The Application Of Hollow Fiber Centrifugal Ultrafiltration In Pharmacokinetics Research

Drugs are bound reversiblly to plasma proteins to a certain extent in thecirculation of blood, in which only unbound or free fraction could be able topermeate cell membrane and get in touch with the related receptor, and alongwith this to induce pharmacological activity. Under a certain condition, thereis a constant between drug and plasma protein. When free fraction wasdecreased by transfer and elimination, some drugs was released fromdrug-protein compound, in order to maintain a centain drug concentration inplasma and site and action. In addition, a saturate binding between drug andplasma protein existed, due to the limited binding capability of plasma protein.When drug concentration exceeded protein binding capability, a saturation todrug-protein binding occurred, leading to an sudden increase to free fraction.Moreover, co-medication was common in clinical, and protein-binding degreewould change, owing to competition of plasma protein binding sites by otherdrugs. As the typies of co-medication increased, the competition would bemore stronger.Plasma protein-binding rate indicates the ratio of drug-proteincompounds to total concentration, which is employed to represent the extentof drug-protein binding. Protein binding rate would influence disposition,metabolism and excretion in vivo, and further the level and time ofpharmacological action, which is an important pharmacokinetics parameter.Therefore, the research of drug-protein binding rate would be significant todevelopment and study of new drug, as well as rational administrationinstruction in clinical.In general, equilibrium dialysis（ED） and ultrafiltration（UF） are commontechniques to determine drug-protein binding rate. ED was tedious andtime-consuming, for example, the temperature was controled at37。C seriously and more than12h were demanded. In addition, a considerable volume ofdialysate would dilute plasma sample, leading to breakdown the originaldrug-protein binding balance, which induced a deviation in assay results. Inrecent years, UF has been a commonly used method to analyze drug-proteinbinding rate, which was simple and rapid compared to ED. However, theultrafiltrate occupied a major percentage to plasma sample, resulting in anincrease to colloid osmotic pressure and protein concentration of plasma. As aresult, the obtained assay result would possibly not represent the actual freefraction in plasma. In addition, the commerical ultrafiltration device wasexpensive, which would increase analysis cost.A mini-hollow fiber centrifugal ultrafiltration（HFCF-UF） was employedto analyze drug-protein binding rate in our research, in which Hollow fiberwas applied to be the medium for separation. Only free fraction of smallmolecule can pass through the hollow fiber while macromolecules were holdup by centrifugal force. More important is that in contrast to plasma sample,the obtained ultrafiltrate volume by HFCF-UF could be ignored. Therefore,the balance of drug-protein binding in plasma was hardly destoryed, whichwould lead to a more accurate result which was extremely near to acualphysiological environment. In addition, the HFCF-UF device indicated easyand inexpensive, which would be popular in practical application. Theproposed method would provid a reliable and practical analysis process fordetermination of plasma protein-binding, especially for co-medication whichwas commonly used.Moreover, biapenem, a new parenteral carbapenem, was reported to be atime-dependent antibiotic. A main evaluation index to PK/PD was AUC>MIC（namely the AUC part above MIC）. In our research, HFCF-UF was employedto carry out plasma sample pre-treatment, and HPLC was applied to determineplasma concentration of biapenem as well as pharmacokinetics. The samplepre-treatment process was simple, time-saving and more sensitive thantraditional protein precipitation. Therefore, the proposed method wasconsidered suitable to analyze plasma concentration and pharmacokinetics of biapenem.PART1Mersurement of protein binding rate of drug by hollow fibercentrifugal ultrafiltration（1） Determination of protein binding rate of chlorogenic acid by hollowfiber centrifugal ultrafiltrationObjective: To separate free fraction of chlorogenic acid in plasm byhollow fiber centrifugal ultrafiltration, followed by HPLC in order toanalyze the protein binding rate of chlorogenic acid.Methods:0.5mL plasm sample was placed to the hollow fibercentrifugal ultrafiltration device, and then centrifuged at1.78×10³g for10min. The ultrafiltrate obtained from hollow fiber was analyzed byHPLC-UV to obtain free fraction, while traditional protein precipitationwas employed to quantitate total concentration.Results: The ultrafiltrate without giant molecule could be injectedinto HPLC directly, and the average absolute recovery was96.4%, withRSDs less than2.0%. A good linear relation was obtained in the range of0.126⁴.04μg·mL^-1for free chlorogenic acid （r=0.9991）. At low, middle,high concentration（0.505μg·mL^-1,2.02μg·mL^-1,4.04μg·mL^-1）, the proteinbinding rate of chlorogenic acid were38.7%,34.7%,35.6%respectively.Conclusion: The proposed method is proved to be simple, fast andrepetitive, which is feasible for the determination of protein binding rate.Moreover, there is a binding of moderate extent between chlorogenic acidand plasma protein, and a concentration-dependent was not seen. （2） The influence of gentamycin on plasma protein-binding rate ofvancomycin by hollow fiber centrifugal ultrafiltrationObjective: We are to study whether the addition of gentamycin would affect the plasma protein-binding rate of vancomycin in vitro.Methods: Hollow fiber centrifugal ultrafiltration followed by HPLCwere employed to analyze free vancomycin fraction in plasma, whereas thecorresponding total concentration was from traditonal protein precipitation.The plasma protein-binding rates of vancomycin before and after the additionof gentamycin were examined in vitro, and paired t-test was adopted to evalutewhether a significant difference exist or not. The separation was carried out ona ZORBAX SB-C_l8（150mm×4.6mm,5μm）, with methanol-50mmol·L-1potassium phosphate （0.3%triethylamine, adjusted to pH3.2by phosphoricacid）=20:80as the mobile phase, at the flow rate of l mL·min-1. The detectionwas236nm, and the injection volume was10μL.Results: At low, middle, high concentrations, plasma protein-bindingrates of vancomycin alone were55.2%,54.8%and53.6%; while togetherwith gentamycin, the corresponding protein-binding rates alter to be50.9%,49.5%and49.7%, respectively. Statistical analysis of two sets of resultsshowed the P-value was <0.001by paired-sample t test, indicating asignificant difference btwteen the vancomycin alone and combination withgentamycin.Conclusion: The protein-binding rate of vancomycin was decreased inassociation with gentamycin, indicating an increasing tendency in terms offree vancomycin concentration. PART2Quantification of biapenem concentration in human plasma by asimple hollow fiber centrifugal ultrafiltration as the pretreatment methodObjective: Hollow fiber centrifugal ultrafiltration （HFCF-UF） wasdeveloped to separate biapenem from plasma, and HPLC-UV was employedto determin plasma concentration, and then the pharmacokinetic research wascarried out. Methods:450μL plasma sample containing1M MOPS （pH7.0）（1:1） asstabilizer was placed into the HFCF-UF device. The ultrafiltrate was withdrawfrom the hollow fiber after centrifugalization at3.60×10³g for10min, and10μL was injected directly into HPLC to obtain biapenem concentrations inplasma.12volunteers （6males and6females） was accepted a singleintervenous drop infusion of300mg and600mg biapenem, and then3P97software was applied to evalute compartment model and pharmacokineticparameter.Results:（1） The proposed method possessed a linear curve ranging from0.10μg·mL^-1to50μg·mL^-1（r=0.999）, which was20times as sensitive astraditional protein precipitation. At0.400,10.0and25.0μg·mL^-1, the averageabsolute recovery was as high as99.7%, with all intra-and inter-dayprecisions（RSD） were less than5.0%.（2） The physiological disposition ofbiapenem consistented with two-compartment model. After a singleintervenous infusion of300mg and600mg biapenem, C_<sub>max（μg·mL） was15.02and31.40; t1/2（h） was1.32±0.18and1.28±0.20; AUC0^∞（mg·h·L^-1）was11.64±3.16and23.98±6.17, CLs（L·h^-1） was24.6±3.18and22.0±4.12.Conclusions: Compared to traiditional protein precipitation, theproposed method improved sensitivity obviously, and only a simple10mincentrifugalization would accomplish a sample pretreatment. The HFCF-UFmethod was considered to be a simple and feasible sample pretreatment toanalyze biapenem concentrations in plasma and pharmacokinetic research. Agood human tolerance was revealed after a single intervenous infusion of300mg and600mg. In addition, the main pharmacokinetic parameters such asC_maxand AUC₀^∞presented correlate, indicating the physiological dispositionof biapenem was a linear pharmacokinetics.