Pharmacokinetics Evaluation and Degradation Products Profiling of Therapeutic Peptide P3V8 in Rats
Refereed conference paper presented and published in conference proceedings


Full Text

Other information
AbstractPurpose
Although P3V8 (amino acids sequence: Ac-DGKSKGIAYIEFK-NH2) is identified as a therapeutic peptide targeting for the polyglutamine neurodegenerative diseases, its pharmacokinetics profile is not known yet. The current study aims to evaluate the pharmacokinetics profile of P3V8 as well as identify its potential degradation products in rat plasma and brain.
Methods
For pharmacokinetics study, after intravenous administration of 2.5 mg/kg P3V8 in rats (n=3), plasma samples were collected at 2, 5, 8, 10, 15, 20, 30, 45, 60, and 90 min post-dosing and brain samples were also collected at 90 min post-dosing after sacrificing the rats. In order to further identify of its potential degradation products profiling, P3V8 at 2 μg/mL and 20 μg/mL were incubated in rat plasma and rat brain homogenate at 37 °C for 60 min in presence and absence of proteases inhibitors. All the collected biological samples were extracted by protein precipitation using two volumes of acetonitrile and subjected to LC/MS/MS analysis performed on a triple quadruple mass spectrometer equipped with electrospray ionization source. Multiple reaction monitoring (with m/z 499.6 → 129.1 in positive ion mode) was used to quantify P3V8 and selected ion monitoring was used to identify the potential degradation products.
Results
After intravenous administration of P3V8 at 2.5 mg/kg in rats, P3V8 was quickly eliminated in plasma in 8 min and not even detectable in brain 90 min post-dosing. The area under the plasma concentration vs time curve (AUC0-90min) was 830.77 ± 193.27 ng×min/mL. To investigate the reasons for such quick elimination, degradation profiles of P3V8 in plasma and brain were further studied. The in vitro stability test showed that under physiological condition (37 °C, pH = 7.4), P3V8 was stable in buffer solution, but not stable in plasma and brain. After 60-min incubation at the concentration of 2 μg/mL, only 20.7 % and 3.3 % of P3V8 remained in rat plasma and in brain homogenate, respectively. Subsequently, three hydrolysis products of P3V8 (between lysine and glycine; between tyrosine and isoleucine) were identified in plasma and one hydrolysis product (between lysine and glycine) was identified in brain homogenate. Degradation of P3V8 and formation of its hydrolysis products in both plasma and brain homogenate were significantly inhibited by serine proteases inhibitor (PMSF, 2 mM) and metalloproteases inhibitor (EDTA, 5 mM), indicating potential involvement of both serine proteases and metalloproteases in the degradation of P3V8.
Conclusion
The pharmacokinetic study demonstrated the quick elimination of P3V8 in plasma and brain, which was further identified to be due to its extensive degradation mediated by serine proteases and metalloproteases. Based on the structures of four degradation products, two cleavage sites on P3V8 were identified. Chemical modification on the structure of the cleavage sites on P3V8 may improve its stability in plasma and brain, in order to optimize its pharmacokinetic properties for its further drug development.
Acceptance Date11/06/2016
All Author(s) ListMengbi Yang, Qianwen Wang, Qian Zhang, Ho Yin Edwin Chan, Zhong Zuo
Name of Conference2016 American Association of Pharmaceutical Scientists (AAPS) Annual Meeting and Exposition
Start Date of Conference13/11/2016
End Date of Conference17/11/2016
Place of ConferenceDenver, Colorado
Country/Region of ConferenceUnited States of America
Year2016
LanguagesEnglish-United States

Last updated on 2018-11-05 at 11:38