Apurva R. Patel

AlgiMatrix™ Based 3D Cell Culture System as an In-Vitro Tumor Model for Anticancer Studies

Background Three-dimensional (3D) in-vitro cultures are recognized for recapitulating the physiological microenvironment and exhibiting high concordance with in-vivo conditions. Taking the advantages of 3D culture, we have developed the in-vitro tumor model for anticancer drug screening. Methods Cancer cells grown in 6 and 96 well AlgiMatrix™ scaffolds resulted in the formation of multicellular spheroids in the size range of 100–300 µm. Spheroids were grown in two weeks in cultures without compromising the growth characteristics. Different marketed anticancer drugs were screened by incubating them for 24 h at 7, 9 and 11 days in 3D cultures and cytotoxicity was measured by AlamarBlue® assay. Effectiveness of anticancer drug treatments were measured based on spheroid number and size distribution. Evaluation of apoptotic and anti-apoptotic markers was done by immunohistochemistry and RT-PCR. The 3D results were compared with the conventional 2D monolayer cultures. Cellular uptake studies for drug (Doxorubicin) and nanoparticle (NLC) were done using spheroids. Results IC50 values for anticancer drugs were significantly higher in AlgiMatrix™ systems compared to 2D culture models. The cleaved caspase-3 expression was significantly decreased (2.09 and 2.47 folds respectively for 5-Fluorouracil and Camptothecin) in H460 spheroid cultures compared to 2D culture system. The cytotoxicity, spheroid size distribution, immunohistochemistry, RT-PCR and nanoparticle penetration data suggested that in vitro tumor models show higher resistance to anticancer drugs and supporting the fact that 3D culture is a better model for the cytotoxic evaluation of anticancer drugs in vitro. Conclusion The results from our studies are useful to develop a high throughput in vitro tumor model to study the effect of various anticancer agents and various molecular pathways affected by the anticancer drugs and formulations.

Formulation, characterization and pulmonary deposition of nebulized celecoxib encapsulated nanostructured lipid carriers

The aim of the current study was to encapsulate celecoxib (Cxb) in the nanostructured lipid carrier (Cxb-NLC) nanoparticles and evaluate the lung disposition of nanoparticles following nebulization in Balb/c mice. Cxb-NLC nanoparticles were prepared with Cxb, Compritol, Miglyol and sodium taurocholate using high-pressure homogenization. Cxb-NLC nanoparticles were characterized for physical and aerosol properties. In-vitro cytotoxicity studies were performed with A549 cells. The lung deposition and pharmacokinetic parameters of Cxb-NLC and Cxb solution (Cxb-Soln) formulations were determined using the Inexpose system and Pari LC star jet nebulizer. The particle size and entrapment efficiency of the Cxb-NLC formulation were 217+/-20nm and >90%, respectively. The Cxb-NLC released the drug in controlled fashion, and in-vitro aerosolization of Cxb-NLC formulation showed an FPF of 75.6+/-4.6%, MMAD of 1.6+/-0.13microm and a GSD of 1.2+/-0.21. Cxb-NLC showed dose and time dependent cytotoxicity against A549 cells. Nebulization of Cxb-NLC demonstrated 4 fold higher AUC(t)/D in lung tissues compared to the Cxb-Soln. The systemic clearance of Cxb-NLC was slower (0.93l/h) compared to the Cxb-Soln (20.03l/h). Cxb encapsulated NLC were found to be stable and aerodynamic properties were within the respirable limits. Aerosolization of Cxb-NLC improved the Cxb pulmonary bioavailability compared to solution formulation which will potentially lead to better patient compliance with minimal dosing intervals.

Skin permeating nanogel for the cutaneous co-delivery of two anti-inflammatory drugs.

The aim of this study was to develop an effective drug delivery system for the simultaneous topical delivery of two anti-inflammatory drugs, spantide II (SP) and ketoprofen (KP). To achieve this primary goal, we have developed a skin permeating nanogel system (SPN) containing surface modified polymeric bilayered nanoparticles along with a gelling agent. Poly-(lactide-co-glycolic acid) and chitosan were used to prepare bilayered nanoparticles (NPS) and the surface was modified with oleic acid (NPSO). Hydroxypropyl methyl cellulose (HPMC) and Carbopol with the desired viscosity were utilized to prepare the nanogels. The nanogel system was further investigated for in vitro skin permeation, drug release and stability studies. Allergic contact dermatitis (ACD) and psoriatic plaque like model were used to assess the effectiveness of SPN. Dispersion of NPSO in HPMC (SPN) produced a stable and uniform dispersion. In vitro permeation studies revealed increase in deposition of SP for the SP-SPN or SP+KP-SPN in the epidermis and dermis by 8.5 and 9.5 folds, respectively than SP-gel. Further, the deposition of KP for KP-SPN or SP+KP-SPN in epidermis and dermis was 9.75 and 11.55 folds higher, respectively than KP-gel. Similarly the amount of KP permeated for KP-SPN or SP+KP-SPN was increased by 9.92 folds than KP-gel. The ear thickness in ACD model and the expression of IL-17 and IL-23; PASI score and TEWL values in psoriatic plaque like model were significantly less (p < 0.001) for SPN compared to control gel. Our results suggest that SP+KP-SPN have significant potential for the percutaneous delivery of SP and KP to the deeper skin layers for treatment of various skin inflammatory disorders.

Anticancer activity of Noscapine, an opioid alkaloid in combination with Cisplatin in human non-small cell lung cancer

The purpose of this study was to examine the efficacy of Noscapine (Nos) and Cisplatin (Cis) combination treatment in vitro in A549 and H460 lung cancer cells, in vivo in murine xenograft model and to investigate the underlying mechanism. The combination index values (< 0.6) suggested synergistic effects of Nos+Cis and resulted in the highest increase in percentage of apoptotic NSCLC cells and increased expression of p53, p21, caspase 3, cleaved caspase 3, cleaved PARP, Bax, and decreased expression of Bcl₂ and surviving proteins compared with treatment with either agent. Nos+Cis treatment reduced tumor volume by 78.1 ± 7.5% compared with 38.2 ± 6.8% by Cis or 35.4 ± 6.9% by Nos alone in murine xenograft lung cancer model. Nos+Cis treatment decreased expression of pAkt, Akt, cyclin D1, survivin, PARP, Bcl₂, and increased expression of p53, p21, Bax, cleaved PARP, caspase 3, cleaved caspase 3, cleaved caspase 8, caspase 8, cleaved caspase 9 and caspase 9 compared to single-agent treated and control groups. Our results suggest that Nos enhanced the anticancer activity of Cis in an additive to synergistic manner by activating multiple signaling pathways including apoptosis. These findings suggest potential benefit for use of Nos and Cis combination in treatment of lung cancer.

Topical delivery of anti-TNFα siRNA and capsaicin via novel lipid-polymer hybrid nanoparticles efficiently inhibits skin inflammation in vivo.

The barrier properties of the skin pose a significant but not insurmountable obstacle for development of new effective anti-inflammatory therapies. The objective of this study was to design and evaluate therapeutic efficacy of anti-nociception agent Capsaicin (Cap) and anti-TNFα siRNA (siTNFα) encapsulated cyclic cationic head lipid-polymer hybrid nanocarriers (CyLiPns) against chronic skin inflammatory diseases. Physico-chemical characterizations including hydrodynamic size, surface potential and entrapment efficacies of CyLiPns were found to be 163±9nm, 35.14±8.23mV and 92% for Cap, respectively. In vitro skin distribution studies revealed that CyLiPns could effectively deliver FITC-siRNA up to 360μm skin depth. Further, enhanced (p<0.001) Cap permeation from CyLiPns was observed compared to Capsaicin-Solution and Capzasin-HP. Therapeutic efficacies of CyLiPns were assessed using imiquamod-induced psoriatic plaque like model. CyLiPns carrying both Cap and siTNFα showed significant reduced expression of TNFα, NF-κB, IL-17, IL-23 and Ki-67 genes compared to either drugs alone (p<0.05) and were in close comparison with Topgraf®. Collectively these findings support our notion that novel cationic lipid-polymer hybrid nanoparticles can efficiently carry siTNFα and Cap into deeper dermal milieu and Cap with a combination of siTNFα shows synergism in treating skin inflammation.

Antitumor Activity of Noscapine in Combination with Doxorubicin in Triple Negative Breast Cancer

Background The aim of this study was to investigate the anticancer activity and mechanism of action of Noscapine alone and in combination with Doxorubicin against triple negative breast cancer (TNBC). Methods TNBC cells were pretreated with Noscapine or Doxorubicin or combination and combination index values were calculated using isobolographic method. Apoptosis was assessed by TUNEL staining. Female athymic Nu/nu mice were xenografted with MDA-MB-231 cells and the efficacy of Noscapine, Doxorubicin and combination was determined. Protein expression, immunohistochemical staining were evaluated in harvested tumor tissues. Results Noscapine inhibited growth of MDA-MB-231 and MDA-MB-468 cells with the IC50 values of 36.16±3.76 and 42.7±4.3 µM respectively. The CI values (<0.59) were suggestive of strong synergistic interaction between Noscapine and Doxorubicin and combination treatment showed significant increase in apoptotic cells. Noscapine showed dose dependent reduction in the tumor volumes at a dose of 150–550 mg/kg/day compared to controls. Noscapine (300 mg/kg), Doxorubicin (1.5 mg/kg) and combination treatment reduced tumor volume by 39.4±5.8, 34.2±5.7 and 82.9±4.5 percent respectively and showed decreased expression of NF-KB pathway proteins, VEGF, cell survival, and increased expression of apoptotic and growth inhibitory proteins compared to single-agent treatment and control groups. Conclusions Noscapine potentiated the anticancer activity of Doxorubicin in a synergistic manner against TNBC tumors via inactivation of NF-KB and anti-angiogenic pathways while stimulating apoptosis. These findings suggest potential benefit for use of oral Noscapine and Doxorubicin combination therapy for treatment of more aggressive TNBC.

Approaches to Improve the Oral Bioavailability and Effects of Novel Anticancer Drugs Berberine and Betulinic Acid

Background The poor bioavailability of Berberine (BBR) and Betulinic acid (BA) limits the development of these promising anticancer agents for clinical use. In the current study, BBR and BA in spray dried (SD) mucoadhesive microparticle formulations were prepared. Methods A patented dual channel spray gun technology established in our laboratory was used for both formulations. Gastrointestinal (GI) permeability studies were carried out using Caco-2 cell monolayer grown in in-vitro system. The oral bioavailability and pharmacokinetic profile of SD formulations were studied in Sprague Dawley rats. A549 orthotopic and H1650 metastatic NSCLC models were utilized for the anticancer evaluations. Results Pharmacokinetic studies demonstrated that BBR and BA SD formulations resulted in 3.46 and 3.90 fold respectively, significant increase in plasma Cmax concentrations. AUC levels were increased by 6.98 and 7.41 fold in BBR and BA SD formulations, respectively. Compared to untreated controls groups, 49.8 & 53.4% decrease in the tumor volumes was observed in SD formulation groups of BBR and BA, respectively. Molecular studies done on excised tumor (A549) tissue suggested that BBR in SD form resulted in a significant decrease in the survivin, Bcl-2, cyclin D1, MMP-9, HIF-1α, VEGF and CD31 expressions. Cleaved caspase 3, p53 and TUNEL expressions were increased in SD formulations. The RT-PCR analysis on H1650 tumor tissue suggested that p38, Phospho-JNK, Bax, BAD, cleaved caspase 3&8 mRNA expressions were significantly increased in BA SD formulations. Chronic administration of BBR and BA SD formulations did not show any toxicity. Conclusions Due to significant increase in oral bioavailability and superior anticancer effects, our results suggest that spray drying is a superior alternative formulation approach for oral delivery of BBR and BA.

Inhalation delivery of Telmisartan enhances intratumoral distribution of nanoparticles in lung cancer models.

The purpose of the present study was to evaluate the effect of Telmisartan (Tel) and Losartan (Los) on nanoparticle intratumoral distribution and anticancer effects in lung cancer. A549 lung tumor cells were orthotopically and metastatically administered to Nu/nu mice. Fluorescent polystyrene nanoparticles (FPNPs, size ~200 nm) beads were used to study their intratumoral distribution after Tel and Los treatments. Animals were administered with FPNPs and after 2h, FPNPs intratumoral distribution was studied by fluorescent microscopy. Tel (~1.12 mg/kg) and Los (~4.5mg/kg) were administered by inhalation delivery at alternative days for 4 weeks to tumor bearing animals. Collagen-1, transforming growth factor beta 1 (TGF-β1), cleaved caspase-3, Vimentin and E-Cadherin expressions were studied by western blotting. To correlate the AT1 receptor blockage to anticancer effects, VEGF levels and microvessel densities (MVD) were quantified. Los and Tel treated group resulted in the 5.33 and 14.33 fold increase respectively in the FPNPs intratumoral distribution as compared to the controls. Tel treatment attenuated 2.23 and 1.70 fold Collagen 1 expression compared to untreated control and Los groups, respectively. Further, in Tel and Los treated groups, the TGF-β1 active levels were significantly (p<0.05) decreased. Tel (at four times less dose) was 1.89 and 1.92 fold superior in anticancer activity to Los respectively in A549 orthotopic and metastatic tumor models (p<0.05) when given by inhalation route. Tel, by virtue of its dual pharmacophoric nature could be an ideal candidate for combination therapy to improve the nanoparticle intratumoral distribution and anticancer effects.

Inhalation Delivery of a Novel Diindolylmethane Derivative for the Treatment of Lung Cancer

The purpose of this study was to determine the anticancer efficacy of 1,1-bis (3′-indolyl)-1-(p-biphenyl) methane (DIM-C-pPhC6H5) by inhalation delivery alone and in combination with i.v. docetaxel in a murine model for lung cancer. An aqueous DIM-C-pPhC6H5 formulation was characterized for its aerodynamic properties. Tumor-bearing athymic nude mice were exposed to nebulized DIM-C-pPhC6H5, docetaxel, or combination (DIM-C-pPhC6H5 plus docetaxel) using a nose-only exposure technique. The aerodynamic properties included mass median aerodynamic diameter of 1.8 ± 0.3 μm and geometric SD of 2.31 ± 0.02. Lung weight reduction in mice treated with the drug combination was 64% compared with 40% and 47% in mice treated with DIM-C-pPhC6H5 aerosol and docetaxel alone, respectively. Combination treatment decreased expression of Akt, cyclin D1, survivin, Mcl-1, NF-κB, IκBα, phospho-IκBα, and vascular endothelial growth factor (VEGF) and increased expression of c-Jun NH2-terminal kinase 2 and Bad compared with tumors collected from single-agent treatment and control groups. DNA fragmentation was also enhanced in mice treated with the drug combination compared with docetaxel or DIM-C-pPhC6H5 alone. Combination treatment decreased expressions of VEGF and CD31 compared with single-agent treated and control groups. These results suggest that DIM-C-pPhC6H5 aerosol enhanced the anticancer activity of docetaxel in a lung cancer model by activating multiple signaling pathways. The study provides evidence that DIM-C-pPhC6H5 can be used alone or in combination with other drugs for the treatment of lung cancer using the inhalation delivery approach. Mol Cancer Ther; 9(11); 3003–14. ©2010 AACR.

Tenofovir-associated renal dysfunction in clinical practice: An observational cohort from western India.

BACKGROUND Tenofovir (TDF) is preferred nucleoside reverse transcriptase inhibitors (NRTI) for the treatment of human immunodeficiency virus infection because of its potency and safety. Renal toxicity with TDF use is low and comparable with other NRTI in clinical trials, but there are many case studies and small case series of renal dysfunction with TDF. MATERIALS AND METHODS This is an observational longitudinal cohort of patients started on a TDF-based regimen from January 2007 to April 2010. Patients were evaluated at baseline and with every follow-up visit for serum creatinine and calculated creatinine clearance (Cockroft-Gault formula). In addition to this, the patients were also subjected to test for serum potassium, phosphorous and urine examinations as and when indicated. Renal dysfunction was defined as rise in serum creatinine to more than the upper level of normal (>1.2 mg%). RESULTS Of 1,271 patients started on a TDF-containing antiretroviral treatment (ART) 83 (6.53%) developed renal dysfunction, of which 79 had impaired serum creatinine and five had Fanconis syndrome. Renal dysfunction was more common with boosted a protease inhibitor (PI) (9.44%)-based regimen as compared to a non- nucleoside reverse transcriptase inhibitors (NNRTI) (5.01%)-based regimen (P = 0.003). The mean decline in creatinine clearance from baseline was 22.27 ml/min. The median time to develop renal dysfunction was 154 (15-935) days. Serum creatinine returned to normal in all the patients after stopping TDF. Five patients presented with features suggestive of Fanconis syndrome without alteration in serum creatinine. CONCLUSION TDF-based treatment is associated with mild but reversible renal dysfunction. Patients receiving PI/r are at a higher risk of renal dysfunction compared to those receiving NNRTI-based ART. Clinicians should be adviced to have intensive renal monitoring, including creatinine clearance, urine examination, K+ and phosphate levels at baseline and during treatment with TDF.