Selective anticancer activity of hydroxyapatite/chitosan-poly(d,l)-lactide-co-glycolide particles loaded with an androstane-based cancer inhibitor.

In an earlier study we demonstrated that hydroxyapatite nanoparticles coated with chitosan-poly(d,l)-lactide-co-glycolide (HAp/Ch-PLGA) target lungs following their intravenous injection into mice. In this study we utilize an emulsification process and freeze drying to load the composite HAp/Ch-PLGA particles with 17ß-hydroxy-17α-picolyl-androst-5-en-3ß-yl-acetate (A), a chemotherapeutic derivative of androstane and a novel compound with a selective anticancer activity against lung cancer cells. 1H NMR and 13C NMR techniques confirmed the intact structure of the derivative A following its entrapment within HAp/Ch-PLGA particles. The thermogravimetric and differential thermal analyses coupled with mass spectrometry were used to assess the thermal degradation products and properties of A-loaded HAp/Ch-PLGA. The loading efficiency, as indicated by the comparison of enthalpies of phase transitions in pure A and A-loaded HAp/Ch-PLGA, equaled 7.47wt.%. The release of A from HAp/Ch-PLGA was sustained, neither exhibiting a burst release nor plateauing after three weeks. Atomic force microscopy and particle size distribution analyses were used to confirm that the particles were spherical with a uniform size distribution of d50=168nm. In vitro cytotoxicity testing of A-loaded HAp/Ch-PLGA using MTT and trypan blue dye exclusion assays demonstrated that the particles were cytotoxic to the A549 human lung carcinoma cell line (46±2%), while simultaneously preserving high viability (83±3%) of regular MRC5 human lung fibroblasts and causing no harm to primary mouse lung fibroblasts. In conclusion, composite A-loaded HAp/Ch-PLGA particles could be seen as promising drug delivery platforms for selective cancer therapies, targeting malignant cells for destruction, while having a significantly lesser cytotoxic effect on the healthy cells.

In vivo and in vitro metabolism of the designer anabolic steroid furazadrol in thoroughbred racehorses.

Furazadrol ([1',2']isoxazolo[4',5':2,3]-5α-androstan-17ß-ol) is a designer anabolic androgenic steroid that is readily available via the internet. It contains an isoxazole fused to the steroid A-ring which offers metabolic stability and noteworthy anabolic activity raising concerns over the potential for abuse of this compound in equine sports. The metabolism of furazadrol was studied by in vivo and in vitro methods for the first time. Urinary furazadrol 17-sulfate and furazadrol 17-glucuronide metabolites were detected in vivo after a controlled administration and compared with synthetically-derived reference materials in order to confirm their identities. They were quantified to establish the excretion profile and a suitable limit of detection. Minor metabolites were also detected, including epifurazadrol, hydroxylated furazadrol, and hydroxylated and oxidised furazadrol, present as the sulfate and glucuronide conjugates. Phase II metabolites were subjected to enzymatic hydrolysis by Escherichia coli ß-glucuronidase and Pseudomonas aeruginosa arylsulfatase to further confirm the identity of the corresponding phase I metabolites. The metabolism profile was compared to the products obtained from an in vitro phase I metabolism study, with all but two of the minor in vivo phase I metabolites observed in the in vitro system. These investigations identify the key urinary metabolites of furazadrol following oral administration, which can be incorporated into anti-doping screening and confirmation procedures.

In vitro and in vivo evaluation of a 3ß-androsterone derivative as inhibitor of 17ß-hydroxysteroid dehydrogenase type 3.

17ß-Hydroxysteroid dehydrogenase type 3 (17ß-HSD3 or HSD17B3) catalyzes the last step in the biosynthesis of the potent androgen testosterone (T), by stereoselectively reducing the C17 ketone of 4-androstene-3,17-dione (4-dione), with NADPH as cofactor. Since T plays an important role in androgen-sensitive diseases, this enzyme is thus an interesting therapeutic target. In an attempt to design compounds to lower the level of T, we synthesized androsterone derivatives substituted at position 3 as inhibitors of 17ß-HSD3, and selected one of the most potent compounds for additional studies. In an enzymatic assay in homogenized and whole HEK-293 cells overexpressing 17ß-HSD3, the inhibitor RM-532-105 efficiently inhibited the conversion of natural substrate 4-dione (50nM) into T with an IC50 of 26nM and 5nM, respectively. Moreover, the inhibitor RM-532-105 (10mg/kg) reached a plasma concentration of 250ng/mL at 7h (AUC 24h: 3485ngh/mL) after subcutaneous (s.c.) injection in the rat. In order to mimic the human situation in which 4-dione is converted to T in the testis, we used intact rats. Treatment for 7 days with 17ß-HSD3 inhibitor RM-532-105 by s.c. injection or oral gavage exerted no effect on the testis, prostate and seminal vesicle weight and no modification in the levels of plasma steroids. However, after this treatment, the concentration of inhibitor in plasma increased depending on the dose. We thereafter determined the concentration of inhibitor in the testis and we discovered that the compound was slightly present. In fact, at 10mg/kg, the inhibitor RM-532-105 seems to have difficulty penetrating inside the testis and was found to be concentrated in the testicular capsule, and therefore unable to inhibit the 17ß-HSD3 located inside the testis. However, with a higher dose of 50mg/kg injected s.c. in rats, RM-532-105 significantly decreased the level of T and dihydrotestosterone measured in plasma at 2h.

In vitro and in vivo aspects of N-acyl-phosphatidylethanolamine-containing liposomes.

Incorporation of the phospholipid, N-acyl-phosphatidylethanolamine (NAPE), has shown to increase the liposomal stability towards plasma components in vitro. Besides increasing the circulation-time, NAPE has been shown to contain fusiogenic properties. Hence, fusion between NAPE-liposomes and target cells may be expected, resulting in a favorable delivery of drug to the target cell. In this study, NAPE has been tested as a potential liposomal component of phosphatidylcholine-liposomes. The liposomes were characterized by size, long-term stability and phase transition temperature (T(m)). In vivo behavior of NAPE-liposomes was determined by the blood-circulation half-life in mice. A characterization of the liposomes revealed that high content of NAPE resulted in liposomes of increased size compared to pure phosphatidylcholine-liposomes. However, the liposomes showed only a slight increase in size during storage for 5 weeks. Determination of T(m) for NAPE-liposomes showed increasing values of T(m) by increasing percentage of NAPE in the liposomal bilayer, due to the higher T(m) of NAPE compared to phosphatidylcholine. Blood-clearance studies showed an initial increase in blood-circulation of liposomes containing high amounts of NAPE. Thus, these results suggest that liposomes containing high percentage of NAPE may be a promising candidate for long-circulating liposomes, possibly in combination with other stabilizing components, e.g. cholesterol.