| Objective: Some fractions of drugs are bound to plasma proteins, some fractions exist in the form of unbound in blood. It is well known that only the free fraction of a drug is able to penetrate to the target site and is pharmacologically active. Therefore, free drug concentration(Cf) is the recognized main basis for pharmacodynamics in vivo and safety assessment. However, the monitoring of Cf is not gain popularity. One of the most important reasons is the analytical method of Cf is not consummate, which thwarts monitoring and further study the change of pharmacodynamics in therapeutic drug monitoring with Cf. Therefore, the analytical method of Cf as well as the relationship of Cf and pharmacodynamics has become the focus in clinical pharmacist practice.The analytical methods of Cf mainly include equilibrium dialysis, ultrafiltration(UF), ultracentrifugation, gel filtration. UF is the most simple and fast method, centrifugal ultrafiltration(CF-UF) and hollow fiber centrifugal ultrafiltration(HFCF-UF) are the most commonly used method. The inconsistent on ultrafiltrate volume(Vu) by CF-UF has been reported in literature, the large Vu may destroy the balance of protein binding, affect the accuracy and precision for analysis of Cf. The dependency of Vu on centrifugation force and centrifugation time has been demonstrated in pooled normal plasma. Therefore, some reports attempted to control centrifugation time and centrifugation force to obtain uniformity and appropriate Vu when CF-UF was applied. The Vu by HFCF-UF could be controlled by the inner diameters of both the glass tube and hollow fiber. However, those studies were all carried out in pooled plasma from healthy volunteers, and were rarely assessed in clinical plasma samples. The change of plasma condition of healthy volunteers is small. But for clinical samples, the plasma conditions(e.g., protein levels, viscosity, cholesterol levels, osmotic pressure, etc.) of the patients vary with individuals12,13 and are significantly different from that of healthy subjects, owing to the physiological and pathological conditions of patients is very complex. However, whether UF could accurately characterize Cf in disease conditions has not been studied.The albumin concentrations of patients are significantly different with individuals, due to the effect of disease states. It is well known that albumin concentration can affect protein binding(PB),and influence Cf. Therefore, the focus of studies is the effect of albumin on Cf for a long time. The premise of characterize the real Cf in plasma is the accurate method under different albumin concentrations. However, the effect and regularity of albumin concentration on the methodology for analysis of Cf has not been studied.In routine TDM, for drugs with large individual differences and toxic effect, the suboptimal treatment or toxic effect of drugs may occur, though the total concentration was suitable. It mainly due to the free fraction of a drug is pharmacologically active, and it determines the curative and toxic effect. PB remains constant in healthy state, Cf and Ct have a good correlation, therefore, Ct can Cf predict at certain level. But in severe or confounding disease states(e.g., uremia, renal failure or hypoalbuminemia), the correlation of Cf and Ct was poor, the results for predicting or evaluating the pharmacodynamics with Ct were one-side, and using Cf instesd of Ct was more scientific and effective. The relationship of Cf with disease conditions is the key for safe and effective medication in clinic. Therefore, the study for the relationship of Cf with pharmacodynamics, and investigate the mechanism of individual different of drug pharmacodynamics are more important clinical reference value.Methods: This study use vancomycin(VCM) as model drug, first validated CF-UF and HFCF-UF in normal plasma, when the validated results are all meet the requirements, further studied the accuracy on the analysis of Cf concentration by ultrafiltration in various disease states. The method was collecting plasma with different disease states and recording the physiological and pathological conditions of those patients. The plasma was subjected CF-UF and HFCF-UF, respectively, recorded the Vu, and ultrafiltrate was used for HPLC analysis.This study use valproic acid(VPA) as model drug, first validated CF-UF and HFCF-UF in normal plasma, when the validated results are all meet the requirements, further studied the effect and regularity of albumin concentration on the methodology for analysis of Cf. the method was simulated quality control of plasma samples at different albumin concentration(10、20、40、50、60 g·L-1), the plasma was subjected CF-UF and HFCF-UF, respectively, for UF-CF, the Vu was controlled at 100 μL, and ultrafiltrate was used for HPLC analysis.through collecting plasma of patients treated with VCM, analysis of Cf and Ct, and calculating free fraction of VCM, to investigate the relationship between Cf and disease states.Results: The results demonstrated that plasma conditions had a significant impact on Vu/Vs by centrifugal ultrafiltration(CF-UF). The Vu by CF-UF ranged from 97 μL to 279 μL among different individuals under the same centrifugation conditions. There was a larger Vu in patients with hyporproteinemia or chonic renal disease than in patients with diabetes or hypertension. Total protein levels and the osmotic pressure of plasma were the main influence factors of Vu in disease states. Moreover, HFCF-UF was successfully applied to free VCM in TDM, and provided a reliable plat for further study the relationship of free VCM and its pharmacodynamics.The results indicated that there was no significant difference of free drug concentrations by HFCF-UF and CF-UF when the plasma albumin levels were above 40 g·L-1. But at low albumin levels(< 40 g·L-1), a considerable difference was discovered and the difference value increased with the decrease of plasma albumin level. The accuracy of analyzing free drug with CF-UF is more easily affected by the different plasma protein concentrations. The effect could be ignored with HFCF-UF. Therefore, the Vu should be more controlled for samples with low albumin levels to avoid erroneous assessment when CF-UF was applied. HFCF-UF was successfully applied for free VPA in TDM, to provide a reliable plat for analysis of VPA in clinic.Conclusion : This study has been developed an accurate plat for analysis of Cf. using VCM as representative drugs, Analyze the free fraction of VCM based on the pathophysiological conditions of the patients, to investigate the effect of pathophysiological conditions on free fraction, as well as to clarify the correlation and regularity of pharmacodynamic differences, and to provide certain theoretical basis for making reasonable VCM dosage regimen. |
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