Purpose: Transient receptor potential vanilloid 1 (TrpV1) has emerged as a promising therapeutic target for neuropathic pain. Resiniferatoxin (RTX) is a daphnane diterpene isolated from the latex of Euphorbia resinifera, is a potent activator of TrpV1, with a potency 103-105 times greater than pure capsaicin. It increases the conductance of cations especially Ca+2 and TrpV1 of sensory nerves which leads to desensitization and analgesia. Intravenous administration of RTX at very low concentration improves urodynamic parameters in patients with neurogenic detrusor overactivity and reduces bladder pain in patients. Herein, a sensitive method for determination of RTX using acetonitrile to prevent RTX destruction has been developed and validated for quantification of RTX in rat plasma.
Methods: A simple, rapid, selective and sensitive method was developed to quantify RTX in vivo pharmacokinetic studies on Sprague Dawley rats (2.5 µg/kg dose; i.v.). Blood samples (200 µl) were collected at a pre-determined time interval and plasma was separated. The drug was extracted from plasma using protein precipitation method using acetonitrile and was analyzed using LC-MS/MS. The Multiple reactions monitoring (MRM) analysis was performed with a Waters Xevo TQ-S and optimized to detect the lowest levels of RTX. The mass spectrometer was operated in positive electrospray ionization ((+) ESI) mode. MassLynx software (Waters Corporation, USA) was used for instrument control and data analysis.
Results: A simple sample preparation using acetonitrile was applied to avoid RTX destruction and to achieve 87% recovery from both matrices spiked with RTX and silydianin as an internal standard. This rapid, sensitive, simple and selective assay enabled the quantification of RTX within the calibration range of 1-50 ng/mL. The lower limit of quantification of RTX was achieved at 0.5 ng/mL in plasma. The precision, accuracy, and recovery values were found to be <15% (<20 % at LLOQ), 100 ± 15%(<20 % at LLOQ), and 87.7–94 %, respectively. This method was successfully applied to measure the concentrations of RTX in plasma samples following the intravenous administration in rats. Pharmacokinetic results in rats have demonstrated AUC0-∞ of 990.6±133.75 h*ng/ml from RTX drug. The controlled release of RTX from niosomes was sustained, hence the clearance was reduced by 0.61 times (ClRTX-2.56±0.35 ml/h/kg) and plasma half-life was enhanced by 2.65-fold (t1/2 RTX-2.56±0.35 ml/h/kg) as compared to RTX respectively, and AUC 0-∞ was increased by 1.97-fold when compared to RTX drug.
Conclusion: This method was successfully applied to in vivo rat pharmacokinetics after intravenous administration with 2.5 µg/kg dose of RTX. This ultra-sensitive method promises the quantification of the ultra-low concentration of RTX release from nanocarrier like niosomes, allowing determination of the kinetic profile in plasma, which is an essential parameter to validate the use of niosomes in drug delivery for RTX.
Apoorva Panda– PhD Candidate, University of Mississippi, University
Bharathi Avula– Senior Research Scientist, National Center for Natural Products Research, University of Mississippi, Oxford, MS
Yan-Hong Wang– University of Mississippi
Ikhlas Khan– Director of the National Center for Natural Products Research, Research Professor - Research Institute of Pharmaceutical Sciences, Professor of Pharmacognosy, Program Director of FDA Programs, University of Mississippi
Narasimha Murthy– Professor-Pharmaceutics and Drug Delivery, University of Mississippi, University, Mississippi