Percutaneous delivery of α-melanocyte-stimulating hormone for the treatment of imiquimod-induced psoriasis.

PURPOSE: α-Melanocyte-stimulating hormone (α-MSH) is an endogenous peptide hormone with anti-inflammatory responses. We developed topical formulation(s) of α-MSH to reduce psoriasis-related inflammation. METHODS: Transcutol (TC) and n-methyl 2-pyrrolidone (NMP) were used to formulate a gel for α-MSH. Skin permeation and dermal microdialysis of the solution and optimized gel were performed. The inflammatory response of α-MSH gel was investigated in imiquimod-induced psoriasis mouse model. Histology and immunohistochemistry were then performed on treated skin. RESULTS: Solution comprising 50%w/w TC and 10%w/w NMP showed higher (p < 0.05) skin retention (0.27 ± 0.024 µg of α-MSH/mg of skin) than solutions containing either 50% w/w TC or 10% w/w NMP at 24 h. Dispersion of α-MSH in Carbopol Ultrez 10 produced a uniform dispersion. α-MSH gel showed pseudoplastic flow with thixotropic behavior. Dermal microdialysis results suggested that skin permeation of gel after 5 h was 1.9-folds higher than the solution. Further, gel-treated psoriatic-like plaque skin sections showed significant (p < 0.05) decrease in the expression of a melanocortin receptor, in the psoriasis area and severity index score and transepidermal water loss compared to the solution. CONCLUSION: TC, NMP and Carbopol Ultrez 10 form a stable gel with improved skin permeation of α-MSH for a reduction in psoriasis-associated inflammation.

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

OBJECTIVES: 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). METHODS: KF formulations comprising a combination of lipophilic and hydrophilic chemical enhancers with varied per cent were prepared and evaluated for skin permeation studies. In vitro skin permeation of KF formulations was performed using Franz diffusion cell. Stability studies of KF formulations were performed according to the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use guideline, and the therapeutic efficacy of KF formulation was evaluated using allergic contact dermatitis animal model. KEY FINDINGS: The efficacy of KF formulations to improve skin delivery of KF was sequenced in the order of: formulation #4 > formulation #2 > formulation #1 > formulation #3, where formulation #4 contains labrasol (40% w/v), ethyl oleate (5% w/v) and span 80 (5% w/v) along with transcutol (40% w/v) and ethanol (10% w/v). Further, all the formulations were stable for 1 month when stored at 30°C/65% relative humidity. CONCLUSIONS: The results of present study suggest that therapeutically effective concentrations of KF can be delivered in the skin using combination of lipophilic and hydrophilic chemical enhancers.

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

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.

(31)P 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.

Efficacy of aerosolized celecoxib encapsulated nanostructured lipid carrier in non-small cell lung cancer in combination with docetaxel.

PURPOSE: Evaluation of in-vivo anticancer activity of aerosolized Celecoxib encapsulated Nanolipidcarriers (Cxb-NLC) as a single therapeutic agent and combined with intravenously administered Docetaxel (Doc) against non-small cell lung cancer. METHODS: Cxb-NLC were prepared by high-pressure homogenization and were characterized for its physicochemical characteristics. Metastatic A549 tumor model in Nu/Nu mice was used to evaluate response of aerosolized Cxb-NLC & Doc. Isolated lung tumor samples were analyzed for: a) DNA fragmentation and cleaved caspase-3 by immunohistochemistry, b) apoptotic and angiogenic protein markers by western blot, c) global proteomic alterations by an isobaric labeling quantitative proteomic method and d) toxicity studies of NLC. RESULTS: The particle size of Cxb-NLC was 217 ± 20 nm, while entrapment efficiency was more than 90%. Cxb-NLC and Doc alone and in combination showed 25 ± 4%, 37 ± 5%, and 67 ± 4% reduction in tumor size respectively compared to control. Proteomic analysis with combination treatment further revealed significantly decreased expression of multiple pro-survival and pro-metastasis proteins as well as tumor invasion markers and the expression of S100 family proteins, such as S100A6 and S100P were decreased by 2.5 and 1.6 fold. CONCLUSIONS: Combination therapy with Cxb-NLC and Doc showed significant reduction in tumor growth which was further confirmed by proteomic analysis.

Dermal microdialysis technique to evaluate the trafficking of surface-modified lipid nanoparticles upon topical application.

PURPOSE: To evaluate the skin pharmacokinetics and tissue distribution of cell penetrating peptides (CPP) modified nano-structured lipid carrier (NLC) using an in vivo dermal microdialysis (MD) technique. METHODS: Celecoxib (Cxb) encapsulated NLCs (CXBN), CPP modified CXBN (CXBN-CPP) and Cxb-Solution (CXBS) formulations were prepared and tested for in vitro skin distribution. MD was used to assess pharmacokinetic parameters of Cxb after topical application of Cxb formulations. The effect of pre-treatment with Cxb formulations was evaluated for expression of prostaglandin-E2 (PGE(2)) and Interleukin-6 (IL-6) after exposure of xylene using MD. Allergic contact dermatitis (ACD) model was used to confirm in vivo therapeutic response of Cxb formulations. RESULTS: The cumulative permeation of Cxb in MD dialysate after 24 h for CXBN-CPP was significantly higher (p < 0.001) than CXBN and CXBS. Further, pre-treatment with CXBN-CPP significantly inhibited PGE(2) and IL-6 expression compared to CXBS and CXBN (p < 0.001). In ACD model, CXBN-CPP showed significant reduction (p < 0.001) in ear thickness compared to controls. CONCLUSIONS: Surface modification of NLC with CPPs can enhance the skin permeation of Cxb and MD can be used to investigate pharmacokinetics of Cxb nanoparticles in the skin.

Interaction of nanoparticles and cell-penetrating peptides with skin for transdermal drug delivery.

Topical or transdermal drug delivery is challenging because the skin acts as a natural and protective barrier. Therefore, several methods have been examined to increase the permeation of therapeutic molecules into and through the skin. One approach is to use the nanoparticulate delivery system. Starting with liposomes and other vesicular systems, several other types of nanosized drug carriers have been developed such as solid lipid nanoparticles, nanostructured lipid carriers, polymer-based nanoparticles and magnetic nanoparticles for dermatological applications. This review article discusses how different particulate systems can interact and penetrate into the skin barrier. In this review, the effectiveness of nanoparticles, as well as possible mode of actions of nanoparticles, is presented. In addition to nanoparticles, cell-penetrating peptide (CPP)-mediated drug delivery into the skin and the possible mechanism of CPP-derived delivery into the skin is discussed. Lastly, the effectiveness and possible mechanism of CPP-modified nanocarriers into the skin are addressed.

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.

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.

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

Targeting of plasmid DNA-lipoplexes to cells with molecules anchored via a metal chelator lipid.

BACKGROUND: The ability to deliver plasmid DNA (pDNA) to specific cells in vivo is crucial for achieving efficient targeted transfection with nonviral vectors. We previously used stealth liposomes containing the chelator lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA(3)-DTDA) to target delivery of antigen and cytokines to immune cells in vivo. In the present study, we utilized liposomes containing NTA(3)-DTDA and the ionizable aminolipid 1,2-dioleoyl-3-dimethyl-ammonium-propane (DODAP) to incorporate pDNA into complexes for targeting to cells. METHODS: Liposomes containing DODAP, NTA(3)-DTDA and helper lipids were acidified (pH 5.5) and mixed with pDNA to form complexes. These lipoplexes were neutralized and engrafted with His-tagged molecules for targeting to extracellular receptors. Targeted transfection efficiency was assessed using the enhanced green fluorescent protein reporter gene. RESULTS: Initial transfections of HEK-293 cells using a His-tagged peptide (T2) related to the Arg-rich motif of HIV-1 TAT protein resulted in a low transfection efficiency (<2.5%). Optimization of the lipid formulation and use of an endosome-destabilizing peptide and inhibitor of DNase II, increased transfection approximately 20-fold. These lipoplexes are approximately 250 nm in diameter, and transfection efficiencies were: approximately 50% for HEK-293 cells targeted with lipoplexes containing pEGFP-N1 and engrafted with T2, and 30-40% for HepG2 cells targeted with lipoplexes engrafted with a peptide specific for the VEGF receptor Flt-1. CONCLUSIONS: The results show that DODAP-containing lipoplexes incorporating NTA(3)-DTDA enable the engraftment of targeting molecules and the effective targeting of pDNA to cells in serum-containing media, resulting in efficient transgene expression. The strategy may provide a convenient approach for targeting pDNA to cells in vivo in therapeutic applications.

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.

Folate-targeted etoposide-encapsulated lipid nanospheres.

Lipid nanospheres (LN) are simple colloidal drug delivery systems, which are proven to be useful for the systemic delivery of lipophilic anticancer drugs. Our previous work shows that the encapsulation of etoposide in LN improved the anticancer activity and a further inclusion of polyethylene glycol-distearoylphosphatidylethanolamine (DSPE-PEG) increased the circulation time and stability of LN. The present study is focused on the targeting ability of LN using Folate-PEG-DSPE. Folate-targeted (Fol-LNE) and non-targeted (SLNE) etoposide-encapsulated lipid nanospheres were prepared with the help of soybean oil, egg phosphatidylcholine, and PEG-DSPE with and without Folate-PEG-DSPE. The anticancer activity of these formulations was assessed in KB cell line. Cell uptake studies were carried out in KB cell lines using fluorescent-labeled targeted (Fol-LN) and non-targeted (SLN) lipid nanospheres without etoposide. Confocal microscopy and flow cytometry results found that, Fol-LN was selectively taken up by the KB cells and the addition of 1 mM folic acid completely blocked this uptake. Cytotoxicity results support the above finding, the IC50 values of etoposide solution, Fol-LNE, and Fol-LNE-comp (competition with 1 mM folic acid) were 33, 5, and 19 muM, respectively. Tissue distribution of Fol-LNE was compared with that of SLNE and etoposide commercial formulation (ETP) in normal mice. The studies show that in the kidney etoposide concentration was higher following Fol-LNE administration than SLNE and ETP.

Antitumor activity of noscapine in human non-small cell lung cancer xenograft model.

PURPOSE: An antitussive plant alkaloid, Noscapine HCl (Nos) displays anticancer activity and has a safe pharmacological profile in humans. The current study was aimed to investigate the in vitro and in vivo anti tumor activity of Nos to determine possible mechanisms of anti tumor activity for treatment of non-small cell lung cancer (NSCLC). METHODS: In vitro cytotoxicity of Nos was studied in H460 cells treated with different doses of Nos (10-160 microM) for 72 h and cell viability was determined using crystal violet assay. Apoptosis in H460 cells was evaluated by TUNEL assay after treatment of cells for 72 h with 30 and 40 microM doses of Nos. For in vivo studies, female athymic Nu/nu mice were xenografted with H460 tumors and on day 4 onwards Nos was administered orally at dose of 300, 450 and 550 mg/kg/day for 24 days. As a control, xenografted tumors were separately treated with Docetaxel (10 mg/kg i.v. bolus on day 5, 11, 17, 23). The tumor volumes were measured every five days. Expression of PARP, Bcl(2, )Bax, and caspase-3 families of proteins was measured by Western Blotting (WB), while TUNEL and Immunohistochemical methods were utilized to determine DNA fragmentation and cleaved caspase-3 levels respectively. RESULTS: Nos inhibited growth of H460 cells with the IC50 values of 34.7 +/- 2.5 microM. Nos at 30 and 40 microM doses caused apoptosis as evidenced by nuclear condensation in treated H460 cells. Nos caused 49, 65 and 86% reduction in the xenografted tumor volumes at a dose of 300 (P < 0.05), 450 (P < 0.01), 550 mg/kg/day (P < 0.01), respectively, when compared to controls. Nos-dependent suppression of xenografted tumor growth involved up regulation of PARP, Bax, caspase-3 and repression of Bcl(2) expression. An increase in Bax/Bcl(2) ratio suggests involvement of a mitochondrial mediated apoptotic processes. Our studies revealed a non significant (P > 0.05) increase in Bax/Bcl(2) ratio with Nos at a dose of 300 mg/kg/day, while a significant (P < 0.001) increase in Bax/Bcl(2) ratio was observed with Nos doses of 450 and 550 mg/kg/day. Further, Nos caused elevated apoptosis in tumor xenografts as evidenced by enhanced expression of caspase-3 and positive TUNEL staining in regressed tumor tissues, thus suggesting induction of apoptosis by mitochondrial pathway. CONCLUSION: Our studies suggest that potent antitumor activity of Nos against NSCLC cells. Oral administration of Nos showed significant reduction in tumor volume in human non-small cell lung tumor xenograft in nude mice in a dose dependant manner. Thus, Nos is a promising novel chemotherapeutic agent for the treatment of human lung cancer.

Pharmacokinetics and tissue distribution of etoposide delivered in long circulating parenteral emulsion.

PURPOSE: The aim of the study is to ascertain the influence of pegylation of parenteral emulsion (PE) on their long circulating property. METHODS: Etoposide encapsulated parenteral emulsion (EPE) was prepared using soybean oil, egg lecithin and cholesterol. Etoposide encapsulated long circulating parenteral emulsion (PEG-EPE) was prepared using PEG (2000)-DSPE as a stealth agent. The effect of monovalent and divalent electrolytes on the stability of EP was assessed by measuring the fixed aqueous layer thickness (FALT) and flocculation rate. Pharmacokinetics and tissue distribution pattern of PE following i.v. (bolus) were assessed in Wistar rats and Swiss albino mice. RESULTS: FALT of PEG-EPE was larger than that of EPE. In case of PEG-EPE, as the concentration of pegylated lipid (PEG) increased from 0.15 to 0.45% w/v the flocculation rate decreased asymptomatically in the presence of monovalent and divalent electrolytes. The increased circulation time of PEG-EPE (0.3%) after intravenous injection to rats confirms the presence of FALT around globules. PEG-EPE showed improved pharmacokinetic parameters with 5.5 times higher AUC than etoposide commercial formulation (ETP). Tissue distribution results show that etoposide levels in all tissues except in brain and heart were lower in case of PEG-EPE than ETP. The percentage of tumor growth suppression rate (%T/C) in Lewis lung carcinoma bearing mice was 63.23, 62.83 and 33.78% in EPE, PEG-EPE and ETP treated mice, respectively. The improved activity of PEG-EPE is due to enhanced permeability and retention effect (EPR). CONCLUSION: Encapsulation of etoposide in PEG-coated PE produced improved pharmacokinetic profile than that of EPE and ETP.