Fully automated chip-based nanoelectrospray ionization-mass spectrometry as an effective tool for rapid and high-throughput screening of 5α-reductase inhibitors.

The 5α-reductase converts testosterone to dihydrotestosterone (DHT), and excess DHT could cause androgen-related diseases such as androgenetic alopecia and benign prostatic hyperplasia (BPH). To discover new 5α-reductase inhibitors, effective drug screening method with high throughput is thus essential. In this study, fully automated chip-based nanoelectrospray ionization-mass spectrometry (nano-ESI-MS) was innovatively utilized as a screening tool for 5α-reductase inhibitory assay in direct infusion mode, which simplified sample pretreatment and greatly improved experimental efficiency. The preliminary data indicated that curcumin, a natural anti-inflammatory compound, exhibited notably 5α-reductase inhibition activity. Moreover, the obtained results of the chip-based nano-ESI-MS were well consistent with those of HPLC-MS, which suggested that the chip-based nano-ESI-MS could be treated as a rapid and high-throughput drugs screening strategy in pharmaceutical development. Graphical abstract.

Quality evaluation of the Finasteride polymorphic forms I and II in capsules.

Finasteride (FNS) is a specific competitive inhibitor of steroid type-II 5α-reductase and is widely used for the treatment of benign prostatic hyperplasia, prostate cancer, and androgenetic alopecia. FNS has two polymorphic forms identified as Form I and Form II. It is known that polymorphism can cause significant differences in the physicochemical properties of a compound such as melting point, density, morphology, solubility, and color. Thus, proper qualitative and quantitative monitoring of the solid-state forms is crucial to ensure high-quality products. There are no published papers studying the influence of the FNS polymorphs on the physicochemical quality of capsules. Furthermore, the available analytical methods are time-consuming, expensive, use buffer or do not demonstrate stability-indicating capacity. The aim of this work was to validate a rapid high-performance liquid chromatography (HPLC) method to evaluate FNS in capsules and to study the physicochemical properties of polymorphic forms, evaluating their possible influence in the dissolution profile and stability of FNS in capsules. Capsules containing Forms I and II of FNS were prepared and subjected to quality control studies, dissolution profiles and a stability study at 50°C. A significant effect of polymorphism on the FNS solubility and dissolution properties was observed. These results suggest that changes in the effects of FNS can occur if a suitable control study is not performed on the raw material used to produce the capsules.

[Establishment of an in vitro screening model for steroid 5 alpha-reductase inhibitors with the microplate reader].

OBJECTIVE: To establish an in vitro screening model for steroid 5 alpha-reductase inhibitors using the microplate reader. METHODS: Steroid 5 alpha-reductase was obtained from the liver of female rats, an in vitro screening model for steroid 5 alpha-reductase inhibitors established using the 96-well plate and microplate reader after determination of the enzymatic activity, and the reliability of the model verified with the known 5 alpha-reductase inhibitors epristeride and finasteride. Added to the 96-well plate were the final concentrations of testosterone (0-40 micromol/L), NADPH (22 micromol/L), epristeride (0-60 nmol/L) or finasteride (0-60 nmol/ L) and steroid 5 alpha-reductase (20 microl), the total volume of each well adjusted to 200 microl with Tris-Hcl buffer. The 96-well plate was placed in the microplate reader, mixed and incubated at 37 degrees C, followed by detection of the A340nm value at 0 and 10 min and analysis of the data. RESULTS: The Km value of steroid 5 alpha-reductase was 3.794 micromol/L, with a Vmax of 0.271 micromol/(L. min). The Ki of epristeride was 148.2 nmol/L, with an IC50 of 31.5 nmol/L, and the enzymatic reaction kinetic curve suggested that epristeride was an uncompetitive enzyme inhibitor. The Ki of finasteride was 158. 8 nmol/L, with an IC50 of 13.6 nmol/L. The enzymatic reaction kinetic curve showed that both epristeride and finasteride were competitive enzyme inhibitors, similar to those reported in the published literature. CONCLUSION: A screening model was successfully established, which could rapidly and effectively screen steroid 5 alpha-reductase inhibitors in vitro.