A Convenient Method to Measure BloodPlasma Concentration Ratio Using Routine Plasma Collection in In Vivo Pharmacokinetic Studies

LEONID M. BEREZHKOVSKIY, XIAOLIN ZHANG, JONATHAN CHEONG Genentech Inc., 1 DNA Way, South San Francisco, California 94080 Received 2 May 2011; revised 18 June 2011; accepted 29 June 2011 Published online 20 July 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22709 ABSTRACT: A practical time-saving method of determination of equilibrium bloodplasma concentration ratio is described. The method is based on the analysis of compound plasma concentrations in regular blood sample and the blood sample diluted with blank plasma. Since only plasma concentrations are analyzed, the method can be conveniently applied in routine pharmacokinetic studies with minimal additional work for obtaining bloodplasma ratio. The method can also be easily used in in vitro experiment. The results obtained by suggested method are in good agreement with that obtained by common in vitro measurements of bloodplasma ratio. 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:52935298, 2011 Keywords: bloodplasma ratio; blood cell partitioning; protein binding; pharmacokinetics; distribution; thermodynamics; bioanalysis

INTRODUCTION
The bloodplasma concentration ratio, r, is dened as the ratio of total drug concentration in blood, Cb , to its total concentration in plasma, Cp , at equilibrium r = Cb /Cp (1)

This parameter is needed for interpretation of pharmacokinetic data. For instance, the maximum value of hepatic clearance calculated with respect to plasma concentration is equal to rQh (but not Qh ), where Qh is the liver blood ow. If the total body clearance, CL, obtained following intravenous administration of drug dose D, CL = D/AUC > rQh , this suggests a possibility of extrahepatic elimination pathways. In physiologically based pharmacokinetic (PBPK) modeling, the quantity of drug in the organ A(t) is determined by the general mass balance equation dA/ dt = rQCp,in rQCp,out E(t), where Q is the rate of blood ow to the organ, Cp,in and Cp,out are, respectively, the drug plasma concentrations in blood entering and exiting the organ, and E(t) is the drug
Correspondence to: Leonid M. Berezhkovskiy (Telephone: +650-225-1798; Fax: +650-467-4836; E-mail: berezhkovskiy.leo @gene.com)
Journal of Pharmaceutical Sciences, Vol. 100, 52935298 (2011) 2011 Wiley-Liss, Inc. and the American Pharmacists Association

elimination rate of the organ. Thus we need to know r in order to perform PBPK modeling. The contribution of blood to the total steady-state volume of distribution, Vss , is equal to rVb , where Vb is the blood volume of the body (Vb 0.4 L/kg for human).Thus, for relatively large bloodplasma ratio, the contribution of blood to Vss may be quite significant. The values of r for some drugs may be rather high, r > 100.1 Typically, bloodplasma ratio is obtained in vitro by dissolving compound in blood or using blood and plasma samples collected in vivo after routine compound dosing. In both cases the analysis of blood and plasma concentrations is performed in order to calculate r by Eq. 1. In routine pharmacokinetic studies the collected blood samples are centrifuged to separate plasma, consequently measure its compound concentration, and eventually obtain the time course of total compound concentration in plasma Cp (t). This article describes the method that allows determining bloodplasma ratio from the measurements of plasma concentrations only using the plasma samples obtained in a regular and specically collected way. By applying this method we avoid the analysis of whole blood. Thus, the suggested method provides noticeable time saving and convenience for the highthroughput screening by skipping the measurements of blood concentrations. Since the calibration curve for
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plasma concentrations is generated for routine sample analysis anyway, the suggested method allows to obtain r very efciently with minimal experimental efforts, and can be easily applied in each routine pharmacokinetic study.

DESCRIPTION OF THE METHOD

In general, the equilibrium distribution of compound between two phases (A and B) is determined by the partion coefcient PAB = CA /CB (intensive parameter) and the volumes of each phase (extensive parameters), where CA and CB are the equilibrium compound concentrations in phase A and B, respectively. PAB can be obtained by measuring the concentrations CA and CB straightforward. The other way to get PAB is to dissolve the known amount of compound in AB system, which has different volume ratios of phase A and B. The concentrations CA and CB will be different for such systems, which allows obtaining PAB by measuring the compound concentrations in one phase only (in A or in B). This idea was applied for the determination of bloodplasma ratio. Here the compound distribution occurs between plasma and blood cells. The common assumption that r is concentration independent (linear range of distribution) is applied further. The following procedure was applied. At a given time point after compound dosing, a regular blood sample was collected. Part of the collected blood (known volume) was transferred to the other tube, which contained a certain volume of blank plasma of the studied species prepared beforehand. This tube was shaked to mix blood and plasma and was kept at room temperature for about 1015 min to allow compound equilibration between blood cells and plasma. Then both samples (regular blood sample and sample of blood diluted with plasma) were processed in a ordinary way by centrifugation and decanting of supernatant plasma. The concentrations of plasma were analyzed (along with plasma samples from other time points) using the calibration curve routinely generated for plasma analysis. Thus we obtained the compound concentration of the regular plasma sample, and the concentration of plasma sample that was prepared from blood diluted with plasma. Let us derive the equation that allows calculating the bloodplasma ratio using these concentrations. The distribution of compound in blood is characterized by its partition coefcient, p, between blood cells (99% of which are the red blood cells1 ) and plasma, which is dened as p= Cbc Cbc = Cup Cp fup (2)

tion in plasma, Cp is the total drug concentration in plasma, and fup is the unbound drug fraction in plasma. The linear range of drug concentration is considered, so that the dependence of fup on drug concentration is negligible and fup is assumed constant.2 To meet this assumption, the concentration of compound in plasma should be much less than that of binding proteins. The concentration of unbound proteins can then be taken as constant equals to its total concentration. This could be especially of concern for basic compounds that bind "1 -acid glycoprotein (AAG). Its concentration in plasma is about 1520 :M, whereas the albumin concentration is around 600700 :M. Drug binding to AAG is also often characterized by one or two binding sites.3 In practice, it is desirable to have compound concentration at least 20-fold less than that of binding protein in order to assume that fup is constant.4,5 As considered further, the concentrations of compounds used in the experiments were in the range of approximately 0.030.3 :M, so that the condition for assuming constant fup was satised. Thought is important to keep in mind a possibility of relatively unusual case of anomalous protein, when fup exhibits strong concentration dependence at low drug concentration.68 The blood cellplasma partition coefcient p (Eq. 2) is also considered as concentration independent because the plasma concentrations of compounds are relatively low.1 The blood is characterized by the volume of blood cells Vbc = Vb H and the volume of plasma Vp = Vb (1 H), where Vb is the total blood volume and H is the hematocrit (typically 0.4 H 0.6). Then the concentration of drug in blood can be provided as Cb = Cbc Vbc + Cp Vp = Cbc H + Cp (1 H) Vb (3)

Thus substituting Eq. 3 in Eq. 1 and using Eq. 2 yields the following expression for bloodplasma ratio r = 1 H + pfup H (4)

The dilution factor, d, for the blood sample that was diluted with plasma was dened straightforward as d= Vadd Vbl + Vadd =1+ Vbl Vbl (5)

where Cbc is the equilibrium drug concentration in blood cells and Cup is the unbound drug concentraJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011

where Vbl is the volume of blood sample and Vadd is the volume of added blank plasma. Let us calculate the drug plasma concentration in the diluted blood sample. This will further allow to obtain bloodplasma ratio by comparing the compound plasma concentrations of diluted and undiluted blood samples. We can calculate the diluted blood concentration and bloodplasma ratio in diluted blood sample to obtain the plasma concentration in diluted
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blood sample using Eq. 1. The concentration of compound in diluted blood, Cb,dil , according to Eq. 5 is equal to Cb,dil = rCp Cb Vbl Cb = = Vbl + Vadd d d (6)

the following equations for the calculation of blood plasma ratio in this case r=1+ d2 B d1 B 1 (12)

Similarly, the hematocrit of the diluted blood sample, Hdil , is calculated as Hdil = Vbl H H = Vbl + Vadd d (7)

Substituting Eq. 7 in Eq. 4 and taking into account that p and fup are assumed concentration independent provides the bloodplasma ratio of the diluted blood sample rdil = 1 Hdil + pfup Hdil = 1 H H + pfup d d (8)

Here d1 and d2 are dilution factors for the split sample and the concentration ratio B = Cp,d1 /Cp,d2 , where Cp,d1 and Cp,d2 are the plasma concentrations of blood samples diluted d1 and d2 fold, respectively. If sample 1 is not diluted (d1 = 1), Eq. 12 transfers into Eq. 11 (with d2 = d). The concentration of compound in plasma can be calculated from the measured plasma concentrations in diluted sample according to Eq. 10 as Cp = BCp,dil . The parameter B is provided by Eq. 11 (with the bloodplasma ratio calculated by Eq. 12) B = 1 + (d 1)/r (13)

Applying Eqs. 1, 6, and 8 yield the drug plasma concentration of the diluted blood sample Cp,dil = rCp Cb,dil = rdil d H + pfup H (9)

Eventually, we obtain the compound concentration in plasma Cp = Cp,d1 1 + d1 1/r = Cp,d2 1 + d2 1/r (14)

The drug plasma concentrations in regular and diluted samples, Cp and Cp,dil are analyzed, so that we get their ratio B= Cp Cp,dil (10)

This is especially applicable for the serial bleeding studies in mice. The blood samples (about 1020 :L) can be collected in blank plasma to avoid the whole blood analysis and analyze plasma only. The bloodplasma ratio and plasma concentrations of compound will be calculated using the described approach (Eqs. 12 and 14).

Eventually, using Eqs. 9, 10, and 4 result in a very simple equation for the calculation of the blood plasma ratio r= d 1 B1 (11)

EXPERIMENTAL RESULTS
The 1 h time point samples from routine rat i.v. bolus pharmacokinetic studies (at 0.5 or 1 mg/kg dose of compound) were used to obtain the bloodplasma ratio. Typically, these samples had relatively high compound plasma concentrations (0.030.3 :M) that could be reliably detected. We had to gure out what dilution factor would be better to use in experiments in order to have reliable calculation of bloodplasma ratio (Eq. 11). Obviously the higher dilution would lead to larger value of concentration ratio B (Eq. 13). On the other hand, if we dilute too much, Cp,dil may become too low and cannot be accurately measured. Also minimizing the quantity of plasma used for dilution reduces the experimental expenses. Actually, Eq. 11 provides the insight on the optimal value of dilution factor for routine screening studies. Most often, the bloodplasma ratio stays in the interval 0.5 r 2.5. The calculated value of r (Eq. 11) as a function of measures ratio B = Cp /Cp,dil is shown in Figure 1 for dilution factors d = 2 and d = 10. As seen in Figure 1, if r 1 and the dilution factor taken as d = 2, the corresponding ratio Cp /Cp,dil 2. Therefore, if we diluted
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The dilution factor d is determined by the experimental setting, and the concentration ratio B is obtained from the routine analysis of plasma sample concentrations. Thus, Eq. 11 is applied to obtain the bloodplasma ratio using the analysis of compound concentrations in plasma only. Interestingly, this equation does not contain the hematocrit H, which is expedient in terms of minimizing the experimental procedure. The described method is quite convenient in drug discovery setting. The blood sample may have a small volume if many time points are taken or if the mouse study is performed with blood samples taken from each mouse for the whole time course (serial bleeding). In this case, the collected blood sample can be split into two parts and each one can be diluted with plasma using different dilution factors. Similar deviations provide
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Table 1. Comparison of BloodPlasma Ratios Obtained from In Vivo Pharmacokinetic Studies by the Described Method with That Measured by Common In Vitro Assay Compound Number 1 2 3 4 5 6 7 8 9 10 11 12 13 BloodPlasma Ratio Obtained In Vivo (Eq. 11) 0.728 0.080 0.769 0.140 0.845 0.019 0.885 0.164 0.957 0.004 1.00 0.330 1.03 0.064 1.06 0.019 1.25 0.327 1.07 0.284 1.10 0.281 1.17 0.220 1.61 0.398 BloodPlasma Ratio Obtained In Vitro 0.689 0.862 0.826 0.807 0.966 0.918 1.00 0.999 1.06 0.962 1.30 1.40 1.48

Figure 1. The dependence of the calculated bloodplasma ratio, r, on the measured ratio of compound plasma concentrations in regular blood sample and blood sample diluted with plasma Cp /Cp,dil (Eq. 11): calculated for dilution factor d = 2, and calculated for dilution factor d = 10.

the blood samples 2-fold (Eq. 5), we would get quite 2 for well detectable concentration ratio Cp /Cp,dil the typical values of r 1. Thus for routine analysis, a 2-fold dilution is sufcient to get accurate values of r in the interval 0.5 r 2.5. As shown in Figure 1, for larger values of r, the ratio Cp /Cp,dil is getting smaller and approaching 1 with the increase of r. In this case, the calculated value of r strongly depends on the accuracy of the measured ratio Cp /Cp,dil . For instance, if r = 10 and d = 2, we have Cp /Cp,dil = 1.1, the accurate detection of which could be problematic just because of the instrumental uctuations in measuring concentrations. Though if we used a 10-fold dilution (d = 10), we would have Cp /Cp,dil = 1.9 (for r = 10), which can be accurately detected and consequently r would be determined with fair precision. Eventually, it could be concluded that for routine screening studies a 2-fold dilution is optimal. In relatively rare cases when the measured ratio Cp /Cp,dil turns out to be close to 1 (which indicates a large value of blood plasma ratio, r 10), an additional experiment can be performed to obtain r accurately. It could be either a common in vitro assay or application of the suggested dilution method, for instance, with d = 10 and d = 20 to check the consistency of the result. The following procedure was used for determination of the bloodplasma ratio in routine rat pharmacokinetic studies. A 50 :L aliquot taken from the 1 h blood sample of 200 :L was transferred to the tube containing 50 :L of blank rat plasma to get a 2-fold dilution (Vbl = Vadd = 50 :L in Eq. 5). The remaining 150 :L of blood was used for the analysis of plasma concentration at this time point (along with blood samples taken at other time points). The tube with
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diluted blood aliquot was shaked thoroughly to mix blood and plasma and left at room temperature for 1015 min for equilibration. After that it was treated the same way as all other blood samples, that is, placed on ice, centrifuged, and plasma was decanted. Plasma samples were analyzed by mass spectrometry using liquid chromatographytandem mass spectrometry method. A detailed description of the analytical procedure is provide in a recent publication.9 The comparison of bloodplasma ratio obtained by the described method (which used in vivo blood samples from routine screening studies in rats) with regular in vitro method is provided in Table 1. For in vivo study, the data were obtained using blood samples from three rats, and for in vitro assay the blood plasma ratio was measured in triplicate by spiking the compounds into blood. The agreement between the values of bloodplasma ratio obtained by different methods appears quite good. To explore the applicability of the suggested method to compounds with high bloodplasma ratio, we measured in vitro bloodplasma ratio of chlorthalidone in rat blood at 37 C using various dilution factors d = 2, 5, 15, and 50. This drug is known to have a high bloodplasma ratio in human blood, r = 30.7 obtained in vitro at 37 C.10 We performed the experiment using 5 :g/mL (14.8 :M) blood concentration of chlorthalidone, so that its expected concentration in plasma (assuming r 20) would not exceed 0.8 :M, which is much less than plasma concentration of AAG. Thus, the protein binding may be considered as concentration independent (which was assumed for the derivation of the equations). The direct measurement by comparison of blood and plasma concentrations (after 30 min equilibration at 37 C) yielded r = 30.3 1.5. Blood diluted with plasma was also incubated for 30 min at 37 C and centrifuged to separate plasma. The concentration of plasma samples were compared to calculate the
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Table 2. BloodPlasma Concentration Ratio of Chlorthalidone in Rat Blood Measured In Vitro by the Described Dilution Method Dilution Factor Bloodplasma ratio d=2 Not calculated d=5 44.1 0.80 d = 15 28.2 0.12 d = 50 23.9 0.40

ratios Cp /Cp,dil and apply Eq. 11 to nd r. The results obtained by the described dilution method are presented in Table 2. An adequate agreement between the direct method and the described novel method was observed for dilution factors d = 15 and d = 50. The measured values of Cp /Cp,dil for d = 2 were 0.91 0.04, which did not allow to calculate r because Cp /Cp,dil should exceed 1. For the given value of r = 30.3, we would expect to obtain Cp /Cp,dil = 1.033, so that Eq. 11 would yield r = 30.3. To measure such a small difference between Cp and Cp,dil could hardly be achieved because, besides the error introduced by pipetting, the uctuations in mass spectrometer response are around 10%. Even if we measured Cp /Cp,dil > 1, the result would not be reliable. Similarly, for dilution factor d = 5, the values of Cp and Cp,dil are rather close, so that we would have to measure Cp /Cp,dil = 1.132 in order to calculate r = 30.3 using Eq. 11. Therefore, the measured value (r = 44.1) was rather inaccurate. Thus the described method may provide reasonably accurate and consistent values of bloodplasma ratio for compounds that are highly distributed into blood cells. Using several dilution factors allows to observe the consistency of the results. It appears that the described dilution method is well suited for routine screening pharmacokinetic studies, whereas it is better to apply the direct measurement of bloodplasma ratio to obtain more accurate values, especially for compounds that enormously partition into blood cells. It does not seem rational to apply two dilutions (d = 2 and d = 10) in routine screening studies because for the majority of compounds r does not exceed 2.5, so that for the 2-fold dilution the measured ratio would be Cp /Cp,dil > 1.4 and could be detected rather reliably. If the observed value Cp /Cp,dil were close to 1, it would be an indication of high bloodplasma ratio. An additional measurement would be required in this uncommon case to obtain r accurately.

DISCUSSION
The suggested method of measuring the blood plasma ratio is quite convenient and allows obtaining this parameter with minimal experimental effort (just collecting and processing an additional sample of blood diluted with plasma). Bloodplasma ratio is the needed parameter even at earliest step of drug development. The assumption r = 1 may lead to misinterpretation of pharmacokinetic data, particularly regarding the clearance routes. Thus the addition of
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bloodplasma ratio measurement to routine pharmacokinetic studies could be quite benecial. The considered method can be applied for mouse pharmacokinetic studies that use serial bleeding technique, when the time course of compound is determined from each mice. The blood samples (about 1020 :L, depending on the number of time points) can be collected in mouse blank plasma to analyze plasma only (but not blood), and calculate blood plasma ratio and plasma concentrations using the described approach (Eqs. 1214). The blood samples (typically of 20 :L) can be collected, for instance, in 40 :L of blank plasma, which would provide enough plasma for concentration analysis. An additional blood sample can be collected in 80 :L of blank plasma. The samples need to be shaked, equilibrated for 1015 min, and then processed in a regular way to obtain plasma and measure compound plasma concentrations. Bloodplasma ratio will be determined in this case using two different dilutions (Eq. 12). For a regular sample collected in 40 :L of plasma, the dilution factor will be d1 = 3 (Vbl = 20 :L Vadd = 40 :L in Eq. 5), and for additional sample collected in 60 :L of plasma the dilution factor will be d2 = 5 (Vbl = 20 :L Vadd = 80 :L in Eq. 5). For a typical value of r = 1, the ratio B = Cp,d1 /Cp,d2 provided by Eq. 12 is equal to Cp,d1 /Cp,d2 = 1.66 (d1 = 3, d2 = 5). Such a difference in concentrations can be detected rather accurately. The described method of measuring bloodplasma ratio can be conveniently used as the in vitro assay. The tested compound just needs to be dissolved in blood, and the plasma obtained from undiluted blood and from the blood diluted with blank plasma should be analyzed. As the blood volume would not be as limited as in the in vivo study, several dilution levels can be applied (for instance d = 2 and d = 10) to check the consistency of obtained value of r. The in vitro application of this method may yield even more accurate value of r if we use the blank plasma prepared from the same blood that is applied in the assays. In this case, a possible inaccuracy due to the fact that the generic blank plasma used for dilution of blood samples may have slightly different level of protein binding than the plasma of blood, will be avoided. The other advantage of using this method in vitro is that we do not have to build the calibration curve for the analysis of compound concentration in plasma. Once we need only the ratio of plasma concentrations to calculate r (Eq. 11 or 12), the ratio of instrument
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responses (possibly normalized by the response of internal standard) will yield the concentration ratio of plasma samples. In general, drug protein binding and blood cell partitioning are pH and temperature dependent. The described method controls pH because the blank plasma used for dilutions is prepared from fresh plasma and is defrosted shortly before use for blood sample dilution. There is no temperature control in obtaining the bloodplasma ratio, as well as in measuring the sample plasma concentrations. Both correspond to the room temperature rather than to physiological temperature of 37 C. On the other hand, having the total plasma concentration Cp (t) and the bloodplasma ratio r at the same (room) temperature, we would correctly calculate the total blood concentrations of the samples Cb (t) = rCp (t). Consequently, we can calculate the CL with respect to blood concentration and compare it with hepatic blood ow Qh to estimate a possibility of extrahepatic elimination. This could be good enough for screening purposes. At advanced stage, in order to obtain the sample plasma concentrations at 37 C, we would need to arrange the blood sample collection at 37 C until plasma is separated from blood, or at least (which seems easier) check if the blood plasma ratio measured at room temperature is close to that obtained at 37 C. The temperature dependence of r is determined by the temperature dependence of fup and partition coefcient p (Eq. 4). The dependence of fup on temperature is known to exist, and is determined by the enthalpy change of the binding reaction. In certain cases, it is accounted in clinical practice (when fup is measured at room temperature) to calculate the total concentration of drug in plasma that would maintain the certain level of unbound drug plasma concentration Cup .11 For highly protein bound compounds fup may increase 5-fold at 37 C compared with that at 20 C.12,5 The blood cell partition coefcient p (Eq. 2) is actually the equilibrium constant of Dbc , where Dup denotes the unthe reaction Dup bound drug in plasma and Dbc denotes the drug in blood cell. Thus a negative enthalpy change of this reaction (which leads to higher partitioning into blood cells) would result in the decrease of p with the increase of temperature. Accordingly, if we measured the certain value of bloodplasma ratio at room temperature, r(20 C), the corresponding value of r at 37 C could be either greater or smaller than r(20 C) depending on the increase of fup and the decrease of p in the product pfup that determines the bloodplasma ratio (Eq. 4). The temperature dependence of bloodplasma ratio may be quite signicant. For instance,

for cyclosporine G, the bloodplasma ratio is 1.45, 1.2, and 0.9 at 5 C, 26 C, and 37 C, respectively.13

ACKNOWLEDGMENTS
The author would like to express his gratitude to Dr. Cornelis E.C.A. Hop for discussion and helpful suggestions on the improvement of the quality of the manuscript.

REFERENCES
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