Punit P. Shah

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.

Enhanced skin permeation using polyarginine modified nanostructured lipid carriers.

The objective of the present study was to investigate the effect of polyarginine chain length on topical delivery of surface modified NLCs. Design of experiments (DOE) was used to optimize number of arginines required to deliver active drug into deeper skin layers. The NLCs were prepared by hot-melt technique and the surface of NLCs was modified with six-histidine tagged cell penetrating peptides (CPPs) or YKA. In vivo confocal microscopy and Raman confocal spectroscopy studies were performed using fluorescent dye encapsulated NLCs and NLC-CPPs. Spantide II (SP) and ketoprofen (KP) were used as model drugs for combined delivery. In vitro skin permeation and drug release studies were performed using Franz diffusion cells. Inflammatory response corresponding to higher skin permeation was investigated in allergic contact dermatitis (ACD) mouse model. NLCs had a particle size of 140±20nm with higher encapsulation efficiencies. The negative charge of NLC was reduced from -17.54 to -8.47 mV after surface modification with CPPs. In vivo confocal microscopy and Raman confocal spectroscopy studies suggested that a peptide containing 11 arginines (R11) had significant permeation enhancing ability than other polyarginines and TAT peptides. The amount of SP and KP retained in dermis after topical application of NLC-R11 was significantly higher than solution and NLC after 24 h of skin permeation. SP was not found in receiver compartment. However, KP was found in receiver compartment and the amount of KP present in receiver compartment was increased approximately 7.9 and 2.6 times compared to the control solution and NLCs, respectively. In an ACD mouse model, SP+KP-NLC-R11 showed significant reduction (p<0.05) in ear thickness compared to SP+KP solution and SP+KP-NLC. Our results strongly suggest that the surface modification of NLC with R11 improved transport of SP and KP across the deeper skin layers and thus results in reduction of inflammation associated with ACD.

Investigation of follicular and non-follicular pathways for polyarginine and oleic acid modified nanoparticles

PurposeTo investigate the percutaneous permeation pathways of cell penetrating peptide modified lipid nanoparticles and oleic acid modified polymeric nanoparticles.MethodsConfocal microscopy was performed on skin cultures (EpiDermFT™) for modified and un-modified nanoparticles. Differential stripping was performed following in vitro skin permeation of Ibuprofen (Ibu) encapsulated nanoparticles to estimate Ibu levels in different skin layers and receiver compartment. The hair follicles (HF) were blocked and in vitro skin permeation of nanoparticles was then compared with unblocked HF. The surface modified nanoparticles were investigated for response on allergic contact dermatitis (ACD).ResultsSurface modified nanoparticles showed a significant higher (p < 0.05) in fluorescence in EpiDermFT™ cultures compared to controls. The HF play less than 5% role in total nanoparticle permeation into the skin. The Ibu levels were significantly high (p < 0.05) for surface modified nanoparticles compared to controls. The Ibu levels in skin and receiver compartment were not significantly different when HF were open or closed. Modified nanoparticles showed significant improvement in treatment of ACD compared to solution.ConclusionsOur studies demonstrate that increased skin permeation of surface modified nanoparticles is not only dependent on a follicular pathway but also occur through non-follicular pathway(s).

Design, Synthesis of Novel Lipids as Chemical Permeation Enhancers and Development of Nanoparticle System for Transdermal Drug Delivery

In the present study, we designed and developed novel lipids that include (Z)-1-(Octadec-9-en-1-yl)-pyrrolidine (Cy5T), 1, 1-Di-((Z)-octadec-9-en-1-yl)pyrrolidin-1-ium iodide (Cy5), (Z)-1-(Octadec-9-en-1-yl)-piperidine (Cy6T), and 1, 1-Di-((Z)-octadec-9-en-1-yl) piperidin-1-ium iodide (Cy6) to enhance the transdermal permeation of some selected drugs. Firstly, we evaluated the transdermal permeation efficacies of these lipids as chemical permeation enhancers in vehicle formulations for melatonin, ß-estradiol, caffeine, α-MSH, and spantide using franz diffusion cells. Among them Cy5 lipid was determined to be the most efficient by increasing the transdermal permeation of melatonin, ß-estradiol, caffeine, α-MSH, and spantide by 1.5 to 3.26-fold more at the epidermal layer and 1.3 to 2.5-fold more at the dermal layer, in comparison to either NMP or OA. Hence we developed a nanoparticle system (cy5 lipid ethanol drug nanoparticles) to evaluate any further improvement in the drug penetration. Cy5 lipid formed uniformly sized nanoparticles ranging from 150–200 nm depending on the type of drug. Further, Cy5 based nanoparticle system significantly (p<0.05) increased the permeation of all the drugs in comparison to the lipid solution and standard permeation enhancers. There were about 1.54 to 22-fold more of drug retained in the dermis for the Cy5 based nanoparticles compared to OA/NMP standard enhancers and 3.87 to 66.67-fold more than lipid solution. In addition, epifluorescent microscopic analysis in rhodamine-PE permeation studies confirmed the superior permeation enhancement of LEDs (detection of fluorescence up to skin depth of 340 μm) more than lipid solution, which revealed fluorescence up to skin depth of only 260 μm. In summary the present findings demonstrate that i) cationic lipid with 5 membered amine heterocyclic ring has higher permeating efficacy than the 6 membered amine hertocyclic ring. ii) The nanoparticle system prepared with Cy5 showed significant (p<0.05) increase in the permeation of the drugs than the control penetration enhancers, oleic acid and NMP.

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.

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.

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

Abstract 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.

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 5660: Proteomic comparative assessment of flash-frozen and OCT embedded breast cancer tissues for utilization in precision oncology discovery studies

Proteomic analysis is an important tool in classifying molecular architecture of tissue samples for subtyping and precision oncology applications. Tissues are commonly either flash frozen (FF) or embedded in optimal cutting temperature (OCT) compound for long-term storage. OCT provides a convenient specimen matrix for cryostat sectioning but contains glycols and resins which are incompatible with mass spectrometric analysis of proteins. In contrast, flash frozen preservation of tissue is historically considered to be more suitable and compatible with mass spectrometry applications. As a pilot study, we evaluated different tissue preservation methods, sample processing methods, and protein extraction methods, and their impact on quantitation and coverage of the proteome. In this pilot study, 30 breast cancer cases with matching flash frozen and OCT-embedded samples were used for proteomic analysis. Tissues were procured from the Clinical Breast Care Project (CBCP) in partnership with the Chan Soon-Shiong Institute of Molecular Medicine at Windber, and the Walter Reed National Military Medical Center (WRNMMC). Breast cancer subtypes were determined by clinical and expanded immunohistochemical panels. These subtypes were Her2, Luminal A (LA), Luminal B (LB) and Triple Negative (TN). Flash frozen tissues were cryosectioned to isolate 60-100 mg total tissue per case, and proteins were extracted using an 8M urea lysis buffer. The OCT specimens were laser microdissected to isolate primarily tumor cells from the samples. Proteins were extracted using the illustra TriplePrep kit. Digested proteins were then multiplexed using Tandem-Mass-Tag (TMT) 10plex isobaric labeling reagents, and chromatographically resolved using a Waters 2D nanoAcquity liquid chromatographer (LC) coupled to a Thermo Q Exactive Plus mass spectrometer (MS). Data was then analyzed using Proteome Discoverer. In total, 6130 proteins were identified using a 1% peptide FDR confidence filter in the 30 samples analyzed. Unsupervised hierarchical clustering of proteins across the samples resulted in two primary clusters, OCT and FF. Within the primary clustering, there were sub-clusters of Luminal, Her2, and Triple Negatives. At 2-fold threshold, 246 proteins were differentially expressed between OCT and FF tissues. However, the differential proteins between ER+ (LA and LB) and ER- (Her2 and TN) tumors correlate irrespective of the cryopreservation and processing methods. In conclusion, proteome analysis of samples stored under two different conditions provide subtyping information; however, there is differential expression of proteins between the two types of sample handling techniques which can impact biological interpretation of breast cancer precision oncology studies. Citation Format: Kiki Panagopoulos, Punit Shah, Albert Kovatich, Michael A. Kiebish, Jeffrey Hooke, Leigh Campbell, Mary Lou Cutler, Audrey Kovatich, Hai Hu, Brenda Deyarmin, Lori Sturtz, Praveen Kumar Raj Kumar, Stella Somiari, Emily Y. Chen, Elder Granger, Viatcheslav Akmaev, Rangaprasad Sarangarajan, Niven Narain, Craig D. Shriver. Proteomic comparative assessment of flash-frozen and OCT embedded breast cancer tissues for utilization in precision oncology discovery studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5660.

Abstract 3139: Nurr1, a novel target of 1,1-bis(3’-indolyl)-1-(p-chlorophenyl) methane for inhibition of the initiation and progression of skin cancer tumorigenesis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The objective of this study was to demonstrate the chemopreventive potential of 1,1-bis(3′-indolyl)-1-(p-chlorophenyl methane) (DIM-D) in skin cancer using an in vitro and in vivo models. In vitro cell cytotoxicity and viability assays were carried out in A431 human epidermoid carcinoma cell line (A431) and normal human epidermal keratinocytes (NHEK) respectively. Apoptosis induction and accumulation of ROS following UVB exposure in DIM-D pretreated NHEK cells (2 hr prior) was also evaluated. Immunocytochemistry and western blot analysis were performed to determine proapoptotic and proinflammatory markers expressions in DIM-D treated A431 cells and in UVB irradiated NHEK cells. For in vivo experiments, DIM-D was encapsulated within nanocarriers by hot melt homogenization using Nano DeBEE. Nanocarriers were surface modified with oleic acid and incorporated into a gel using established procedures (G-NOD) and characterized for particle size, zeta potential, entrapment efficiency, rheology, skin permeation and drug release. The chemo-preventive efficiency of G-NOD was evaluated using UVB-induced skin cancer model in SKH hairless mice and skin samples were collected and investigated for lipid peroxidation, protein carbonyls, and molecular markers by western blot. The IC50 values of DIM-D were 24.5±2.6, 17.2±3.6and 4.1±1.1 whilst for EGCG were 192.1±3.8, 85.3±2.4 and 26.0 ±1.4 for 24, 48 and 72 hr treatments respectively. 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). Nanocarriers containing DIM-D were 188.00±8.00 nm in size with polydispersity of 0.59±0.01. The zeta potential and entrapment efficiency were 37.17±0.90mV and 89±0.50%, respectively. There was significant (p<0.05) reduction in tumor size/number for G-NOD pretreated group (2 tumors/mouse, average diameter, 2±0.50 mm) compared to DIM-PG (4 tumors/mouse, average diameter, 4±0.45mm) and EGCG PEG-gel (EGCG-PG; 6 tumors/mouse, average diameter, 5±0.25mm). IHC and western blot results indicated significant increase (p<0.05) in expression of Nurr1 in G-NOD pretreated group compared to EGCG-PG. There was however pronounced reduction in expression of STAT3, NFkB, cleaved Caspase-3, BCl2 and 8-OHdG for G-NOD pretreated group compared to DIM-PG and EGCG-PG. Lipid peroxidation assay revealed decrease in amount of Malondialehyde (MDA) produced in G-NOD pretreated group prior to UV exposure by 2 fold compared to DIM-PG and 1.3 fold compared to EGCG-PG treatments. Therefore, findings in both in vitro and in vivo studies suggest that the enhanced percutaneous delivery of DIM-D reduces UVB-induced damage to skin lipids and protein and inhibits initiation and progression of skin photocarcinogenesis in SKH mice via the transactivation of Nurr1. Citation Format: Ravi Doddapaneni, Cedar Boakye, Punit Shah, Apurva R. Patel, Chandraiah Godugu, Stephen Safe, Santosh Katiyar, Mandip Sachdeva. Nurr1, a novel target of 1,1-bis(3’-indolyl)-1-(p-chlorophenyl) methane for inhibition of the initiation and progression of skin cancer tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3139. doi:10.1158/1538-7445.AM2014-3139