Pharmaceutical studies of Amphotericin-B : b oral formulation using nanotechnology

PhD ThesisAmphotericin B (AmB) is the gold standard treatment of systemic fungal infections. It is the secondary treatment of leishmaniasis with 97% cure rate with no reported resistance. However, the only dosage form of AmB in the market is parenteral formulations as intermittent iv infusion. Sodium deoxycholate (Fungizone) was the first conventional formulation followed by several new lipid formulations since 1990 (Ablecet, Amphocil and Ambisone). These formulations were proved to be very effective, less toxic and require shorter course of therapy compared to Fungizone®. Unfortunately, all these parenteral formulations suffer from the inconvenience of intravenous (iv) administration, required hospitalization, more importantly, induce a number of side effects, and are very expensive. Therefore, the need for oral formulations of AmB is a crucial concern. During the last four decades there have been several published efforts representing different strategies in formulating oral delivery system of AmB with reduced toxicity. None of these formulations has been introduced into the market yet. In this study a novel stealth nanotechnology formulations containing AmB have been developed for an oral administration in order to improve its solubility, bioavailability an with lower toxicity. The selection of the copolymer used PEGylated poly-lactic- co- glycolic acid (PLGA-PEG) was based on the hypothesis that FDA has approved each individual component of the copolymers for oral and parenteral administrations of poorly soluble drugs. The development of these oral formulations of AmB went through several optimization steps, including method of preparation, polymer selection, stirring rate, cosolvency, pH, particle size reduction, stabilizers, surfactants, absorption enhancer, and HPLC assay for in vitro analysis. Novel oral biodegradable stealth polymeric nanoparticles have been successfully fabricated using the commercially available PLGA-PEG copolymer for AmB by emulsion diffusion method. In vitro characterizations of the lyophilized formulations showed that iv main particles size (MPS) of AmB loaded to NPs ranged from 23.8 ±4.8 to 1068± 489.8 nm. An increased stirring rate favored AmB NPs with smaller MPS. There was a significant reduction in MPS (P < 0.05) and significant increment in the drug content (P < 0.05) and the amount of drug released (P < 0.05), when AmB-NPs were prepared using the diblock copolymer PLGA–PEG with 15% PEG compared to other copolymers investigated. The addition of three emulsifying agents poly vinyl pyrrolidone (PVP), Vitamin E (TPGS) and pluronic F-68 to AmB formulations led to significant reduction in particle size and increase in drug entrapment efficiency (DEE) compared to addition of PVP alone. Fourier transform infrared spectroscopy demonstrated a successful loading of AmB to stealth PLGA– PEG copolymers. PLGA–PEG copolymer entrapment efficiency of AmB was increased up to 56.7%, with 92.7% drug yield. The physicochemical characterization done by SEM and TEM images indicate spherical particle in nanometer size in isolated and agglomerated state. The FTIR changes obtained from different AmB-NPs formulations suggesting some sort of interaction between the drug and the polymer. A simple, fast, accurate and reliable HPLC method for the in vitro investigations (drug content and drug release from the NPs) of AmB has been developed. Intra-day and inter-day variability were ≤ 6.2% with excellent linearity(R2=0.9982) and reproducibility. All tested formulations have shown slow initial release with 20% to 54% released within 24 h in phosphate buffer containing 2% sodium deoxycholate. The release rates of AmB from these formulations were best fit to the Korsmeyer–Peppas model. Therefore, a diffusion release model is the best to characterize the in vitro release of AmB from these formulations. A new selective, sensitive and precise LC MS/MS method was developed to measure AmB concentrations via electrospray ionization source with positive ionization mode. The precision and accuracy of the developed LC MS/MS method in the concentration range of 100–4000 ng/ml show no significant difference among inter- and-intra-day analysis (p > 0.05). Linearity was observed over the investigated range with correlation coefficient, R2> 0.9943 (n = 6/day). The assay was able to detect all the drug v concentrations for pharmacokinetics and bioavailability estimation after oral dosing of AmB loaded to PLGA-PEG compared to its parenteral administration in rats. AmB solutions or NPs were kept in refrigerator during the study. After PO administration AmB loaded to PLGA-PEG (C6), AmB was rapidly absorbed, distributed, and then slowly eliminated. The effect of glycyrrhizic acid (GA) as a natural absorption enhancer added during formulation or before administration was investigated. The addition of GA significantly (P 790 % than Fungizoneis observed after GA Addition. AmB formulations have been screened for toxicity using several in vitro assays; hemolysis, antifungal activity and analysis of the cell viability by 3-(4,5dimethylthiazoly)-2,5-diphenyl-tetrazolium bromide(MTT) assay. Fungizone was used as the reference standard. AmB loaded to PLGA-PEG of the selected formulations showed a significant reduction (P< 0.05) in the in vitro hemolytic activity as compared to Fungizone. On cellular level, AmB loaded to PLGA-PEG showed a potent activity against the in vitroC. albicans with a parallel decrease in the cytotoxicity against in the in vitro tested cell lines. Additionally, no pathological abnormalities were observed in rats given iv administration of AmB protocol except after Fungizone administration. The in vivo nephrotoxicity and histopathological evaluation of AmB were performed in both rats' kidneys and liver using forty eight rats divided into eight groups after single and multiple iv administrations of 1mg/kg of the selected formulations or Fungizonefor a week. AmB induced nephrotoxicity was also estimated in these rats by measuring blood urea nitrogen and plasma creatinine (biomedical parameters). A significant (P<0.05) nephrotoxicity was observed after Fungizone® administration compared to all AmB loaded to PLGA-PEG formulations. The histopathological study of the isolated kidney tissues confirmed this finding. Furthermore, the incidence of the liver toxicity due to the selected AmB loaded PLGA-PEG formulations has been reduced significantly as investigated through in vivo analysis (histopathological and biochemical tests) in comparison with Fungizone. vi In conclusion, a successful novel AmB oral formulation with improved efficacy and bioavailability and with less toxicity to the kidney and the liver was developed. Further investigations are needed in the near future for this formulation to be in the market as an oral delivery system of AmB

Oral Formulations of diclofenac beads and their Characterization

Diclofenac sodium (DS) is an effective non-steroidal anti-inflammatory drug (NSAID) agent. However, DS has short half life and adverse effects (e.g., ulcer bleeding or perforation of intestinal wall). The objectives of this study were to improve the oral bioavailability by loading DS in sodium alginate beads. The feasibility of different concentration and stabilizers on the mean particle size (MPS) and entrapment efficiency were also investigated. Materials and methods: DS-floating alginate or pectin beads were prepared by extrusion congealing technique. Physicochemical properties and particle size characterization were evaluated using Fourier Transform Infra-Red spectroscopy (FTIR), differential scanning calorimetry. Moreover, in vitro dissolution profiles were performed for all formulated DS loaded beads. Results: MPS of the prepared spherical beads of DS ranged from 568.3 ± 193 to 1791.3 ± 592 nm. and decreasing in sodium alginate or pectin concentration to the hydroxylpropylmethlycellulose ratio favored DS beads with a smaller MPS. There was a significant reduction in MPS, increment in drug content and drug release, with reduction of sodium alginate or pectin concentrations in the formulated beads. Both DSC and FTIR spectroscopy demonstrated a some sort of interaction between the drug and polymer used. Under conditions mimicking those in the stomach, a small amount of drug was released. The DS beads showed a release behavior dependent on pH value and alginate or pectin to hydroxypropylmethylcellulose ratio

Development and validation of a new HPLC analytical method for the determination of diclofenac in tablets

A new selective and sensitive high-performance liquid chromatography (HPLC) method was developed for the quantification of diclofenac sodium (DS) in pharmaceutical dosage form using lidocaine as internal standard (IS). Chromatographic separation was achieved on a symmetry C18 column (4.6 mm × 150 mm, 3 μm spherical particles) using 0.05 M orthophosporic (pH 2.0) 35% and acetonitrile as 65%, as the mobile phase at a flow rate of 2.0 mL/min and monitored at 210 nm. The run time was 2 min.The method was validated to fulfill International Conference on Harmonisation (ICH) requirements and this validation included specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision and robustness. The calibration curve was linear over the concentration range from 10 to 200 µg/ml, and lower limit of detection of 12.5 ng/ml. The accuracy and precision of the method were within the acceptable limit of ±20% at the lower limit of quantitation and ±15% at other concentrations. Diclofenac was unstable at room temperature it showed more than 25% loss after 24 h. While, DS is very stable at refrigerator 4 °C auto-sampler, freeze/thaw cycles and 30 days storage in a freezer at −35 ± 2 °C.All results were acceptable and this confirmed that the method is suitable for its intended use in routine quality control and assay of drugs. Keywords: Diclofenac, Reverse phase high performance liquid chromatography, HPL

Optimization and evaluation of Lisinopril mucoadhesive sustained release matrix pellets: In-vitro and ex-vivo studies

Lisinopril (LIS) is antihypertensive drug, classified as a class III drug with high water solubility and low permeability. To overcome the low permeability, 32 factorial designs aimed to formulate LIS as a sustained-release (LIS-SR) matrix pellet by extrusion/spheronization. Matrix pellets were composed of wet mass containing Avicel® and polymeric matrix polymers (sodium alginate (SA) and chitosan (CS)). Evaluation of the effect of two independent variables, matrix-forming units (SA and CS) on mean line torque, on pellet size, dissolution rate after 6 h, and mucoadhesion strength of the pellets were assessed using Statgraphics software. The tested formulations (F1-F9) showed that mean line torque ranged from 1.583 to 0.461 Nm, with LIS content in the LIS-SR pellets ranged from 87.9 to 103%, sizes varied from 1906 to 1404 µm and high percentages of drug released from pellets formulations (68.48 to 74.18 %), while the mean zeta potential value of mucoadhesive range from −17.5 to –22.9 mV.The selection of optimized formulation must have the following desirability: maximum peak torque, maximum pellets’ particle size, and minimum % LIS release after 6hr. LIS optimized sustained release pellet formula composed of 2,159 % SA and 0.357 % CS was chosen as optimized formula. It’s showed a 1.055 Nm mean line torque was responsible for the increased pellet size to 1830.8 μm with decreased release rate 56.2 % after 6 hr, and −20.33 mV average mucin zeta potential.Ex-vivo mucoadhesion studies revealed that that the optimize formulation, exhibited excellent mucoadhesive properties, after 1 h, about 73% of the pellets were still attached to the mucus membrane. Additionally, ex-vivo permeation determination of LIS from the optimized LIS-SR formulation was found to be significantly higher (1.7-folds) as compared to free LIS.In conclusion: LIS-SR matrix pellets, prepared with an extrusion/spheronization have desirable excellent characteristics in-vitro and ex-vivo sustained-release pellet formulation of LIS-SR was able to sustain the release of LIS for up to 8 h

Pharmacokinetics of ketorolac loaded to polyethylcyanoacrylate nanoparticles using UPLC MS/MS for its determination in rats

Polyethylcyanoacrylate (PECA) nanoparticles (NPs) have been employed as biodegradable polymeric carriers for oral (PO) delivery of ketorolac. The nanoparticles were prepared by polymerization technique at room temperature in a continuous aqueous phase at pH 2.5. This polymerization technique was able to hold 76-96% of ketorolac and the drug loading was a monomer concentration dependent. The feasibility of PECA NPs as PO controlled drug delivery systems of ketorolac was investigated in two groups of rats which were given orally either ketorolac tromethamine solution (1.5 mg/kg) or the selected ketorolac NPs aqueous dispersion (1.6 mg/kg). Ketorolac plasma concentrations were measured by a new fully validated specific, precise and accurate ultra-performance liquid chromatography tandem mass spectrometry (UPLC MS/MS) assay. The detection was performed on Waters TQ detector via negative electrospray ionization in a multiple reaction monitoring mode. Linear response (r(2) >= 0.995) was observed over the range of 10-10,000 ng/ml of ketorolac, with the lower limit of quantification of 5 ng/ml with 1 mu l injection volume. The intra- and inter-day precision (relative standard deviation, R.S.D.) values were <10% and the accuracy (relative error) was <= 8 for ketorolac concentrations. The drug solution is rapidly absorbed, distributed, and eliminated and shows a monophasic elimination phase. The assay was sensitive to follow pharmacokinetics of ketorolac in rats up to 24 h after a PO dose of its aqueous solution or NPs suspension. After NPs administration the mean Cmax, 5.0 +/- 1.3 mg/l, was attained at 1 h. The drug was successfully maintained around this elevated plasma drug concentration up to 6 h (>2t(1/2)). in rats. The AUC was significantly higher after the NPs suspension than the solution of ketorolac. This present study provides evidence that the sorption of ketorolac to PECA NPs could control the drug release/elimination in rats. (C) 2010 Elsevier B.V. All rights reservedResearch center, female medical and science, king saud universit

Oral administration of amphotericin B nanoparticles: antifungal activity, bioavailability and toxicity in rats

Amphotericin B (AMB) is used most commonly in severe systemic life-threatening fungal infections. There is currently an unmet need for an efficacious (AMB) formulation amenable to oral administration with better bioavailability and lower nephrotoxicity. Novel PEGylated polylactic-polyglycolic acid copolymer (PLGA-PEG) nanoparticles (NPs) formulations of AMB were therefore studied for their ability to kill Candida albicans (C. albicans). The antifungal activity of AMB formulations was assessed in C. albicans. Its bioavalability was investigated in nine groups of rats (n = 6). Toxicity was examined by an in vitro blood hemolysis assay, and in vivo nephrotoxicity after single and multiple dosing for a week by blood urea nitrogen (BUN) and plasma creatinine (PCr) measurements. The MIC of AMB loaded to PLGA-PEG NPs against C. albicans was reduced two to threefold compared with free AMB. Novel oral AMB delivery loaded to PLGA-PEG NPs was markedly systemically available compared to Fungizone® in rats. The addition of 2% of GA to the AMB formulation significantly (p  790% that of Fungizone®. The novel AMB formulations showed minimal toxicity and better efficacy compared to Fungizone®. No nephrotoxicity in rats was detected after a week of multiple dosing of AMB NPs based on BUN and PCr, which remained at normal levels. An oral delivery system of AMB-loaded to PLGA-PEG NPs with better efficacy and minimal toxicity was formulated. The addition of glycyrrhizic acid (GA) to AMB NPs formulation resulted in a significant oral absorption and improved bioavailability in rats

Pharmaceutical evaluation of different shampoo brands in local Saudi market

Shampooing is the most common form of hair treatment. Shampoos are primarily products aimed at cleansing the hair and scalp. There are many brands of shampoos in Saudi Arabia, available from different sources, locally and imported from other countries. This study aims to investigate whether such brands comply with the Saudi standard specifications for shampoos, issued by the National Center for Specifications and Standards, and to what extent these specifications are applied. Six shampoo brands were randomly collected from Riyadh market (Pantene®, Sunsilk®, Herbal essences®, Garnier Ultra Doux®, Syoss® and L'Oreal Elvive®). The selected shampoos were evaluated according to their physicochemical properties, including organoleptic characterization, pH measurement, percentage of solid content, rheological measurements, dirt dispersion level, foaming ability and foam stability, and surface tension. All shampoos had a good percentage of solids, excellent foam formation with stable foam and a highly viscous nature. Regarding the pH measurement, all shampoo samples were within the specified range with good wetting ability. Keywords: Shampoo, Physicochemical approach, Evaluatio

Retinal Delivery of the Protein Kinase C-β Inhibitor Ruboxistaurin Using Non-Invasive Nanoparticles of Polyamidoamine Dendrimers

Ruboxistaurin (RBX) is an anti-vascular endothelial growth factor (anti-VEGF) agent that is used in the treatment of diabetic retinopathy and is mainly given intravitreally. To provide a safe and effective method for RBX administration, this study was designed to develop RBX nanoparticles using polyamidoamine (PAMAM) dendrimer generation 5 for the treatment of diabetic retinopathy. Drug loading efficiency, and in vitro release of proposed complexes of RBX: PAMAM dendrimers were determined and the complexation ratio that showed the highest possible loading efficiency was selected. The drug loading efficiency (%) of 1:1, 2.5:1, and 5:1 complexes was 89.2%, 96.4%, and 97.6%, respectively. Loading capacities of 1:1, 2.5:1, and 5:1 complexes were 1.6%, 4.0%, and 7.2% respectively. In comparison, the 5:1 complex showed the best results in the aforementioned measurements. The in vitro release studies showed that in 8 h, the RBX release from 1:1, 2.5:1, and 5:1 complexes was 37.5%, 35.9%, and 77.0%, respectively. In particular, 5:1 complex showed the highest drug release. In addition, particle size measurements showed that the diameter of empty PAMAM dendrimers was 214.9 ± 8.5 nm, whereas the diameters of loaded PAMAM dendrimers in 1:1, 2.5:1, 5:1 complexes were found to be 461.0 ± 6.4, 482.4 ± 12.5, and 420.0 ± 7.1 nm, respectively. Polydispersity index (PDI) showed that there were no significant changes in the PDI between the free and loaded PAMAM dendrimers. The zeta potential measurements showed that the free and loaded nanoparticles possessed neutral charges due to the presence of anionic and cationic terminal structures. Furthermore, the safety of this formulation was apparent on the viability of the MIO-M1 cell lines. This nanoformulation will improve the therapeutic outcomes of anti-VEGF therapy and the bioavailability of RBX to prevent vision loss in patients with diabetic retinopathy