Mathematical Model-Based Accelerated Development of Extended-release Metformin Hydrochloride Tablet Formulation.

A computational fluid dynamic (CFD) model was developed to predict metformin release from a hydroxypropylmethylcellulose (HPMC) matrix-based extended-release formulation that took into consideration the physical and chemical properties of the drug substance, composition, as well as size and shape of the tablet. New high dose strength (1000 mg) tablet geometry was selected based on the surface area/volume (SA/V) approach advocated by Lapidus/Lordi/Reynold to obtain the desired equivalent metformin release kinetics. Maintaining a similar SA/V ratio across all extended-release metformin hydrochloride (Met XR) tablet strengths that had different geometries provided similar simulations of dissolution behavior. Experimental dissolution profiles of three lots of high-strength tablets agreed with the simulated release kinetics. Additionally, a pharmacokinetic absorption model was developed using GastroPlus™ software and known physicochemical, pharmacokinetic, and in vitro dissolution properties of metformin to predict the clinical exposure of the new high strength (1000 mg) tablet prior to conducting a human clinical bioequivalence study. In vitro metformin release kinetics were utilized in the absorption model to predict exposures in humans for new 1000-mg Met XR tablets, and the absorption model correctly projected equivalent in vivo exposure across all dose strengths. A clinical bioequivalence study was pursued based on the combined modeling results and demonstrated equivalent exposure as predicted by the simulations.

Activation of mechanosensitive ion channel TRPV4 normalizes tumor vasculature and improves cancer therapy.

Tumor vessels are characterized by abnormal morphology and hyperpermeability that together cause inefficient delivery of chemotherapeutic agents. Although vascular endothelial growth factor has been established as a critical regulator of tumor angiogenesis, the role of mechanical signaling in the regulation of tumor vasculature or tumor endothelial cell (TEC) function is not known. Here we show that the mechanosensitive ion channel transient receptor potential vanilloid 4 (TRPV4) regulates tumor angiogenesis and tumor vessel maturation via modulation of TEC mechanosensitivity. We found that TECs exhibit reduced TRPV4 expression and function, which is correlated with aberrant mechanosensitivity towards extracellular matrix stiffness, increased migration and abnormal angiogenesis by TEC. Further, syngeneic tumor experiments revealed that the absence of TRPV4 induced increased vascular density, vessel diameter and reduced pericyte coverage resulting in enhanced tumor growth in TRPV4 knockout mice. Importantly, overexpression or pharmacological activation of TRPV4 restored aberrant TEC mechanosensitivity, migration and normalized abnormal angiogenesis in vitro by modulating Rho activity. Finally, a small molecule activator of TRPV4, GSK1016790A, in combination with anticancer drug cisplatin, significantly reduced tumor growth in wild-type mice by inducing vessel maturation. Our findings demonstrate TRPV4 channels to be critical regulators of tumor angiogenesis and represent a novel target for anti-angiogenic and vascular normalization therapies.

Endogenous production of leukotriene D4 mediates autocrine survival and proliferation via CysLT1 receptor signalling in intestinal epithelial cells.

The cysteinyl leukotriene1 (CysLT1) receptor (CysLT1R) enhances survival and proliferation of intestinal cells via distinct pathways. Here, we have demonstrated that there is significant endogenous production of CysLTs from both non-tumour- and tumour-derived intestinal epithelial cells. Treatment of two non-tumour cell lines, Int 407 and IEC-6, with CysLT1R antagonists led to shrinkage and detachment of cells, confirmed as apoptotic cell death, and a dose-dependent reduction in proliferation. However, in the tumour intestinal cell lines Caco-2, SW480, HCT-116 and HT-29, treatment with CysLT1R antagonists significantly reduced proliferation, but had no effect on apoptosis. A unique characteristic of intestinal cancer cells is the presence of nuclear CysLT1Rs, which are inaccessible to receptor antagonists. In these cells, inhibition of the endogenous production of CysLTs indirectly, by 5-lipoxygenase inhibition, impaired CysLT1R signalling throughout the cell, and resulted in apoptosis of the tumour cells. These data reveal the existence of constitutive CysLT1R signalling that mediates both survival and proliferation in intestinal cells. Importantly, we propose that tumour-derived intestinal cells are resistant to CysLT1R antagonist-induced apoptosis, a phenomena that could be explained by nuclear CysLT1R signalling.

Effect of hydroxypropyl cellulose (HPC) on dissolution rate of hydrochlorothiazide tablets.

Hydrochlorothiazide (HCTZ) 60 mg strength tablets containing commonly used excipients and hydroxypropyl cellulose, marketed as either Klucel-EF (HPC, NF from Hercules, USA) or HPC-L (HPC, NF from Nippon Soda, Japan), as a binder were manufactured using identical aqueous wet granulation process. The tablets containing Klucel-EF as a binder exhibited higher dissolution rates than those manufactured using HPC-L. The granulations containing Klucel-EF or HPC-L showed no significant differences in compressibility and compactibility based on analysis performed using the Instron-Stress-Strain Analyzer. Both HPC grades met NF specifications and there were no differences for the NF test results in the certificates of analysis by their respective vendors. Further evaluation of both HPC grades indicated that the cloud point values for Klucel-EF and HPC-L in water were 39(+/- 1) and 48 degrees C, respectively. The differences in cloud points of Klucel-EF and HPC-L were correlated to the differences in the percent hydroxypropoxy content and the degree of molecular substitution, which were higher for Klucel-EF than for HPC-L. These structural features make Klucel-EF less hydrophilic. Since the cloud point of Klucel-EF was similar to the dissolution medium temperature of 37(+/- 2) degrees C, it may present a less viscous layer surrounding the HCTZ granules enabling faster dissolution of the drug.

The epsilon isoform of protein kinase C is involved in regulation of the LTD(4)-induced calcium signal in human intestinal epithelial cells.

We investigated the potential roles of specific isoforms of protein kinase C (PKC) in the regulation of leukotriene D(4)-induced Ca(2+) signaling in the intestinal epithelial cell line Int 407. RT-PCR and Western blot analysis revealed that these cells express the PKC isoforms alpha, betaII, delta, epsilon, zeta, and mu, but not betaI, gamma, eta, or theta;. The inflammatory mediator leukotriene D(4) (LTD(4)) caused the TPA-sensitive PKC isoforms alpha, delta, and epsilon, but not betaII, to rapidly translocate to a membrane-enriched fraction. The PKC inhibitor GF109203X at 30 microM but not 2 microM significantly impaired the LTD(4)-induced Ca(2+) signal, indicating that the response involves a novel PKC isoform, such as delta or epsilon, but not alpha. LTD(4)-induced Ca(2+) signaling was significantly suppressed in cells pretreated with TPA for 15 min and was abolished when the pretreatment was prolonged to 2 h. Immunoblot analysis revealed that the reduction in the LTD(4)-induced calcium signal coincided with a reduction in the cellular content of PKCepsilon and, to a limited extent, PKCdelta. LTD(4)-induced Ca(2+) signaling was also markedly suppressed by microinjection of antibodies against PKCepsilon but not PKCdelta. These data suggest that PKCepsilon plays a unique role in regulation of the LTD(4)-dependent Ca(2+) signal in intestinal epithelial cells.