177

12 TECHNIQUES FOR DETERMINING PROTEIN BINDING IN DRUG DISCOVERY AND DEVELOPMENT

Tom Lloyd

12.1 INTRODUCTION

Plasma protein binding plays an important role in the whole - body disposition of drugs. Pharmacokinetic ( PK ) properties such as hepatic metabolism rate, renal excre-tion rate, membrane transport rate, and distribution volume are functions of the ratio of free fraction. As such, drug protein binding data can be useful in allome-tric scaling of PK parameters from animal studies to human subjects. From a pharmacodynamic viewpoint, knowing the ratio of free fraction of drugs in plasma is important because unbound drugs can easily reach the target organ, whereas bound drugs pass the blood capil-lary wall with great diffi culty. This principle can often be extended to other compartments that are in equilibrium with plasma as long as there is no active transport. Therefore, precise information on the free drug fraction is essential for drug development and for determining the safety of a drug in clinical trials [1 – 12] .

Intersubject variations in plasma protein concentra-tions have been documented [13 – 16] . However, the importance of these differences is minimized by the predominance of two protein components in plasma. Albumin and α1 - acid glycoprotein (AGP) comprise 60% of the total protein in plasma and account for the majority of drug binding [17] . Still, cases where specifi c drugs have exhibited varied inter - and intraindividual subject binding characteristics include instances of high - affi nity binding site saturation, disease - induced varia-tions, genetically determined modifi cations of proteins,

diurnal variation in concentration of transport proteins, metabolite protein binding effects, and even the pres-ence of exogenous contaminants from plasticizers and smoking. There may also be stereoselective differences as a consequence of chiral discriminative properties of the binding sites of human serum albumin and AGP. These can account for signifi cant differences between species [18] .

Most drugs bind to proteins in a reversible manner by means of weak chemical bonds such as ionic, van der Waals, hydrogen, and hydrophobic bonds with the hydroxyl, carboxyl, or other reversible sites available in the amino acids that constitute the protein chain [19] . Albumin makes up roughly half of the total plasma proteins (normal human albumin range is 34 – 54 mg/mL) with a molecular weight ranging from 65,000 to 69,000 Da. Acidic drugs are known to bind tightly to human serum albumin. AGP has a concentration in plasma of between 0.4 and 1 mg/mL and a molecular weight of roughly 40,000 Da. AGP primarily binds to basic and neutral drugs [20] .

Literature protein binding values for selected drugs in human plasma are presented in Table 12.1 [21] . These are typically calculated as

PPB /total free total% ( ) ,= − ×C C C 100

where PPB% is the percentage of binding to plasma proteins, Cfree is the concentration of the free drug (measured at equilibrium in buffer correcting for any

ADME-Enabling Technologies in Drug Design and Development, First Edition. Edited by Donglu Zhang and Sekhar Surapaneni.© 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

178 TECHNIQUES FOR DETERMINING PROTEIN BINDING IN DRUG DISCOVERY AND DEVELOPMENT

their place as tools for screening or rapid rank ordering of compounds in terms of their protein binding. They can also be useful for understanding binding interac-tions with specifi c components, but are generally not used to represent the overall protein environment. Also, due to challenges in sensitivity, they do not always allow for measurements at the therapeutically relevant con-centration. Spectroscopic tools can also play an impor-tant role addressing specifi c binding issues.

Other parameters to control that have been shown to alter the in vitro fractional binding of a ligand to plasma components regardless of the technique being applied include assay temperature, drug concentration, ligand stability, buffer source (pH, concentration, and composi-tion), plasma pH, and freshness of the plasma source [24 – 26] . Whenever possible, incubation with 10% CO 2should be used for the assay of plasma protein binding (especially for basic drugs that may become more ionized with a rising pH) since it produces pH values close to physiological conditions (pH 7.35 – 7.42) while leaving plasma composition unchanged [27, 28] . Alternatively, a 10 - fold dilution of plasma with isotonic buffer could be used, as long as the concentration of drug remains at least 10 times lower than that of the proteins.

Pooling sample matrix to reduce the number of cali-bration curves and protect against analyte adsorption during processing is commonly applied. A free fraction sample is added to a well already loaded with an equiva-lent volume of control plasma. Similarly, plasma sample aliquots are diluted with an equivalent volume of buffer. The standards also are prepared in a 50% plasma matrix so that all the generated unknown samples match up with a single mixed matrix curve [29 – 31] . The same approach has been applied in determinations of drug protein binding in microsomes [29, 32] and homoge-nized brain tissue [29] .

An extension of this approach has been applied in cases where substantial compound loss has been incurred due to nonspecifi c binding during ultrafi ltra-tion and the volume of fi ltrate is not known up front. In this case, the collection tubes are weighed twice to measure the volume of fi ltrate. A control plasma sample is prepared in parallel. Then the sample and control retentates are crossed over with the respective fi ltrates. The result is a sample representing the drug in the fi l-trate and another representing the drug in the retentate. By comparing the total drug recovery of the fi ltrate and retentate samples with the starting plasma concentra-tion, one determines the degree of nonspecifi c binding. The volume of fi ltrate produced is calculated from the weights of the collection vials measured both pre - and postfi ltrate generation assuming a density of 1.0. The concentration of the drug in the fi ltrate is then deter-mined by correcting for dilution of the fi ltrate with

dilutions or protein - free plasma), and Ctotal is the initial total concentration of drug in plasma. Conversely, the free fraction ( funbound ) can be expressed as

funbound PPB /= −1 100( % ).

The fraction unbound often relates directly to the pharmacological activity of the drug. It is determined by the drug ’ s affi nity for the protein, the concentration of the binding protein, and the concentration of the drug relative to the binding protein. Since the binding of drugs to plasma proteins is rather nonselective, many drugs with similar physicochemical characteristics compete with each other and with endogenous sub-stances for these binding sites. This can lead to a type of drug interaction based on one drug displacing another, particularly signifi cant for highly bound drugs. Exam-ples include tolbutamide and warfarin, where the free concentration can be signifi cantly elevated in the pres-ence of nonsteroidal anti - infl ammatory agents. Warfarin activity can also be elevated leading to an increased risk of bleeding in the presence of sulfonamides such as furosemide. Risk of adverse effect is greatest if the dis-placed drug has a limited volume of distribution, if the competition extends to the drug bound in tissues, if elimination of the drug is also reduced, or if the displac-ing drug is administered in high dosage by rapid intra-venous injection [21] .

12.2 OVERVIEW

There are many different techniques that have been used to assess the free fraction of drugs in the presence of proteins. The most common are equilibrium dialysis, ultrafi ltration, and ultracentrifugation [18, 22, 23] . Such in vitro techniques can then be verifi ed in vivo by micro-dialysis to answer specifi c questions. Other chromatog-raphy and capillary electrophoresis techniques have

TABLE 12.1. Human Protein Binding Measurements for Selected Drugs

Drug Bound in Plasma (%)

Ibuprofen > 99 Warfarin 99 Verapamil 90 ± 2 Propranolol 93.3 ± 1.2 Digoxin 25 ± 5 Caffeine 36 ± 7 Furosemide 98.8 ± 0.2

Selected from Goodman and Gilman ’ s The Pharmacological Basis of Therapeutics [21] .

EQUILIBRIUM DIALYSIS 179

for an acidic solution of Coomassie Brilliant Blue shifts from 465 nm to 595 nm when binding occurs [36] . This assay is sensitive in measuring protein concentration from 0.2 to 1.5 mg/mL (linear range of the assay). An immunoglobulin G (IgG) protein standard solution (e.g., bovine gamma globulin, lyophilized IgG, Bio - Rad, Hercules, CA) is used to generate a standard curve for protein determination. When mixed with the protein assay dye reagent, absorbance measurements can be made using a spectrophotometer at 595 nm. Unknown protein concentration is calculated by plotting a stan-dard curve , calculating the best fi t for that curve using a least squares linear regression and reading the protein content from the standard curve. Such a technique can be used to assess the actual protein concentration of ostensi-bly protein free fractions from various techniques.

12.3 EQUILIBRIUM DIALYSIS

Equilibrium dialysis is the most frequently used method and is often considered the gold standard for determin-ing drug protein binding. In such an experiment, two compartments are separated by a semipermeable mem-brane typically with an 8000 – 12,000 - Da molecular weight cutoff. The membrane allows the analyte being studied to pass but retains the protein content on one side (Figure 12.1 ). A time course experiment predeter-mines the amount of time required to reach equilibrium in the system. This can vary by compound and is typi-cally carried out at 37 ° C. Reported equilibration times range from 2 to 24 h depending on the setup. The volume - to - surface area ratio on the dialysis membrane governs the time required for the analyte to reach equi-librium between the bound fraction, protein sample side, and the free fraction, dialysate buffer side. Vertical alignment of the dialysis membrane reduces the poten-tial for trapped air pockets. Such air pockets would reduce the surface - to - volume ratio and slow the time to reach equilibrium. The capability to shake the apparatus can speed the time to reach equilibrium. The individual wells or chambers are covered to prevent evaporation and pH change. Measurements of the compound stabil-ity in matrix at 37 ° C for the duration of the incubation typically accompany such experiments. High - throughput formats with 24 - , 48 - and 96 - well designs accessible by automated liquid handlers have been introduced in recent years to increase the capacity to perform such determinations [17, 29, 30, 37] .

Most devices for equilibrium dialysis are constructed out of Tefl on ™ (DuPont, Wilmington, DE, USA). Although this offers an improvement over plastics used in disposable ultrafi ltration devices with respect to nonspecifi c binding, it does not eliminate the concern.

control retentate back to the total sample volume applied initially [33] .

As the application area moves upstream into the drug discovery stage, pooling analytes in protein binding experiments has been shown to increase throughput [31] . One example involved ultrafi ltration in a 96 - well format, pooling four different compounds and assessing their binding at 10 μ M in the presence of each other. Because of the much greater protein concentrations relative to analyte, this technique was validated showing strong agreement with individual analyte controls as well as the literature values for these analytes [34] . Simi-larly, pooling of as many as 25 compounds in homoge-nized brain tissue drug protein binding studies has been reported [35] .

Analysis can be accomplished by liquid scintillation counter for radiolabeled compounds or by light chromatography - tandem mass spectrometry (liquid chromatography [LC] - MS/MS). In the case of radiola-beled compounds with either tritium or carbon isotope labeling, one needs to ensure the purity and stability of the label. This is especially important with tritium, which while easier to synthesize, may not be as stable to exchange. Liquid chromatography (LC) analysis with radiochemical detection may be required to assess com-pound stability for radiolabeled analytes. Although requiring additional extraction and separation steps, LC - MS/MS affords more selective detection.

The most common techniques, equilibrium dialysis, ultrafi ltration, and ultracentrifugation have all been reformatted in recent years to reduce matrix volumes consumed and facilitate the use of automation. Many, if not all, of the process steps can incorporate liquid handler technology and even robotic handling to increase throughput and reliability. Such steps may include diluting stocks, spiking analytes into the matrix, loading matrix and possibly buffer into the desired format, sampling free fraction and matrix, diluting or pooling samples after collection, and fi nally, microplate extraction processing in the case of LC - MS/MS analysis. With increased capacity to execute such studies comes the ability to assess binding at different concentrations, across different species, and in different matrices (e.g., microsomes and different tissue homogenates).

For ultrafi ltration and equilibrium dialysis devices, one must monitor for the possibility of protein leakage, which would compromise the validity of the experi-ments. In the case of ultracentrifugation, one must simi-larly monitor the protein content of the free fraction sampled after centrifugation.

The Bradford assay offers an easily applied, spectro-photometric method for the determination of unknown protein concentration. The Bradford protein assay is based on the observation that the absorbance maximum

180 TECHNIQUES FOR DETERMINING PROTEIN BINDING IN DRUG DISCOVERY AND DEVELOPMENT

teins [37 – 41] . Extra osmotic pressure may need to be corrected for in a case where a Donnan effect is observed. In equilibrium dialysis, with a nondiffusible, charged protein on one side of the membrane, Donnan equilibrium leads to a pH difference across the mem-brane. With an ionizable drug, the concentration dis-solved in water will be different on the two sides of the membrane to an extent dependent on the pH difference and the p Ka of the drug. This must be allowed for in calculating the concentration bound to protein [42] . Poor aqueous solubility of an analyte can also pose problems for this technique.

12.4 ULTRACENTRIFUGATION

A simpler approach involves ultracentrifugation where the plasma sample is separated into three distinct layers by a centrifugal fi eld at very high speeds (typically 40,000– 100,000 rpm generating several hundred thou-sand g ). The centrifuge is operated under a vacuum to reduce friction spinning at such high speeds. This also allows for maintaining a constant temperature. At the very surface after ultracentrifugation, one fi nds chylo-microns and very low - density lipoproteins [43] . Just beneath this surface layer is the protein - free layer with

Nonspecifi c binding can also take place on the mem-brane itself. There is particular concern over nonspecifi c binding for lipophilic compounds. Measuring concentra-tions from both sides of the membrane and utilizing the ratio will further reduce the effect of nonspecifi c binding. The magnitude of the nonspecifi c binding can be inferred as a part of total recovery measurements comparing matrix stability concentration with the sum of the recov-eries from either side of the membrane after dialysis. Nonspecifi c binding can also be evaluated by spiking the compound into dialysate buffer alone, adding it to both sides of the dialysis apparatus and measuring the con-centration recovered.

Frequently, although not in all cases [30] , the mem-branes require a series of soaking pretreatments to remove preservatives and prepare it for use. An example procedure would be soaking the membrane in water for 20 min, then in 30% ethanol for 15 min, rinsing with water three times and then soaking in isotonic sodium phosphate buffer until the time of use [37] . Once wetted, the membrane must not be allowed to dry out.

Such devices have the potential to incur volume shifts from the dialysate buffer to the plasma side due to differences in osmotic pressure. This shifting results in plasma protein dilution and has been shown to reduce the in vitro fractional binding of ligand to plasma pro-

FIGURE 12.1. Wyeth (formerly Collegeville, PA, USA) custom equilibrium dialysis assembly with 500 - μ L chambers.

ULTRAFILTRATION 181

centrifugal force or positive pressure) is applied to force the solution through the membrane.

One parameter to pay attention to in comparing various devices and formats for ultrafi ltration is the molecular weight cutoff of the fi lter being used and the amount of protein it allows to pass. One fi nds cutoff ranges in the literature from 10,000 to 30,000 Da being employed. In cases where one is measuring very highly bound compounds, obviously using a membrane that allows 1% or perhaps even 5% protein breakthrough is going to bias measurements of free fraction. Conversely, using a fi ner fi ltration membrane will reduce the fi ltrate yield in terms of the volume as well as require a longer centrifugation step. The volume of fi ltrate harvested will also vary according to the g - force being applied across an array of tubes or microplate well positions as the radius varies.

As an example, Amicon centrifree micropartition devices with a fi lter membrane of 30,000 molecular weight cutoff from Millipore (Billerica, MA) were used in one instance. To achieve equilibrium between the drug and plasma proteins, the spiked protein samples were incubated at 4 ° C for 20 min prior to ultrafi ltration. Samples of 1 mL volume were transferred to Amicon centrifree micropartition devices and centrifuged at 3000g (4 ° C) for 15 min. Approximately 200 μ L of the ultrafi ltrates were then collected [4] .

The primary disadvantage of the ultrafi ltration method is nonspecifi c binding of the drug to the fi ltra-tion apparatus. Another is concentration of the plasma proteins during centrifugation.

Online, automated versions of these techniques include a system for continuous ultrafi ltration, affording calculation of the protein binding over a wide range of different drug/protein ratios in one single experiment [51] . Examples of protein binding determinations by online equilibrium dialysis have also been described [52, 53] . Solid - phase microextraction, which uses an extrac-tion phase that dissolves or adsorbs the drug of interest and rejects proteins, is another technique recently reported that measures the protein binding at equilib-rium. Advantages of this approach include requiring a relatively small sample size, short analysis time, and the ability to directly study complex samples such as whole blood [27] .

All three of these most commonly used in vitro tech-niques yield protein binding values that closely agree for the majority of drugs assessed with each methodol-ogy having associated shortcomings as noted. Of course, the best way to corroborate these determinations is to measure in vivo directly within the system being studied. The only tool currently available that can inter-cede and explicitly provide information on the extracel-lular free fraction is microdialysis. However, this too

the protein - containing phase found at the bottom. Any concerns over nonspecifi c binding are reduced as there is no membrane - separated compartment. The assump-tions of this technique are that there is no protein in the zone being sampled (which can be measured as well as qualitatively characterized) and that there is no altera-tion of the binding equilibrium during ultracentrifuga-tion (this has been typically demonstrated indirectly by comparing values with other techniques). There have been a few reports of a protein - contaminated fraction that was used for the determination of free drug con-centration so this fraction should be tested [44, 45] . There are reports in the literature of physical phenom-ena such as sedimentation, back diffusion, and viscosity leading to differences between free drug concentrations determined by ultracentrifugation compared with equi-librium dialysis [42, 46] . In one report, the error due to sedimentation of the drug can be as large as 10% for drugs at 300 Da and up to 40% for high - mass drugs such as suramin (1297 Da), so comparisons across techniques would be indicated [47] .

In addition to the need to have such a specialized centrifuge, disadvantages of this technique included lengthy centrifugation times of 4 – 6 h, consumption of larger volumes of sample (e.g., 2 mL), and smaller capacity rotors (e.g., 20 – 40 tubes) [48] . Recent develop-ments have scaled down these requirements with faster speeds requiring 1.5 – 3.5 h of centrifugation, smaller sample volumes (e.g., 200 μ L), and enlarged rotor capacity up to 72 tubes (e.g., available from Beckman Coulter Inc., Fullerton, CA) [49, 50] . Even in these scaled - down examples, studies to assess the protein content found in the free fraction samples suggested a negligible effect for compounds ≤ 99% protein bound. Samples must be gathered carefully and in a timely manner as jostling of the tubes or diffusion over time will undo the protein concentration gradient. This step is well suited for a robotic liquid handler that can quickly and reproducibly sample at exactly the same depth each time. This is particularly true in the scaled - down version of this technique where a sample of 20 – 35 μ L from a reinforced polycarbonate wall ultracentrifugation tube containing a total of 200 μ L is required.

12.5 ULTRAFILTRATION

Ultrafi ltration also involves the use of a molecular weight cutoff membrane. Protein samples are typically incubated with shaking at 37 ° C for 30 – 60 min to allow for equilibration of the drug protein binding. Matrix stability samples are usually collected before and after this incubation. The equilibrated protein sample is then applied to a fi ltration device, and pressure (typically

182 TECHNIQUES FOR DETERMINING PROTEIN BINDING IN DRUG DISCOVERY AND DEVELOPMENT

optical rotary dispersion, and circular dichroism. Such measurements can be performed in solution and monitor changes of the electronic and spectroscopic energy levels caused by the binding event with the ligand or protein. Such methods provide a better understanding of the binding mechanism and insight into three - dimensional protein structure [57] . They do not charac-terize binding with multiple components however, and such methods often lack suffi cient sensitivity to allow study in the therapeutic range.

Surface plasmon resonance has allowed the interac-tion of drugs with albumin and AAG to be character-ized both thermodynamically and kinetically [58] . Such a system monitors changes in the refractive index that occur as molecule complexes form or break during the binding reaction at the sensor surface anchoring one of the interaction partners [59] . In this case, a solution of the drug would be passed over the surface protein. However, the immobilization linkage to the surface may alter the binding activity of some proteins. Still, espe-cially for protein – protein interactions (which cannot be measured by conventional methods such as equilibrium dialysis, ultracentrifugation, and ultrafi ltration that rely on gross differences in molecular size), surface plasmon resonance, which senses changes proportional to the mass of the bound material on the sensor chip, may be a sound alternative [57] .

For early discovery screening purposes seeking to rank - order compounds on the basis of drug protein binding, other in vitro and chromatographic tools have been applied. One such in vitro technique is the parallel artifi cial membrane permeability assay ( PAMPA ) recently adapted to measure serum protein binding con-stants [60] . Either a hexadecane or 1 - octanol membrane is used to separate two compartments allowing free drug to pass while measuring rates with and without human serum albumin in the donor chamber. The assay is in a 96 - well format, requires no equilibration period, self - corrects for nonspecifi c adsorption, and has no potential for volume shift as the chemical membranes are not water permeable. Another approach is the erythrocyte partition method, which consists of measur-ing the partitioning of the drug between plasma and blood cells and the partitioning between buffer and blood cells. The ratio of the two partition coeffi cients yields the free fraction [7] . A recent in vitro microplate format involves human serum albumin immobilized on Transil ™ beads (Nimbus Biotechnology, Leipzig, Germany) [61] . The process involves a 2 - min incubation in the presence of the beads followed by a centrifuga-tion or fi ltration step to separate out the beads. The resulting supernatant can then be read by a plate reader. Alternatively, one can instead purchase attached egg yolk phosphatidylcholine to these silica beads, which

would require some application specifi c validation as there are numerous cases where the absolute concentra-tion values differ between in vitro and in vivo free frac-tion determinations. These can be due to microdialysis experimental challenges that may vary according to drug class and tissue region [54] .

12.6 MICRODIALYSIS

Microdialysis involves the implantation of a small probe into a specifi c region of a tissue - or fl uid - fi lled space [55] . A variety of probe designs have been used, including linear, U - shaped, or concentric geometries. Semiperme-able membrane materials used in probe construction range from low to high molecular weight cutoff. During microdialysis, a physiologically compatible perfusion fl uid is delivered through the probe at a low and con-stant fl ow rate (typically between 0.1 and 5 μ L/min). This sampling technique is volume neutral in most applications so there are no volume limitations even when working with small animals. In static mode, sample volume is limited to 0.1 – 0.2 μ L/min. Experimental con-ditions that affect probe recovery include the perfusion rate, temperature, probe membrane composition and surface area, nature of the dialyzed tissue, physicochem-ical properties of the analyte, and other factors that infl uence molecular diffusion characteristics [56] .

As microdialysis probes are usually perfused with aqueous solutions, the technique is conceptually limited to the study of water - soluble drugs. In order to enable the measurement of lipophilic compounds routinely, invitro experiments have demonstrated the usefulness of lipid emulsion as perfusate instead of aqueous solution. Microdialysate samples consist primarily of relatively small hydrophilic analytes in highly ionic aqueous samples. Typical sample volume is a few microliters or less with analyte concentrations in the picomolar or nanomolar range. However, microdialysis samples are generally protein free requiring no sample preparation prior to high - performance liquid chromatography (HPLC) analysis. Since microdialysis is labor intensive and requires specialized skills, it is not suitable for high - throughput screening of large numbers of compounds. Rather it is used to address specifi c questions and confi rm in vitro screening models [54] .

12.7 SPECTROSCOPY

Other approaches are available to study drug – protein interactions in greater detail such as various spectro-scopic techniques including ultraviolet (UV) - visible, fl uorescence, infrared, nuclear magnetic resonance,

SUMMARY DISCUSSION 183

limits often do not allow for measurements at therapeu-tic levels. Typically, these applications are performed with UV detection, which allows for operating at physi-ological conditions (buffer pH and ionic strength). One can turn to mass spectrometry (MS) detection for greater sensitivity; however, this greatly restricts the buffers that can be used [57] .

12.9 SUMMARY DISCUSSION

Although such abbreviated in vitro methods and chro-matography techniques can be applied in early screen-ing for the purposes of ranking the binding affi nity and quantitative structure – activity relationship modeling, when the protein binding data is intended for in vivoscaling, a single compound measurement using equilib-rium dialysis is strongly recommended [12] . This is par-ticularly true for highly bound compounds where small absolute differences in percent unbound become ampli-fi ed. Although the main drug binding proteins are albumin and AGP, plasma contains many other proteins. There is a high probability that many small molecules will exhibit some levels of binding. Therefore, to deter-mine the extent of plasma protein binding, the molecule should be tested directly in a protein binding assay using plasma or serum [27] .

Having compared over the past decade eight differ-ent formats and variations of the three most widely accepted techniques (equilibrium dialysis, ultrafi ltra-tion, and ultracentrifugation) for determining drug protein binding, despite all the caveats in their applica-tion noted previously, we found considerable agreement for most compounds studied. At therapeutic concentra-tions in plasma and microsomes, a combination of equi-librium dialysis and ultracentrifugation techniques matched with liquid scintillation counting or LC - MS/MS detection offered a powerful combination of tools. For the defi nitive determinations to be used in early drug development for allometric PK scaling, custom equilibrium dialysis devices similar to those described by Banker et al. [37] , but having a larger 500 μ L volume on either side of the membrane (Figure 12.1 ) offered suffi cient working volume to be able to accurately measure free fraction even for potent, high binding compounds. These devices were reusable and proved very reliable and cost - effective. A complementary tech-nique was ultracentrifugation particularly useful for the outlying instances where nonspecifi c binding rendered equilibrium dialysis data questionable.

As demands for in vitro data on drug protein binding in plasma and microsomes expanded upstream to the compound optimization stage, a scaled - down version of the ultracentrifugation format (Figure 12.2 ) [29, 49,

will serve as a lipid membrane. The solid - supported lipid membrane can then be used as a substitute for the blood cells in the aforementioned erythrocyte partition method [62] .

12.8 CHROMATOGRAPHIC METHODS

In a similar approach, affi nity chromatography uses immobilized biopolymer (enzymes, receptors, ion chan-nels, or antibodies) protein stationary phases. The stabil-ity and constant binding behavior of such columns provide a tool for studying interactions between small ligands and biomacromolecules. However, nonphysio-logical experimental conditions (pH adjustment, pres-ence of organic modifi ers) may alter the conformation and natural binding behavior of the attached protein (similar to the attachment in surface plasmon reso-nance). This approach may provide information on the relative affi nity of ligand binding and on the area(s) where the interaction takes place [18] . Again most of these efforts have involved human serum albumin [63, 64] , with limited success studying binding to AGP [65] and at least one instance of evaluating binding to high - density lipoproteins [66] .

High - performance size exclusion chromatography is based on packing that has sorbent only inside the pores. Equilibrated analytes in plasma are injected onto the column. Proteins are too large to enter these pores and interact with the sorbent, thereby passing unretained through the column. Free drug, however, is able to diffuse into the pores and interact with the sorbent resulting in later elution. Zonal elution involves a small - plug injection where the retention time is used to obtain the association constants. In frontal analysis, a large - plug injection is made and quantifi cation is based on plateau height. Drug and protein are mixed together and injected into either the mixture of drug and protein (direct separation method), into one of the interaction partners while the other species is dissolved in the eluent (Hummel – Dreyer method) [67] , or into buffer while both components are dissolved in the mobile phase (vacancy peak method) [68] .

Capillary electrophoresis is also used to study drug protein binding, providing the possibility to evaluate interactions in free solution [69] . Capillary electropho-retic methods have been applied successfully for both 1:1 and 1: n combination ratios between receptor and ligand, but they are restricted to a single protein at a time and do not allow precise control of the tempera-ture [27] . This technique offers fast separations and analysis while consuming small amounts of sample and reagent. However, there is the potential for protein adsorption on the capillary walls and the detection

184 TECHNIQUES FOR DETERMINING PROTEIN BINDING IN DRUG DISCOVERY AND DEVELOPMENT

observations (Figure 12.3 ), was also applied. These values, combined with the drug concentrations deter-mined in plasma and brain, provided a much more com-plete comparison of the drug candidates being considered. As the free drug principle suggests, only the collision of free drug with the target is likely to contrib-ute to binding [11] . Traditional brain - to - plasma concen-trations alone can be misleading, especially in cases where the compounds are P - glycoprotein effl ux sub-strates (can be assessed based on Caco - 2 cell permeabil-ity asymmetry) [70] . Drug delivery to the brain can be

50] met the data quality and throughput requirements feeding into the human PK modeling and candidate selection process.

At an even earlier drug discovery stage supporting central nervous system therapeutic area research efforts, the equilibrium dialysis device shown in Figure 12.1 was applied to assess free fractions in plasma and homoge-nized brain tissue collected from rodent PK studies. In some instances, a commercial version, deemed not suffi ciently reliable for defi nitive drug development stage experiments due to occasional membrane leakage

FIGURE 12.2. Miniaturization of the ultracentrifugation format.

FIGURE 12.3. Automated liquid handling for the Thermo Scientifi c Pierce RED Device for Rapid Equilibrium Dialysis (Rockford, IL, USA).

REFERENCES 185

ACKNOWLEDGMENT

This chapter is dedicated to the memory of Joe McDe-vitt, who was the lead in this application area at Wyeth over the past decade and a guiding force.

REFERENCES

1. Kwong TC ( 1985 ) Free drug measurements: Methodology and clinical signifi cance . Clin Chim Acta 151 : 193 .

2. Levy RH , Moreland TA ( 1984 ) Rational for monitoring free drug level . Clin Pharmacokinet 9 ( Suppl 1 ): 1 .

3. Svensson CK , Woodruff MN , Baxter JG , Lalka D ( 1986 ) Free drug concentration monitoring in clinical practice: Rationale and current status . Clin Pharmacokinet 11 : 450 .

4. Tang Y , Zhu H , Zhang Y , Huang C ( 2006 ) Determination of human plasma protein binding of baicalin by ultrafi l-tration and high - performance liquid chromatography . Biomed Chromatogr 20 : 1116 – 1119 .

5. Greenblatt DJ , Sellers EM , Koch - Weser J ( 1982 ) Impor-tance of protein binding for the interpretation of serum and plasma drug concentrations . J Clin Pharmacol 22 : 259 – 263 .

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7. Schuhmacher J , Buhner K , Witt - Laido A ( 2000 ) Determi-nation of the free fraction and relative free fraction of drugs strongly bound to plasma proteins . J Pharm Sci 89 : 1008 – 1021 .

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comprehensively described by the concentration ratio of unbound drug in brain to blood, permeability clear-ance into the brain, and intrabrain distribution [71] .

All of these formats can be run with fairly high throughput utilizing automation to homogenize tissue samples in parallel with an isotonic solution (e.g., Auto-gizer™ , Tomtec ® , Hamden, CT) and to automate liquid handling steps (Figures 12.3 and 12.4 ). The effect of diluting tissue concentration as part of the homogeniza-tion and dialysis process was studied and found to be consistent up to at least a 20 - fold tissue dilution [35] .

There are allometric PK scaling software tools such as Simcyp ™ (Simcyp Ltd., Sheffi eld, UK) that apply not only plasma protein binding but also values for micro-somal and hepatocyte free fraction into the models pre-dicting human PK and pharmacodynamics from animal and in vitro data. These predictions include potential drug– drug interactions. Microsomal binding (specifi c, nonspecifi c, or a combination of both) may have a major effect on estimation of inhibitory potency of P450 inhib-itors [32] .

The extent to which a compound is bound to plasma proteins is a critical component to predicting how a potential drug will interact with its intended target invivo and with the clearance mechanisms of the organism [6] . The literature suggests that plasma protein binding controls the free drug concentrations in plasma and in those compartments in steady - state equilibrium with plasma. As such, it is a key factor in the translation of in vitro biochemical activity into in vivo pharmacologi-cal activity [11] .

FIGURE 12.4. Automated liquid handling for ultracentrifugation or equilibrium dialysis with plasma, microsomes, and homog-enized brain tissue using the Tecan Freedom (Tecan, Durham, NC, USA).

186 TECHNIQUES FOR DETERMINING PROTEIN BINDING IN DRUG DISCOVERY AND DEVELOPMENT

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