better spectrum of activity against various cancer cell lines.     

Synthesis of potent lymphocyte function‐associated antigen‐1 inhibitors labeled with carbon‐14 and deuterium,part 1

The lymphocyte function‐associated antigen‐1 (LFA‐1) is an essential component in normal immune system function and is a target for drug discovery for its broad therapeutic potential in treating inflammatory diseases. Here, we report the synthesis of three potent antagonists of LFA‐1 labeled with carbon‐14 and deuterium to support drug metabolism and pharmacokinetics studies. Carbon‐14 labeled (R)‐1‐acetyl‐5‐(4‐bromobenzyl)‐3‐(3,5‐dichlorophenyl)‐5‐methyl‐imidazolidine‐2,4‐dione (1) was prepared in 27% radiochemical yield in two steps and with a specific activity of 2.1 GBq/mmol by using [¹⁴C]‐phosgene. Carbon‐14 labeled 5‐bromopyrimidine was used to prepare (R)‐5‐(1‐piperazinylsulfonyl)‐1‐(3,5‐dichlorophenyl)‐3‐[4‐(5‐pyrimidinyl)benzyl]‐3‐methyl‐1‐H‐imidazo[1,2a]imidazol‐2‐one (2) and (R)‐1‐[7‐(3,5‐dichlorophenyl)‐5‐methyl‐6‐oxo‐5‐(4‐pyrimidin‐5‐yl‐benzyl)‐6,7‐dihydro‐5H‐imidazo[1,2‐a]imidazole‐3‐sulfonyl]piperidin‐4‐carboxylic acid amide (3) via a Suzuki reaction with the corresponding boronic acid esters in 42% and 67% radiochemical yield and specific activities of 1.85 GBq/mmol and 1.95 GBq/mmol, respectively. Deuterium labeled piperazine was reacted with the sulfonyl chloride derivative (7), followed by a Suzuki coupling to the pyrimidine boronic ester to give deuterium labeled (2) in 47% yield. Deuterium labeled isonipecotamide was reacted in a similar way with the sulfonyl chloride derivative (14) to furnish deuterium labeled (3) in one step and in 94% yield. Copyright © 2011 John Wiley & Sons, Ltd.