Optimization of an in vitro method for assessing pulmonary permeability of inhaled drugs using alveolar epithelial cells.

INTRODUCTION: Inhalation of drugs for the treatment of pulmonary diseases has been used since a long time. Due to lungs' larger absorptive surface area, delivery of drugs to the lungs is the method of choice for different disorders. Here we present the establishment of a comprehensive permeability model using Type II alveolar epithelial cells and Beclomethasone Dipropionate (BDP) as a model drug delivered by pressurized metered dose inhaler (pMDI). METHODS: Using Type II alveolar epithelial cells, the method was standardized for parameters viz., cell density, viability, incubation period and membrane integrity. The delivery and deposition of drug were using the pMDI device with a Twin Stage Impinger (TSI) modified to accommodate cell culture insert having monolayer of cells. The analytical method for simultaneous estimation of BDP and Beclomathasone-17-Monopropionate (17-BMP) was validated as per the bioanalytical guidelines. The extent and rate of absorption of BDP was determined by quantifying the amount of drug permeated and the data represented by calculating its apparent permeability. RESULTS: Type II alveolar epithelial cells cultured at 0.55 × 105 cells/cm2 for 8-12 days under air-liquid interface were optimized for conducting permeability studies. The data obtained for absorptive transport showed a linear increase in the drug permeated against time for both BDP and 17-BMP along with proportional permeability profile. DISCUSSION: We have developed a robust in vitro model to study absorptive rate of drug transport across alveolar layer. Such models would create potential value during formulation development for comparative studies and selection of clinical candidates.

Ketorolac modulates Rac-1/HIF-1α/DDX3/ß-catenin signalling via a tumor suppressor prostate apoptosis response-4 (Par-4) in renal cell carcinoma.

Renal cell carcinoma (RCC) is the most difficult-to-treat form of kidney cancer with a median 5-year survival of 10% under metastatic setting. In RCC, although cytoreductive nephrectomy is common, approximately 20-30% of patients will develop recurrent cancer after surgery, which highlights the need for an effective therapy. Rho-GTPases viz, Rac-1 and Cdc42 are the central regulators of cancer cell migration and invasion and thus metastasis in multiple cancer types. Hence, we elucidated the role of Ketorolac, a modulator Rho-GTPases against RCC through potentiation of tumor suppressor Par-4. The effect of Ketorolac alone and in combination on proliferation, apoptosis, cell-cycle progression, migration, tumor inhibition and their related markers were studied. Moreover, Ketorolac's impact on metastasis by influencing Rac-1/HIF-1α/DDX3/ß-catenin signalling was studied with respect to its ability to modulate the expression of tumor suppressor Par-4, and this mechanism was confirmed by siRNA knockdown studies. Ketorolac induced cytotoxicity in a panel of renal cells including patient derived tumor cells with IC50 2.8 to 9.02 mM and 0.28 to 3.8 mM in monolayer and anchorage independent clonogenic assays respectively. Ketorolac caused significant down regulation of proliferation (Ki-67, Cyclin D1, pRB and DDX3), migration/invasion (Rac-1, Cdc42, and Tiam1), and angiogenesis (HIF-1α and VEGF) markers as studied by gene and protein expression. Moreover, it caused a significant upregulation of tumor suppressor Par-4 known to be downregulated in RCC. This mechanism was further confirmed by using siRNA knockdown studies where we could demonstrate a negative relation between the expression of Par-4 and Rac-1/Cdc42. Importantly, Ketorolac alone and in combination with Sunitinib showed tumor growth inhibition (TGI) of 73% and 86% respectively in xenograft model. This anti-tumor activity was further corroborated by down regulation of Rac-1/Cdc42/HIF-1α/DDX3/ß-catenin signalling. This is the first report which implicates the role of Ketorolac against RCC by acting as a small molecule secretagogue causing upregulation of Par-4 in autocrine and paracrine manner. Consequently, these findings suggest that Par-4 can serve as a valuable therapeutic target and a prognostic marker for the treatment of RCC.

Prostate Apoptosis Response-4 (Par-4): A Novel Target in Pyronaridine-Induced Apoptosis in Glioblastoma (GBM) Cells.

Glioblastoma (GBM) is an aggressive form of brain tumor with a median survival of approximately 12 months. With no new drugs in the last few decades and limited success in clinics for known therapies, drug repurposing is an attractive choice for its treatment. Here, we examined the efficacy of pyronaridine (PYR), an anti-malarial drug in GBM cells. PYR induced anti-proliferative activity in GBM cells with IC50 ranging from 1.16 to 6.82 µM. Synergistic activity was observed when PYR was combined with Doxorubicin and Ritonavir. Mechanistically, PYR triggered mitochondrial membrane depolarization and enhanced the ROS levels causing caspase-3 mediated apoptosis. PYR significantly decreased markers associated with proliferation, EMT, hypoxia, and stemness and upregulated the expression of E-cadherin. Interestingly, PYR induced the expression of intracellular as well as secretory Par-4, a tumor suppressor in GBM cells, which was confirmed using siRNA. Notably, Par-4 levels in plasma samples of GBM patients were significantly lower than normal healthy volunteers. Thus, our study demonstrates for the first time that PYR can be repurposed against GBM with a novel mechanism of action involving Par-4. Herewith, we discuss the role of upregulated Par-4 in a highly interconnected signaling network thereby advocating its importance as a therapeutic target.

Characterization of peptide 20-30 of follicle stimulating hormone receptor as an antagonist of receptor activity: significance of charged residues.

We had previously reported the region (20-30) from follicle stimulating hormone receptor as being an immunodominant epitope and the smallest reported peptide capable of inhibiting hormone binding. We now report it to be an effective antagonist of ligand-induced cAMP signalling as well. The region (20-30) of follicle stimulating hormone receptor has three charged residues, namely, E(22), D(26) and R(29) that are specific to follicle stimulating hormone receptor and are conserved in mammals. This study aimed to verify whether the charged residues contribute to the activity of the follicle stimulating hormone receptor peptide (20-30). This was done using analogs of follicle stimulating hormone receptor peptide (20-30), each having an alanine substitution for a corresponding charged residue. The analog peptides displayed a loss of activity and could not inhibit hormone binding or the subsequent signal transduction. The ability of follicle stimulating hormone receptor peptide (20-30) to bind antipeptide antibodies against follicle stimulating hormone receptor peptide (9-30) was either decreased or abolished with the alanine substituted analog peptides of follicle stimulating hormone receptor peptide (20-30). The loss of function led us to verify whether there was a conformational change as well. CD spectral analysis did not reveal a significant change. These observations indicate that the charged aminoacids present in follicle stimulating hormone receptor peptide (20-30) are crucial for the observed follicle stimulating hormone antagonistic activity. This information could form the basis for the design of novel compounds capable of functioning as follicle stimulating hormone antagonists.

Detailed analysis of the region 9-30 of rat follicle stimulating hormone receptor: identification of peptide 20-30 as a potential hormone binding inhibitor.

The extracellular domain (ECD) of the follicle stimulating hormone receptor (FSHR) has been shown to be a major determinant of hormone selectivity. The N-terminal 9-30 region, the sequence of which is unique to FSHR, has been extensively studied earlier and has been proposed to be an FSHR neutralizing epitope. In this study antipeptide antibodies specific to the peptide 9-30 were generated and used for identifying a specific immunodominant region within it. Overlapping peptides corresponding to the regions 9-19, 15-25 and 20-30 were synthesized. The ability of the antipeptide antibodies to 9-30 of FSHR to bind to different peptides was checked. The results indicated that the antibodies mainly recognized the peptide 20-30 and not the other two overlapping peptides. Further, the effect of the peptide 20-30 on the binding of radiolabeled FSH to its receptor was monitored. This peptide showed FSH-binding inhibitory activity with an IC(50) value of 0.598 x 10(-4)M and was more effective than the peptide 9-30 itself. Binding kinetics revealed that the observed effect of the peptide 20-30 is due to mixed type of inhibitory mechanism. This is the smallest peptide from the rat FSHR sequence having ability to inhibit FSH binding to its receptor by more than 90%.