Chinmoy S Dey

P-glycoprotein inhibitors and their screening: a perspective from bioavailability enhancement

Drug efflux pumps like P-glycoprotein (P-gp) and multidrug resistance (MDR) proteins were recognized to possess functional role in determining the pharmacokinetics of drugs administered by peroral as well as parenteral route. Advancements in molecular biology, to some extent, had revealed the structure, localization and functional role of P-glycoprotein and its mechanism of drug efflux. Broad substrate recognition by this protein and clinical implications of its inhibition has revolutionized cancer chemotherapy leading to design and development of novel P-glycoprotein inhibitors. In the recent times, the application of these inhibitors in improving peroral drug delivery has gained special interest. Inhibition of P-glycoprotein improves intestinal absorption and tissue distribution while reducing the substrate metabolism and its elimination. Eventually, various screening methodologies have been developed for determining the activity of P-glycoprotein, kinetics of drug transport and identification of substrates and inhibitors. In the present review, techniques used for screening P-glycoprotein inhibitors and the scope of these inhibitors in optimizing peroral drug absorption and pharmacokinetics are discussed along with a brief introduction to P-glycoprotein, its physiological function and active role in extrusion of drugs.

Development of insulin resistance and reversal by thiazolidinediones in C2C12 skeletal muscle cells.

AIM/HYPOTHESISnThe aim of this study was to develop an insulin-resistant cell culture model in skeletal muscle cell line by chronic presence of insulin in serum-free medium and to determine the effect of thiazolidinediones on insulin signaling.nnnMETHODSnWe differentiated C2C12 in a combination of serum-free medium in presence or absence of insulin and determined differentiation by creatine kinase activity, myogenin and MyoD expression. The development of insulin resistance was determined by tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1, phosphatidylinositol 3-kinase activity associated with insulin receptor substrate-1 and glucose uptake. We treated the cells with 50 microM of thiazolidinediones to determine the effect on these parameters.nnnRESULTSnC2C12 cells were differentiated normally in the serum-free medium in the absence or presence of insulin. Chronic treatment of insulin resulted in reduced tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1; activation of phosphatidylinositol 3-kinase was impaired and insulin-stimulated glucose uptake was reduced. The treatment of insulin-resistant cells with thiazolidinediones resulted in the enhancement of insulin signaling pathway by increasing tyrosine phosphorylation of insulin receptor, insulin receptor substrate-1, phosphatidylinositol 3-kinase activity and glucose uptake.nnnCONCLUSION/INTERPRETATIONnThese results indicate that insulin resistance can be developed in C2C12 skeletal muscle cell line. These findings implicate a direct mechanism of action of thiazolidinediones on skeletal muscle.

PPAR‐γ expression modulates insulin sensitivity in C2C12 skeletal muscle cells

Peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) expression is very low in skeletal muscle cells, which is one of the most important target tissues for insulin and plays a predominant role in glucose homeostasis. It has recently been shown that muscle‐specific PPAR‐γ deletion in mouse causes insulin resistance. However, it is likely that the observed effects might be due to secondary interaction in whole animal. The aim of the study was to explore the role of muscle PPAR‐γ in insulin sensitivity. We stably transfected C2C12 skeletal muscle cells with plasmids containing sense or antisense constructs of PPAR‐γ and examined the effect of modulation of PPAR‐γ expression in terms of glucose uptake. Effect was also examined in insulin‐resistant C2C12 skeletal muscle cells. In transfected C2C12 cell line, the inhibition of PPAR‐γ expression (23.0±0.005%) was observed to induce insulin resistance as determined by functional assessment of 2‐deoxyglucose incorporation. Overexpression of PPAR‐γ (28.5±0.008%) produced an additional effect on insulin (100 nM) and Pioglitazone (50 μM), resulting in 42.7±3.5% increase in glucose uptake as against 29.2±2.8% in wild‐type C2C12 skeletal muscle cells differentiated under normal (2% horse serum) condition. Under similar treatment, PPAR‐γ overexpressing cells resistant to insulin exhibited enhanced glucose uptake upto 60.7±4.08%, as compared to 23.8±5.1% observed in wild‐type C2C12 skeletal muscle cells. These data demonstrate a direct involvement of PPAR‐γ in insulin sensitization of TZD action on skeletal muscle cells, and suggest that pharmacological overexpression of muscle PPAR‐γ gene in skeletal muscle might be a useful strategy for the treatment of insulin resistance.

Altered PPARγ expression inhibits myogenic differentiation in C2C12 skeletal muscle cells

Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily known to regulate adipocyte differentiation. However, its role in skeletal muscle differentiation is not known. To investigate possible involvement of PPARγ in skeletal muscle differentiation, we modulated its expression in C2C12 mouse skeletal muscle cells by stable transfection with sense or antisense plasmid constructs of PPARγ cDNA. Phenotypic observations and biochemical analysis of different myogenic markers showed that altered expression of PPARγ inhibited the formation of myotubes, as well as expression of muscle-specific myogenic proteins including myogenin, MyoD and creatine kinase activity. Together, we show that critical expression of PPARγ is required for skeletal muscle cells differentiation.

Transdermal iontophoresis of insulin. Part 1: A study on the issues associated with the use of platinum electrodes on rat skin

We have studied the issues associated with the use of platinum electrodes for transdermal iontophoretic delivery of peptides, using insulin as a model peptide. Insulin permeation was studied using full‐thickness rat skin by varying the donor solution pH as a function of electrode polarity. The stability of insulin under the iontophoretic conditions was studied using TLC, SDS‐polyacrylamide gel electrophoresis and HPLC. Large pH shifts were observed during anodal iontophoresis (AI), when the donor solution pH was above the isoelectric point of insulin and in cathodal iontophoresis (CI), when the donor solution pH was below the isoelectric point of insulin. The direction and magnitude of electroosmotic flow was influenced by pH of the donor solution and the electrode polarity. On the other hand, the buffer used to maintain the pH governed the contribution of electrorepulsion to the overall transport of insulin. Electrochemical degradation of insulin was significant during Al at pH 7.4. Among the pH investigated, Al of insulin at pH 3.6 and Cl at pH 8.35 were better, as the pH shift was relatively less and electrochemically more stable during iontophoresis as compared with other pH. In summary, the pH shift caused by platinum electrodes had a significant influence on the permeation and stability of insulin.

Differential activation of ERK and JNK by arsenite in mouse muscle cells.

We studied the activation of MAPKs, such as ERK and JNK, by arsenite in C2C12 mouse skeletal muscle cells as a function of proliferation and differentiation. Data showed that both ERK and JNK were activated by arsenite in proliferated and differentiated cells in a differential manner. The activation of the enzymes was not due to alteration in their concentration. The activities were independent of each other. ERK activation was possibly partly through the activity of Ras, Raf and the MEK cascade, and due to oxidative stress, which possibly led to the activation of the transcription factor, Elk-1. In contrast, the activation of JNK was solely due to the generation of free radicals, resulting in activation of c-Jun and perhaps Elk-1. These results show for the first time that, in skeletal muscle, stress caused by arsenite involves the MAP-kinase signal transduction cascade, perhaps in a cell type-specific regulatory pathway.

Restoration of impaired p38 activation by insulin in insulin resistant skeletal muscle cells treated with thiazolidinediones

We have previously reported that thiazolidinediones (TZDs) are able to restore the tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1, activation of phosphatidyl inositol 3-kinase and glucose uptake in insulin resistant skeletal muscle cells [21]. In this study, we investigated the effects of insulin stimulation and TZDs on the role of mitogen-activated protein kinase (MAPK) in insulin resistant skeletal muscle cells. All the three MAPKs [extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK] were activated by insulin in the sensitive skeletal muscle cells. In contrast, activation of p38 MAPK was impaired in insulin resistant cells, where as ERK and JNK were activated by insulin. Treatment with TZDs resulted in the restoration of p38 MAPK activity in insulin resistant cells. The treatment of cells with p38 MAPK inhibitor, SB203580, blocked the insulin stimulated glucose uptake in sensitive as well as resistant cells and it also prevented the activation of p38 by insulin. These results suggest the potential involvement of p38 as well as the mechanistic role of TZDs in insulin resistance.

Differential regulation of map kinase isoforms by H2O2 in neuronal cells

The mitogen-activated protein kinase (MAPK) family is involved in the regulation of cellular proliferation, differentiation and stress signals. In this study, we investigated the role of MAPK in response to H2O2, an oxidative stress, in neuronal cells. Activation of c-Jun-N-terminal kinase (JNK) was sustained, where as extracellular signal-regulated kinase (ERK) and p38 MAPK were transiently activated in response to H2O2 treatment. Inhibition of ERK and p38 resulted in higher activation of JNK in response to H2O2 treatment. The treatment with H2O2 led to apoptosis. The results implicate potential role of MAP kinases in neurodegenerative disorders mediated by oxidative stress.