Study On The Pharmacokinetics Of Euphorbiasteroid

Background and ObjectiveThe genus Euphorbia is the largest one in Euphorbiaceae. Many species of Euphorbia are used as medicinal plants. Euphorbia plants are rich resources in R&D of bioactive diterpenoids. Euphorbia Lathyris Linn belonging to Euphorbia is widely distributed in China and has been used in the treatment of cancer and warts as Chinese traditional medicine. A series of diterpenoids based on the lathyrane skeleton have been isolated from Euphorbia Lathyris Linn. Biological activity of these lathyrane-type diterpenoids have been carried out showing powerful anti-cancer activity and ability of reversing multi-drug resistance (MDR)。Euphorbiasteroid(Euphorbia factor L1) is one of the main constituents isolated from Euphorbia lathyris. It was reported to have significant growth-inhibitory effects on human cervical cancer HeLa cells in vitro, reversal activity against P-glycoprotein (P-gp) mediated MDR in resistant cells such as KBv200, MCF-7/adr and MES-SA/Dx5cell lines. In our preceding researches, we found that euphorbiasteroid was potential against HIV in vitro, for its inhibitory activity in the fusion between H9cells chronically infected by HIV-1ⅢB with the target MT-2cells with a half inhibitory concentration (IC50) and therapeutic index (TI)0.8μM, and600respectively. Aikeqing-2, developed by the Tropical Medicine Institute of Guangzhou University of Traditional Chinese Medicine, is a combinatorial medicine of compound Chinese herbal medicine Aikeqing with euphorbiasteroid for the treatment of acquired immunodeficiency syndrome (AIDS). In our previous researches, Aikeqing-2exerted certain effect on increasing peripheral CD4+T lymphocytes count and the ratio of CD4+/CD8+So euphorbiasteroid is expected to be developed as a lead compound for the treatment of AIDS and cancer. Profiles including pharmacological activities mentioned above and results related to content determination, toxic action were reported, as far as we know the knowledge regard to pharmacokinetics and metabolism of euphorbiasteroid is not clear. In order to provide a meaningful basis in pharmacokinetics for the new drug development we carried out a pharmacokinetic study on the compound euphobiasteroid. This paper describes the establishment and validation of a method based on high performance liquid chromatography-tandem mass spectrometric method (HPLC-MS/MS) for the determination of euphorbiasteroid in biological sample, which was used subsequently to determine the pharmacokinetics of euphorbiasteroid in rats, healthy rhesus macaques and rhesus macaques infected with simian immunodeficiency virus (SIV), namely SAIDS model monkeys.Methods1. A high performance liquid chromatography-tandem mass spectrometric method (HPLC-MS/MS) for the determination of euphorbiasteroid in biological sample was established and validated, which was used subsequently to determine the pharmacokinetics of euphorbiasteroid in rats, rhesus macaques and SAIDS rhesus macaques.2. Epoxylathyrol was isolated from alkaline hydrolysis of euphorbiasteroid by Waters2767Sample Manager. On the basis of spectroscopic data including those of MS,1H-NMR, and13C-NMR spectra, the structure was confirmed.3. Liquid chromatography-ion trap mass spectrometry in the multi-stage MS full scan mode (LC/MSn) was used to analyze metabolites of euphorbiasteroid in rats.4. Study on the binding of euphorbiasteroid to plasma proteins from different biological species by ultrafiltration.Results1. An HPLC-MS/MS method for the determination of euphorbiasteroid in plasma of rats, healthy and SAIDS monkeys was established and validated. Eupborbiasteroid was extracted with ethyl acetate from biological samples. A Waters Symmetry Shield C8(4.6mm×150mm,5.0μm) column was used with a mobile phase consisting of methanol (A)-lOmM ammonium formate containing0.1%formic acid (B) gradient elution. The determination was performed with electrospray ionization (ESI) source in the positive mode by a triple quadrupole tandem mass spectrometer. Quantification was performed by multiple reaction monitoring (MRM) of the transitions of m/z553～m/2297for euphorbiasteroid and m/z285→m/270for IS(wogonin). The linear calibration curves were obtained in the concentration range of1～10000μg·L-1(r=0.9975). The lower limit of determination (LLOQ) was1μg·L-1. The run time for each sample was7. Omin. The intra-and inter-day precision at three quality control (QC) levels were within2.6%-10.7%. The accuracy of the assay was within1.6%-5.1%.The method was proved suitable for the preclinical pharmacokinetics of euphorbiasteroid, which offered advantages of high sensitivity and selectivity.2. The pharmacokinetics of euphorbiasteroid in rats, healthy and SAIDS monkeys were investigated using the established HPLC-MS/MS method.An HPLC-MS/MS method for the determination of euphorbiasteroid in biological sample was used to determine the pharmacokinetics of euphorbiasteroid in rats and rhesus macaques following a single oral dose and intravenous administration, the pharmacokinetics of euphorbiasteroid in SAIDS monkeys following an oral administration. The non-compartmental pharmacokinetics parameters were processed by DAS2.1software program (Chinese Pharmacological Society).The mean maximum concentration of drug (Cmax) was3418.6μg· L-1after intravenous administration at a dose of10mg·kg-’and87μg· L-1after an oral administration of euphorbiasteroid to SD rats at a dose of100mg· kg-1respectively.The mean values of Cmax were1359.5μg·L-1after intravenous administration at a dose of5mg· kg-1and139.1μg· L-1after an oral administration of euphorbiasteroid to rhesus macaques at a dose of30mg·kg-1respectively.After oral and intravenous administration, the elimination of euphorbiasteroid in rats and rhesus macaques were both rapid. Apparently individual difference in the absorption of euphorbiasteroid existed both in rats and rhesus macaques.The mean value of Cmax was56.9μg· L-1to SAIDS rhesus macaques after oral administration at the same dose with rhesus macaques. Apparently differences were found existing in the absorption of euphorbiasteroid in both healthy and SAIDS rhesus macaques.The mean bioavailability of euphorbiasteroid in rats, rhesus macaques and SAIDS rhesus macaques were2.0%,4.6%and1.8%, respectively.Z. Under a waler bath at50℃, euphorbiasteroid was dissolved in THF and hydrolyzed with mol· L-1NaOH for48h. The hydrolysate was detected by photodiode array detector (DAD), analyzed by Liquid chromatography electrospray ion trap mass spectrometry in the multi-stage MS full scan mode(LC/MS"), and isolated by preparative HPLC to give a pure product with a yield rate of5.42%. The chemical structure was confirmed as epoxylathyrol on the basis of its spectroscopic data including those of MS,1H-NMR and13C-NMR spectra.4. Electrospray ionization mass spectrometry method was applied to analyze the possible structures of metabolites of euphorbiasteroid in plasma and urine of rats.In positive mode, a total of sixteen euphorbiasteroid derivatives were detected by use of electrospray ion trap mass spectrometry in the multi-stage MS full scan mode (LC/MS") in rats plasma and urine after oral or intravenous administration. Seven euphorbiasteroid derivatives in the plasma were deduced as products related esterases hydrolysis, methylation, oxidation and reduction after intravenous administration. Four of the five metabolites in oral administration were the same as intravenous administration. The main metabolites in rat plasma was phase Ⅰ metabolism of euphorbiasteroid, whereas phase Ⅱ metabolites was found in urine. Four euphorbiasteroid derivatives were deduced as glucuronidated and methyl-glucuronidated metabolites in rat urine after oral administration.5. The plasma protein binding (PPB) of euphorbiasteroid in rat, rhesus macaque, human plasma were studied by ultrafiltration. Amicon Ultra-0.5centrifugal filter devices(Nominal Molecular Weight Limit, NMWL10K) were used for the nonspecific binding(NSB) and PPB measurements. The mean NSB of euphorbiasteroid in PBS is60.5%. All of the mean PPB in rat, rhesus macaque, human plasma under the three levels of concentration (1000,100,20μg·L-1) were as high as76.0%~98.0%, and independent of the concentration. The PPB values were independent of the concentration. The PPB of euphorbiasteroid in human and rhesus macaque plasma are higher(94.6%~98.0%) than in rat plasma (76.0%~79.8%).There was no statistical difference between the PPB of euphorbiasteroid in human plasma and the PPB in rhesus macaque plasma at three dose levels. The PPB values with NSB correction in human and rhesus macaque plasma were still more than86%.Conelusion1. An HPLC-MS/MS method with ESI source in MRM mode was developed and validated for determination of euphorbiasteroid in biological sample. The lower limit of determination (LLOQ) was1μg· L-1The linear calibration curves were obtained in the concentration range of1～10000μg·L-1. The established method was proved suitable for preclinical pharmacokinetics of euphorbiasteroid. The pharmacokinetics of euphorbiasteroid in rhesus macaques and SAIDS rhesus macaques were investigated by an HPLC-MS/MS method for the first time. The non-compartmental pharmacokinetics parameters in rats, healthy rhesus macaques and SAIDS rhesus macaques were determined and compared after oral and intravenous administrations. The results showed that times to reach the peaks of blood concentrations for rats, healthy or SIV-infectious rhesus macaques in oral route were measured as0.3-0.5h. The elimination of euphorbiasteroid in rats or monkeys after oral administration were both rapid. The oral bioavailabilities of euphorbiasteroid in rats, healthy or SIV-infectious rhesus macaques were as low as2.0%,4.6%and1.8%respectively implying procedures which are capable of increasing bioavailability may be crucial for the oral administration.2.Epoxylathyrol was prepared from alkaline hydrolysates of euphorbiasteroid for the first time. The LC-DAD/MSn method was used in monitoring the process of hydrolysis.3. Electrospray ionization mass spectrometry method was applied to analyze the possible structures of metabolites of euphorbiasteroid in plasma and urine of rats. More than ten euphorbiasteroid derivatives in plasma or urine were deduced as metabolites of euphorbiasteroid related the products of esterase hydrolysis, methylation, oxidation and reduction.4.The plasma protein binding (PPB) of euphorbiasteroid in rat, rhesus macaque, and human plasma were studied by ultrafiltration for the first time. The PPB of euphorbiasteroid in human and rhesus macaque plasma are higher (94.6%~98.0%) than those in rat plasma (6.0%~79.8%). There was no statistical difference for PPB of euphorbiasteroid in human and rhesus macaque at the three dose levels. The PPB values with NSB correction in human and rhesus macaque plasma were still more than86%.