Celastrol (CL), a bioactive compound isolated from Tripterygium wilfordii, has demonstrated bioactivities against a variety of diseases including cancer and obesity. However, its poor water solubility and rapid in vivo clearance limit its clinical applications. To overcome these limitations, nanotechnology has been employed to improve its pharmacokinetic properties. Nanoparticles made of biological materials offer minimal adverse effects while maintaining the efficacy of encapsulated therapeutics. Silk fibroin (SF) solution was prepared successfully by extraction from the cocoons of silkworms, and a final concentration of 2 mg/mL SF solution was used for the preparation of CL-loaded SF nanoparticles (CL-SFNP) by the desolvation method. A stirring speed of 750 rpm and storage time of 20 h at −20 °C resulted in optimized product yield. A high-performance liquid chromatography (HPLC) method was developed and validated for the analysis of CL in rat plasma in terms of selectivity, linearity, intra-/inter-day precision and accuracy, and recovery. No interference was observed in rat plasma. Linearity in the concentration range of 0.05–5 µg/mL was observed with R2 of 0.999. Precision and accuracy values were below the limit of acceptance criteria, i.e., 15% for quality control (QC) samples and 20% for lower limit of quantification (LLOQ) samples. Rats were given intravenous (IV) administration of 1 mg/kg of pure CL in PEG 300 solution or CL-SFNP. The pharmacokinetic profile was improved with CL-SFNP compared to pure CL. Pure CL resulted in a maximum concentration (Cmax) value of 0.17 µg mL−1 at 5 min following administration, whereas that for CL-SFNP was 0.87 µg mL−1 and the extrapolated initial concentrations (C0) were 0.25 and 1.09 µg mL−1, respectively, for pure CL and CL-SFNP. A 2.4-fold increase in total area under the curve (AUC0-inf) (µg h mL−1) was observed with CL-SFNP when compared with pure CL. CL-SFNP demonstrated longer mean residence time (MRT; 0.67 h) than pure CL (0.26 h). In conclusion, the preparation of CL-SFNP was optimized and the formulation demonstrated improved pharmacokinetic properties compared to CL in solution following IV administration.

Celastrol (CL), a compound isolated from Tripterygium wilfordii, possesses various bioactivities such as antitumor, anti-inflammatory and anti-obesity effects. In previous studies, we developed CL-encapsulated silk fibroin nanoparticles (CL-SFNP) with satisfactory formulation properties and in vitro cancer cytotoxicity effect. For further in vivo oral bioavailability evaluation, in this study, a simple and reliable LC-MS/MS method was optimized and validated to determine CL concentration in rat plasma. The separation of CL was performed on a C18 column (150 by 2 mm, 5 µm) following sample preparation using liquid–liquid extraction with the optimized extraction solvent of tert-butyl methylether. The assay exhibited a good linearity in the concentration range of 0.5–500 ng/mL with the lower limit of quantification (LLOQ) of 0.5 ng/mL. The method was validated to meet the requirements for bioassay with accuracy of 91.1–110.0%, precision (RSD%) less than 9.1%, extraction recovery of 63.5–74.7% and matrix effect of 87.3–101.2%. The developed method was successfully applied to the oral bioavailability evaluation of CL-SFNP. The pharmacokinetic results indicated the AUC0-∞ values of CL were both significantly (p < 0.05) higher than those for pure CL after intravenous (IV) or oral (PO) administration of equivalent CL in rats. The oral absolute bioavailability (F, %) of CL significantly (p < 0.05) increased from 3.14% for pure CL to 7.56% for CL-SFNP after dosage normalization. This study provides valuable information for future CL product development.

Celastrol (CL), a compound isolated from Tripterygium wilfordii, possesses various bioactivities such as anti-tumor, anti-inflammatory and anti-obesity effects. Many new drug delivery systems of CL have been designed to overcome its poor water solubility and low bioavailability. However, few studies are available concerning its in vivo pharmacokinetics. In this study, a simple and sensitive LC-MS/MS method was developed and validated to determine CL in rat plasma using 18α-glycyrrhetinic acid as the internal standard. The separation of CL was performed on a Phenomenex Gemini C18 column (150 mm × 2 mm, 5 µm) following sample preparation using liquid-liquid extraction with the optimized extraction solvent of tert-butyl methyl ether and a small plasma sample volume of 50 μL. The analyte was quantitated under positive electrospray ionization and multiple reactions monitoring mode. The assay exhibited a good linearity in the concentration range of 0.5-500 ng/mL. The lower limit of quantification (LLOQ) was 0.5 ng/mL. The method was fully validated to meet the requirements for bioassay with accuracy of 91.1-110.0%, precision (RSD %) of less than 9.1 %, extraction recovery of 63.5-74.7%, and matrix effect of 87.3-101.2%. The developed method was successfully applied to the pharmacokinetic evaluation of CL-encapsulated silk fibroin nanoparticles (CL-SFNP). The results show, after intravenous (IV) and oral (PO) administration of equivalent CL (1 mg/kg and 3 mg/kg, respectively) in rats, the AUC values of CL were 8646.1±1998.9 ng/mL·h after IV dosing and 1065.5±494.6 ng/mL·h after PO dosing for CL-SFNP, which were significantly (P&lt;0.05) higher than those for pure CL (4697.7±723.0 ng/mL·h and 441.9±82.6 ng/mL·h, respectively). The dose-normalized oral bioavailability of CL significantly (P&lt;0.05) increased from 3.1% for pure CL to 7.6% for CL-SFNP. Citation Format: Shuyu Zhan, Amy Paik, Felicia Onyeabor, Baoyue Ding, Sunil Prabhu, Jeffrey Wang. Development and validation of an LC/MS/MS method for the determination of celastrol in rat plasma and its application to the pharmacokinetic evaluation of celastrol-encapsulated silk fibroin nanoparticles [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6252.

Background Oral vardenafil (VDF) tablet is an effective treatment for erectile dysfunction (ED), but intranasal administration with a suitable formulation can lead to a faster onset of action and offer more convenient planning for ED treatment. Aim The primary purpose of the present pilot clinical study was to determine whether intranasal VDF with an alcohol-based formulation can result in more “user-friendly pharmacokinetics” as compared with oral tablet administration. Methods This single-dose randomized crossover study was conducted in 12 healthy young volunteers receiving VDF as a 10-mg oral tablet or 3.38-mg intranasal spray. Multiple blood concentrations were obtained, and VDF concentrations were determined with a liquid chromatography–tandem mass spectrometry assay. Pharmacokinetic parameters following each treatment were compared and adverse events assessed. Outcomes Pharmacokinetic parameters were obtained: apparent elimination rate constant, elimination half-life, peak concentration, peak time, total area under the curve, and relative bioavailability. Results Although mean apparent elimination rate constant, elimination half-life, peak concentration, and total area under the curve were similar between intranasal and oral administration, the median peak time from intranasal was much shorter (10 vs 58 minutes, P &lt; .001, Mann-Whitney U test). The variability of the pharmacokinetic parameters was also less with intranasal than oral administration. The relative bioavailability of intranasal to oral was 1.67. Intranasal VDF caused transient but tolerable local nasal reactions in 50% of subjects. Other adverse events (eg, headache) were similar between the treatments. The incidence of adverse events was, however, significantly less in the second treatment after initial exposure to VDF. No serious adverse events were noted. Clinical Implications Intranasal VDF potentially offers a more timely and lower dose for the treatment of ED in patients who can tolerate the transient local adverse reactions. Strengths and Limitations The strength of this study is its randomized crossover design. Because the study was conducted in 12 healthy young subjects, the results may not reflect those observed in elderly patients who may be likely taking VDF for ED. Nevertheless, the changes of pharmacokinetic parameters in the present study are likely a reflection of the differences between intranasal and oral administration of the formulations. Conclusion Our study indicated that the present VDF formulation, when administered intranasally, can achieve a more rapid but similar plasma concentration with only about one-third dose when compared with the oral administration.