Springer Science and Business Media LLC

Protein therapeutics often require repeated administrations of drug over a long period of time. Protein instability is a major obstacle to the development of systems for their controlled and sustained release. We describe a surface modification of nanoporous silicon particles (NSP) with an agarose hydrogel matrix that enhances their ability to load and release proteins, influencing intracellular delivery and preserving molecular stability. We developed and characterized an agarose surface modification of NSP. Stability of the released protein after enzymatic treatment of loaded particles was evaluated with SDS-page and HPLC analysis. FITC-conjugated BSA was chosen as probe protein and intracellular delivery evaluated by fluorescence microscopy. We showed that agarose coating does not affect NSP protein release rate, while fewer digestion products were found in the released solution after all the enzymatic treatments. Confocal images show that the hydrogel coating improves intracellular delivery, specifically within the nucleus, without affecting the internalization process. This modification of porous silicon adds to its tunability, biocompatibility, and biodegradability the ability to preserve protein integrity during delivery without affecting release rates and internalization dynamics. Moreover, it may allow the silicon particles to function as protein carriers that enable control of cell function.

The pharmacokinetics of platinum was investigated in 10 cancer patients treated with a 1-hr infusion of 300 mg/m2 of carboplatin which was given 2–4 days after the administration of 100 mg/kg (20-mg/kg bolus and 80-mg/kg intravenous infusion) of methotrexate. Platinum was analyzed in the samples by flameless atomic absorption spectrophotometry. The concentration vs time data for total platinum in plasma followed a two-compartment model and the mean (and SE) values for β, TBC, V c, and RC were 0.0827 (0.22) hr−1, 2.355 (0.252) liters/hr · m2, 10.74 (0.62) liters/m2, and 2.405 (0.228) liters/hr · m2, respectively. There was no significant change in the creatinine clearance or TBC with repeated treatment. The ultrafilterable platinum which was measured in the plasma of two patients constituted 82 and 11.3% of the total platinum at 1 and 24 hr, respectively, and the data conformed to the one-compartment model. The mean (SE) values for t β, TBC, and V d for free platinum were 1.844 (0.208) hr, 4.583 (1.059) liters/hr · m2, and 11.88 (1.45) liters/m2, respectively. The above data are in good agreement with those reported earlier for platinum following the administration of carboplatin as a single agent. These results suggest that high-dose methotrexate therapy, when administered 2–4 days before carboplatin, does not affect the pharmacokinetics of platinum in the plasma.

To formulate a xanthan gum–containing linezolid ophthalmic solution (LZD-XG) as a new antibiotic treatment against ocular bacterial infection. LZD-XG was prepared and evaluated for its in vitro/in vivo ocular tolerance, in vitro/in vivo antibacterial activity, and in vivo ocular penetration. The optimized LZD-XG exhibited good in vitro/in vivo eye tolerance. A prolonged ocular surface residence time of LZD-XG was observed after topical instillation, and the ocular permeation was significantly better for LZD-XG than fora linezolid (LZD) ophthalmic solution. The in vitro antimicrobial activity was significantly better with LZD-XG than with LZD. In vivo evaluation also confirmed a strong therapeutic treatment effect of LZD-XG, as it significantly improved the clinical symptoms, ameliorated the damage of Staphylococcus aureus to ocular tissues, lowered the colony forming unit counts in the cornea, and decreased the myeloperoxidase activity in the cornea. LZD-XG was deemed a viable ophthalmic solution against ocular bacterial infection due to its excellent in vitro and in vivo characterizations.

This study aims at developing poly(ethylene oxide)-modified poly(epsilon-caprolactone) (PEO-PCL) nanoparticulate system as an intracellular delivery vehicle for saquinavir, an anti-HIV protease inhibitor. Saquinavir-loaded PEO-PCL nanoparticles were prepared by a solvent displacement process. The formed nanoparticles were characterized for size, surface charge, and surface presence of PEO chains. Cellular uptake and distribution of the nanoparticle was examined in THP-1 human monocyte/macrophage (Mo/Mac) cell line. Intracellular saquinavir concentrations were measured as a function of dose and duration of incubation. The PEO-PCL nanoparticles had a smooth surface and spherical shape and showed a relatively uniform size distribution with a mean particle diameter of approximately 200 nm. The surface presence of PEO chains was confirmed by an increase in the –C–O–(ether) signature of the C1s spectra in electron spectroscopy for chemical analysis. Rapid cellular uptake of rhodamine-123 encapsulated PEO-PCL nanoparticles was observed in THP-1 cells. Intracellular saquinavir concentrations when administered in the nanoparticle formulation were significantly higher than from aqueous solution. This study shows that PEO-PCL nanoparticles provide a versatile platform for encapsulation of saquinavir and subsequent intracellular delivery in Mo/Mac cells.

Cultured L1210 murine lymphocytic leukemia cells were used to compare metabolic activation and cytotoxicity of 5-fluorouracil (FU), Ftorafur (FT), and three novel FU-sulfur analogues. These analogues, l-(2′-tetrahydrothienyl)-5-fluorouracil (FUS), l-(2′-tetrahydrothienyl)-5-fluorouracil-l′-oxide (FUSO), and 1-(2′-tetrahydrothienyl)-5-fluorouracil-l′-l′-dioxide (FUSO2), have yet to be fully evaluated for potential therapeutic value based on in vitro cytotoxicity. The role of these FU analogues as prodrugs was evaluated by comparing metabolism of normal pyrimidine pathways and activation by hepatic mixed function oxidases (MFO). Significant differences in biochemical activity and cytotoxicity were measured between FU and FU analogues. FU and FU analogues were cytotoxic to L1210 cells (63–92% growth inhibition of 100 µM concentrations after 72 hr of incubation). However, at equimolar concentrations cytotoxicity of the FU analogues after MFO activation (56–66% growth inhibition) was greater than FU (47% growth inhibition). Hypoxanthine, a purine precursor, did not significantly alter fluoropyrimidine cytotoxicity with or without MFO. Thymidine and uridine, pyrimidine precursors, reduced FT and FUS cytotoxicities in the presence (27, 40%) and absence (25, 15%) of MFO but did not modify FU, FUSO, or FUSO2 cytotoxicities.

The aim of this study is to determine the effects of saccharide-containing excipients on the surface composition of spray-dried protein formulations and their matrix heterogeneity. Spray-dried formulations of myoglobin or bovine serum albumin (BSA) were prepared without excipient or with sucrose, trehalose, or dextrans. Samples were characterized by solid-state Fourier-transform infrared spectroscopy (ssFTIR), differential scanning calorimetry (DSC), size exclusion chromatography (SEC) and scanning electron microscopy (SEM). Protein surface coverage was determined by X-ray photoelectron spectroscopy (XPS), while conformational differences were determined by solid-state hydrogen/deuterium exchange with mass spectrometry (ssHDX-MS). Structural differences were exhibited with the inclusion of different excipients, with dextran formulations indicating perturbation of secondary structure. XPS indicated sucrose and trehalose reduced protein surface concentration better than dextran-containing formulations. Using ssHDX-MS, the amount of deuterium incorporation and populations present were the largest in the samples processed with dextrans. Linear correlation was found between protein surface coverage and ssHDX-MS peak area (R2 = 0.853) for all formulations with saccharide-containing excipients. Lower molecular weight species of saccharides tend to enrich the particle surface and reduce protein concentration at the air-liquid interface, resulting in reduced population heterogeneity and improved physical stability, as identified by ssHDX-MS.

Purpose. The current animal model generally accepted by the pharmaceutical industry and the FDA for assessment of muscle damage following intramuscular injection (IM) is the rabbit lesion volume model (RbLV). However, this model is resource intensive. The goal of this study was to find a resource sparing alternative to the rabbit lesion model for assessing injection site toleration in IM formulation screening. Methods. Short term animal model alternatives to RbLV for evaluating IM formulations were examined. In addition to RbLV, myeloperoxidase (MPO), p-nitrophenyl N-acetyl-β-glucosaminide (NAβG) and/or plasma creatine phosphokinase (CK) activities were determined in rabbits (Rb) and rats (Rt) after injection of formulations (digoxin, azithromycin and danofloxacin). The edema from these formulations 24 hr after subcutaneous injection into the rat footpad (RFE) was also determined. Results. MPO and NAβG were not considered very useful as biochemical predictors of muscle damage for these formulations. Histology generally correlated with RbLV values. Compared to saline, RbLV was marked for all formulations within 1–3 days of injection. After day 3, lesions quickly resolved, and no significant differences were found. For these formulations, all CK animal models and RFE were generally predictive of RbLV. A formulation with RtCK > 1000 U/L or RbCK > 3000 U/L, was predicted to be poorly, tolerated. Conclusions. Due to ease, number of animals, time and intrinsic mechanism, we concluded that for most formulations, 2 and 4 hr RtCK data alone should be reasonably predictive of muscle damage.

To determine the transscleral permeability of chemotherapeutic drugs vinblastine and doxorubicin for treatment of intraocular tumors, and to compare the use of doxorubicin encapsulated in PLGA and liposome nanoparticles. Human sclera was isolated and mounted in a Lucite chamber. Fluorescently tagged vinblastine (VIN), innately fluorescent free doxorubicin (DOX), PLGA doxorubicin (PLGA-DOX), or Doxil (Tibotec Therapeutics) were added to the episcleral donor chamber. The choroidal side was perfused with Balanced Salt Solution. Perfusate fractions were collected over 24 h and measured for fluorescence. Following the experiment, tissue sections were imaged, underwent a drug wash out procedure, and tissue drug content was analyzed using an LC–MS/MS method. Within 24 h, a total of 68%, 74%, 29%, and 1.9% of the drug dose from VIN, DOX, PLGA-DOX, and Doxil, respectively, diffused across the sclera. VIN and DOX scleral tissue showed strong fluorescence after 24 h. PLGA-DOX displayed scattered fluorescence, and Doxil indicated minimal fluorescence. LC–MS/MS revealed strong tissue binding of DOX. This study suggests both vinblastine and doxorubicin are able to diffuse across human sclera. In addition, PLGA nanoparticles delivered doxorubicin at a slower rate across the sclera, and the liposome preparation resulted in the slowest delivery of drug.