Sarala Pamujula

Oral delivery of spray dried PLGA/amifostine nanoparticles

Amifostine (Ethyol, WR‐2721) is a cytoprotective drug approved by the US Food & Drug Administration for intravenous administration in cancer patients receiving radiation therapy and certain forms of chemotherapy. The primary objective of this project was to develop orally active amifostine nanoparticles using spray drying technique. Two different nanoparticle formulations (Amifostine‐PLGA (0.4:1.0 and 1.0:1.0)) were prepared using a Buchi B191 Mini Spray Dryer. A water‐in‐oil emulsion of amifostine and PLGA (RG 502) was spray dried using an airflow of 600 Lh−1 and input temperature of 55°C. A tissue distribution study in mice was conducted following oral administration of the formulation containing drug‐polymer (0.4:1.0). The efficiency of encapsulation was 90% and 100%, respectively, for the two formulations while the median particle sizes were 257 and 240 nm, with 90% confidence between 182 and 417 nm. Since amifostine is metabolized to its active form, WR‐1065, by intracellular alkaline phosphatase, the tissue levels of WR‐1065 were measured, instead of WR‐2721. WR‐1065 was detected in significant amounts in all tissues, including bone marrow, jejunum and the kidneys, and there was some degree of selectivity in its distribution in various tissues. This work demonstrates the feasibility of developing an orally effective formulation of amifostine that can be used clinically.

Radioprotection in mice following oral delivery of amifostine nanoparticles

Purpose: Amifostine (Ethyol®) is an approved cytoprotective agent prescribed to reduce certain side-effects in the chemotherapy of ovarian or non-small cell lung cancer, or in radiation treatment of head-and-neck cancer. The usefulness of this drug is further hampered, because it is not effective when given orally. The objective of this part of the project was to evaluate the radioprotective efficacy of orally active amifostine nanoparticles. Materials and methods: Radioprotective efficacy was evaluated by measuring the ability of the amifostine nanoparticles (equivalent to 500 mg/Kg) to inhibit whole-body gamma irradiation -induced injury in mice. All mice received acute whole-body gamma irradiation from a Cesium-137 source and the radioprotective efficacy of the formulation was determined by measuring 30-day survival at 9 Gy, bone marrow hemopoeitic progenitor cell survival at 9 Gy and 8 Gy, and intestinal crypt cell survival at 11 Gy. Results: Thirty-day survival, hemopoietic progenitor cell survival, as well as the jejunal crypt cell survival were all significantly enhanced when the mice were treated orally with the amifostine nanoparticles 1 h prior to irradiation. Conclusions: These results clearly and unequivocally demonstrate that the amifostine nanoparticles developed in our laboratory provides significant protection from acute whole-body gamma irradiation injury in mice.

Spray-Dried Chitosan as a Direct Compression Tableting Excipient

The objective of this study was to prepare and evaluate a novel spray-dried tableting excipient using a mixture of chitosan and lactose. Three different grades of chitosan (low-, medium-, and high-molecular-weight) were used for this study. Propranolol hydrochloride was used as a model drug. A specific amount of chitosan (1, 1.9, and 2.5 g, respectively) was dissolved in 50 mL of an aqueous solution of citric acid (1%) and later mixed with 50 mL of an aqueous solution containing lactose (20, 19.1, and 18.5 g, respectively) and propanolol (2.2 g). The resultant solution was sprayed through a laboratory spray drier at 1.4 mL/min. The granules were evaluated for bulk density, tap density, Carr index, particle size distribution, surface morphology, thermal properties, and tableting properties. Bulk density of the granules decreased from 0.16 to 0.13 g/mL when the granules were prepared using medium- or high-molecular-weight chitosan compared with the low-molecular-weight chitosan. The relative proportion of chitosan also showed a significant effect on the bulk density. The granules prepared with 1 g of low-molecular-weight chitosan showed the minimum Carr index (11.1%) indicating the best flow properties among all five formulations. All three granules prepared with 1 g chitosan, irrespective of their molecular weight, showed excellent flow properties. Floating tablets prepared by direct compression of these granules with sodium bicarbonate showed 50% drug release between 30 and 35 min. In conclusion, the spray-dried granules prepared with chitosan and lactose showed excellent flow properties and were suitable for tableting.

Preparation and in vitro characterization of amifostine biodegradable microcapsules

The purpose of this project was to develop sustained release microcapsules of amifostine. The microcapsules were prepared using solvent evaporation technique. The effect of several formulation variables on the characteristics of the microcapsules was studied. The formulation variables studied were drug loading, polymer (polylactide-co-glycolide) (PLGA) concentration, and the amount of gelatin in the initial aqueous phase. The drug loading was studied at three different levels (5, 10, and 25 mg); the PLGA concentration was studied at two levels (500 and 1000 mg); and the amount of gelatin used ranged from 2 to 14 mg. In general, the microcapsules were less than 155 microm in diameter with median size between 50 and 80 microm. While the use of higher amounts of PLGA significantly increased the median size of the microcapsules, using higher amounts of amifostine had no significant effect, irrespective of the amount of PLGA. The use of gelatin, within the range 2-14 mg, did not show any significant effect on the particle size distribution. Scanning electron microscopy (SEM) of the microcapsules revealed that all nine formulations yielded spherical particles. The use of 500 mg PLGA with 10 or 25 mg amifostine yielded microcapsules with porous surfaces. The surface pores, however, were not present in microcapsules prepared using 1000 mg PLGA. The efficiency of encapsulation decreased significantly from 63 to 24% when the amount of amifostine increased from 5 to 25 mg in the formulations using 500 mg PLGA. Similarly, the efficiency of encapsulation decreased from 87 to 23% when the amount of PLGA was doubled to 1000 mg. An increase in the amount of amifostine in the formulation using 500 mg PLGA also resulted in a significant increase in initial drug release (from 20 to 62%) within the first hour. These results were consistent with the porous morphology of these microcapsules. In general, all batches of microcapsules showed 24-96 h sustained drug release.

Radioprotection in mice following oral administration of WR-1065/PLGA nanoparticles

Purpose: N-(2-mercaptoethyl)1,3-diaminopropane (WR-1065), is the active metabolite of amifostine, a broad spectrum cytoprotective agent used in conjunction with both chemo- and radiotherapy of certain cancers. This report describes for the first time an oral formulation of WR-1065 and follows on from our earlier report of a similar oral formulation of amifostine. Materials and methods: The nanoparticles of WR-1065 were prepared by spray drying technique using poly lactide-co-glycolide (PLGA) as the polymer matrix. Radioprotection was determined by measuring reductions in radiation-induced: (i) 30-day survival; (ii) bone marrow suppression; and (iii) intestinal injury following 9 Gray (Gy) whole body gamma irradiation in mice. All treatments were given 1 hour pre-irradiation and WR-1065 was tested at the dose of 500 mg/kg. Results: The WR-1065/PLGA nanoparticles were smooth and spherical with the average diameter of 206 nm and contained 21.7% (w/w) WR-1065. While irradiation markedly reduced 30-day survival in non-treated control mice, and caused significant bone marrow suppression and intestinal injury in surviving mice, oral administration of WR-1065/PLGA nanoparticles resulted in significant radioprotection as evidenced by a marked reduction in all three of the above mentioned parameters of radiation injury. Conclusions: These findings clearly demonstrate the feasibility of developing an effective oral formulation of WR-1065 as a radioprotective agent.

Development and in vitro evaluation of a nanoemulsion for transcutaneous delivery

Abstract Objective: The purpose of this study is to develop a nanoemulsion formulation for its use as a transcutaneous vaccine delivery system. Materials and methods: With bovine albumin-fluorescein isothiocyanate conjugate (FITC-BSA) as a vaccine model, formulations were selected with the construction of pseudo-ternary phase diagrams and a short-term stability study. The size of the emulsion droplets was furthered optimized with high-pressure homogenization. The optimized formulation was evaluated for its skin permeation efficiency. In vitro skin permeation studies were conducted with shaved BALB/c mice skin samples with a Franz diffusion cell system. Different drug concentrations were compared, and the effect of the nanoemulsion excipients on the permeation of the FITC-BSA was also studied. Results: The optimum homogenization regime was determined to be five passes at 20 000 psi, with no evidence of protein degradation during processing. With these conditions, the particle diameter was 85.2 nm ± 15.5 nm with a polydispersity index of 0.186 ± 0.026 and viscosity of 14.6 cP ± 1.2 cP. The optimized formulation proved stable for 1 year at 4 °C. In vitro skin diffusion studies show that the optimized formulation improves the permeation of FITC-BSA through skin with an enhancement ratio of 4.2 compared to a neat control solution. Finally, a comparison of the skin permeation of the nanoemulsion versus only the surfactant excipients resulted in a steady state flux of 23.44 μg/cm2/h for the nanoemulsion as opposed to 6.10 μg/cm2/h for the emulsifiers. Conclusion: A novel nanoemulsion with optimized physical characteristics and superior skin permeation compared to control solution was manufactured. The formulation proposed in this study has the flexibility for the incorporation of a variety of active ingredients and warrants further development as a transcutaneous vaccine delivery vehicle.

Preparation of polylactide-co-glycolide and chitosan hybrid microcapsules of amifostine using coaxial ultrasonic atomizer with solvent evaporation

The objective of this study was to evaluate the effect of various processing and formulation factors on the characteristics of amifostine hybrid microcapsules. Amifostine‐loaded hybrid microcapsules were prepared using PLGA and chitosan. In short, amifostine powder was dissolved in de‐aerated water with or without chitosan. The amifostine solution was later emulsified into PLGA solution in dichloromethane containing phosphatidylcholine. The resultant emulsion was fed through the inner capillary of a coaxial ultrasonic atomizer. The liquid fed through the coaxial outer capillary was either water or chitosan solution. The atomized droplets were collected into PVA solution and the droplets formed microcapsules immediately. The hybrid microcapsules prepared with chitosan solution only as an outer layer liquid showed the maximum efficiency of encapsulation (30%). The median sizes of all three formulations were 33–44 μm. These formulations with chitosan showed positive zeta‐potential and sustained drug release with 13–45% amifostine released in 24 h. When chitosan was incorporated into inner as well as outer liquid layers, the drug release increased significantly, 45% (compared with other formulations) released in 24 h and almost 100% released in 11 days. Hybrid microcapsules of amifostine showed moderately high efficiency of encapsulation. The cationic charge (due to the presence of chitosan) of these particles is expected to favour oral absorption and thus overall bioavailability of orally administered amifostine.

Abstract 5531: Preparation and cellular delivery of doxorubicin nanoemulsion for cancer therapy

Purpose: The aim of this study is to formulate a novel nanoemulsion of doxorubicin to enhance the therapeutic efficacy of the drug. Method: Four different multiple emulsions (W 1 /O/W 2 ) were prepared (A, B, C, and D) by homogenization of a mixture of aqueous and oil phases. The resultant primary emulsions were later pass through a high pressure homogenization to further reduce the globules size to nanometer range. The formulation A is a control nanoemulsion, consisted of only deionized water as W 1 phase. The W 1 phase of the other three formulations (B, C, and D) consisted of aqueous solution of BSA (as an emulsion stabilizer), doxorubicin, and a mixture of BSA and doxorubicin, respectively. For all four formulations, the oil phase (O) consisted of a 2:1 mixture of soyabean oil and Span 80, and the outer aqueous phase (W 2 ) consisted of prolaxamer P-85 and Vitamin E TPGS. The nanoemulsions were evaluated for stability, polydispersity index, size, and viscosity. Anticancer efficacy of these formulations was evaluated by Alamar Blue Assay and Colony Forming Assay using MCF-7 cells as a model malignant breast cancer cell line. Results: The stability of nanoemulsions was found to be significantly better for the formulations containing BSA. Presence of BSA or doxorubicin resulted in a significant increase in viscosity from 2.09 to 10.6 cP. Cell culture studies showed that doxorubicin nanoemulsions had higher localization in cytoplasm as compared to the nucleus. In regard to cellular uptake (intracellular quantification of doxorubicin), the following results were obtained at 2h, 24h, 48h, and 120h post-incubation, respectively: 42%, 30%, 37%, and 22% with doxorubicin solution; 12%, 19%, 21%, 33% with formulation C; and 0.25%, 6%, 10%, 19% with formulation D. The results of Alamar Blue Assay showed IC 50 values of 0.97μM (solution), 1.62μM (formulation C) and 1.91μM (formulation D). The results of Colony Forming Assay (12days-post-treatment with equivalent to 0.86 μM doxorubicin) showed 74% (solution), 86% (formulation C) and 92.6% (formulation D) growth inhibitions. Conclusions: The presence of BSA within the aqueous phase enhances the stability of nanoemulsions. In comparison to doxorubicin solution, the nanoemulsions produce higher anticancer efficacy. This exploratory study will be followed by more detailed studies to confirm this preliminary finding. Acknowledgements: This work was funded in part by the NASA grant # NCC3-946-S4, Louisiana Board of Regents RC/EEP (2007-10), NIH grant# 5P20CA118768-02, and Louisiana Cancer Research Consortium. We are thankful to Dr. Thomas Wiese of the College of Pharmacy, XULA, for providing the Cell culture lab facilities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5531.