Plasma Protein Binding of Highly Bound Drugs Determined With Equilibrium Gel Filtration of Nonradiolabeled Compounds and LC-MS/MS Detection

Accurate determination of the free fraction of a drug in plasma can be challenging when it falls below 1% and even more so when below 0.1%. Equilibrium dialysis with diluted plasma has been used to determine unbound fraction below 1%, but some analytes are not amenable to this method. One robust alternative for accurately measuring very highly bound compounds is equilibrium gel filtration; however, radiolabeled compounds have been used with this technique to quantify the low analyte concentrations. This report examined results obtained using radiolabeled compounds with liquid scintillation detection and those obtained using their nonradiolabeled analogs with liquid chromatography–tandem mass spectrometry detection. The 2 methods provided comparable results over the range of 0.005%-4% free, with a slope of 1.0 and a R² = 0.93. These results demonstrate that equilibrium gel filtration with liquid chromatography–tandem mass spectrometry detection can be used earlier in the drug discovery process to determine the unbound fraction of highly bound drugs and may help obviate the need for radiolabeled compound.     

Quantitative Estimation of Plasma Free Drug Fraction in Patients With Varying Degrees of Hepatic Impairment: A Methodological Evaluation

Quantitative prediction of unbound drug fraction (f_u) is essential for scaling pharmacokinetics through physiologically based approaches. However, few attempts have been made to evaluate the projection of f_u values under pathological conditions. The primary objective of this study was to predict f_u values (n = 105) of 56 compounds with or without the information of predominant binding protein in patients with varying degrees of hepatic insufficiency by accounting for quantitative changes in molar concentrations of either the major binding protein or albumin plus alpha 1-acid glycoprotein associated with differing levels of hepatic dysfunction. For the purpose of scaling, data pertaining to albumin and α1-acid glycoprotein levels in response to differing degrees of hepatic impairment were systematically collected from 919 adult donors. The results of the present study demonstrate for the first time the feasibility of physiologically based scaling f_u in hepatic dysfunction after verifying with experimentally measured data of a wide variety of compounds from individuals with varying degrees of hepatic insufficiency. Furthermore, the high level of predictive accuracy indicates that the inter-relation between the severity of hepatic impairment and these plasma protein levels are physiologically accurate. The present study enhances the confidence in predicting f_u in hepatic insufficiency, particularly for albumin-bound drugs.     

Determination of the Plasma Protein Binding of Liraglutide Using the EScalate* Equilibrium Shift Assay

The plasma protein binding capability of drug substances represents an important assay parameter in drug discovery and development. For very strong plasma protein binding molecules, however, the free fraction in plasma f_u is very small and therefore difficult to determine with standard methods. To solve this problem, the EScalate equilibrium shift in vitro assay was developed. Escalating concentrations of plasma were found to shift the binding equilibrium in solution between the test item and immobilized human serum albumin. Following liquid chromatography coupled to mass spectrometry analysis of the samples, the test compound’s f_u in plasma is calculated with a 2-dimensional fitting procedure. Comparability of EScalate assay results was demonstrated for 4 extensively studied small molecule drugs (carbamazepine, desipramine, pyrimethamine, and warfarin) as well as for liraglutide, a fatty acid-conjugated peptide drug with very strong plasma protein binding. The results were in good agreement with published data. A free fraction of 0.51% was determined for liraglutide. Our results confirm the compound’s very strong plasma protein binding properties in a novel and robust assay system.     

In vitro protein binding of propafenone in normal and uraemic human sera

Summary The protein binding of propafenone, a Class I antiarrhythmic agent, was studied in vitro using a selective and sensitive electron-capture detection gas-liquid capillary chromatographic assay method developed in our laboratory.The concentration-dependency of the serum protein binding of propafenone was confirmed in vitro by equilibrium dialysis, using serum obtained from healthy human subjects and patients with chronic renal failure.In normal serum the unbound fraction of propafenone was 0.027 at a propafenone concentration of 0.25 µg · ml^–1, 0.041 within the therapeutic concentration range (0.5–2 µg · ml^–1), 0.138 at a propafenone concentration of 25 µg · ml^–1, and 0.187 when the propafenone concentration was increased to 100 µg · ml^–1.There was no evidence of significant concentration-dependent changes in unbound fraction within the propafenone concentration range of 0.5–1.5 µg · ml^–1. However, concentration-dependent binding was demonstrated at concentrations greater than 1.5 µg · ml^–1.A high-affinity, low-capacity binding site (K₁=6.53×10⁵ l · mol^–1; n₁P₁=1.73×10^–4 mol · l^–1) and a low-affinity, high-capacity binding site (K₂=8.77×10³ l · mol^–1; n₂P₂=8.57×10^–3 mol · ×l^–1) were identified.In pooled uraemic serum the unbound fraction of propafenone was approximately 50% of that of normal serum throughout the concentration range studied (1–5 µg · ml^–1). In sera from patients with chronic renal failure the increase in propafenone binding ratio or the decrease in unbound fraction was associated with the increase in alpha₁-acid glycoprotein concentrations, and there was a correlation (r=0.8302) between alpha₁-acid glycoprotein concentration and the propafenone binding ratio.     

Improving Prediction of Metabolic Clearance Using Quantitative Extrapolation of Results Obtained From Human Hepatic Micropatterned Cocultures Model and by Considering the Impact of Albumin Binding

The objective was to compare, with the same data set, the predictive performance of 3 in vitro assays of hepatic clearance (CL), namely, micropatterned cocultures (also referring to HepatoPac^®) and suspension as well as monolayer hepatocytes to define which assay is the most accurate. Furthermore, existing in vitro-to-in vivo extrapolation (IVIVE) methods were challenged to verify which method is the most predictive (i.e., direct scaling method without binding correction, conventional method based either on the unbound fraction in plasma (fu_p) according to the free-drug hypothesis, or based on an fu_p value adjusted for the albumin [ALB]-facilitated hepatic uptake phenomenon). Accordingly, the role of ALB binding was specifically challenged, and consequently, the ALB production was monitored in parallel to the metabolic stability. The ALB concentration data were used to compare the in vitro assays and to adjust the value of fu_p of each drug to mimic the ALB-facilitated hepatic uptake phenomenon. The results confirmed that the direct and conventional IVIVE methods generally overpredicted and underpredicted the CL in vivo in humans, respectively. However, the underprediction of the conventional IVIVE method based on fu_p was significantly reduced from data generated with the HepatoPac^® system compared with the 2 other in vitro assays, which is possibly because that system is producing ALB at a rate much closer to the in vivo condition in liver. Hence, these observations suggest that the presence of more ALB molecules per hepatocyte in that HepatoPac^® system may have facilitated the hepatic uptake of several bound drugs because their intrinsic CL was increased instead of being decreased by the ALB binding effect. Accordingly, the IVIVE method based on the fu_p value adjusted for the ALB-facilitated uptake phenomenon gave the lowest prediction bias from the statistical analyses. This study indicated that the HepatoPac^® system combined with the adjusted value of fu_p was the most reliable IVIVE method and revealed the importance of quantifying the in vitro-to-in vivo variation of ALB concentration to improve the CL predictions, which would help any future physiologically based pharmacokinetics modeling exercise.     

A Novel Method for Preventing Non-specific Binding in Equilibrium Dialysis Assays Using Solutol® as an Additive

Accurate determination of fraction unbound in plasma is required for the interpretation of pharmacology and toxicology data, in addition to predicting human pharmacokinetics, dose, and drug-drug interaction potential. A trend, largely driven by changing target space and new chemical modalities, has increased the occurrence of compounds beyond the traditional rule of 5 physicochemical property space, meaning many drugs under development have high lipophilicity. This can present challenges for ADME assays, including non-specific binding to labware, low dynamic range and solubility. When determining unbound fraction, low recovery, due to non-specific binding, makes bioanalytical sensitivity limiting and prevents determination of free fraction for highly bound compounds. Here, mitigation of non-specific binding through the addition of 0.01% v/v of the excipient Solutol® to an equilibrium dialysis assay has been explored. Solutol® prevented non-specific binding to the dialysis membrane and showed no significant binding to plasma proteins. A test set of compounds demonstrates that this method gives comparable values of fraction unbound. In conclusion, the use of Solutol® as an additive in equilibrium dialysis formats could provide a method of mitigating non-specific binding, enabling the determination of fraction unbound values for highly lipophilic compounds.     

Applying Two Orthogonal Methods to Assess Accuracy of Plasma Protein Binding Measurements for Highly Bound Compounds

Significant advances have been made over the years to accurately measure plasma protein binding (PPB) of highly bound compounds. However, because of perceived uncertainty based on historical suboptimal methods and limitation of radiochemical purity of radiolabeled materials, current regulatory guidelines recommend using an arbitrary cutoff fraction unbound (f_u) of 0.01 as the lower limit for drug-drug interaction (DDI) prediction. This can result in significant overprediction of DDI for highly bound compounds, unnecessary DDI clinical trials and more restrictive drug product labels. To build confidence in the accuracy of PPB measurement for highly bound compounds, 2 orthogonal methods, equilibrium dialysis and ultracentrifugation, are assessed in this study to measure PPB of 10 highly bound drugs (f_u < 0.01). The results show that the 2 very different methods yield comparable f_u values, generally within 2-fold of each other. The data suggest that PPB of highly bound compounds can be measured accurately using current state-of-art methods, and the experimental f_u should be used for DDI prediction to provide a more realistic evaluation of DDI risk in the clinic.     

Method for Simultaneous Determination of Free Concentration,Total Concentration,and Plasma Binding Capacity in Clinical Samples

Most quantitative research methods are based on measuring either the total or the free concentration of an analyte in a sample. However, this is often insufficient for the study of complex biological systems. The main objective of this research was to develop new methods for providing more information from samples: the free concentration (C_f), the total concentration (C_t), and the plasma binding capacity (PBC). Samples were processed using microextraction and ultrafiltration. For each of these techniques, two quantification procedures were used: addition of isotopically labeled standard and repeated analysis of the same sample. The new methods were validated by analyzing clinical samples and samples with known concentrations. Methods based on addition of labeled compound were found to be the fastest, and most reproducible. For analysis of clinical samples, methods based on microextraction were more sensitive and more accurate than those based on ultrafiltration. For analysis of pooled plasma samples, the overall accuracy of all approaches to determine PBC, testosterone C_f, and testosterone C_t was between 94 and 109%, 87–113%, and 94–122% respectively. The new approach goes beyond a simple concentration measurement, giving more information from clinical samples, with great potential for personalizing drug dosage and therapy to the needs of individual patients.