Ravi Doddapaneni

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.

Topical administration of dual siRNAs using fusogenic lipid nanoparticles for treating psoriatic-like plaques

AIM Psoriasis is a chronic autoimmune skin disorder with substantial negative impact on the patients quality of life. The present study was carried out to demonstrate the efficiency of a novel topical delivery system in the transport of two siRNAs for the treatment of psoriatic-like plaques. MATERIALS & METHODS We designed and developed a novel fusogenic nucleic acid lipid particle (F-NALP) system containing two therapeutic nucleic acids, anti-STAT3 siRNA (siSTAT3) and anti-TNF-α siRNA (siTNF-α). Novel cationic amphiphilic lipid with oleyl chains was synthesized and used in the nanocarrier system. Therapeutic efficacies of F-NALPs were assessed using an imiquimod-induced psoriatic-like plaque model. RESULTS Hydrodynamic size and surface potential of F-NALPs were 102 ± 6 nm and 32.14 ± 6.21 mV, respectively. F-NALPs delivered fluorescein isothiocyanate-siRNA to a skin depth of 360 µm. F-NALPs carrying siSTAT3 and siTNF-α significantly (p < 0.05) reduced expression of STAT3 and TNF-α mRNAs and IL-23 and Ki-67 proteins compared with solution, and was superior in comparison with Topgraf(®) (GlaxoSmithKline Pharmaceuticals Limited, Maharashtra, India). CONCLUSION Our observations demonstrate that F-NALPs can efficiently carry siSTAT3 and siTNF-α into the dermis and combination of the two nucleic acids can synergistically treat psoriatic-like plaques.

Piperlongumine for Enhancing Oral Bioavailability and Cytotoxicity of Docetaxel in Triple-Negative Breast Cancer

Very low oral bioavailability due to extensive pre-systemic metabolism and P-gp efflux has constrained the oral metronomic chemotherapy of docetaxel (DTX). There is tremendous need of compounds facilitating oral delivery of DTX. The research was aimed to investigate the effect of piperlongumine (PPL) on human liver microsomal metabolism, Caco-2 permeability, and cytotoxicity of DTX in triple-negative breast cancer cell lines. Reduction in testosterone and DTX metabolism (twofold increase in half-life) by PPL was comparable to the standard CYP3A4 inhibitor, cyclosporine A. P-gp efflux ratio of DTX across caco-2 monolayer was reduced from 2.37 to 1.52 on co-incubation with PPL. The IC50 value of DTX was reduced three to five times and combination index values in all the cell lines were below 0.6. PPL at non-cytotoxic concentration showed significant enhancement of the antimigration effect of DTX. Expression of tumor markers such as survivin, bcl2, C-myc, and cyclin D1 were downregulated to a great extent with enhanced p53 expression when treated with combination instead of individual drug. Co-treatment with PPL led to 1.68-fold enhancement in DTX bioavailability in SD rats. PPL could be a potential candidate in overcoming the obstacles associated with oral DTX delivery with synergistic anticancer activity.

Novel amphiphilic lipid augments the co-delivery of erlotinib and IL36 siRNA into the skin for psoriasis treatment.

Abstract In this study, we demonstrate for the first time the concurrent transdermal delivery of erlotinib and IL36&agr; siRNA as a potential dual therapy for psoriasis. The objectives were to develop and evaluate lipid nanocarriers (CYnLIP) using a novel pyrrolidinium lipid to disrupt the skin barrier for enhanced transdermal delivery. CYnLIP (132.00 ± 6.23 nm) had encapsulation efficiency of 49.04 ± 2.54% for erlotinib. DSC confirmed encapsulation of erlotinib within CYnLIP. Atomic Force Microscopy demonstrated notable topographical changes in the stratum corneum of skin permeated with CYnLIP that were absent in skin hydrated with water. Peak force distance curves also exhibited a more permeable membrane for CYnLIP‐incubated skin than hydrated skin. Permeation studies showed enhanced (p < 0.01) skin retention of erlotinib by CYnLIP (40.76‐fold) than solution and more pronounced fluorescence at deeper layers of the skin for fluorescein‐labeled siRNA‐CYnLIP than solution. The enhanced co‐transdermal delivery of erlotinib and IL36&agr; siRNA by CYnLIP efficaciously treated psoriatic‐like plaques in C57BL/6 mice (PASI score of 1) compared to imiquimod‐only treatment (PASI score of 4). IHC and western blotting revealed reduction in epidermal hyperplasia (Ki67) and in the dermal infiltration of inflammatory cytokines (IL36&agr;, pSTAT3, TNF&agr;, NF&kgr;B, IL23 and IL17) for erlotinib/IL36&agr; siRNA‐CYnLIP (p < 0.05) comparable to Tacrolimus but markedly less than imiquimod‐only treatment. Graphical abstract Figure. No Caption available.

NR4A1 Antagonists Inhibit β1-Integrin-Dependent Breast Cancer Cell Migration

ABSTRACT Overexpression of the nuclear receptor 4A1 (NR4A1) in breast cancer patients is a prognostic factor for decreased survival and increased metastasis, and this has been linked to NR4A1-dependent regulation of transforming growth factor β (TGF-β) signaling. Results of RNA interference studies demonstrate that basal migration of aggressive SKBR3 and MDA-MB-231 breast cancer cells is TGF-β independent and dependent on regulation of β1-integrin gene expression by NR4A1 which can be inhibited by the NR4A1 antagonists 1,1-bis(3′-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) and a related p-carboxymethylphenyl [1,1-bis(3′-indolyl)-1-(p-carboxymethylphenyl)methane (DIM-C-pPhCO2Me)] analog. The NR4A1 antagonists also inhibited TGF-β-induced migration of MDA-MB-231 cells by blocking nuclear export of NR4A1, which is an essential step in TGF-β-induced cell migration. We also observed that NR4A1 regulates expression of both β1- and β3-integrins, and unlike other β1-integrin inhibitors which induce prometastatic β3-integrin, NR4A1 antagonists inhibit expression of both β1- and β3-integrin, demonstrating a novel mechanism-based approach for targeting integrins and integrin-dependent breast cancer metastasis.

Tumor neovasculature-targeted cationic PEGylated liposomes of gambogic acid for the treatment of triple-negative breast cancer

Abstract Gambogic acid (GA) is a naturally derived potent anticancer agent with extremely poor aqueous solubility. In the present study, positively charged PEGylated liposomal formulation of GA (GAL) was developed for parenteral delivery for the treatment of triple-negative breast cancer (TNBC). The GAL was formulated with a particle size of 107.3 ± 10.6 nm with +32 mV zeta potential. GAL showed very minimal release of GA over 24 h period confirming the non-leakiness and stability of liposomes. In vitro cytotoxicity assays showed similar cell killing with GA and GAL against MDA-MB-231 cells but significantly higher inhibition of HUVEC growth was observed with GAL. Furthermore, GAL significantly (p < 0.05) inhibited the MDA-MB-231 orthotopic xenograft tumor growth with >50% reduction of tumor volume and reduction in tumor weight by 1.7-fold and 2.2-fold when compared to GA and controls, respectively. Results of western blot analysis indicated that GAL significantly suppressed the expression of apoptotic markers, bcl2, cyclinD1, survivin and microvessel density marker-CD31 and increased the expression of p53 and Bax compared to GA and control. Collectively, these data provide further support for the potential applications of cationic GAL in its intravenous delivery and its significant role in inhibiting angiogenesis against TNBC.

Chemoprevention of Skin Cancer with 1,1-Bis (3′-Indolyl)-1-(Aromatic) Methane Analog through Induction of the Orphan Nuclear Receptor, NR4A2 (Nurr1)

Background The objective of this study was to demonstrate the anti-skin cancer and chemopreventive potential of 1,1-bis(3′-indolyl)-1-(p-chlorophenyl methane) (DIM-D) using an in vitro model. Methods In vitro cell cytotoxicity and viability assays were carried out in A431 human epidermoid carcinoma cell line and normal human epidermal keratinocytes (NHEK) respectively by crystal violet staining. Apoptosis induction in A431 cells (DIM-D treated) and NHEK cells pretreated with DIM-D (2 hr) prior to UVB irradiation, were assessed. The accumulation of reactive oxygen species (ROS) in DIM-D pretreated NHEK cells (2 hr) prior to UVB exposure was also determined. Immunocytochemistry and western blot analysis was performed to determine cleaved caspase 3 and DNA damage markers in DIM-D treated A431 cells and in DIM-D pretreated NHEK cells prior to UVB irradiation. Results The IC50 values of DIM-D were 68.7±7.3, 48.3±10.1 and 11.5±3.1 μM whilst for Epigallocatechin gallate (EGCG) were 419.1±8.3, 186.1±5.2 and 56.7±3.1 μM for 24, 48 and 72 hr treatments respectively. DIM-D exhibited a significantly (p<0.05) greater induction of DNA fragmentation in A431 cells compared to EGCG with percent cell death of 38.9. In addition, DIM-D induced higher expression in A431 cells compared to EGCG of cleaved caspase 3 (3.0-fold vs. 2.4-fold changes), Nurr1 (2.7-fold vs. 1.7-fold changes) and NFκB (1.3-fold vs. 1.1-fold changes). DIM-D also exhibited chemopreventive activity in UVB-irradiated NHEK cells by significantly (p<0.05) reducing UVB-induced ROS formation and apoptosis compared to EGCG. Additionally, DIM-D induced expression of Nurr1 but reduced expression of 8-OHdG significantly in UVB-irradiated NHEK cells compared to EGCG and UV only. Conclusion Our results suggest that DIM-D exhibits Nurr1-dependent transactivation in the induction of apoptosis in A431 cells and it protects NHEK cells against UVB-induced ROS formation and DNA damage.

Tumor stromal disrupting agent enhances the anticancer efficacy of docetaxel loaded PEGylated liposomes in lung cancer.

AIM Therapeutic efficacy of anticancer nanomedicine is compromised by tumor stromal barriers. The present study deals with the development of docetaxel loaded PEGylated liposomes (DTXPL) and to investigate the effect of tumor stroma disrupting agent, telmisartan, on anticancer efficacy of DTXPL. METHODS DTXPL was prepared using proprietary modified hydration method. Effect of oral telmisartan treatment on tumor uptake of coumarin-6 liposomes and anticancer efficacy of DTXPL was evaluated in orthotopic xenograft lung tumor bearing mice. RESULTS DTXPL (105.7 ± 3.8 nm) showed very high physical stability, negligible hemolysis, 428% enhancement in bioavailability with significantly higher intratumoral uptake. Marked reduction in collagen-I, MMP2/9 and lung tumor weight were observed in DTXPL+telmisartan group. CONCLUSION Combination of DTXPL with telmisartan could significantly enhance clinical outcome in lung cancer.

Noscapine chemosensitization enhances docetaxel anticancer activity and nanocarrier uptake in triple negative breast cancer

Chemosensitization and enhanced delivery to solid tumor are widely explored strategies to augment the anticancer efficacy of existing chemotherapeutics agents. The aim of current research was to investigate the role of low dose Noscapine (Nos) in potentiating docetaxel cytotoxicity and enhancing tumor penetration of nanocarriers. The objectives are; (1) To evaluate the chemo-sensitizing effect of Nos in combination with docetaxel (DTX), and to elucidate the possible mechanism (2) To investigate the effect of low dose Nos on tumor stroma and enhancing nanocarrier uptake in triple negative breast cancer (TNBC) bearing nude mice. Cytotoxicity and flow cytometry analysis of DTX in Nos (4µM) pre-treated MDA-MB-231 cells showed 3.0-fold increase in cell killing and 30% increase in number of late apoptotic cells, respectively. Stress transducer p38 phosphorylation was significantly upregulated with Nos exposure. DTX showed remarkable downregulation in expression of bcl-2, survivin and pAKT in Nos pre-treated MDA-MB-231 cells. Nos pre-sensitization significantly (p<0.02) enhanced the anti-migration effect of DTX. In vivo studies in orthotopic TNBC tumor bearing mice showed marked reduction in tumor collagen-I levels and significantly (p<0.03) higher intra-tumoral uptake of coumarin-6 loaded PEGylated liposomes (7-fold) in Nos treated group. Chemo-sensitization and anti-fibrotic effect of Nos could be a promising approach to increase anticancer efficacy of DTX which can be used for other nanomedicinal products.