Ram R. Patlolla

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.

Translocation of Cell Penetrating Peptide Engrafted Nanoparticles Across Skin Layers

The objective of the current study was to evaluate the ability of cell penetrating peptides (CPP) to translocate the lipid payload into the skin layers. Fluorescent dye (DID-oil) encapsulated nano lipid crystal nanoparticles (FNLCN) were prepared using Compritol, Miglyol and DOGS-NTA-Ni lipids by hot melt homogenization technique. The FNLCN surface was coated with TAT peptide (FNLCNT) or control YKA peptide (FNLCNY) and in vitro rat skin permeation studies were performed using Franz diffusion cells. Observation of lateral skin sections obtained using cryotome with a confocal microscope demonstrated that skin permeation of FNLCNT was time dependent and after 24h, fluorescence was observed upto a depth of 120 microm which was localized in the hair follicles and epidermis. In case of FNLCN and FNLCNY formulations fluorescence was mainly observed in the hair follicles. This observation was further supported by confocal Raman spectroscopy where higher fluorescence signal intensity was observed at 80 and 120 microm depth with FNLCNT treated skin and intensity of fluorescence peaks was in the ratio of 2:1:1 and 5:3:1 for FNLCNT, FNLCN, and FNLCNY treated skin sections, respectively. Furthermore, replacement of DID-oil with celecoxib (Cxb), a model lipophilic drug showed similar results and after 24h, the CXBNT formulation increased the Cxb concentration in SC by 3 and 6 fold and in epidermis by 2 and 3 fold as compared to CXBN and CXBNY formulations respectively. Our results strongly suggest that CPP can translocate nanoparticles with their payloads into deeper skin layers.

Evaluation of EpiDerm full thickness-300 (EFT-300) as an in vitro model for skin irritation: studies on aliphatic hydrocarbons.

The aim of this study was to understand the skin irritation effects of saturated aliphatic hydrocarbons (HCs), C9-C16, found jet fuels using in vitro 3-dimensional EpiDerm full thickness-300 (EFT-300) skin cultures. The EFT-300 cultures were treated with 2.5microl of HCs and the culture medium and skin samples were collected at 24 and 48h to measure the release of various inflammatory biomarkers (IL-1alpha, IL-6 and IL-8). To validate the in vitro results, in vivo skin irritation studies were carried out in hairless rats by measuring trans epidermal water loss (TEWL) and erythema following un-occlusive dermal exposure of HCs for 72h. The MTT tissue viability assay results with the EFT-300 tissue show that 2.5microl/tissue ( approximately 4.1microl/cm(2)) of the HCs did not induce any significant changes in the tissue viability for exposure times up to 48h of exposure. Microscopic observation of the EFT-300 cross-sections indicated that there were no obvious changes in the tissue morphology of the samples at 24h, but after 48h of exposure, tridecane, tetradecane and hexadecane produced a slight thickening and disruption of stratum corneum. Dermal exposures of C12-C16 HCs for 24h significantly increased the expression of IL-1alpha in the skin as well as in the culture medium. Similarly, dermal exposure of all HCs for 24h significantly increased the expression of interleukin-6 (IL-6) and IL-8 in the skin as well as in the culture medium in proportion to the HC chain length. As the exposure time increased to 48h, IL-6 concentrations increased 2-fold compared to the IL-6 values at 24h. The in vivo skin irritation data also showed that both TEWL and erythema scores increased with increased HCs chain length (C9-C16). In conclusion, the EFT-300 showed that the skin irritation profile of HCs was in the order of C9C10C11C12

Dermal microdialysis of inflammatory markers induced by aliphatic hydrocarbons in rats

In the present study we made an attempt to understand the skin irritation cascade of selected aliphatic hydrocarbons using microdialysis technique. Microdialysis probes were inserted into dermis in the dorsal skin of hairless rats. After 2h of probes insertion, occlusive dermal exposure (2h) was carried out with 230 microl of nonane, dodecane and tetradecane, using Hill top chambers((R)). Inflammatory biomarkers such as substance P (SP), alpha-melanocyte stimulating hormone (alpha-MSH) Interleukin 6 (IL-6) and prostaglandin E2 (PGE(2)) were analyzed in the dialysis samples by enzyme immunoassay (EIA). SP, alpha-MSH and IL6 were released in significant amounts following the dermal exposure of nonane and dodecane, whereas tetradecane did not induce any of these markers in significant amounts compared to control. Nonane increased the PGE(2) levels in significant amounts within 2h of chemical exposure compared to dodecane and tetradecane. IL-6 response was found to be slow and 2-3-fold increase in IL-6 levels was observed after 5h following nonane and dodecane application. The magnitude of skin irritation exerted by all three chemicals was in the order of nonane>or=dodecane>or=tetradecane. The results demonstrate that microdialysis can be used to measure the inflammatory biomarkers in the skin irritation studies and irritation response of chemicals was quantifiable by this method. In conclusion, microdialysis was found to be an excellent tool to measure several inflammatory biomarkers as a function of time after dermal exposures with irritant chemicals.

31P Solid-state NMR based monitoring of permeation of cell penetrating peptides into skin

The main objective of the current study was to investigate penetration of cell penetrating peptides (CPPs: TAT, R8, R11, and YKA) through skin intercellular lipids using (31)P magic angle spinning (MAS) solid-state NMR. In vitro skin permeation studies were performed on rat skin, and sections (0-60, 61-120, and 121-180μm) were collected and analyzed for (31)P NMR signal. The concentration-dependent shift of 0, 25, 50, 100, and 200mg/ml of TAT on skin layers, diffusion of TAT, R8, R11, and YKA in the skin and time dependent permeation of R11 was measured on various skin sections using (31)P solid-state NMR. Further, CPPs and CPP-tagged fluorescent dye encapsulate liposomes (FLip) in skin layers were tagged using confocal microscopy. The change in (31)P NMR chemical shift was found to depend monotonically on the amount of CPP applied on skin, with saturation behavior above 100mg/ml CPP concentration. R11 and TAT caused more shift in solid-state NMR peaks compared to other peptides. Furthermore, NMR spectra showed R11 penetration up to 180μm within 30min. The results of the solid-state NMR study were in agreement with confocal microscopy studies. Thus, (31)P solid-state NMR can be used to track CPP penetration into different skin layers.

Epithelial transport of Noscapine across cell monolayer and influence of absorption enhancers on in vitro permeation and bioavailability: implications for intestinal absorption

Abstract The purpose of this study was to investigate the permeation of Noscapine (Nos) across the Caco-2 and Madin–Darby canine kidney (MDCK) cell monolayers and to evaluate the influence of absorption enhancers on in vitro and in vivo absorption of Nos. The bidirectional transport of Nos was studied in Caco-2 and MDCK cell monolayers at pH 5.0–7.8. The effect of 0.5% w/v chitosan (CH) or Captisol (CP) on Nos permeability was investigated at pH 5.0 and 5.8. The effect of 1–5% w/v of CP on oral bioavailability of Nos (150 mg/kg) was evaluated in Sprague–Dawley rats. The effective permeability coefficients (Peff) of Nos across Caco-2 and MDCK cell monolayers was found to be in the order of pH 5.0 > 5.8 > 6.8 > 7.8. The efflux ratios of Peff < 2 demonstrated that active efflux does not limit the absorption of Nos. The use of CH or CP have shown significant (***, p < 0.001) enhancement in Peff of Nos across cell monolayer compared with the control group. The CP (1–5% w/v) based Nos formulations resulted in significant (***, p < 0.001) increase in the bioavailability of Nos compared with Nos solution. The use of CP represents viable approach for enhancing the oral bioavailability of Nos and reducing the required dose.

Effect of combination of hydrophilic and lipophilic permeation enhancers on the skin permeation of kahalalide F.

The purpose of this study was to investigate the influence of combination of various lipophilic and hydrophilic chemical enhancers on skin delivery of kahalalide F (KF).

Abstract 5511: Multifunctional CREKA peptide conjugated lipid nanocarriers of synergistically acting Noscapine and Doxorubicin for breast cancer therapy

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Estrogen receptor negative (ER−) breast cancers (∼40%) are clinically aggressive with poor clinical outcome. Combination of different mechanism based antimicrotubular Noscapine and DNA intercalating Doxorubicin may lead to additive/synergistic activity against ER− breast cancer. Clinical utility of safer oral Noscapine (poor bioavailability and short half life) and Doxorubicin (cardiotoxicity and myelosuppression) has been limited. Encapsulation of Noscapine and Doxorubicin in nanocarriers whose surface is modified with pegylated CREKA peptide (MNCs) will significantly deliver nanocarriers to tumors by homing to tumor stroma, vessel wall and thereby releasing both drugs in controlled manner to exert anticancer activity. The purpose of this study was to encapsulate synergistically acting Noscapine and Doxorubicin in nanocarrier and modify the nanocarrier surface with CREKA and evaluate for anticancer activity in MDA-MB 231 ER− breast cancer cells. Isobolographic method and TUNEL assay were used to study Noscapine (10, 20 and 30 µM) and Doxorubicin interaction in MDA-MB 231 cells. For preparation of MNCs, Noscapine and Doxorubicin (molar ratio of 1:400) were dissolved in lipophilic phase composed of Miglyol (6% w/v), Compritol (3% w/v) and DOGS-NTA-Ni (0.2 % w/v). Lipophilic phase was poured to aqueous phase containing Polaxamer 188 (1.2 % w/v in water) and subjected to high-pressure homogenization to yield DOGS-NTA-Ni engrafted nanocarriers. Six-Histidine tagged PEG (1K)-CREKA (0.01-0.04 % w/v) was incubated with nanocarriers for 30 min for conjugation of DOGS-NTA-Ni engrafted nanocarrier with Histidine tagged peptide to yield MNCs. MNCs were characterized for size, drug release, antiproliferative and clot binding efficiency. Noscapine and Doxorubicin alone showed IC50 of 42 ± 4 µM and 0.25 ± 0.02 µM against MDA-MB cells respectively. In presence of Noscapine solution (20 µM), the IC50 of Doxorubicin solution was reduced to 0.05 µM (5-fold). Further, the combination Index values (< 0.6) and higher apoptotic cells (P 96 % of encapsulation and controlled release of both drugs (8 hr∼15 % and 48 hr∼ 60 %). A significantly (P < 0.01) higher binding of MNCs (CREKA concentration 0.045 % w/v) to the clotted plasma proteins showed the targeting ability of nanocarriers. MNCs showed similar IC50 values (20 µM Noscapine + 0.05 µM Doxorubicin) to that of solution combination. In conclusion, Noscapine acts synergistically with Doxorubicin and combination delivery of Noscapine and Doxorubicin using nanocarriers conjugated with CREKA showed significant increase in cytotoxicity with controlled drug release and significant binding efficiency. Multifunctional nanocarriers can effectively inhibit breast cancer and may reduce limitations associated with chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5511.