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Title: Pharmaceutical studies of Amphotericin-B : $$b oral formulation using nanotechnology
Authors: ALQuadeib, Bushra
Issue Date: 2016
Publisher: Newcastle University
Abstract: Amphotericin 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<0.05) increased AmB AUC and Cmax (2 - 3 folds). There was no significant change (P=0.09) in adding GA during formulation or before administration. A tremendous improvement of AmB oral absorption in rats by > 790 % than Fungizoneis 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 Fungizonefor 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.
Description: PhD Thesis
Appears in Collections:School of Chemical Engineering and Advanced Materials

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