Current Drug Delivery (v.14, #4)

Meet Our Editor-in-Chief by Xing-Jie Liang (445-445).

Cholinergic Anti-inflammatory Pathway and Stroke by Kamil Duris, Jolana Lipkova, Michal Jurajda (449-457).
Background: Stroke is devastating cerebrovascular event which is responsible for 6.7 million deaths each year worldwide. Inflammation plays an important role in the pathophysiology of stroke. Targeting inflammation after stroke is highly actual topic for both experimental and clinical research.

Methods: Research articles related to cholinergic anti-inflammatory pathway (CHAIP) and stroke were reviewed. The first part of review describes the basic characteristics of inflammatory response after stroke, main components and function of CHAIP. The second part reviews studies focused on CHAIP as a therapeutic target for ischemic and hemorrhagic stroke. Both pharmacological stimulation of α7 nAChR and vagus nerve stimulation after stroke are reviewed.

Results: Cholinergic anti-inflammatory pathway (CHAIP) is a physiological mechanism by which central nervous system regulates immune response and controls inflammation. Vagus nerve, spleen and α7 nicotinic acetylcholine receptor (α7 nAChR) are the main components of CHAIP.

Conclusion: Targeting cholinergic anti-inflammatory pathway is a promising way of immunomodulation which attenuates inflammation in a complex manner without causing immunosuppression.

Traversing the Skin Barrier with Nano-emulsions by Cornel Burger, Yasser Shahzad, Alicia Brummer, Minja Gerber, Jeanetta du Plessis (458-472).
Background: In recent years, colloidal delivery systems based on nano-emulsion are gaining popularity; being used for encapsulation and delivery of many drugs. This review therefore aims at summarizing various methods of nano-emulsion formulation and their use as a topical and transdermal delivery vehicle for a number of active pharmaceutical ingredients from different pharmacological classes.

Methods: This article represents a systematic review of nano-emulsions for topical and transdermal drug delivery. A vast literature was searched and critically analysed.

Results: Nano-emulsions are thermokinetically stable dispersion systems, which have been used in topical and transdermal delivery of a number of pharmaceutically active compounds. Nano-emulsions have a narrow droplet size range with tuneable surface properties, which make them an ideal delivery vehicle. Nanoemulsions have a number of advantages over conventional emulsions, including easy preparation using various low and high energy methods, optical transparency, high solubilisation capacity, high stability to droplet aggregation and the ability to penetrate the skin; thus allowing the transdermal delivery of drugs.

Conclusion: This review indicated that nano-emulsions are promising vehicle for entrapping various drugs and are suitable for traversing the skin barrier for systemic effects.

Background: There is a general consensus that sleep-wake cycle is controlled by neuroanatomical, neurochemical and molecular systems as well as by homeostatic and circadian complex networks. The research has shown that a molecular element that could be displaying a relevant role in the modulation of sleep is the peroxisome proliferator-activated receptor alpha (PPARα), which belongs to the family of nuclear receptor ligand-activated transcription factors that includes PPARβ/δ and PPARγ. A growing body of evidence supports the notion that PPARα is activated by natural ligands such as the anorexic lipid mediator oleoylethanolamide (OEA) or synthetic compounds including Wy14643 whereas antagonists like MK-886 block the neurobiological outcomes of PPARα. More recently, studies have reported the permissive role of PPARα by modulating diverse neurobiological functions such as inflammation, metabolic disorders, learning, degenerative diseases and sleep. Remarkably, this nuclear receptor has been described in sleep-related brain regions leading to the hypothesis that PPARα might be involved in sleep modulation inasmuch as activation of this protein promotes a robust enhancement of wakefulness while reduces sleep.

Objective: In this mini review, the emerging evidence of the putative role of PPARα in sleep control is highlighted. Even though the data are derived from new areas of research, there are many reasons to believe that understanding and appreciation of PPARα functions may provide knowledge of possible mechanisms of action activated by this nuclear receptor in sleep modulation.

Conclusion: Novel insights of therapeutic intervention for sleep disorders might be visualized targeting the function of PPARα in sleep abnormalities.

Background: Ursodeoxycholic acid, usually used to dissolve cholesterol gallstones in clinic, is a typical hydrophobic drug with poor oral bioavailability due to dissolution rate-limited performance. The objective of this study was to increase the dissolution of ursodeoxycholic acid by amorphous nanosuspensions.

Methods: Nanoprecipitation based on acid-base neutralization was used to prepare the nanosuspensions with central composite design to optimize the formula. The nanosuspensions were characterized by particle size, morphology, crystallology and dissolution.

Results: The ursodeoxycholic acid nanosuspensions showed mean particle size around 380 nm with polydispersion index value about 0.25. Scanning electron microscope observed high coverage of HPMC-E50 onto the surface of the nanosuspensions. Differential scanning calorimetry and powder X-ray diffractometry revealed amorphous structure of the ursodeoxycholic acid nanosuspensions. A significant increase of dissolution in acidic media was achieved by the amorphous nanosuspensions compared with the physical mixture.

Conclusion: It can be predicted that the amorphous nanosuspensions show great potential in improving the oral bioavailability of ursodeoxycholic acid.

Stimulation of Tumor-Specific Immunity by p5 HER-2/neu Generated Peptide Encapsulated in Nano-liposomes with High Phase Transition Temperature Phospholipids by Mona Yazdani, Seyed Amir Jalali, Ali Badiee, Sheida Shariat, Mercedeh Mansourian, Leila Arabi, Azam Abbasi, Zahra Saberi, Mahmoud Reza Jaafari (492-502).
Background: The aim of this study is to evaluate the possible advantages of liposomes with high transition temperature (Tm) in the function of a vaccine for P5 HER2/neu-generated peptide and its adjuvant action to elicit CD8+ T cell response and its efficacy in TUBO in vivo tumor mice model, which over expresses the HER2/neu oncogene. P5, a hydrophobic peptide, was encapsulated in the nanoliposomes consisting of DSPC/DSPG/Chol(Tm 54A°C) with a chaotropic loading system via 7M urea and described by size, zeta potential, encapsulation efficiency, and the structural stability of SDS-PAGE.

Methods: We immunized the mice for three times subcutaneously based on a two-week intervals using encapsulated peptide in the nanoliposomes, empty liposome, P5 in PBS, and PBS. Enzyme-linked immunospot assay, cytotoxicity test, and flow cytometric studies followed by the size of tumor and survival time measurements, which were done in TUBO tumor mice version.

Results: Findings of ELISpot and flow cytometric analysis showed that immunization with Lip-p5 nanoliposomes has enhanced the antigen-specific IFN-γ response of CD8+ T cells and induced CTL response, which resulted in a smaller tumor and longer survival time. In addition to increase in amounts of IFN-γ-CD8+ T cells in a group, which was immunized with Lip-P5, our findings also revealed a Th1 shift in the group immunized with an empty liposome with reduced frequencies of IL-4-producing cells and increase of IFN-γ-producing cells.

Conclusion: The results indicated that simple liposomes consisting of phospholipids with high transition temperature could be an effective vaccine vehicle for tumor-associated antigens for inducing cell mediate immunity.

Development of Salbutamol Sulphate Sublingual Films in Pullulan Matrix for Enhanced Bioavailability & Clinical Efficacy by Nahed Mohamed Sallam, Rania Abdel-Basset Sanad, Rasha Mohamed kharshoom, Mokhles Abdelfadil Zeneldin (503-515).
Background: Salbutamol sulphate (SS) is a model short-acting β2-adrenergic receptor agonist used for the relief of bronchospasm having poor bioavailability (50%) due to its extensive first-pass effect.

Objective: Formulation of SS as fast dissolving sublingual films (FDSFs) using a biocompatible and biodegradable Pullulan polymer to bypass its metabolism in liver and offers a fast relieve of asthma.

Method: Films were prepared by solvent casting technique adopting 32 factorial design to study the effect of two independent variables namely; polymer type (HPMC E5, Pullulan, and L-HPC) and polymer to Maltodextrin ratio (3:0, 3:1 or 5:1). Films physicomechanical properties, disintegration time and in-vitro dissolution of the prepared formulations were evaluated.

Results: Formula F3 containing Pullulan with Maltodextrin in a ratio 3:1 has the least disintegration time (26A±2.6 seconds) and the highest dissolution rate (100%A±0.5%) after four minutes. Pharmacokinetic study of the optimum formula F3 in healthy human volunteers revealed a shorter tmax (0.75A±0.25 hour) compared to Ventolin® tablet 2 mg (2.1A±0.37 hour). Clinical evaluation of F3 in 14 male asthmatic patients using ZAN Spirometry showed clinical improvement in lung function parameters when compared with Ventolin® tablets.

Conclusion: FDSFs significantly improved the bioavailability of SS and resulted in dramatic improvement in its clinical efficacy.

Ex vivo Skin Permeation Evaluation of An Innovative Transdermal Vehicle Using Nimesulide and Piroxicam as Model Drugs by Rafaela de Oliveira Pereira, Taize Carla Costa Pelisson e Silva, Anderson de Oliveira Ferreira, Marcos Antonio Fernandes Brandao, Nadia Rezende Barbosa Raposo, Hudson Caetano Polonini (516-520).
Background: The transdermal dosage forms presented a limited usage for a long time, for it was believed that the stratum corneum, the outermost layer of epidermis, made it impracticable the permeation of medications through the skin. Studies exploring this area came up with strategies to overcome this barrier; for example, creating a transdermal vehicle to facilitate the drug absorption.

Objective: This study aimed to evaluate a new transdermal vehicle through the comparison of its permeation profile and the profile of commercial products, using nimesulide and piroxicam, non steroidal anti-inflammatory drugs.

Methods: Four different products were evaluated: nimesulide and piroxicam compounded with the new vehicle (emulsion) and commercial nimesulide and piroxicam gels. Ex vivo permeation experiments using Franz-type diffusion cell equipment were conducted, using human skin as membrane. For evaluation of permeated active pharmaceutical ingredients concentrations, we performed quantification from the receptor solution, stratum corneum and viable epidermis + dermis, through high-performance liquid chromatography analyses.

Results: The new vehicle promoted increased permeation of active pharmaceutical ingredients through the viable epidermis and dermis, when compared to commercial products, but the stratum corrneum continued to keep the highest retention.

Conclusion: The innovative vehicle was capable of enhancing the transdermal absorption of active pharmaceutical ingredients from the compounded formulations, thus, demonstrating the capability thereof to improve the permeability of active pharmaceutical ingredients by transdermal use.

Background: Tacrolimus, an immunosuppressive drug has a variable pharmacokinetic and poor oral bioavailability due to poor solubility. The aim of the present study was enhancing the solubility and oral bioavailability of this drug.

Methods: Tacrolimus nanoparticles were prepared by precipitation anti-solvent evaporation method. The effect of different parameters including: surfactant type, solvent to nonsolvent ratio and drug to surfactant ratio were studied on the particle size, saturated solubility and dissolution rate of the drug. The solid state characterization was done by fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD) and scanning electron microscopy. The untreated drug (5, nanoparticles or physical mixture of the drug and the stabilizers were administered to rats and blood levels of tacrolimus were assessed by electrochemiluminence method. The oral drug administration was done for 10 days then the changes in white blood cells (WBC) and percent lymphocyte count were determined before drug administration, 5 and 10 days after drug administration.

Results: FTIR spectroscopy showed no interaction between the drug and stabilizers. XRPD and DSC studies indicated the amorphous state of the drug in nanosuspensions. The solvent to nonsolvent ratio of 1:20 and drug to surfactant ratio of 1:3 enhanced 185 fold the saturated solubility and 17 fold dissolution rate of the drug. In vivo studies also showed tacrolimus nanoparticles significantly reduced the lymphocyte and WBC, enhanced 66 and 34 fold the AUC0-24 and Cmax of the drug, respectively.

Conclusion: The precipitation anti-solvent evaporation is a nano-crystalization technique which showed to be an effective approach for enhancing water solubility and bioavailability of tacrolimus.

The Efficiency of Tat Cell Penetrating Peptide for Intracellular Uptake of HIV-1 Nef Expressed in E. coli and Mammalian Cell by Somayeh Kadkhodayan, Azam Bolhassani, Seyed Mehdi Sadat, Shiva Irani, Fatemeh Fotouhi (536-542).
Background: Cell penetrating peptides (CPPs) or protein transduction domains (PTDs) have been known as a new field in cargo delivery. These peptides such as Tat, Pep-1 and Cady-2 are able to deliver genes and biologically active proteins to cytoplasmic compartments via the plasma membrane.

Methods: In current study, the efficiency of pEGFP-N1 eukaryotic vector for expression of HIV-1 Tat- Nef fusion was evaluated in HEK-293T cells using TurboFect transfection reagent. In addition, the recombinant GST-Tat-Nef protein was generated in E. coli and transfected using two amphipathic CPPs (Pep-1 and Cady-2) into mammalian cells. The size and morphology of the CPP/GST-Tat-Nef complexes were evaluated by scanning electron microscopy (SEM), Zetasizer, and SDS-PAGE. The transfection of HIV-1 GST-Tat-Nef protein was also analyzed using SDS-PAGE and western blotting.

Results: Our data indicated that the recombinant GST-Tat-Nef protein generated in BL21 strain migrated as a clear band of ~ 52 kDa in SDS-PAGE. The results of SEM and Zetasizer confirmed the formation of protein/ Pep-1 or protein/ Cady-2 nanoparticles less than 200 nm in diameter. Tat CPP fused to Nef protein could deliver the recombinant Nef protein alone and notably by forming the noncovalent complexes with TurboFect, Pep-1 and Cady-2 as detected in western blotting. Moreover, intracellular uptake of Tat-Nef gene and subsequently its expression in mammalian cells was considerably higher than that for Nef gene.

Conclusion: This data indicated that the Tat gene sequence could also increase the transfection of Nef gene in vitro. Generally, the Tat-Nef interaction led to enhance further gene expression and also protein delivery.

Background: Recent immunologic data implicates involvement of mucosal immune cells of the intestine like eosinophils and mast cells to be functionally involved in the pathogenesis of UC. Mast cell activation is followed by increased secretion and elevated tissue concentration of histamine. Inhibition of mucosal histamine release in colon may be an effective therapeutic approach to treat UC. Some studies report that intestinal inflammation associated with acute and chronic colitis has been ameliorated by fexofenadine in mice.

Objective: In the present work, we investigated the effect of colon- specific prodrugs of antihistaminic fexofenadine on TNBS- induced colitis in Wistar rats, applying the principle of drug repositioning.

Method: Amino acid- appended amide prodrugs of fexofenadine were designed and characterized spectrally. In vitro kinetics and protective effect of prodrugs were studied on TNBS-induced colitis in Wistar rats.

Results: Conjugation with amino acids improved the hydrophilicity of fexofenadine (log P: 0.037 to 0.082) to enable efficient delivery to colon. Prodrugs were chemically and enzymatically stable in aqueous buffers (pH 1.2 and7.4) and stomach homogenates/intestinal homogenates, respectively. Prodrugs were substantially cleaved to release 60-70% of fexofenadine in homogenates of colon in 12 h. Prodrug of fexofenadine with L-glutamine additively and significantly suppressed TNBS-induced colitis showing comparable effects to orally administered 5-aminosalicylic acid.

Conclusion: The outcome of this preliminary work emphasizes involvement of mast cells that release histamine as one of the important pathological inducers of UC. These promising, dual acting, colontargeting fexofenadine prodrugs could be explored further for repositioning fexofenadine in the treatment of UC and its relapse.

Background: Glaucoma, a chronic eye condition caused by progressive degeneration of retinal ganglion cells may result in permanent blindness. The restricted efficiency of currently available glaucoma treatments has awakened a global need for designing a novel anti-glaucoma drug delivery mechanism that targets the drug to the site of action in a sustained manner with less toxicity and side effects. With reference to this global problem in the present study, we have fabricated a biodegradable transparent polymeric material loaded with minimum amount of anti-glaucoma drug, acetazolamide.

Methods: The drug was modified using a suitable nanocarrier for its sustained drug release. The efficient modification of nanocarrier encapsulated drug as well as the drug loaded polymeric film was studied using various characterization techniques such as UV-visible spectrophotometry, spectroflurimetry, polarizing optical microscope, Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), X-ray diffraction (XRD), thermogravimetric analysis and differential scanning calorimetry (TGA/DSC).

Results: The characterization techniques indicated efficient transformation of the crystalline drug in to amorphous during nanocarrier encapsulation. The in-vitro drug release study in simulated tear fluid (STF) showed prolonged release of the drug from the nano drug complex for 3 h with the complete degradation of the polymer matrix within 5 min.

Conclusion: The fabricated biomaterial showed excellent potential to be developed in to a drug loaded contact lens for sustained glaucoma drug delivery with benefits of low drug content and fewer drug induced side effects.

Liposomes Containing Gadodiamide: Preparation, Physicochemical Characterization, and In Vitro Cytotoxic Evaluation by Ana Luiza Chaves Maia, Christian Fernandes, Cynthia Nara Pereira de Oliveira, Claudia Salviano Teixeira, Mariana Silva Oliveira, Daniel Cristian Ferreira Soares, Gilson Andrade Ramaldes (566-574).
Background: The aim of this study was to develop, characterize and assess the cytotoxic activity of pHsensitive (pHL-Gd), stealth pH-sensitive (SpHL-Gd), and conventional (convL-Gd) liposomes containing gadodiamide (Gd-DTPA-BMA).

Methods: Formulations were prepared by reverse-phase evaporation method and their physicochemical properties were evaluated by means of particle size, zeta potential, and Gd-DTPA-BMA entrapment. SpHL-Gd was considered being the most promising liposome, since it combines stealth and fusogenic characteristics that might contribute to achieve higher therapeutic efficiency. Their drug encapsulation percentages have been optimized satisfactorily. The addition of Gd-DTPA-BMA at 125 μmol/mL in the SpHL-Gd preparation allowed obtaining liposomes with appropriate encapsulation percentage (20.3 A± 0.1%) and entrapment (25.4 A± 0.1 μmol/mL).

Results: The cytotoxic studies on the 4T1 breast cancer cell line demonstrated that liposomes-loaded with Gd-DTPA-BMA inhibited cancer cell. pHL-Gd and SpHL-Gd liposomes showed higher activity than convL-Gd and free Gd-DTPA-BMA, indicating that the pH-sensitive characteristic was important to improve intracellular delivery.

Conclusion: The presence of polyethylene glycol (PEG) in the SpHL-Gd formulation did not affect the pH-sensitivity and internalization. Therefore, the results of this study suggest the feasibility of liposomes containing Gd-DTPA-BMA as a new promising controlled delivery system.

Safety Assessment of Tretinoin Loaded Nano Emulsion and Nanostructured Lipid Carriers: A Non-invasive Trial on Human Volunteers by Saman Ahmad Nasrollahi, Hurnaz Hassanzade, Azadeh Moradi, Mahsa Sabouri, Aniseh Samadi, Mansour Nassiri kashani, Alireza Firooz (575-580).
Background and Aim: Topical application of tretinoin (TRE) is followed by a high incidence of side effects. One method to overcome the problem is loading TRE into lipid nanoparticles. The potential safety of the nanoparticle materials has been always considered as a major concern. In this in vivo study, changes in human skin biophysical parameters including hydration, TEWL, erythema, and pH have been used to determine the safety of tretinoin loaded nano emulsion (NE) and nanostructured lipid carriers (NLC).

Method: TRE loaded NE and NLC were prepared using a high pressure homogenizer. Skin biophysical parameters were measured on the volar forearms of twenty healthy volunteers, before and after applying TRE-NE and TRE-NLC lotions. All the measurements were done using respective probes of MPA 580Cutometer®.

Result: We obtained particles of nanometric size (<130 nm) with narrow distribution and optimal physical stability. None of the formulations made any statistically significant change in any of the measured skin properties. P-values were 0.646, 0.139, 0.386, 0.169 after applying TRE-NE and 0.508, 0.051, 0.139, 0.333 after applying TRE-NLC, respectively.

Conclusion: Both formulations are reasonably safe to apply on human skin and topical application of TRE-NE and TRE-NLC had almost similar effects on skin biophysical parameters.

Background: Pilocarpine hydrochloride is commonly prescribed to patients with dry mouth and eye using a frequent dosing schedule. The aim of this study was to evaluate the sustained effects of this highly soluble drug carried by a gelatin hydrogel, which was administered by an implant mediated drug delivery system (IMDDS).

Methods: The IMDDS was installed in a total of 24 rabbits. After complete healing, pilocarpine hydrochloride was administered as 30 mg as raw powder (Group 1; n = 8), 30 mg in gelatin hydrogel (Group 2; n = 8), and 60 mg in gelatin hydrogel (Group 3; n = 8). The effects were evaluated by tear volume measured using the Schirmer tear test for 2 weeks after administration.

Results: All 3 groups showed an increase in tear volume from the initial measurement at 1 hour. Group 1 exhibited this increase for 24 hours, while Groups 2 and 3 sustained this increase for 5 days and 7.5 days, respectively.

Conclusion: When provided in gelatin hydrogel, highly water-soluble pilocarpine hydrochloride administered through IMDDS resulted in sustained effects with increased tear volume in normal rabbits.