Pharmacokinetics Of Phosphocreatine And Its Metabolite Creatine And Related Atp In Mice By Ion-pair HPLC-UV Assay

Objective: (1) To develop an ion-par HPLC-UV method for simultaneous determination of exogenous phosphocreatine (PCr) and its metabolite creatine (Cr) and Pcr-related ATP in plasma and heart tissue in mice. In order to provide a bio-analytical methodology for study of pharmacokinetics and metabolic disposition of exogenous PCr in mice. (2) To research the characteristics of pharmacokinetics and disposition in mice after iv PCr. (3) To research the characteristics of pharmacokinetics and metabolic disposition in mice after iv Cr.Methods: The PCr, Cr and ATP was chromatographically separated on a Kromasil C18 column using a tertiary gradient mobile phase composed of 0.2% KH₂PO₄ + 0.08% tetrabutylammonium hydrogen sulphate (pH3.0) (A) , A adjusted to pH 7.5 with 1N NaOH (B) and MeOH (C) coupled with a detection wavelength of 210 nm for PCr and Cr and 260nm for ATP. A blank plasma and heart tissue samples was initially run for baseline subtraction. For quantification TMP was used as internal standard. Plasma was deproteinized with 6% PCA, followed by neutralization to pH7.0 with K2CO3 prior to HPLC; heart tissue was deproteinized with 0.4M PCA, followed by neutralization to pH7.5 with K2CO3 prior to HPLC. The peak area rations of analytes to TMP as internal standard vs concentration of analytes to construct calibration curves. Use the method of baseline subtraction to calculate the exogenous PCr, Cr, and ATP. Mice were randomly divided into groups receiveing iv administration via caudal vein of PCr and Cr respectively. Plasma and heart tissue were deproteinized with 6% and 0.4M PCA respectively followed by neutrallyzation for IP-HPLC to determinate the concentration of PCr, Cr, and ATP. The parameter of pharmacokinetics and bioavailability were calculated.Results: The method validation including specificity, linearity, precision, accuracy, recovery and stability was conducted by routine procedures as described in guiding principles of PK study issued by SFDA. It was shown to be specific without interference from matrix, a good linearity was obtained over a wide range of 1-7500μg/ml (plasma) and 7.5-750 ug/ml (heart tissue) for PCr, and 1-1500 ug/ml (plasma) and 7.5-750 ug/ml (heart tissue) for both Cr and ATP (r2>0.99) . The QC samples of 3 analytes showed intra-day and inter-day precisions (RSD) of≤7.42% and≤8.45% (plasma) and≤9.07% and≤9.20% (heart tissue), accuracy of 95.96～101.89% (plasma) and 94.27～102.99% (heart tissue), and in the plasma and heart tissue the extraction recovery of > 90%. The PK behavior of PCr in the plasma after iv PCr could be described by two-compartment model , with t1/2β22.95～25.35 min, Vd 0.96～0.98 L/kg and CL 0.028 L/(kg·min). After iv PCr, in the plasma Cr appered immediately. it could be described by two-compartment model, with t1/2β51.6～53.8 min. Through iv PCr and Cr, the convertion percent from PCr to Cr is f(m) 72.21% caculated with the AUC of Cr. After iv PCr, ATP was not detected in the plasma, but it could be detected in the heart tissue. After iv PCr it did not apper PCr in the heart tissue, but its metabolite Cr and related ATP could be detected. tmax60min, t1/2β347.12³57.62min was for Cr, and tmax 90min, t1/2β685.46⁷00.54min was for ATP in the heart tissue the. After iv Cr 456mg/kg, there is no more PCr and ATP in the plasma, the Cr in the plasma could be described by two-compartment model, with t1/2β,Vd and CL as the same as the condition iv PCr. After iv Cr 456mg/kg, there is no more PCr in the heart tissue, but it could survy Cr and ATP in the heart tissue ,with t1/2β,Vd and CL as the same as the condition iv PCr. After iv Pit 5 min and iv PCr, the PK behavior of PCr in the plasma could be described by two-compartment model , with t1/2β35.22～38.12 min, Vd 0.95～0.97 L/kg and CL 0.018 L/(kg·min), it not apper ATP ,but it apper Cr immediately, it could be described by one-compartment model and first-order kinetics, with tmax 10 min, t1/2k(m)78.9⁸5.7 min, it is longer than PCr. After iv Pit 5 min and iv PCr, in the heart tissue, it not apper PCr, but could find Cr and ATP, Cr :t1/2 293.19²97.47min, tmax 30min; ATP: t1/2k 501.92⁵11.86min, tmax 90min.Conclusion: The method was successfully used to simultaneously determine plasma and heart muscle concentrations of the 3 analytes after iv administration of PCr in mice and yielded a typical biexponential decay C-T curve for PCr and an extra-vascular absorption–like curve for both Cr and ATP. The PK behavior of PCr in the plasma after iv PCr could be described by two-compartment model. After iv administration of PCr, the rapid appearance of the hydrolytic metabolite Cr was evident, the conversion percent is 72.21%. Mean concentrations of creatine in the cardiac muscle were increased significantly after intravenous administration of PCr. ATP concentrations in cardiac muscle of phosphocreatine-treated mice were also increased significantly after 90min compared with controls. PCr was not detected in the cardiac muscle. We concluded that the elevation of ATP and creatine levels in cardiac muscle may be involved in the protective effect provided by exogenous phosphocreatine. The results indicated this method meet the requirement of PCr pharmacokinetics study and metabolic disposition of mice. The conversion percent of ATP in the cardiac muscle after intravenous administration of PCr and Cr is 40.01%.We concluded the ATP concentrations of cardiac muscle increased by two mechanisms: one is from the metabolite Cr, the other is from the exogenous PCr itself. After iv Cr 456mg/kg, there is no more PCr and ATP in the plasma, the Cr in the plasma could be described by two-compartment model, with t1/2β,Vd and CL as the same as that in iv PCr; In the heart tissue, there is no more PCr, but Cr and ATP could be detected, with t1/2β,Vd and CL as the same as that in iv PCr. After iv Pit 5 min and iv PCr, the PK behavior of PCr in the plasma could be described by two-compartment model. ATP did not apper,but Cr appered immediately, it could be described by one-compartment model and first-order kinetics, and t1/2k(m) is longer than PCr; In the heart tissue, PCr did not apper, but Cr and ATP could be found.