Advanced Pharmaceutical Bulletin
Volume:5 Issue: 3, Sep 2015

Cell therapy is a promising intervention for treating liver diseases and liver failure. Different animal models of human liver cell therapy have been developed in recent years. Rats and mice are the most commonly used liver failure models. In fact, rodent models of hepatic failure have shown significant improvement in liver function after cell infusion. With the advent of stem-cell technologies, it is now possible to re-programme adult somatic cells such as skin or hair-follicle cells from individual patients to stem-like cells and differentiate them into liver cells. Such regenerative stem cells are highly promising in the personalization of cell therapy. The present review article will summarize current approaches to liver stem cell therapy with rodent models. In addition, we discuss common cell tracking techniques and how tracking data help to direct liver cell therapy research in animal models of hepatic failure.

Bone marrow microenvironment contains cellular and acellular compartments. The cellular compartment includes hematopoietic stem cells, mesenchymal stem cells and some other stromal cell types, while the acellular compartment is composed of scaffold proteins known as the extra cellular matrix. Direct cell-cell contact as well as cytokines secreted by mesenchymal stem cells during coculture of hematopoietic stem cells and mesenchymal stem cells play a critical role in hematopoiesis, and determines the fate of hematopoietic stem cells. Several studies have demonstrated the impact of mesenchymal stem cells on self-renewal, expansion, proliferation and differentiation of hematopoietic stem cells in vitro, which have shown different and contradictory results. In this paper, we will investigate the effect of mesenchymal stem cells on differentiation of hematopoietic stem cells in vitro.

Lipid nanoparticles (LNPs) have attracted special interest during last few decades. Solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) are two major types of Lipid-based nanoparticles. SLNs were developed to overcome the limitations of other colloidal carriers, such as emulsions, liposomes and polymeric nanoparticles because they have advantages like good release profile and targeted drug delivery with excellent physical stability. In the next generation of the lipid nanoparticle, NLCs are modified SLNs which improve the stability and capacity loading. Three structural models of NLCs have been proposed. These LNPs have potential applications in drug delivery field, research, cosmetics, clinical medicine, etc. This article focuses on features, structure and innovation of LNPs and presents a wide discussion about preparation methods, advantages, disadvantages and applications of LNPs by focusing on SLNs and NLCs.

Hypoxia is a condition of decreased availability of oxygen. When cells are exposed to a low oxygen environment, they impel the hypoxia responses to adapt to new situation. The hypoxia response leads to the activation of various cellular signaling pathways. The aim of this study was to evaluate the effect of ghrelin on angiogenesis, Hypoxia-Inducible-Factor-1α (HIF-1) and Vascular endothelial growth factor (VEGF) levels in normobaric hypoxia situation. Twenty four animals were divided into 4 groups (n=6): control (C), ghrelin (Gh), hypoxia (H), and hypoxic animals that received ghrelin (H+Gh). Hypoxia (11%) was induced by an Environmental Chamber System GO2 Altitude. Animals in ghrelin groups received a subcutaneous injection of ghrelin (150 μg/kg/day) for 14 days. Our results showed that hypoxia significantly (p<0.05) increased angiogenesis without any significant changes on HIF-1 and VEGF levels, whereas ghrelin significantly (p<0.05) decreased angiogenesis, expression of HIF-1 and VEGF in this condition. Ghrelin administration did not show any significant changes in normal conditions. Ghrelin had no effect on angiogenesis, expression of HIF-1 and VEGF in normal oxygen conditions but it reduced angiogenesis process in lung tissue with reducing the level of HIF and VEGF in hypoxic condition. Therefore, effect of ghrelin on angiogenesis could be related to blood oxygen level.

Diabetes mellitus as a main risk-factor of ischemic heart disease may interfere with postconditioning’scardioprotective effects. This study aimed to investigate the involvement of glycogen synthase kinase-3β (GSK-3β) and oxidation status in chronic diabetes-induced loss of cardioprotective effect of ischemic-postconditioning (IPostC) in Wistar rats. After 8 weeks of induction of diabetes by streptozotocin (50mg/kg), hearts of control and diabetic rats were isolated and mounted on a constant-pressure Langendorff system. All hearts were subjected to 30min regional ischemia followed by 60min reperfusion (by occluding and re-opening of left anterior descending coronary artery, respectively). IPostC was applied immediately at the onset of reperfusion. At the end of reperfusion, the infarct size of myocardium was measured via computerized planimetry. Myocardial contents of malondealdehyde and glutathione as indices of oxidative status were assayed spectrophotometrically and the total and phosphorylated forms of myocardial GSK- 3β were quantified through western blotting. IPostC reduced the infarct size of control hearts from 41±2.9% to 28±1.9% (P<0.05), whereas it could not induce significant changes in infarct size of diabetic animals (35±1.8% vs. 39±3.1%). IPostC-induced reduction in malondealdehyde and elevation in glutathione contents were significant only in control not in diabetic hearts. The total forms of GSK-3β were similar in all groups; however, the phosphorylation of GSK-3β (at Ser9) by IPostC was greater in control hearts than diabetics (P<0.01). The failure of cardioprotection by IPostC in diabetic hearts may be attributed to the loss of phosphorylation of GSK-3β and thereby increase in oxidative stress in diabetic states.

Amitriptyline, one of the commonly used tricyclic antidepressants, caused rare but severe hepatotoxicity in patients who received it continuously. Previous findings showed that the intermediate metabolites of amitriptyline produced by CYP450 are involved in hepatic injury. Melatonin is an antiaging and antioxidant hormone synthesized from pineal gland. The aim of present study was to evaluate the protective role of melatonin in an in vitro model of isolated rat hepatocytes. Markers such as cell viability, reactive oxygen species formation, lipid peroxidation, mitochondrial membrane potential, and hepatocytes glutathione content were evaluated every 60 minutes for 180 minutes. Present results indicated that administration of 1mM of melatonin effectively reduced the cell death, ROS formation and lipid peroxidation, mitochondrial membrane potential collapse, and reduced cellular glutathione content caused by amitriptyline. Our results indicated that melatonin is an effective antioxidant in preventing amitriptyline-induced hepatotoxicity. We recommend further in vivo animal and clinical trial studies on the hepatoprotective effects of melatonin in patients receiving amitriptyline.

The study aimed at assessing any association between quinolone resistance, MDR and ESBL production and their relation with the presence of integrons in Esherichia coli and Klebsiella pneumoniae. E.coli and K.pneumoniae isolated from various clinical infections were fully identified and analyzed for being quinolone resistant. These isolates were further tested for ESBL production, multi drug resistance and carriage of integrons. In total, 135 isolates were confirmed as quinolone resistant. K.pneumoniae was observed as potent ESBL producer in comparison to E.coli. Ciprofloxacin resistance in both organisms was related significantly with the presence of integron class 1, co-presence of class 1 and 2 as well as to the presence of ESBL production (p< 0.001). However, nalidixic acid resistance was related significantly (p< 0.01) with only integron class 1 and to the presence of ESBL production. Class 1 and 2 integrons were found in 73.5% of MDR isolates with 13.2% of them possessing both intI1 and intI2 genes. Prevalence of quinolone resistance together with ESBL production and MDR in E.coli and K.pneumoniae has contributed to the emergence of antibacterial resistance burden. The higher integron prevalence in our isolates advocates the potentiality of these isolates as a source for dissemination of resistance determinants.

Particle size of Beclometasone Dipropionate (BDP) was reduced by the rapid expansion of supercritical solution (RESS) process, using CO2 as supercritical solvent. Also, the effect of RESS parameters such as extraction pressure, pre-expansion temperature, and weight fraction of co-solvent on the size and distribution of BDP particles were investigated. The effects of extraction pressure (200-260 bar), pre-expansion temperature 70- 110 °C) and weight fraction of menthol as a co-solvent on mean particle size (MPS) of BDP were investigated by design of experiment (DOE). Particles were characterized using Scanning Electron Microscopy (SEM) and Dynamic Light Scattering (DLS). The average sizes of precipitated BDP were between 64.1 and 294 nm. Analysis of variance (ANOVA) showed that extraction pressure was the most significant parameter and a higher extraction pressure caused production of smaller particles. Also, it was found that higher temperature and weight fraction of co-solvent increased the MPS. The interaction effects of extraction pressure-pre-expansion temperature and pre-expansion temperature cosolvent ratio were significant through the analysis of variance. It was observed that the MPS of precipitated particles was mostly influenced by pressure. The smallest MPS of BDP obtained from the RESS process was 65 nm that revealed a significant size reduction from its original MPS of 9 μm. Moreover, a slight change was observed for precipitated particles of BDP into spherical form while the original particles were irregular in shape. RESS process showed as a promising method for production of BDP nanoparticles that may results in improvement of drug’s physicochemical properties.

Doxorubicin is administrated as a single agent in first-line therapy of breast cancer to induce apoptosis in tumor cells. Bax, Bcl-xL, Caspase-8 and 9 proteins are involved in induction of apoptosis. The present study describes Bax, Bcl-xL gene expression and Caspase-8 and 9 protein levels in MCF-7 cells incubated with doxorubicin at different doses an incubation times. The cytotoxic effects of doxorubicin were studied using MTT assay. MCF-7 cells were treated with three concentrations of doxorubicin (0.1, 0.5, 1 μM) and incubated for 24, 48 and 72 hours then expression levels of Bax and Bcl-xL genes were elucidated by Realtime RT-PCR technique and protein levels of caspase-8 and caspase-9 proteins were measured using ELISA method. Morphological modifications of the cells were also monitored via light microscopic images. Doxorubicin decreased the anti-apoptotic Bcl-xL and increased pro-apoptotic Bax mRNA levels. Doxorubicin induced a significant increase in Bax /Bcl-xL ratio in all doses and incubation times (p<0.05). Highest (more than 10 fold) increase in Bax /Bcl-xL ratio was revealed after 48 h incubation of the cells with in all doses of doxorubicin. Doxorubicin also increased caspase-9 level in a time and dose-dependent manner, while caspase-8 level didnt follow time and dose dependency pattern. Our results confirm that doxorubicin induces mitochondrial-dependent apoptosis by down-regulation of Bcl-xL and up- regulation of Bax and caspase-9 expressions.

A Multiparticulate system of Mebendazole was developed for colon targeted drug delivery by using natural polysaccharides like Chitosan and Sodium-alginate beads. Chitosan microspheres were formulated by using Emulsion crosslinking method using Glutaraldehyde as crosslinking agent. Sodium-alginate beads were formulated by using Calcium chloride as gelling agent. Optimization for Chitosan microspheres was carried out by using 23 full factorial design. 32 full factorial design was used for the optimization of Sodium-alginate beads. The formulated batches were evaluated for percentage yield, particle size measurement, flow properties, percent entrapment efficiency, Swelling studies. The formulations were subjected to Stability studies and In-vitro release study (with and without rat caecal content). Release kinetics data was subjected to different dissolution models. The formulated batches showed acceptable particle size range as well as excellent flow properties. Entrapment efficiency for optimized batches of Chitosan microspheres and sodium alginate beads was found to be 74.18% and 88.48% respectively. In-vitro release of drug for the optimized batches was found to be increased in presence of rat caecal content. The best-fit models were koresmeyer-peppas for Chitosan microspheres and zero order for sodium-alginate beads. Chitosan and Sodium-alginate was used successfully for the formulation of Colon targeted Multiparticulate system.

The Importance and key role of Wnt/β-catenin signaling pathway in spermatogenesis is known. Abnormalities of this pathway in Sertoli and germ cells leads to infertility. Leydig cells play an important role in spermatogenesis and male reproduction. As of now, exact position of the Wnt/β-catenin signaling pathway disorders in the tissue and possible involvement of Leydig cells has not been investigated. Samples of our previous study were used for common Y chromosome microdeletions screening and common CFTR gene mutations.1 β-catenin gene expression were evaluated and compared between testicular tissue obtained by testicular sperm extraction (TESE) in two groups of obstructive (n=10) and non-obstructive (n=10) azoospermic infertile men. Location of β-catenin accumulation was detected by immunofluorescence technic and quantitatively compared in the tissue followed by counterstaining with anti-vimentin antibody. It was used as specific marker of leydig cells to determine and confirm the cells in which this gathering was occurred. β-catenin gene expression does not have a significant difference between the obstructive azoospermia (0.998) and non-obstructive azoospermia group (0.891). β catenin was abnormally aggregated in leydig cell of non-obstructive azoospermic men. Gathering β-catenin in cytoplasm of leydig cells can disrupt spermatogenesis and cause infertility in men.

Insulin resistance plays a key role in the onset and development of type 2 diabetes mellitus (T2DM) and its complications. In this study, we evaluated the effect of swim training on insulin resistance in diabetic rats. Forty male Wistar rats were randomly divided into four groups (n=10): sedentary control (Con), sedentary diabetic (Dia), swim trained control (Exe) and swim trained diabetic (Dia+Exe) rats. Diabetes was induced by high fat diet (HFD) and a low dose of streptozotocin (35 mg/kg, i.p). In trained groups, one week after the induction of diabetes, animals were subjected to swimming (60 min/5 days a week) for 10 weeks. At the end of training, fasting blood sugar (FBS), oral glucose tolerance test (OGTT), fasting/basal insulin, glycosylated hemoglobin (HbA1c) levels, insulin resistance index, homeostasis model assessment method (HOMA-IR), triglycerides (TG,) total cholesterol (TCh), and high density lipoprotein (HDL) levels in blood were measured. Swimming significantly improved OGTT (P<0.01) and HOMA-IR (P<0.01). Swim training also significantly decreased FBS (p<0.01), fasting/basal insulin (P<0.01), HbA1C (p<0.01), TG (P<0.05), and TCh (P<0.05) levels. It also significantly increased HDL (p<0.05) level. Our findings indicate that swim training improved glycemic control and insulin sensitivity in type 2 diabetes caused by high fat diet in male rats.

This paper describes a simple, precise and accurate RP-HPLC method for simultaneous estimation of atorvastatin and ezetimibe in plasma. The chromatographic separation of the drugs were performed on an X-Terra C (4.6 x 150 mm, 3.5 m), with phosphate buffer [pH 3.5 with Ortho Phosphoric Acid] – acetonitrile 40:60 (v/v) as mobile phase. The detection was performed at 235 nm. The flow rate was maintained at 1.2 mL/min. The run time was 8.0 min. The accuracy and reliability of the method was assessed by evaluation of linearity (5-25 μg/mL for both atorvastatin calcium and ezetimibe), precision (intra-day RSD 0.57 % and inter-day RSD 0.02 % for atorvastatin calcium and intra-day RSD 0.56 % and inter day RSD 0.1 % for ezetimibe), accuracy (100.08- 100.84 % for atorvastatin calcium and 100.56- 101.00 % for ezetimibe), and specificity, in accordance with ICH guidelines. The LLOQ obtained by the proposed method were 1.294 and 1.384 μg/mL for atorvastatin and ezetimibe respectively. Overall the proposed method was found to be suitable and accurate for the quantitative determination in plasma. The method was effectively separated the drug from plasma.

Because of the emergence of multi-antibiotic resistant bacteria, a number of infectious diseases have become a major concern to treat in health care services worldwide. This situation is worsened by the fact that very limited progress has been made in developing new and potent antibiotics in recent years. In this context antimicrobial peptides (AMPs) represent new potential therapeutic compounds with bactericidal or bacteriostatic activity against closely related bacterial strains. In this study, a collection of enterococci (n=170) from clinical sources were investigated for their potential to inhibit multiresistant nosocomial enterococci from Iranian hospitals. Four isolates produced antimicrobial peptides that inhibited all the antibiotic resistant enterococci. This included three Enterococcus faecium isolates producing combinations of enterocin A, B and L50 AB. The most potent antagonism was produced by E. faecalis HO91. Purification and subsequent characterization by MALDI-TOF MS, Edman degradation and DNA-sequencing revealed that the antimicrobial compound was Hiracin. The purified Hiracin was evaluated for antibacterial activity against 12 multiresistant enterococcal isolates from clinical samples. The results demonstrated that Hiracin is highly effective towards enterococci which were resistant even to antibiotics from four distinct classes. The present research addresses Hiracin as a promising alternative to conventional antibiotics in treatment of multiresistant enterococcal infections.

The main objective of the current research work was to formulate and evaluate furosemide loaded silica lipid hybrid microparticles for improved oral delivery. A novel silica-lipid hybrid microparticulate system is used for enhancing the oral absorption of low solubility and low permeability of (BCS Class IV) drugs. Silica-lipid hybrid microparticles include the drug solubilising effect of dispersed lipids and stabilizing effect of hydrophilic silica particles to increase drug solubilisation, which leads to enhanced oral bioavailability. The slica lipid hybrid (SLH) microparticles were composed of poorly soluble drug (furosemide), dispersion of oil phase (Soya bean oil and miglyol) in lecithin (Phospholipoid 90H), non-ionic surfactant (Polysorbate 80) and adsorbent (Aerosol 380). Saturation solubility studies were performed in different oils and surfactants with increased concentration of drug revealed increased solubility of furosemide. In vitro dissolution studies conducted under simulated gastric medium revealed 2 4 fold increase in dissolution efficiencies for SLH microparticles compared to that of pure drug (furosemide) and marketed formulation Lasix®. Ex vivo studies showed enhanced lipid digestibility, which improved drug permeability. Solid-state characterization of SLH microparticles by X-ray powder diffraction and Fourier transform infrared spectroscopic analysis confirmed non-crystalline nature and more compatibility of furosemide in silicalipid hybrid microparticles. It can be concluded that the role of lipids and hydrophilic silica based carrier highlighted in enhancing solubility and permeability, and hence the oral bioavailability of poorly soluble drugs.

The aim of this study was to find a relationship between drug solubility and its transdermal permeation and find the best vehicle composition to improve transdermal permeation of Tadalafil. Pure or binary mixtures of commonly used solvents in pharmaceutical sciences including ethanol, glycerin, N-methyl pyrrolidone (NMP), polyethylene glycol (PEG) 400 and propylene glycol (PG) were evaluated for drug solubility and transdermal delivery through the exercised rat skin employing Franz diffusion cells. Tadalafil showed higher solubility in NMP compared to the other solvents. The amount of Tadalafil permeation from the pure vehicles was ranked as follow: Ethanol >glycerin >NMP>PEG 400 >PG. Furthermore, the solubility and transdermal delivery from binary mixtures of NMP and PG were higher than that obtained from pure PG, and accordingly, both increased with increasing NMP concentration in the binary solvent mixtures. The Flux values were determined as following order for Ethanol>NMP>glycerin>PG>PEG 400. Generally, increase in Tadalafil solubility resulted in a decrease in its skin penetration rate and amount. However, NMP exhibited substantial drug skin penetration rate and amount accompanying with appropriate drug solvency. In conclusion, the results of this study introduced NMP as a solvent suitable for application in the formulation of topically applied drug delivery systems.

Citrus is considered as a medically important plant from ancient times and the bioflavonoids of different variety of citrus fruits were well explored for their biological activities. The study aim was to explore the effect of citrus bioflavonoids (CB) to prevent and cure hyperoxaluria induced urolithiasis. Twenty four Wistar rats were segregated into 4 Groups. Group 1: Control; Group 2: Urolithic (EG-0.75%); Group 3: Preventive study (EG+CB, day 1-50); Group 4: Curative study (EG+CB, day 30-50). Animals received CB orally (20mg/kg body weight) after performing a toxicity study. Urinary risk factors and serum renal function parameters were significantly reduced by CB administration in both preventive and curative study (p<0.001). Hematoxylin & Eosin and von Kossa staining demonstrated that renal protection was offered by CB against EG insult. Immunohistochemical analyses revealed over expression and abnormal localization of THP and NF-κB in urolithic rats, while it was effectively regulated by CB supplementation. CB prevented and significantly controlled lithogenic factors and CaOx deposition in rats. We propose CB as a potential therapy in management of urolithiasis.

Integrins are heterodimeric membrane proteins, which are exposed to post translational modifications in eukaryotic cells in contrast to prokaryotic cells. These modifications provide advantages for production of proper nanobody, mono and polyclonal antibody against this surface protein and also in aptamer selection process. Since the majority of diagnostic and therapeutic antibodies, target the surface epitopes, eukaryotic membrane proteins provide an appropriate model for further investigation on therapeutic agents. Escherichia coli strain top 10, was used as host for ITGA-4 expression vector encoding the human integrin α4. The plasmid was extracted and consequently, ITGA-4 vector was digested to make a linear plasmid. Human Embryonic Kidney-293 (HEK-293) cell transfected with linear plasmid and subsequently screened for stable ITGA-4 expressing Cells. Three separated clones were isolated twenty one days after transfection. Chromosomal DNA was extracted from ITGA-4-transfected cells. The presence of ITGA-4 gene in HEK-293 genome was confirmed by PCR. The expression level of ITGA-4 on HEK-293 cells was also analyzed by Flow cytometry. Flow cytometric analysis showed that HEK-293 cells have no expression of integrin α4 on their surface while 95% of transfected HEK-293 cells with ITGA4, expressed different levels of integrin α4 on their surfaces which correlates well with genomic DNA PCR amplification results. The results suggest that we have successfully constructed the integrin α4 expressing HEK293 cell, which will facilitate further research into the production of antibody, nanobody and aptamer against α4 integrin.

Solid dispersions have been efficient in improving the dissolution rate and bioavailability of hydrophobic drugs. The aim of the present study was enhancement of the dissolution profile of Spironolactone using solid dispersion. Spironolactone solid dispersions (1:1, 1:2 and 2:1 drug: carrier weight ratio) were prepared by polyethylene glycol (PEG) 6000 as a carrier by hot melt method. The influence of several amounts of Tween 20, 60, and 80 were also studied. The dissolution profile was evaluated by USP Apparatus II. The results showed that solid dispersions were efficacious to enhance the dissolution rate of Spironolactone in water; and the procedure indicated that there was an increase in dissolution rate for solid dispersions containing the surfactant Tweens. The solid dispersions were evaluated using Fourier-transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) and Differential Scanning Calorimetry (DSC) studies; and the results showed no complex formation or change in crystal shape of drug. It is concluded that solid dispersion technique can be successfully used for improvement of dissolution of Spironolactone.

Scrophularia variegata M. Beib. (Scrophulariaceae) is an Iranian medicinal plant which is used for various inflammatory disorders in traditional medicine. In this study we evaluated the anti-cancer and cytotoxic effects of the Scrophularia variegata (S. variegata) ethanolic extract on the human breast cancer cell line. The cytotoxicity effect of the extract on MCF-7 cells was evaluated by MTT assay. In addition, Caspase activity, DNA ladder and Cell death were evaluated by ELISA, gel electrophoresis and Annexin V-FITC/PI staining, respectively. The S. variegata extract showed significant effect cytotoxicity on MCF-7 human breast cancer cell line. Treatment with the extract induced apoptosis on the breast cancer cells by cell cycle arrest in G2/M phase. The results indicated that cytotoxicity activity was associated with an increase of apoptosis as demonstrated by DNA fragmentation as well as an increase of the amount of caspase 3 and caspase 9. In addition, the phytochemical assay showed that the extract had antioxidant capacity and also flavonoids, phenolic compounds and phenyl propanoids were presented in the extract. Our findings indicated that S. variegata extract induced apoptosis via mitochondrial intrinsic pathway on breast cancer by cell cycle arrest in G2/M phase and an increase of caspase 3 and caspase 9. However future studies are needed.