Discovery of novel ceramide analogs with favorable pharmacokinetic properties and combination with AKT inhibitor against colon cancer.

Ceramides have emerged as potential therapeutic option with novel mechanism to affect the proliferation, differentiation, senescence, and apoptosis of cancer cells through regulation of multiple signal transduction. Aiming at the improvement of the apoptosis activity and pharmacokinetic profiles of ceramides, a novel series of ceramide analogs were developed through structure simplification and conformation restriction. Among them, compound 12 bearing an alkoxyl naphthyl motif, with favorable rat pharmacokinetic properties, showed better anti-proliferative activity against various colon cancer cells (IC50 ∼20 µM) than other ceramide analogues, as well as the synergistic effect combined with AKT inhibitor MK2206. Additionally, we demonstrated that this combination therapy promoted caspase 3-dependent apoptotic pathway and intensified cell cycle arrest in the G0/G1 phase. Furthermore, the combination of compound 12 and MK2206 displayed synergistic anti-tumor effect in vivo.

Rubusoside-assisted solubilization of poorly soluble C6-Ceramide for a pilot pharmacokinetic study.

Although C6-Ceramide has attracted much attention as a possible tumor suppressor, the delivery of C6-Ceramide is still challenging due to its inherent hydrophobicity and insolubility. In this study we explored the use of a natural compound rubusoside (RUB) as a solubilizer to enhance the solubility of a fluorescence-labeled C6-Ceramide (NBD C6-Ceramide) and to characterize its pharmacokinetics and tissue distribution in an animal model. RUB significantly enhanced the solubility of NBD C6-Ceramide by forming nanomicelles, and efficiently delivered NBD C6-Ceramide in rats by oral and intravenous administration. RUB loaded 1.96 % of NBD C6-Ceramide in the nanomicelles and solubilized it to a concentration of 3.6 mg/mL in water. NBD C6-Ceramide in nanomicelles remained stable in aqueous solutions, allowing intravenous administration without the use of any organic solvents or surfactants. After oral administration, NBD C6-Ceramide rapidly rose to peak plasma concentrations within the first 90 min, distributed to tissues, and remained in vivo for more than 24 h. Tissular levels of NBD C6-Ceramide from high to low were associated with heart, lung, cerebellum, testicle, spleen, liver, kidney, and brain. Altogether, our study demonstrated that RUB-assisted nanomicelles can serve as an efficient and convenient delivery system for short-chain C6-Ceramide and enable in vivo evaluation of potential new cancer treatments.

Ceramide-Rubusoside Nanomicelles, a Potential Therapeutic Approach to Target Cancers Carrying p53 Missense Mutations.

Ceramide (Cer) is an active cellular sphingolipid that can induce apoptosis or proliferation-arrest of cancer cells. Nanoparticle-based delivery offers an effective approach for overcoming bioavailability and biopharmaceutics issues attributable to the pronounced hydrophobicity of Cer. Missense mutations of the protein p53, which have been detected in approximately 42% of cancer cases, not only lose the tumor suppression activity of wild-type p53, but also gain oncogenic functions promoting tumor progression and drug resistance. Our previous works showed that cellular Cer can eradicate cancer cells that carry a p53 deletion-mutation by modulating alternative pre-mRNA splicing, restoring wild-type p53 protein expression. Here, we report that new ceramide-rubusoside (Cer-RUB) nanomicelles considerably enhance Cer in vivo bioavailability and restore p53-dependent tumor suppression in cancer cells carrying a p53 missense mutation. Natural RUB encapsulated short-chain C6-Cer so as to form Cer-RUB nanomicelles (∼32 nm in diameter) that substantially enhanced Cer solubility and its levels in tissues and tumors of mice dosed intraperitoneally. Intriguingly, Cer-RUB nanomicelle treatments restored p53-dependent tumor suppression and sensitivity to cisplatin in OVCAR-3 ovarian cancer cells and xenograft tumors carrying p53 R248Q mutation. Moreover, Cer-RUB nanomicelles showed no signs of significant nonspecific toxicity to noncancerous cells or normal tissues, including bone marrow. Furthermore, Cer-RUB nanomicelles restored p53 phosphorylated protein and downstream function to wild-type levels in p53 R172H/+ transgenic mice. Altogether, this study, for the first time, indicates that natural Cer-RUB nanomicelles offer a feasible approach for efficaciously and safely targeting cancers carrying p53 missense mutations.

Oxidized graphene nanoparticles as a delivery system for the pro-apoptotic sphingolipid C6 ceramide.

Sphingolipids such as ceramide have attracted much attention as possible anticancer agents due to their potent pro-apoptotic effects. However, due to their extreme hydrophobicity, there is currently no clinically approved delivery method for in vivo use as a therapeutic agent. To this end, we have developed a novel method for loading the short-chain C6 ceramide onto oxidized graphene nanoribbons (O-GNRs) and graphene nanoplatelets (GNPs). Mass spectrometry revealed loading efficiencies of 57% and 51.5% for C6 ceramide onto O-GNRs and GNPs, respectively. The PrestoBlue viability assay revealed that 100 µg/mL of C6 ceramide-loaded O-GNRs and C6 ceramide-loaded GNPs reduced HeLa cell viability by approximately 93% and approximately 76%, respectively, compared to untreated HeLa cells, while equal concentrations of these nanoparticles without C6 ceramide did not significantly reduce HeLa cell viability. We confirmed that this cytotoxicity was apoptotic in nature via capase-3 activity and Hoechst staining. Using live-cell confocal imaging with the fluorescent NBD-ceramide loaded on O-GNRs, we observed robust uptake into HeLa cells within 30 min while NBD-ceramide on its own was uptaken much more rapidly. Transmission electron microscopy confirmed that C6 ceramide-loaded O-GNRs were actually entering cells. Taken together, these data show that O-GNRs are a promising delivery agent for ceramide. To our knowledge, this study is the first to use such a loading method. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 25-37, 2019.

Oleate increase neutral lipid accumulation, cellular respiration and rescues palmitate-exposed GLP-1 secreting cells by reducing ceramide-induced ROS.

BACKGROUND: Fatty acids (FAs), and especially monounsaturated FAs (MUFAs) stimulate GLP-1 release. However, lipotoxicity is indicated in GLP-1 secreting cells following long-term exposure to elevated levels of saturated FAs (SFAs) in vivo and in vitro, where in vitro studies indicate that cosupplementation with MUFAs confers lipoprotection. SFAs and MUFAs differentially affect the fate of cells in ways that depend on the cell type, concentration and ratio of the FAs. The present study was designed to further elucidate the mechanisms underlying the effects of SFAs/MUFAs on GLP-1-producing cells in terms of lipotoxicity/lipoprotection and GLP-1 secretion. METHODS: Cultured GLP-1 secreting cells were exposed to hyperlipidemia simulated by SFA-albumin complexes where the molar ratio was 2:1. The cellular response to simulated hyperlipidemia was assessed in the presence/absence of MUFA cosupplementation by determining intracellular ceramide, ROS, neutral lipid accumulation, and cellular respiration. The role for cellular respiration in GLP-1 secretion in response to SFAs/MUFAs was assessed. RESULTS: Generation of intracellular ceramide mediate a detrimental increased in ROS production following long term exposure to SFAs in GLP-1-secreting cells. Cosupplementation with MUFAs increases cellular respiration, triglyceride synthesis, and the expression of ceramide kinase, while reducing ceramide synthesis and attenuating ROS production, caspase-3 activity and DNA fragmentation. Further, acute secretory effects of unsaturated FAs are independent of FAO, but mediated by a FFAR1 induced increase in cellular respiration. CONCLUSION: This study demonstrates novel data supporting effects of MUFAs on the ceramide biosynthetic pathway, triglyceride storage respiration and secretion in GLP-1 secreting cells. These findings may be of value for nutritional interventions, as well as for identification of novel targets, to help preserve L-cell mass and potentiate GLP-1 secretion in diabesity.

Development and validation of LC/APCI-MS method for the quantification of oat ceramides in skin permeation studies.

Ceramides (CERs) are the backbone of the intercellular lipid lamellae of the stratum corneum (SC), the outer layer of the skin. Skin diseases such as atopic dermatitis, psoriasis, and aged skin are characterized by dysfunctional skin barrier and dryness which are associated with reduced levels of CERs. Replenishing the depleted epidermal CERs with exogenous CERs has been shown to have beneficial effects in improving the skin barrier and hydration. The exogenous CERs such as phyto-derived CERs (PhytoCERs) can be delivered deep into the SC using novel topical formulations. This, however, requires investigating the rate and extent of skin permeation of CERs. In this study, an LC/APCI-MS method to detect and quantify PhytoCERs in different layers of the skin has been developed and validated. The method was used to investigate the skin permeation of PhytoCERs using Franz diffusion cells after applying an amphiphilic cream containing PhytoCERs to the surface of ex vivo human skin. As plant-specific CERs are not commercially available, well-characterized CERs isolated from oat (Avena abyssinica) were used as reference standards for the development and validation of the method. The method was linear over the range of 30-1050 ng/mL and sensitive with limit of detection and quantification of 10 and 30 ng/mL, respectively. The method was also selective, accurate, and precise with minimal matrix effect (with mean matrix factor around 100%). Even if more than 85% of oat CERs in the cream remained in the cream after the incubation periods of 30, 100, and 300 min, it was possible to quantify the small quantities of oat CERs distributed across the SC, epidermis, and dermis of the skin indicating the method's sensitivity. Therefore, the method can be used to investigate the skin permeation of oat CERs from the various pharmaceutical and cosmeceutical products without any interference from the skin constituents such as the epidermal lipids. Graphical abstract ᅟ.

Quantifying ceramide kinetics in vivo using stable isotope tracers and LC-MS/MS.

Numerous studies have implicated dyslipidemia as a key factor in mediating insulin resistance. Ceramides have received special attention since their levels are inversely associated with normal insulin signaling and positively associated with factors that are involved in cardiometabolic disease. Despite the growing literature surrounding ceramide biology, there are limited data regarding the activity of ceramide synthesis and turnover in vivo. Herein, we demonstrate the ability to measure ceramide kinetics by coupling the administration of [2H]water with LC-MS/MS analyses. As a "proof-of-concept" we determined the effect of a diet-induced alteration on ceramide flux; studies also examined the effect of myriocin (a known inhibitor of serine palmitoyltransferase, the first step in sphingosine biosynthesis). Our data suggest that one can estimate ceramide synthesis and draw conclusions regarding the source of fatty acids; we discuss caveats in regards to method development in this area.

Retarder action of isosorbide in a microemulsion for a targeted delivery of ceramide NP into the stratum corneum.

Ceramide [NP] is an integral component of the stratum corneum (SC) lipid matrix and is capable of forming tough and stable lamellar structures. It was proven, that in skin diseases as psoriasis or atopic dermatitis different ceramide (CER) classes, including [NP], are degraded. It is obvious that topically application of CER on impaired skin is useful for repairing the skin barrier but a tendency for low penetration due to its poor solubility in conventional dosage forms was observed. Therefore, a stable and physiologic compatible colloidal carrier system, a microemulsion (ME), was developed and characterized. The increasing knowledge of the new colloidal systems in this last decade shows their benefits in dermal application. Isosorbide (Polysorb P) was incorporated into the ME developed. It was expected that Polysorb P has a retarder potential in order to accumulate the CER in the SC, the outermost layer of the skin. Thereby the CER [NP] would be able to interact with the affected skin layers to strengthen the skin barrier. The release and penetration behavior of the CER [NP] from the ME was assessed ex vivo in a Franz diffusion cell. The results of the study showed that CER [NP] penetrate largely in the upper layers of the skin (from SC to stratum basale), which was the desired region. A recovery in the acceptor could not be detected that underlines an accumulation in upper layers. Furthermore, significantly increased values for the SC for the ME with retarder were not received. No differences in the concentrations of CER [NP] were observed. However, the toxicity of MEs was investigated using hens egg test chorioallantoic membrane (HET-CAM). For the isosorbide-containing ME no difference was obtained in comparison to the non-containing. The results showed that both MEs are safe to be used on the skin for the controlled penetration of CER [NP] into the skin. The isosorbide had no effect on the irritating effect as well as on the penetration of the used CER.

Nanovesicles of nitrendipine with lipid complex for transdermal delivery: pharmacokinetic and pharmacodynamic studies.

CONTEXT: Vesicular transdermal delivery can enhance the bioavailability of a drug especially affected by first-pass metabolism, e.g. nitrendipine. However effective transdermal delivery employs permeation enhancer, e.g oleic acid (OA) with ceramide 2, stearic acid, behenic acid, and cholesteryl sulfate lipid complex. OBJECTIVE: This study investigated the preparation, characterization of physicochemical properties, ex vivo permeation using human skin, pharmacokinetic parameters and antihypertensive potential in rats, of nitrendipine-loaded nanovesicles of ceramide 2, stearic acid, behenic acid and cholesteryl sulfate containing oleic acid gel (NOVG). MATERIALS AND METHODS: The nanovesicles were made using film hydration method and characterized for physicochemical properties, ex vivo permeation using human skin, pharmacokinetic parameters and antihypertensive potential. RESULTS: Nitrendipine-loaded nanovesicles of ceramide-2 containing oleic acid (NOV-5) have shown fluxes in the range of 4.88-24.72 µg/cm(2)/h nitrendipine oral suspension (NOS) at equal dose. NOVG-5 has shown almost 33% reduction in blood pressure in the first hour and a further decrease of 25% in the second hour to restore the normal pressure. DISCUSSION: The permeation increases with increase in OA content. OA gets integrated in vesicle wall and enhances its permeability, whereas ceramide content makes sure that skin does not become damaged even after permeation. CONCLUSION: NOVG-5 has shown the most favorable physicochemical properties and good permeation through skin providing good management of hypertension during crucial initial hours.

Ceramides with a pentadecasphingosine chain and short acyls have strong permeabilization effects on skin and model lipid membranes.

The composition and organization of stratum corneum lipids play an essential role in skin barrier function. Ceramides represent essential components of this lipid matrix; however, the importance of the individual structural features in ceramides is not fully understood. To probe the structure-permeability relationships in ceramides, we prepared analogs of N-lignoceroylsphingosine with shortened sphingosine (15 and 12 carbons) and acyl chains (2, 4 and 6 carbons) and studied their behavior in skin and in model lipid membranes. Ceramide analogs with pentadecasphingosine (15C) chains were more barrier-perturbing than 12C- and 18C-sphingosine ceramides; the greatest effects were found with 4 to 6C acyls (up to 15 times higher skin permeability compared to an untreated control and up to 79 times higher permeability of model stratum corneum lipid membranes compared to native very long-chain ceramides). Infrared spectroscopy using deuterated lipids and X-ray powder diffraction showed surprisingly similar behavior of the short ceramide membranes in terms of lipid chain order and packing, phase transitions and domain formation. The high- and low-permeability membranes differed in their amide I band shape and lamellar organization. These skin and membrane permeabilization properties of some short ceramides may be explored, for example, for the rational design of permeation enhancers for transdermal drug delivery.

Activity of neutral and alkaline ceramidases on fluorogenic N-acylated coumarin-containing aminodiols.

Ceramidases catalyze the cleavage of ceramides into sphingosine and fatty acids. Previously, we reported on the use of the RBM14 fluorogenic ceramide analogs to determine acidic ceramidase activity. In this work, we investigated the activity of other amidohydrolases on RBM14 compounds. Both bacterial and human purified neutral ceramidases (NCs), as well as ectopically expressed mouse neutral ceramidase hydrolyzed RBM14 with different selectivity, depending on the N-acyl chain length. On the other hand, microsomes from alkaline ceramidase (ACER)3 knockdown cells were less competent at hydrolyzing RBM14C12, RBM12C14, and RBM14C16 than controls, while microsomes from ACER2 and ACER3 overexpressing cells showed no activity toward the RBM14 substrates. Conversely, N-acylethanolamine-hydrolyzing acid amidase (NAAA) overexpressing cells hydrolyzed RBM14C14 and RBM14C16 at acidic pH. Overall, NC, ACER3, and, to a lesser extent, NAAA hydrolyze fluorogenic RBM14 compounds. Although the selectivity of the substrates toward ceramidases can be modulated by the length of the N-acyl chain, none of them was specific for a particular enzyme. Despite the lack of specificity, these substrates should prove useful in library screening programs aimed at identifying potent and selective inhibitors for NC and ACER3.

Milk Phospholipids Enhance Lymphatic Absorption of Dietary Sphingomyelin in Lymph-Cannulated Rats.

Supplementation with sphingomyelin has been reported to have beneficial effects on disease prevention and health maintenance. However, compared with glycerolipids, intact sphingomyelin and ceramides are poorly absorbed. Therefore, if the bioavailability of dietary sphingomyelin is increased, then the dose administered can be reduced. This study was designed to identify molecular species of ceramide in rat lymph after the ingestion of milk sphingomyelin, and to compare the effect of purified sphingomyelin with milk phospholipids concentrate (MPL, 185 mg sphingomyelin/g) on lymphatic absorption of milk sphingomyelin. Lymph was collected hourly for 6 h from lymph-cannulated rats (n = 8/group) after the administration of a control emulsion (triolein, bovine serum albumin, and sodium taurocholate), a sphingomyelin emulsion (control + purified sphingomyelin), or a MPL emulsion (control + MPL). Molecular species of ceramide in lymph were analyzed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Molecular species of ceramide, containing not only d18:1, but also d17:1 and d16:1 sphingosine with 16:0, 22:0, 23:0, and 24:0 fatty acids (specific to milk sphingomyelin), were increased in rat lymph after the administration of milk sphingomyelin. Their molecular species were similar to those of dietary milk sphingomyelin. Recovery of ceramide moieties from dietary sphingomyelin was 1.28- to 1.80-fold significantly higher in the MPL group than in the sphingomyelin group. Our results demonstrated that dietary sphingomyelin from milk was transported to lymph as molecular species of ceramide hydrolyzed from milk sphingomyelin and co-ingestion of sphingomyelin with glycerophospholipids enhanced the bioavailability of dietary sphingomyelin.

EGFR Targeted Theranostic Nanoemulsion for Image-Guided Ovarian Cancer Therapy.

PURPOSE: Platinum-based therapies are the first line treatments for most types of cancer including ovarian cancer. However, their use is associated with dose-limiting toxicities and resistance. We report initial translational studies of a theranostic nanoemulsion loaded with a cisplatin derivative, myrisplatin and pro-apoptotic agent, C6-ceramide. METHODS: The surface of the nanoemulsion is annotated with an endothelial growth factor receptor (EGFR) binding peptide to improve targeting ability and gadolinium to provide diagnostic capability for image-guided therapy of EGFR overexpressing ovarian cancers. A high shear microfludization process was employed to produce the formulation with particle size below 150 nm. RESULTS: Pharmacokinetic study showed a prolonged blood platinum and gadolinium levels with nanoemulsions in nu/nu mice. The theranostic nanoemulsions also exhibited less toxicity and enhanced the survival time of mice as compared to an equivalent cisplatin treatment. CONCLUSIONS: Magnetic resonance imaging (MRI) studies indicate the theranostic nanoemulsions were effective contrast agents and could be used to track accumulation in a tumor. The MRI study additionally indicate that significantly more EGFR-targeted theranostic nanoemulsion accumulated in a tumor than non-targeted nanoemulsuion providing the feasibility of using a targeted theranostic agent in conjunction with MRI to image disease loci and quantify the disease progression.

Ceramide-enriched LDL induces cytokine release through TLR4 and CD14 in monocytes. Similarities with electronegative LDL / La LDL enriquecida en ceramida induce la liberación de citoquinas a través de TLR4 y CD14 en monocitos. Similitudes con la LDL electronegativa

Introduction: In vitro ceramide-enriched LDL (CER-LDL) reproduces most of the properties of electronegative LDL (LDL(-)), a heterogeneous subfraction of LDL found in plasma. LDL(-) comprises several modifications of LDL and has an increased content in ceramide (CER). It promotes cytokine release in monocytes through CD14 and TLR4. CER-LDL also induces cytokine release in these cells but the mechanism is unknown. Aim To evaluate TLR4 andCD14 as the putative receptors involved in cytokine release induced by CER-LDL. Methods CER-LDL was obtained by incubating native LDL with CER-enriched liposomes. CER content in CER-LDL was assessed by thin layer chromatography of lipid extracts. CER-LDL and LDL(-) were incubated for 20 h with human monocytes in the presence or absence of a TLR4 signaling inhibitor. Both LDLs were also incubated with two human monocytic cell lines, normal and THP1 overexpressing CD14 (THP1-CD14) cells. The release of IL-6, IL-10 and MCP-1 was evaluated by ELISA in culture medium. Results: The release of IL-6, IL-10 and MCP-1 induced by CER-LDL in monocytes was inhibited by VIPER (90% inhibition), a specific TLR4 inhibitor. The cytokine release induced by contrast, the induction of cytokine release in THP1-CD14 was high and dependent on the CER content in LDL. Conclusion: CER-LDL induces IL-6, IL-10 and MCP-1 release through the activation of CD14 and TLR4 in monocytes, reproducing the behavior of LDL(-). The increased content of CER in LDL(-) is then related to the inflammatory action of LDL(-)

Introducción: La LDL enriquecida in vitro en ceramida (CER-LDL) reproduce varias características atribuidas a la LDL electronegativa (LDL(-)), subfracción heterogénea de LDL presente en circulación que induce la liberación de citoquinas en monocitos mediante CD14 y TLR4. La CER-LDL estimula también la liberación de citoquinas en monocitos, aunque el mecanismo se desconoce. Objetivo: Evaluar si CD14-TLR4 son receptores activados por la CER-LDL para inducir la liberación de citoquinas. Material y métodos: La CER-LDL se obtuvo in vitro mediante incubación de LDL nativa con liposomas enriquecidos en CER. El contenido en CER de la CER-LDL fue evaluado mediante cromatografía en capa fina. La CER-LDL y la LDL(−) fueron incubadas 20 h con monocitos humanos en presencia o ausencia de un inhibidor de la señalización de TLR4. También se incubaron con 2 líneas de monocitos humanos, THP1 y THP1, que sobreexpresan CD14 (THP1-CD14). Se evaluaron IL-6, IL-10 y MCP-1 en todos los sobrenadantes celulares mediante ELISA. Resultados: La liberación de IL-6, IL-10 y MCP-1 inducida por la CER-LDL en monocitos fue inhibida mediante VIPER (90% de inhibición), inhibidor específico de TLR4. Las citoquinas liberadas por la CER-LDL fueron escasas en THP1, células que presentan baja expresión de CD14. Las citoquinas liberadas por la CER-LDL en THP1-CD14 fueron superiores y dependientes del contenido en CER de la LDL. Conclusión: La LDL-CER induce IL-6, IL-10 y MCP-1 a través de la activación de CD14-TLR4 en monocitos, mimetizando a la LDL(-). La acción inflamatoria de la LDL(-) está relacionada con su contenido aumentado en CER

PLGA/liposome hybrid nanoparticles for short-chain ceramide delivery.

PURPOSE: Rapid premature release of lipophilic drugs from liposomal lipid bilayer to plasma proteins and biological membranes is a challenge for targeted drug delivery. The purpose of this study is to reduce premature release of lipophilic short-chain ceramides by encapsulating ceramides into liposomal aqueous interior with the aid of poly (lactic-coglycolicacid) (PLGA). METHODS: BODIPY FL labeled ceramide (FL-ceramide) and BODIPY-TR labeled ceramide (TR-ceramide) were encapsulated into carboxy-terminated PLGA nanoparticles. The negatively charged PLGA nanoparticles were then encapsulated into cationic liposomes to obtain PLGA/liposome hybrids. As a control, FL-ceramide and/or TR ceramide co-loaded liposomes without PLGA were prepared. The release of ceramides from PLGA/liposome hybrids and liposomes in rat plasma, cultured MDA-MB-231 cells, and rat blood circulation was compared using fluorescence resonance energy transfer (FRET) between FL-ceramide (donor) and TR-ceramide (acceptor). RESULTS: FRET analysis showed that FL-ceramide and TR-ceramide in liposomal lipid bilayer were rapidly released during incubation with rat plasma. In contrast, the FL-ceramide and TR-ceramide in PLGA/liposome hybrids showed extended release. FRET images of cells revealed that ceramides in liposomal bilayer were rapidly transferred to cell membranes. In contrast, ceramides in PLGA/liposome hybrids were internalized into cells with nanoparticles simultaneously. Upon intravenous administration to rats, ceramides encapsulated in liposomal bilayer were completely released in 2 min. In contrast, ceramides encapsulated in the PLGA core were retained in PLGA/liposome hybrids for 4 h. CONCLUSIONS: The PLGA/liposome hybrid nanoparticles reduced in vitro and in vivo premature release of ceramides and offer a viable platform for targeted delivery of lipophilic drugs.

Development of aceclofenac nanovesicular system using biomaterial for transdermal delivery: physical characterization, ex vivo, in vivo, and anti-inflammatory studies.

CONTEXT: Aceclofenac is an important NSAID; however, it causes GI disturbances whereas employing transdermal route would require permeation enhancer for systemic application, thereby causing skin damage. Ceramide 2 is a natural lipid having an important role in the maintenance of skin. OBJECTIVE: Aceclofenac-loaded nanovesicles of ceramide-2, cholesterol, palmitic acid, and cholesteryl sulfate were formulated and analyzed for physical and biological properties. MATERIALS AND METHOD: Film hydration method was used to prepare the vesicles and physical parameters, in vitro drug release and stability were evaluated. Then, they were formulated into gel and evaluated against a commercial formulation (CF) and gel-containing plain drug (CPG) for ex vivo, in vivo drug permeation, and anti-inflammatory activity. RESULTS: The developed formulations showed best physical profile and ACV-1 gave 92.89% drug release in in vitro studies. Ex vivo studies showed drug permeation between 15.32-31.12 µg/cm(2), whereas CPG and CF released 0.47 and 2.81 µg/cm(2), respectively. ACVG-1 and CF showed Cmax of 8.1 and 1.2 µg/ml at 8 and 4 h, respectively. ACVG-1 showed 11.6 times AUC than CF. ACVG-1 inhibited edema by 44% in first hour itself. DISCUSSION: Ceramide 2 and palmitic acid played an important role in the formulation and promotes the drug permeation through stratum corneum and dermis. Ceramide content of the formulation also contributes towards stability and skin protection. CONCLUSION: The composition of the vesicle formulation performs an important role in physical properties and drug permeation, thereby producing an optimum formulation.

Lecithin-based microemulsions for targeted delivery of ceramide AP into the stratum corneum: formulation, characterizations, and in vitro release and penetration studies.

PURPOSE: To improve the solubility and penetration of Ceramide AP (CER [AP]) into the stratum corneum that potentially restores the barrier function of aged and affected skin. METHODS: CER [AP] microemulsions (MEs) were formulated using lecithin, Miglyol® 812 (miglyol) and water-1,2 pentandiol (PeG) mixture as amphiphilic, oily and hydrophilic components, respectively. The nanostructure of the MEs was revealed using electrical conductivity, differential scanning calorimeter (DSC) and electron paramagnetic resonance (EPR) techniques. Photon correlation spectroscopy (PCS) was used to measure the sizes and shape of ME droplets. The release and penetration of the CER into the stratum corneum was investigated in vitro using a multi-layer membrane model. RESULTS: The MEs exhibited excellent thermodynamic stability (>2 years) and loading capacity (0.5% CER [AP]). The pseudo-ternary phase diagrams of the MEs were obtained and PCS results showed that the droplets are spherical in shape and bigger in size. In vitro investigations showed that the MEs exhibited excellent rate and extent of release and penetration. CONCLUSIONS: Stable lecithin-based CER [AP] MEs that significantly enhance the solubility and penetration of CER [AP] into the stratum corneum were developed. The MEs also have better properties than the previously reported polyglycerol fatty acid surfactant-based CER [AP] MEs.

Polyglycerol fatty acid ester surfactant-based microemulsions for targeted delivery of ceramide AP into the stratum corneum: formulation, characterisation, in vitro release and penetration investigation.

Ceramide AP (CER [AP]) is an integral component of the stratum corneum (SC) lipid matrix and is capable of forming tough and super stable lamellae. It may help to restore the barrier function in aged and affected skin. However, its effectiveness from conventional dosage forms is limited due to its poor solubility and penetration into the SC. Therefore, stable polyglycerol fatty acid ester surfactant (SAA)-based CER [AP] microemulsions (MEs) were formulated and characterised to enhance its solubilisation and penetration into the SC. TEGO® CARE PL 4 (TCPL4: polyglycerol-4-laurate), isopropyl palmitate (IPP) and water-1, 2 pentandiol (PeG) were used as amphiphilic, oily and hydrophilic components, respectively. The effects of HYDRIOL® PGMO.4 (HPGMO4: polyglyceryl-4-oleate) as a co-surfactant (co-SAA) and linoleic acid (Lin A) as part of the oil component on the stability and characteristics of the MEs were investigated. EPR results were used for the first time to reveal MEs nanostructures. The release and penetration behaviour of the MEs was assessed in vitro by using a multi-layer membrane model. The results obtained showed that HPGMO4 and Lin A increased stability and expanded the ME region considerably. The formulations were stable for 10 to >24 months. Dynamic light scattering (DLS) results showed that the droplets were bigger and asymmetric, which might be helpful to localise the CER into the upper layers of the epidermis. Release and penetration from the MEs was superior as compared to the hydrophilic cream (DAB). The rate and extent of CER [AP] released and penetrated from O/W MEs was better than W/O MEs.

Hyaluronic acid-ceramide-based optical/MR dual imaging nanoprobe for cancer diagnosis.

Hyaluronic acid-ceramide (HACE)-based nanoprobes for magnetic resonance (MR) and optical imaging were developed for cancer diagnosis. Diethylenetriaminepentaacetic dianhydride (DTPA) was conjugated to HACE for the chelation of gadolinium (Gd) as an MR contrast agent. Cy5.5 was also conjugated to the HACE backbone as a near-infrared fluorescence (NIRF) imaging dye. The self-assembled HACE-based nanoprobe, Cy5.5-HACE-DTPA-Gd, exhibited a uniformly distributed particle size and morphological shape. The HACE-based nanoprobe did not induce serious cytotoxicity in U87-MG (low expression of CD44 receptor) and SCC7 (high expression of CD44 receptor) cells. The cellular uptake efficiency of the HACE-based nanoprobe was higher in SCC7 cells than in U87-MG cells, indicating an HA-CD44 receptor interaction. In vitro MR signal enhancement of the HACE-based nanoprobe was confirmed compared with a commercial formulation (Magnevist). Moreover, in vivo MR contrast enhancement of the HACE-based nanoprobe in the tumor region was verified in an SCC7 tumor xenograft mouse model. The tumor targetability of the developed nanoprobe was monitored by an NIRF imaging study, and improved accumulation of the nanoprobe in the tumor region was observed. Therefore, this HACE-based dual-imaging nanoprobe can be used to make a more accurate diagnosis of cancer based on its passive and active tumor targeting strategies.

Relevance of the salvage pathway to N-hexanoylsphingosine metabolic downregulation in human neurotumor cells: implications for apoptosis.

N-Hexanoylsphingosine (C6-Cer) is currently being evaluated as an antineoplastic agent, after preclinical studies showing its property to reduce tumor growth. Herein it is reported that the cytotoxic effect of C6-Cer, as observed in CHP-100 neurotumor cells, impinges on its continuous uptake from the culture medium, ensuring maintainance of elevated steady-state intracellular levels, in the face of the rapid metabolic removal. C6-Cer metabolism not only does occur by direct glucosylation but is also relevantly driven by utilization via the sphingosine salvage pathway, leading to accumulation of natural ceramide that, in CHP-100 cells, has been demonstrated to lack apoptotic properties. Upon inhibition of glucosylceramide synthase by D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, previously shown to enhance C6-Cer cytotoxic activity, short-chain ceramide metabolism was partly redirected to the salvage pathway, likely attenuating the chemosensitizing effect of the above-mentioned compound. Elucidation of the metabolic machinery driving C6-Cer recycling via the salvage pathway might thus be relevant for optimization of its therapeutic utilization.