Chemical synthesis, NMR characterization, and anticancer activity of androstene derivatives with a C17-side chain.

Cancer remains one of the leading causes of death, worldwide. In addition, the lack of efficacy and selectivity of chemotherapeutic agents for cancer cells is a challenge that needs to be addressed through the development of new drugs. Since aminosteroids are of interest in fighting cancer, our group previously reported antiproliferative activity on several cancer cell lines of two representatives, RM-133 and RM-581. To extend the structure-activity relationship study of aminosteroids, of which RM-133 (androstane) and RM-581 (estrane) are the main candidates, we performed the chemical synthesis and biological evaluation on lung (SHP-77), breast (T-47D) and prostate (DU-145, PC-3 and LAPC-4) cancer cells of four analogues of RM-581. We moved the functionalized side chain from position 2 of the androstane and estrane derivatives to incorporate it into a new chain located at position 17. Chemical synthesis took place in 2 steps from steroidal side-chain carboxylic acids, allowing to obtain 4 steroid derivatives with acceptable yields, which were fully characterized by nuclear magnetic resonance spectroscopy (1H and 13C NMR). After the evaluation of compounds 12-15, lower antiproliferative activities varying from 12 to 54%, 0-33% and 0-63% were observed for SHP-77, DU-145 and PC-3 cell lines, respectively, while higher activities varying from 33 to 62% and 45-84% were observed for T-47D and LAPC-4 cell lines, respectively, when tested at 10 µM. Overall, it was observed that these aminosteroids have a lower cytotoxic activity than that of RM-581 and, that moving the side chain from steroid position C2 to C17 is clearly detrimental for antiproliferative activity. However, this work has enabled us to expand our knowledge of the structural requirements to maintain the anticancer activity of aminosteroid derivatives.

The effect of drug loading on the properties of abiraterone-hydroxypropyl beta cyclodextrin solid dispersions processed by solvent free KinetiSol® technology.

Abiraterone is a poorly water-soluble drug used in the treatment of prostate cancer. In our previous study, we reported that KinetiSol® processed solid dispersions (KSDs) based on hydroxypropyl ß-cyclodextrin (HPBCD) showed improved dissolution and pharmacokinetics of abiraterone. However, the nature of abiraterone-HPBCD interaction within the KSDs or the effect of drug loading on the physicochemical properties and in vivo performance of HPBCD-based KSDs remain largely unknown. We hypothesize that KinetiSol technology can prepare abiraterone-HPBCD complexes within KSDs and that increasing the drug loading beyond an optimal point reduces the in vitro and in vivo performance of these KSDs. To confirm our hypothesis, we developed KSDs with 10-50% w/w drug loading and analyzed them using X-ray diffractometry and modulated differential scanning calorimetry. We found that KSDs containing 10-30% drug were amorphous. Interestingly, two-dimensional solid-state nuclear magnetic resonance and Raman spectroscopy indicated that the abiraterone-HPBCD complexes were formed. At elevated temperatures, the 10% and 20% drug-loaded KSDs were physically stable, while the 30% drug-loaded KSD showed recrystallization of abiraterone. In vitro dissolution and in vivo pharmacokinetic performances improved as the drug loading decreased; we attribute this to increased noncovalent interactions between abiraterone and HPBCD at lower drug loadings. Overall, the 10% drug loaded KSD showed a dissolution enhancement of 15.7-fold compared to crystalline abiraterone, and bioavailability enhancement of 3.9-fold compared to the commercial abiraterone acetate tablet Zytiga®. This study is first to confirm that KinetiSol, a high-energy, solvent-free technology, is capable of forming abiraterone-HPBCD complexes. Furthermore, in terms of in vitro and in vivo performance, a 10% drug load is optimal.

Bioconversion of antifungal viridin to phytotoxin viridiol by environmental non-viridin producing microorganisms.

Biotransformation of viridin, an antifungal produced by biocontrol agent, with non-viridin producing microorganisms is studied. The results show that some environmental non-targeted microorganisms are able to reduce it in the known phytotoxin viridiol, and its 3-epimer. Consequently, this reduction, which happens in some cases by detoxification mechanism, could be disastrous for the plant in a biocontrol of plant disease. However, a process fermentation/biotransformation could be an efficient approach for the preparation of this phytotoxin.

Arimistane: Degradation product or metabolite of 7-oxo-DHEA?

RATIONALE: The instability of androst-5-ene-3,7-dione structures under acidic conditions is known. The formation of arimistane from 7-oxo-DHEA, influenced by the conditions of sample extraction, and mainly derivatization reaction and gas chromatography (GC) injector temperature, was described earlier, potentially leading to misinterpretation of results. By using a liquid chromatography (LC)-mass spectrometry (MS) (LC-MS) we investigated the stability of the 7-oxo-DHEA in two different solvents (methanol and dimethyl sulfoxide [DMSO]), and the arimistane formation after the application common analytical procedures. Additionally, in vitro and in vivo studies of 7-oxo-DHEA were performed. METHODS: The stability of 7-oxo-DHEA was studied in solutions after 60 days storage at -20°C. In vitro studies were performed by incubating 7-oxo-DHEA with human liver microsomes (HLMs). Healthy volunteers collected urine samples before and after the administration of a single dose of 7-oxo-DHEA. Analyses were performed using high-performance LC (HPLC) coupled to a triple quadrupole mass spectrometer (MS/MS) and GC combustion isotope ratio mass spectrometry (GC-C-IRMS) following HPLC purification. RESULTS: 7-oxo-DHEA was stable after 60 days in DMSO while a protic solvent as methanol promotes the degradation of 7-oxo-DHEA to arimistane. HLM incubations showed no formation of arimistane and the sample preparation only influenced the degradation of 7-oxo-DHEA when solvolysis was applied. After the administration study the presence of arimistane also after the hydrolysis with ß-glucuronidase (Escherichia coli) was observed while using ß-glucuronidase/arylsulfatase (Helix pomatia) showed the presence of arimistane already in blank samples collected before administration. CONCLUSIONS: Our results confirm arimistane as a valuable diagnostic marker of 7-oxo-DHEA administration, but also indicate that its formation is due to degradation processes rather than to metabolic biotransformation reactions.

A quick UPLC-MS/MS method for therapeutic drug monitoring of abiraterone and delta(4)-abiraterone in human plasma.

Abiraterone acetate efficacy against prostate cancer is dependent on the circulating levels of abiraterone and its active metabolites, which present significant pharmacokinetic variability among patients. Thus, therapeutic drug monitoring can be performed to improve treatment outcomes. To support such studies, there are only a limited number of bioanalytical methods in current literature. This work presents a fast method to quantify abiraterone and D4A in plasma in 4 min by UPLC-MS/MS. Bioanalytical method validation was performed according to the recommendations of the US Food and Drug Administration. The method was linear within the range of 1-400 ng/ml for abiraterone and 0.2-20 ng/ml for D4A (r2 > 0.99). Based on the analysis of quality control samples at the lower limit of quantification, low, medium and high concentrations, the method was precise (CVabiraterone ≤ 9.72%; CVD4A ≤ 14.64%) and accurate (CVabiraterone 95.51-107.59%; CVD4A 98.04-99.89%). Application of the method to the quantification of abiraterone and D4A in 10 clinical samples revealed important variability in the conversion ratio of abiraterone to D4A (CV 90.85%). Considering the current literature, this is the fastest method to quantify abiraterone and D4A in plasma, allowing for optimization of the analytical routine.

Synthesis of some steroidal mitocans of nanomolar cytotoxicity acting by apoptosis.

Several steroids (abiraterone, prednisone, testosterone, cholesterol) and the BCL-2 inhibitor bexarotene were used as starting materials to synthesize iperazinyl-spacered rhodamine B conjugates. The conjugates were screened for their cytotoxicity in SRB assays against several human tumor cell lines and found to be active in a low µM to nM range. The conjugate derived from testosterone held an EC50 = 59 nM against MCF-7 tumor cells and acted mainly by necrosis. The prednisone conjugate, however, was less cytotoxic but acted mainly by apoptosis and held a moderate selectivity against MCF-7 tumor cells.

Interaction of an abiraterone with calf thymus DNA: Investigation with spectroscopic technique and modelling studies.

Binding of toxic ligands to DNA could result in undesirable biological processes, such as carcinogenesis or mutagenesis. Binding mode of Abiraterone (ABR), a steroid drug and calf thymus DNA (ctDNA) was investigated in this study using fluorescence and ultraviolet-visible spectroscopy. The probable prediction of binding and the type of interaction forces involved in the arrangement between ABR and ctDNA were explored through spectroscopic and molecular docking studies. The results indicated that ABR binds to the ctDNA in the minor groove. The binding constants were in the range of 1.35 × 106-0.36 × 106 L mol-1 at the studied temperatures. Fluorescence and spectrophotometric data suggested static quenching between ctDNA and ABR. The endothermic values of thermodynamic parameters ΔH°=-82.84 kJ mol-1; ΔS°=-161 J mol-1K-1 suggested that hydrogen bonding is the main force involved in binding of ABR with ctDNA. In experimental studies, the free binding energy at 298 K was -34.9 kJ mol-1 with the relative binding energy ≈ -29.65 kJ mol-1 of docked structure. The Ksv obtained for ABR-KI was similar to that for ABR- ctDNA -KI demonstrating no protection by ctDNA against quenching effect of KI. Thus, suggesting involvement of groove binding between ABR and ctDNA. No change in the fluorescence intensity of ABR-ctDNA was observed in presence of NaCl. Thus, ruling out the involvement of electrostatic interaction. These studies could serve as new insights in understanding the mechanisms of toxicity, resistance and side effects of ABR.

A photochemical approach to 18-nor-17ß-hydroxymethyl-17α-methylandrost-13-ene steroids.

A photochemical approach to 18-nor-17ß-hydroxymethyl-17α-methylandrost-13-ene unit of the long-term metabolites of 17-methylated androgenic anabolic steroids (AAS) is reported. It is based on a visible light-promoted radical decarboxylative alkynylation of steroidal redox-active ester. The developed method was used in synthesis of the long-term metabolite of AAS oxymesterone.

The ligand exchange of citrates to thioabiraterone on gold nanoparticles for prostate cancer therapy.

Cancer is one of the leading causes of morbidity and mortality worldwide and nanotechnology has a significant potential to enhance the therapeutic and diagnostic performance of anti-cancer agents. Our work offers a simple and feasible strategy for thiocompound nanomedicines to be used in cancer therapy. Novel gold nanoparticles conjugated with thioabiraterone (AuNP-S-AB) were synthesized and significant new analytical methodologies were developed for their characterization by UV-Vis, TEM, IR, NMR and TGA. Our synthetic approach was based on the ligand exchange of citrates to thioabiraterone on gold nanoparticles. The average particle size of AuNP-S-AB was 14.5 nm with a spherical shape. The identity of thioabiraterone on the gold nanoparticles was proved by NMR and IR spectroscopy. The coverage of the gold nanoparticles with 40.9% (m/m) thioabiraterone was calculated from a TGA analysis. Molecular interactions between the thiol group of thioabiraterone and gold nanoparticles were evaluated through a combined experimental and theoretical study using the density functional theory (DFT). Additionally, an experiment conducted on hepatocytes or human prostate epithelial cells proved that newly synthesized thiol forms of abiraterone, as well as AuNP-S-AB, are more biocompatible than abiraterone. Our proposed idea of delivering abiraterone with our newly designed AuNP-S-AB may constitute a promising and novel prospect in cancer therapy.

Late-stage oxidative C(sp3)-H methylation.

Frequently referred to as the 'magic methyl effect', the installation of methyl groups-especially adjacent (α) to heteroatoms-has been shown to dramatically increase the potency of biologically active molecules1-3. However, existing methylation methods show limited scope and have not been demonstrated in complex settings1. Here we report a regioselective and chemoselective oxidative C(sp3)-H methylation method that is compatible with late-stage functionalization of drug scaffolds and natural products. This combines a highly site-selective and chemoselective C-H hydroxylation with a mild, functional-group-tolerant methylation. Using a small-molecule manganese catalyst, Mn(CF3PDP), at low loading (at a substrate/catalyst ratio of 200) affords targeted C-H hydroxylation on heterocyclic cores, while preserving electron-neutral and electron-rich aryls. Fluorine- or Lewis-acid-assisted formation of reactive iminium or oxonium intermediates enables the use of a mildly nucleophilic organoaluminium methylating reagent that preserves other electrophilic functionalities on the substrate. We show this late-stage C(sp3)-H methylation on 41 substrates housing 16 different medicinally important cores that include electron-rich aryls, heterocycles, carbonyls and amines. Eighteen pharmacologically relevant molecules with competing sites-including drugs (for example, tedizolid) and natural products-are methylated site-selectively at the most electron rich, least sterically hindered position. We demonstrate the syntheses of two magic methyl substrates-an inverse agonist for the nuclear receptor RORc and an antagonist of the sphingosine-1-phosphate receptor-1-via late-stage methylation from the drug or its advanced precursor. We also show a remote methylation of the B-ring carbocycle of an abiraterone analogue. The ability to methylate such complex molecules at late stages will reduce synthetic effort and thereby expedite broader exploration of the magic methyl effect in pursuit of new small-molecule therapeutics and chemical probes.

New steroidal oxazolines, benzoxazoles and benzimidazoles related to abiraterone and galeterone.

Seven new oxazoline, benzoxazole and benzimidazole derivatives were synthesized from 3ß-acetoxyandrosta-5,16-dien-17-carboxylic, 3ß-acetoxyandrost-5-en-17ß-carboxylic and 3ß-acetoxypregn-5-en-21-oic acids. Docking to active site of human 17α-hydroxylase/17,20-lyase revealed that all oxazolines, as well as benzoxazoles and benzimidazoles comprising Δ16 could form stable complexes with enzyme, in which steroid moiety is positioned similarly to that of abiraterone and galeterone, and nitrogen atom coordinates heme iron, while 16,17-saturated benzoxazoles and benzimidazoles could only bind in a position where heterocycle is located nearly parallel to heme plane. Modeling of the interaction of new benzoxazole and benzimidazole derivatives with androgen receptor revealed the destabilization of helix 12, constituting activation function 2 (AF2) site, by mentioned compounds, similar to one induced by known antagonist galeterone. The synthesized compounds inhibited growth of prostate carcinoma LNCaP and PC-3 cells at 96 h incubation; the potency of 2'-(3ß-hydroxyandrosta-5,16-dien-17-yl)-4',5'-dihydro-1',3'-oxazole and 2'-(3ß-hydroxyandrosta-5,16-dien-17-yl)-benzimidazole was superior and could inspire further investigations of these compounds as potential anti-cancer agents.

Estimation of the inhibiting impact of abiraterone D4A metabolite on human steroid 21-monooxygenase (CYP21A2).

Abiraterone D4A metabolite, the product of 3ß-hydroxysteroid dehydrogenase activity toward abiraterone, may serve as a potential antitumor agent for the treatment of prostate cancer. The main adverse effect of abiraterone is the disruption of corticosteroid biosynthesis, and the more pharmacologically active abiraterone D4A metabolite may have the same issues. We therefore estimated the inhibiting impact of the abiraterone D4A metabolite on one of the key corticosteroidogenic enzymes - human steroid 21-monooxygenase (CYP21A2). Molecular docking of D4A into the active site of CYP21A2 has been predicted to be similar to abiraterone binding with the enzyme. Abiraterone D4A metabolite, similar to abiraterone, induces type II spectral changes of CYP21A2. The spectral dissociation constant for the abiraterone D4A metabolite-CYP21A2 complex was calculated as 3.4 ±â€¯0.5 µM. Abiraterone D4A metabolite demonstrates competitive/mixed type CYP21A2 inhibition with an inhibitory constant of 1.8 ±â€¯0.8 µM, as obtained by Dixon plot. These results make it possible to predict the adverse effects of the new perspective candidate compound for antitumor therapy.

Design, synthesis, and biological evaluation of novel androst-17ß-amide structurally related compounds as dual 5α-reductase inhibitors and androgen receptor antagonists.

Prostate cancer (PCa) is the second leading cause of death in men. Apart from androgen receptor, 5α-reductase has also been recognized as a potential target. In this study, a series of androst-17ß-amide compounds have been designed and synthesized targeting both AR and 5α-reductase. Their anti-proliferation activities were evaluated in AR + cell line 22RV1 and AR - cell line PC-3. The results indicated that most of the synthesized compounds inhibited the testosterone-stimulated cell proliferation with good selectivity and safety. Among all the compounds, androst[3,2-c]pyrazole derivatives (9a-9d) displayed the best inhibition activity comparable with flutamide. Moreover, most of the synthesized compounds displayed good 5α-reductase inhibitory activities with IC50 lower than 1 µM. The docking result of 9d-AR indicated that AR was forced to expands its binding cavity and maintain an antagonistic conformation since the steric hindrance of 9d impeded H12 transposition. Overall, compound 9d can be identified as a potential dual 5α-reductase inhibitor and AR antagonist, which might be of therapeutic importance for PCa treatment.

Conformational selection dominates binding of steroids to human cytochrome P450 17A1.

Cytochrome P450 (P450, CYP) enzymes are the major catalysts involved in the oxidation of steroids as well as many other compounds. Their versatility has been explained in part by flexibility of the proteins and complexity of the binding mechanisms. However, whether these proteins bind their substrates via induced fit or conformational selection is not understood. P450 17A1 has a major role in steroidogenesis, catalyzing the two-step oxidations of progesterone and pregnenolone to androstenedione and dehydroepiandrosterone, respectively, via 17α-hydroxy (OH) intermediates. We examined the interaction of P450 17A1 with its steroid substrates by analyzing progress curves (UV-visible spectroscopy), revealing that the rates of binding of any of these substrates decreased with increasing substrate concentration, a hallmark of conformational selection. Further, when the concentration of 17α-OH pregnenolone was held constant and the P450 concentration increased, the binding rate increased, and such opposite patterns are also diagnostic of conformational selection. Kinetic simulation modeling was also more consistent with conformational selection than with an induced-fit mechanism. Cytochrome b5 partially enhances P450 17A1 lyase activity by altering the P450 17A1 conformation but did not measurably alter the binding of 17α-OH pregnenolone or 17α-OH progesterone, as judged by the apparent Kd and binding kinetics. The P450 17A1 inhibitor abiraterone also bound to P450 17A1 in a multistep manner, and modeling indicated that the selective inhibition of the two P450 17A1 steps by the drug orteronel can be rationalized only by a multiple-conformation model. In conclusion, P450 17A1 binds its steroid substrates via conformational selection.

The synthesis of 16-androstene sulfoconjugates from primary porcine Leydig cell culture.

Increased public interest in the welfare of pigs reared for pork production has led to an enhanced effort in finding alternatives to castration for controlling the unpleasant odour and flavour from heated pork products known as boar taint. The purpose of this study was to investigate the testicular metabolism of androstenone, one of the major components of boar taint. Leydig cells were isolated from mature boars and incubated with radiolabeled androstenone for 10 min, 1 h, 4 h, 8 h, and 12 h. Steroid profiles were analyzed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS/MS). Sulfoconjugated, but not glucuronidated steroids were produced by Leydig cells. Approximately 85% of androstenone was converted into sulfoconjugated metabolites in Leydig cell incubations after 8 h. This sulfoconjugate fraction included androstenol-3-sulfate and two major sulfated forms of androstenone. Following removal of the sulfate group, these two sulfated forms of androstenone returned the parent compound androstenone, and not a hydroxylated metabolite. These findings provided direct evidence for the testicular production of sulfoconjugated forms of androstenone and androstenol in the boar. The high proportion of sulfoconjugates produced by the Leydig cells emphasizes the importance of steroid conjugation, which serves to regulate the amount of unconjugated steroid hormones available for accumulation in adipose tissue.

11-Oxygenated C19 Steroids Do Not Decline With Age in Women.

CONTEXT: The ovaries and adrenals are sources of androgens in women. Although dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), and testosterone (T) all decline with age, these C19 steroids correlate poorly with parameters of androgen action in postmenopausal women. OBJECTIVE: To comprehensively compare the androgen profiles of pre- and postmenopausal women. METHODS: We quantified 19 steroids-including DHEA; DHEAS; T; androstenedione (A4); and the following adrenal-specific 11-oxygenated C19 steroids (11oxyandrogens): 11ß-hydroxytestosterone (11OHT), 11-ketotestosterone (11KT), 11ß-hydroxyandrostenedione (11OHA4), and 11-ketoandrostenedione (11KA4)-using liquid chromatography-tandem mass spectrometry in morning serum obtained from 100 premenopausal (age 20 to 40 years) and 100 postmenopausal (age ≥ 60 years) women. Double immunofluorescence of 3ß-hydroxysteroid dehydrogenase type 2 (HSD3B2) with cytochrome b5 (CYB5A) or sulfotransferase 2A1 (SULT2A1) was performed in normal adrenal glands obtained from eight premenopausal and eight postmenopausal women. RESULTS: DHEA, DHEAS, A4, and T were significantly higher in pre- than in postmenopausal women (2.9, 2.8, 2.9, and 1.6-fold, respectively; P < 0.0001). In contrast, the 11-oxyandrogens did not decrease with aging, and the 11OHT/T and 11OHA4/A4 ratios showed strong positive correlations with age (r = 0.5 and 0.8, respectively; P < 0.0001). Double immunofluorescence analysis showed that with the involution of the zona reticularis in the old adrenals, the sharp zonal segregation of HSD3B2 and CYB5A becomes less distinct, and areas of HSD3B2 and CYB5A overlap are observed. CONCLUSIONS: Unlike DHEA, DHEAS, A4, and T, the 11oxyandrogens do not decline in aging women. Structural changes within the adrenal cortex might explain the evolution of androgen profiles in aging women.

Induction of endoplasmic reticulum stress by aminosteroid derivative RM-581 leads to tumor regression in PANC-1 xenograft model.

The high fatality and morbidity of pancreatic cancer have remained almost unchanged over the last decades and new clinical therapeutic tools are urgently needed. We determined the cytotoxic activity of aminosteroid derivatives RM-133 (androstane) and RM-581 (estrane) in three human pancreatic cancer cell lines (BxPC3, Hs766T and PANC-1). In PANC-1, a similar level of antiproliferative activity was observed for RM-581 and RM-133 (IC50 = 3.9 and 4.3 µM, respectively), but RM-581 provided a higher selectivity index (SI = 12.8) for cancer cells over normal pancreatic cells than RM-133 (SI = 2.8). We also confirmed that RM-581 induces the same ER stress-apoptosis markers (BIP, CHOP and HERP) than RM-133 in PANC-1 cells, pointing out to a similar mechanism of action. Finally, these relevant in vitro results have been successfully translated in vivo by testing RM-581 using different doses (10-60 mg/kg/day) and modes of administration in PANC-1 xenograft models, which have led to tumor regression without any sign of toxicity in mice (animal weight, behavior and histology). Interestingly, RM-581 fully reduced the pancreatic tumor growth when administered orally in mice.

An LC-MS/MS method for quantification of abiraterone, its active metabolites D(4)-abiraterone (D4A) and 5α-abiraterone, and their inactive glucuronide derivatives.

Abiraterone acetate (AA) is a prodrug of abiraterone, a selective and potent steroidal cytochrome P450 17alpha- hydroxylase-17,20-lyase (CYP17A1) blocking androgen synthesis in the treatment of advanced prostate cancer. Abiraterone (Abi) is metabolized to D(4)-abiraterone (D4A) directly blocking CYP17A1 and other steroidogenic enzymes and antagonizing the androgen receptor (AR). D4A is converted by 5α-reductase to 3-keto-5α-abiraterone (5α-Abi), an AR agonist. Our recent work suggests phase II biotransformation of Abi, D4A and 5α-Abi conjugated to glucuronic acid in vitro leading to four glucuronides (G). We developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using a 6500 Qtrap mass analyzer coupled with a Shimadzu Nexera system for quantification of Abi, its active metabolites and their G derivatives in human plasma samples with deuterated internal standards. Validation was carried out according to FDA guidelines for bioanalytical method and results were within the acceptance limits. Analytes were extracted from 50 µL of plasma using a solid phase extraction procedure. Multiple reaction monitoring was used with electrospray ionization in a positive mode. Linearity, precision, and accuracy were validated over a large range of concentrations for each compound (range of 0.5-100 ng/mL for Abi and for metabolites and 0.05-10.00 ng/mL for glucuronides). The method could measure all seven analytes with sensitivity, accuracy (87-106%), and reproducibility (CV < 10.7%). Its clinical application was further examined with plasma samples obtained from prostate cancer patients under AA treatment. This reliable and validated LC-MS/MS method could be a useful tool for human biomonitoring studies.

Development of novel PLGA nanoparticles with co-encapsulation of docetaxel and abiraterone acetate for a highly efficient delivery into tumor cells.

Co-encapsulation of abiraterone acetate (AbrA) and docetaxel (Dtx) in polymeric nanoparticles as novel prototypes for prostate cancer treatment combining hormonal and chemotherapy was designed. Nanoparticles (NPs) composed of poly(dl-lactide-co-glycolide) (PLGA) were prepared by single-emulsion solvent evaporation technique and characterized in terms of morphology with atomic force microscopy and transmission electron microscopy. HPLC method for simultaneous determination of AbrA and Dtx encapsulation efficacy was developed. Also differential scanning calorimetry and Fourier-transform infrared spectroscopy were provided. To study the effectiveness of cellular internalization and distribution of NPs with AbrA and Dtx co-encapsulation (NP-AbrA/Dtx), a fluorescence microscopy was utilized. NPs prepared had size 256.3 ±9.4 nm and zeta potential -18.4 ±1.4 mV. Encapsulation efficacy for AbrA was 68.7% and for Dtx was 74.3%. NPs were able to control the AbrA and Dtx release within 24 h. The mathematical model of drug release was performed. The results obtained from confocal microscopy showed the effective accumulation of the NP-AbrA/Dtx in the cytoplasm of cells. Synthesized NPs possessed satisfactory parameters and a biphasic release profile, proceeding by the Fick diffusion mechanism, which provide prolonged release of the drugs and maintenance of their concentration. It was shown that NPs loaded with AbrA and Dtx exhibited a high cytotoxic activity on the LNCaP cell line, similar to the combination of free drugs of AbrA and Dtx, but in contrast to the combination of substances, had a synergistic mechanism of action. Our findings support the potential use of developed NPs in further in vivo studies. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1150-1158, 2019.