In vitro and in vivo effects of phenolic antioxidants against cisplatin-induced nephrotoxicity

We have investigated the effect of phenolic antioxidants on cisplatin-induced cytotoxicity in vero (African Green Monkey Kidney) cells and in rat renal cortical slices in vitro, and on cisplatin-induced nephrotoxicity in rats in vivo. Incubation of cisplatin with vero cells resulted in time- and concentration-dependent cytotoxicity, as characterized by decreased tryphan blue exclusion (TBE) and increased release of lactate dehydrogenase (LDH) into the medium. Cisplatin also caused reduction of glutathione (GSH) in a concentration-dependent manner. In the rat renal cortical slices model, incubation of cisplatin for 120 min caused an increase in malondialdehyde (MDA), a decrease in GSH and inhibited p-aminohippurate (PAH) uptake in a concentration-dependent manner. Among phenolic antioxidants, isoeugenol (IG) was found to be more active against cisplatin-induced cytotoxicity in vero cells as well as in rat renal cortical slices than eugenol (EG) and dehydrozingerone (DZ). However none of the test compounds were able to arrest the reduction of the GSH content induced by cisplatin in either the vero cells or the renal cortical slice model. Administration of cisplatin (3 mg/kg) i.p. to rats resulted in significant reduction of body weight, and elevation of blood urea nitrogen (BUN) and serum creatinine. Treatment with IG 10 mg/kg i.p. 1 h before cisplatin resulted in partial but significant protection against the cisplatin-induced reduction of body weight, and elevation of BUN and serum creatinine, the protection being 34, 46, and 62%, respectively. EG and DZ (10 mg/kg, i.p.) were found to be inactive in vivo. Because IG is a potent free radical scavenger and protects against cisplatin-induced toxicitiy, the present results have many clinical implications in chemotherapy and thus warrants further investigation.     

A physical approach to reduce nonspecific adhesion in molecular recognition atomic force microscopy

Atomic force microscopy is one of the few techniques that allow analysis of biological recognition processes at the single-molecule level. A major limitation of this approach is the nonspecific interaction between the force sensor and substrate. We have modeled the nonspecific interaction by looking at the interaction potential between a conical Si3N4 tip with a spherical end face and a mica surface in solution, using DLVO (Derjaguin, Landau, Verwey, Overbeek) theory and numerical calculations. Insertion of the tip-sample potential in a simulation of an approach-retract cycle of the cantilever gives the well-known force-distance curve. Simulating a force-distance curve at low salt concentration predicts a discrete hopping of the tip, caused by thermal fluctuations. This hopping behavior was observed experimentally and gave rise to a novel approach to making measurements in adhesion mode that essentially works in the repulsive regime. The distance between tip and sample will still be small enough to allow spacer-involved specific interactions, and the percentage of nonspecific interactions of the bare tip with the mica is minimized. We have validated this physical model by imaging intercellular adhesion molecule 1 (ICAM-1) antigen with a tip functionalized with anti-ICAM-1 antibody. The measurement demonstrated that a significant decrease in the number of nonspecific interactions was realized, and the topographical image quality and the specific bonding capability of the tip were not affected.     

Calculating the Risk of Batch Failure in the Manufacture of Drug Products

A statistical tool has been developed to estimate the risk of batch failure in commercial production from the results of the development scale-up campaigns. This tool serves to indicate the robustness of the manufacturing procedure and its ability to meet the specifications set. Other uses include assistance in the resetting of specifications in the light of actual scale-up batch manufacturing experience and assessing the impact of changes in specifications proposed by the registration authorities. A template is given that simplifies the calculations required.     

Elevated levels of the IGF-binding protein protease MMP-1 in asthmatic airway smooth muscle

We characterized the changes in nitric oxide (NO) levels in the brain during global forebrain ischemia and reperfusion and tested the ability of the natural flavonoid, quercetin, and a synthetic flavonoid, FB277, to increase the amount of available NO by elimination of the superoxide radicals produced during reperfusion. In Sprague-Dawley rats, we used a four-vessel occlusion model of forebrain ischemia (15 min) and reperfusion (30 min). Brain NO was measured on samples of cerebral cortex and cerebellum ex vivo by electron paramagnetic resonance (EPR) spectroscopy. The spin trap used was diethyldithiocarbamate sodium salt (DETC) associated with ferrous citrate. The complex Fe(DETC)2NO was detected at 77 K as a triplet signal at g = 2.035. Groups of animals were treated with quercetin or FB277 (3-morpholinomethyl-3',4',5,7tetramethoxyflavone) or polyethylene glycol-conjugated superoxide dismutase (PEG-SOD). In control (intact anesthetized animals), the signal was about 3 times greater in the cortex than in the cerebellum. During ischemia, the signal rose to 110% in cortex (NS) and 283% in cerebellum (P < 0.05). In reperfusion, it fell again to 91% of control in cerebellum (NS) and 35% in cortex (P < 0.05). Treatment by quercetin (5 mg/kg i.v.) of intact and ischemia-reperfusion groups did not significantly change the signal amplitude in the cerebellum, but did double it in the cortex (to 76% of control) for the ischemia-reperfusion group (P < 0.05). In contrast, FB277 (3.75 mg/kg i.v.) did not increase the signal in the cortex during ischemia-reperfusion, but did do so in the cerebellum (to 152% of control, P < 0.05). The results obtained for PEG-SOD (10,000 U/kg i.v.) were similar to those for FB277. In separate in vitro measurements, we found that quercetin but not FB277 efficiently scavenged superoxide. We hypothesize that quercetin but not FB277 scavenged superoxide anions released in the cortex during reperfusion, thus diminishing the amount of NO removed by the formation of peroxynitrite. The lack of effect of PEG-SOD may be related to the need for chronic treatment to obtain protection.     

Deletion of cell division cycle 2-like 1 gene locus on 1p36 in non-Hodgkin lymphoma

Our laboratories have documented a significantly high occurrence of chromosome 1p36 rearrangements in non-Hodgkin lymphoma (NHL). The cell division cycle 2-like 1(CDC2L1) (also known as TP58 or PITSLRE) gene, a protein kinase implicated in apoptotic signaling, is located at the very distal region of chromosome 1p36 and is likely to be disrupted by structural rearrangements involving 1p36. To determine the molecular consequences of the recurrent involvement of the 1p36 region, we examined metaphases containing 1p36 abnormalities from 31 specimens derived from 26 patients for the possible deletion of CDC2L1 by fluorescence in situ hybridization (FISH) using the TP58clk-1 DNA probe. Twenty-three cases exhibited the loss of CDC2L1 from the abnormal chromosome 1. In 2 of 26 cases, the gene locus was translocated to the partner chromosome, and in four specimens, all derived from one case, CDC2L1 was not deleted. This pilot investigation suggests that 1p36 rearrangements, and consequently the loss of the CDC2L1 gene locus, is important in NHL. This work also opens avenues for further molecular studies and prognostic correlations.     

The early active mobilization in the treatment of old lesions in muscles-flexors-tendons of the fingers of the hand in critical surgery zone

The results of application of an early active mobilization while 107 patients treatment with inveterate affection of the muscles-flexors tendons of the hand in critical zone were followed up from 5 mos till 2.5 yrs. Excellent and good results were obtained in 70.8% of observations.