Methods

UNIT 7.5Determination of Compound Binding toPlasma ProteinsThe pharmacokinetic and pharmacodynamic properties of a compound are profoundlyaffected by the extent of its binding to plasma proteins. Consequently, the determinationof the plasma protein binding properties of a compound is essential during drug develop-ment and is increasingly required during lead prioritization.

The protocols described in this unit detail the techniques of ultrafiltration and equilibriumdialysis for determination of plasma protein binding. Equilibrium dialysis is the morerobust of the two techniques, whereas ultrafiltration (see Basic Protocol 1) is the morerapid. Two versions of the equilibrium dialysis technique are detailed in these protocols,the traditional approach (see Basic Protocol 2) and a higher throughput 96-well plateversion (see Alternate Protocol).

CAUTION: Human and animal plasma should be handled with appropriate care anddisposed of responsibly. Specifically, gloves, labcoats, and safety glasses should be worn,spills cleaned and sterilized promptly, and waste disposed of as appropriate for abiohazard.

CAUTION: Radioactive materials require special handling. Refer to the institutionalRadiation Safety Office for guidelines concerning proper handling and disposal.

BASICPROTOCOL 1

DETERMINATION OF PLASMA PROTEIN BINDING BYULTRAFILTRATION

The technique of ultrafiltration relies on centrifugal force to drive the unbound test article(compound of interest) through a size-selective membrane. A representation of the deviceis shown in Figure 7.5.1. Ideally, the degree of nonspecific binding of the test article tothe apparatus should be determined prior to investigating plasma protein binding. If thenonspecific binding exceeds 5%, ultrafiltration is not recommended for the determinationof plasma protein binding. An alternative technique such as equilibrium dialysis shouldbe investigated. It is not advisable to attempt to subtract nonspecific binding when

Supplement 18

Contributed by Natasha DowCurrent Protocols in Pharmacology (2002) 7.5.1-7.5.14Copyright © 2002 by John Wiley & Sons, Inc.

stopper

membranefilter

upper portion

lower portion

Figure 7.5.1 Micropartition device for determination of plasma protein binding by ultrafiltration.

7.5.1

Pharmacokinetics

determining plasma protein binding as the presence of plasma may alter its extent. Plasmafrom at least three donors should be pooled by species to ensure true representation of thespecies under investigation. Plasma may be stored frozen at −20°C prior to use.

Materials

Test article(s)Solvent (e.g., acetonitrile, methanol, DMSO)0.01 M phosphate-buffered saline (PBS), pH 7.4 (see recipe)Plasma samples from all species to be investigated, fresh or frozenSodium phosphate monobasic (NaH2PO4), solidSodium phosphate dibasic (Na2HPO4), solidFixed-angle rotorMicropartition devices containing membrane filters with a 10 to 30 kDa MWCO

(e.g., Centrifree; Millipore)

Additional reagents and equipment for liquid scintillation counting (LSC;radiolabeled test articles only), determining protein concentration (APPENDIX 3B),and HPLC or LC/MS analysis

Determine nonspecific binding (NSB)1. Set centrifuge and fixed-angle rotor to warm to 37°C.

This may require centrifugation of the rotor at a high centrifugal force.

2. Prepare a stock solution or solutions of the test article or articles in a suitable solvent(e.g., acetonitrile, methanol, DMSO) at concentrations 100 times those required forinvestigation. If a radiolabeled test article is used, dilute with unlabeled test articleto achieve a maximum radioactive concentration of 0.5 µCi/ml, thus conservingexpensive radiolabelled material and reducing disposal costs.

The preparation of the concentrated stock ensures that the maximum final solvent concen-tration is only 1% (v/v).

Typical concentrations for investigation are the anticipated maximum plasma concentra-tion (Cmax) and 10× Cmax, or 10 and 100 �M when Cmax is unknown. Preferentially thelowest concentration should be investigated to determine the maximal percentage nonspe-cific binding.

3. Aliquot 990 µl of 0.01 M PBS, pH 7.4, into two 2-ml microcentrifuge tubes (i.e.,duplicate) per concentration of each test article. Add 10 µl of 100× test article stocksolution to each. Vortex mix and incubate ∼20 min in an ∼37°C water bath.

Preparation of individual samples instead of a pool is preferred for loading of the Centrifreemicropartition units, as a Pasteur pipet can be used for transfer into the centrifuge tubesinstead of the wider pipet tip required for delivering a specific volume.

4. Following ∼20 min of incubation, remove the samples from the water bath. If the testarticle is radiolabeled, remove appropriate duplicate aliquots for determination of theactual dosed concentration by liquid scintillation counting (LSC). Using a long glassPasteur pipet, transfer remaining sample directly to the upper portion of a micropar-tition device containing a membrane filter with a 10 to 30 kDa MWCO (e.g.,Centrifree).

The 20 min incubation time allows the sample to reach physiological temperature and isconvenient when performing multiple batches, as a new set of incubations can be initiatedonce centrifugation of the previous set is started.

Take care not to introduce air bubbles while adding the sample to the unit. If an air bubbleis noted, gently tap the entire unit on the bench until the bubble is removed.

Supplement 18 Current Protocols in Pharmacology

7.5.2

Determination ofCompoundBinding to

Plasma Proteins

5. Centrifuge the samples 20 min at ∼1000 × g, 37°C.

6. Remove and appropriately discard the upper portion of the unit.

7. For radiolabelled test articles, remove duplicate aliquots of the ultrafiltrate (lowerportion) and determine the radioactive concentration by LSC. For nonlabeled testarticles, determine the test article concentration of the ultrafiltrate and of a 1:100 PBSdilution of the original 100× stock solution by an appropriate analytical technique.

Quantification against a standard curve is not strictly necessary as the percentage recoverycan be determined by comparison of the responses instead of absolute concentrations.

Ultrafiltrate portions may be capped and stored frozen prior to analysis. The temperatureand duration of storage depends on the stability of the test article. If the stability of the testarticle is not characterized, storage should be up to 3 months at −70°C.

8. Determine the percentage nonspecific binding (NSB) according to the equationbelow:

Prepare plasma for determination of plasma protein binding9. Defrost frozen plasma from all species to be investigated at room temperature until

thawed.

10. Centrifuge defrosted plasma 5 min at ∼2000 × g, room temperature. Decant and retainthe supernatant.

Freeze-thaw of plasma can produce a fibrous precipitate.

11. Pool plasma by species and measure pH.

12. Adjust the pH to 7.4 by adding small amounts (i.e., a few grains) of either solidNaH2PO4 (decrease pH) or Na2HPO4 (increase pH).

Variation in plasma pH can dramatically affect the extent of plasma protein binding of atest article, as exemplified by propranolol (Paxton and Calder, 1983).

13. Retain an aliquot of each plasma sample for determination of total protein concen-tration (APPENDIX 3B) and if required, specific plasma protein concentrations.

This step is to confirm that the plasma used contains protein concentrations representativeof the species and strain. This can be achieved using a clinical chemistry analyzer such asthe Hitachi 912 and a suitable diagnostic kit (e.g., JAS Diagnostics). Note that themethodology is not described in this unit.

Determine plasma protein binding14. Determine plasma protein binding in the same manner as described for determination

of nonspecific binding, replacing PBS with pooled plasma (see steps 1 to 6).

15. Calculate the percentage plasma protein binding according to the equation:

concentration in ultrafiltrateNSB(%) =100 100

concentration added

− ×

concentration in ultrafiltrateplasma protein binding (%) 100 100

concentration added

= − ×

Current Protocols in Pharmacology Supplement 18

7.5.3

Pharmacokinetics

16. If required, subject a portion of the ultrafiltrate to analysis by HPLC or liquidchromatography/mass spectroscopy (LC/MS) to confirm the identity of the freefraction. This step is particularly important if the test article contains impurities, isprone to degradation (or if the plasma stability is unknown), and/or is highly proteinbound.

The volume of ultrafiltrate should not exceed 40% of the initial volume to avoid dissociationof bound compound due to removal of the free compound (Whitlam and Brown, 1981).

BASICPROTOCOL 2

DETERMINATION OF PLASMA PROTEIN BINDING BY TRADITIONALEQUILIBRIUM DIALYSIS

The technique of equilibrium dialysis is conducted with two Teflon cells, one containingplasma and the other an equal volume of buffer, separated by a size-selective membrane.The unbound test article is allowed to reach equilibrium between the two compartments.Thus, measurement of the test article concentration in each compartment allows thedegree of plasma protein binding to be calculated.

Equilibrium dialysis is theoretically the most accurate method for determination ofplasma protein binding because the equilibrium is not shifted when aliquots are takenfrom either side of the membrane (Sebille et al., 1990). Some advantages of equilibriumdialysis are that reproducible results can be obtained even for low affinity compounds,and that meaningful results can be determined even for compounds bound nonspecificallyto the apparatus. Disadvantages of equilibrium dialysis include degradation of thecompound due to the duration of exposure at 37°C, and volume shifts due to osmoticequilibrium. It is recommended that the equilibration time be determined in the absenceof plasma to ease analysis, preserve plasma, allow determination of nonspecific binding,and to simply measure the time required for equilibration of free drug between compart-ments, avoiding any complication presented by protein interactions.

Materials

PBS, pH 7.4 (see recipe)Test article or articlesSolvent (e.g., acetonitrile, methanol, DMSO)Plasma samples from all species to be investigated, either fresh or frozenSodium phosphate monobasic (NaH2PO4), solidSodium phosphate dibasic (Na2HPO4), solid

47-mm-diameter precut Spectra/Por 4 membrane discs with 12 to 14 kDa MWCO(Spectrum Laboratories)

Spectra/Por equilibrium dialyzer with stopper plugs and semi-micro Teflon cells(Spectrum Laboratories) labeled 1 to 20 in indelible ink

37°C acrylic water bath (e.g., Spectrum Laboratories or equivalent)Blunt-nose 22-G needles and syringes

Determine equilibration time and NSB1. For each concentration of test article, soak ten 47-mm-diameter precut Spectra/Por

4 membrane discs with 12 to 14 kDa MWCO in a dish of room-temperature deionizedwater for at least 30 min. Replace the water with PBS, pH 7.4, and incubate a further30 min.

This step is required to remove preservatives and impurities in the membrane discs and toequilibrate them to dialysis conditions. The dish used for soaking should be sufficientlylarge to allow the membranes to soak as a single layer.


7.5.4


Plasma Proteins

2. Prepare a 100× stock solution or solutions of test article or articles in suitable solventas described for ultrafiltration investigations (see Basic Protocol 1, step 2).

3. Add 120 µl each test article stock solution to 11.88 ml PBS and vortex mix to preparedosing solutions.

4. Assemble the first set of five semi-micro Teflon cells of the Spectra/Por equilibriumdialyzer according to the manufacturer’s instructions, placing the larger half of eachcell on the bottom of the guide rod and aligning the filling ports. Plug the single porton each cell half with the supplied stopper. Maintain temperature with a 37°C acrylicwater bath.

5. Using a blunt-nose 22-G needle and syringe, fill the larger half of each cell with 1 mlPBS through one of the double filling ports.

This cell half will be referred to as the buffer compartment.

6. Similarly, fill the other cell half with the appropriate PBS solution spiked with testarticle (step 3).

This cell half will be referred to as the dosing compartment.

7. Stopper all filling ports and place the complete cell set in the drive unit maintainedin the temperature controlled environment (37°C). Set the drive unit to 20 rpm andobserve to ensure the stoppers remain in place during rotation. Record the start timeas when the rotation commences.

8. Repeat steps 4 to 7 as necessary, such that 10 cells are prepared for each concentrationof each test article.

9. Following 1, 2, 4, 6, and 20 hr of dialysis, completely remove the liquid from bothcompartments of two cells per test article concentration, using blunt-nosed 22-Gneedles and syringes through the single port. Use a different filling port to removethe sample than the one used to add the sample, thus avoiding contamination. Removea stopper from both cell halves to allow air flow and successful sample removal.

10. Determine the concentration of test article in both compartments and in the PBSdosing solution by an appropriate method, such as liquid scintillation counting (LSC)for radiolabeled test articles or LC/MS for nonlabeled test articles.

11. Plot the concentration of test article in each compartment against duration of dialysisand determine the time at which equilibrium is achieved.

12. Calculate the percentage nonspecific binding (NSB) at equilibration time accordingto the equation:

where dose concentration is the test article concentration (e.g., disintegrations perminute, nanomoles, micrograms) expressed per milliliter and amount recoveredequals the concentration in the dosing compartment plus the concentration in buffercompartment.

This equation is valid since the compartment volumes are 1 ml.

��

��

− = − ×


7.5.5

Pharmacokinetics

Determination of plasma protein binding13. For each concentration of each test article, soak two Spectra/Por 4 membrane discs

in a dish of deionized water followed by PBS buffer, as described for the nonspecificbinding determination (step 1).

14. Prepare test article stock solutions as described for ultrafiltration investigations (seeBasic Protocol 1, step 2).

15. As described for ultrafiltration (see Basic Protocol 1, steps 9 to 13), thaw (ifnecessary), centrifuge, and pool plasma samples from all species to be tested. Adjustthe pH of the plasma for investigation and retain a portion for determination of totalprotein content.

16. Add 25 µl of each 100× test article stock solution to 2.475 ml plasma and vortex tomix.

17. Set up the equilibrium dialysis equipment as described in steps 4 to 8, such that twocells are prepared for each test article concentration per species. Note the identity ofeach cell.

18. Following the duration of dialysis previously determined as necessary to attainequilibrium (step 11), remove both the dosing and buffer compartments. Note thevolume recovered from each compartment and record any variation >15%.

If a >15% variation in volume recovered from each compartment is noted, the volume shiftmay affect the final results. The experiment should be repeated, measuring the finalcompartment volumes and accounting for any differences when calculating percentagebound (Tozer et al., 1983).

19. Determine the concentration of test article in both compartments and in the plasmadosing solution by an appropriate method.

If the test article is radiolabeled, aliquots of plasma and buffer samples should be subjectedto LSC to determine the test article concentration.

If the test article is not radiolabeled, any plasma samples should be extracted with a suitablesolvent (such as 1 vol acetonitrile) to precipitate and remove the protein prior to analysisby HPLC with UV, fluorescence, or MS detection. The efficiency of this extraction must beconfirmed, by comparison of the response of an extracted plasma curve to that of aprotein-free buffer curve treated in the same manner.

20a. For reactions not requiring compensation for volume change: Calculate the percent-age recovery and plasma protein binding not accounting for volume changesaccording to the following expressions:

Where Cpe is the test article concentration in the plasma compartment after dialysis,Cbe is its concentration in the buffer compartment after dialysis, and Cpi is itsconcentration in plasma compartment prior to dialysis.

��

��

− ×

��

��×


7.5.6


Plasma Proteins

20b. For reactions in which volume change must be accounted: Calculate the percentagerecovery and plasma protein binding, this time accounting for volume changesaccording to the following expressions:

Where Vpe is the volume in the plasma compartment after dialysis, Vbe is the volumeof the buffer compartment after dialysis, and Vpi is the volume of plasma loaded intocell (1 ml). All volumes are expressed in milliliters and all concentrations areexpressed in units per milliliter (e.g., disintegrations per minute, nanomoles, micro-grams per milliliter).

If the concentration of test article in the buffer compartment is below the limit of detection,the minimum percentage protein binding may be estimated by substituting the limit ofdetection in place of the buffer compartment concentration.

22. If required, analyze a portion of the buffer compartment to determine the nature ofthe unbound material.

ALTERNATEPROTOCOL

DETERMINATION OF PLASMA PROTEIN BINDING BY 96-WELL PLATEEQUILIBRIUM DIALYSYS

The Dialyzer-96 is a 96-well plate with each well bisected horizontally by a size-selectivemembrane. The plate allows multiple equilibrium dialysis investigations to be performedsimultaneously and utilizes less plasma. The determination of equilibration time whenmultiple compounds are investigated on the plate is impractical, thus this step is omittedand the dialysis performed over-night to ensure equilibration is achieved. This method-ology is not recommended for precise determination of plasma protein binding ofcompounds in development, but is useful during compound screening and selection. Notethat plate membranes are compromised upon exposure to liquid and thus cannot bere-used.

Additional Materials (also see Basic Protocol 2)

Equilibrium Dialyzer-96 plates with 10-kDa MWCO membrane (HarvardApparatus)

1. Prepare the test article stock solutions and pooled human plasma as described (seeBasic Protocol 1, step 2 and steps 9 to 13, respectively).

2. Spike 990 µl pooled plasma, pH adjusted as described (see Basic Protocol 1, step 14),with 10 µl each 100× test article stock solution. Prepare spiked PBS in the samefashion.

3. Aliquot 0.25-ml portions of the spiked plasma and PBS into duplicate wells of anEquilibrium Dialyzer-96 plate with 10-kDa MWCO membrane. Cap the plasmacompartments.

(Cpe Cbe) (Vpe/ Vpi)protein binding (%) = 100

[(Cpe Cbe) (Vpe/ Vpi)] + Cbe

− × ×− ×

(Cpe Vpe) + (Cbe Vbe)recovery (%) = 100

(Cpi Vpi)

× × ××


7.5.7

Pharmacokinetics

4. Invert the plate and aliquot 0.25 ml PBS into the underside of all wells to beinvestigated. Cap the buffer compartments.

5. Incubate at ∼37°C with shaking, as per manufacturer’s instructions.

6. Following ∼20 hr of incubation, remove the plasma and buffer compartments to clean96-well plates with a multichannel pipet.

7. Determine the concentration of test article in both compartments as previouslydescribed.

8. Calculate the percentage recovery and percentage protein binding according to theequations detailed previously (see Basic Protocol 2, step 20a).

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, seeAPPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Phosphate-buffered saline (PBS), 0.01 M, pH 7.4Dissolve the required number of phosphate-buffered-saline tablets (Sigma) inHPLC-grade water to achieve 0.01 M phosphate buffer, pH 7.4/0.0027 M potassiumchloride/0.137 M sodium chloride. Store up to 3 months at ∼4°C.

COMMENTARY

Background InformationThe determination of the plasma protein

binding of a compound is critical for interpre-tation of the associated pharmacodynamic,pharmacokinetic, and toxicity profiles. It isgenerally accepted that the pharmacologicaleffects of a drug are regulated by the unbound(free) concentration in plasma. This view fol-lows the assumption that the unbound com-pound in plasma is in equilibrium with that inthe extravascular regions where the drug targetis situated (Sansom and Evans, 1995). Theeffect of the fraction of unbound drug on thepharmacokinetic profile of a compound is in-fluenced by many parameters, such as thephysicochemical properties of the compoundand the extraction ratios of the metabolizingorgans. Typical effects are summarized as fol-lows (McEnlay and D’Arcy, 1983):1. Absorption is favored if a compound ishighly bound due to the large concentrationgradient between the free compound at theabsorption site and in the plasma.2. Drug distribution is decreased if a com-pound is highly protein bound, since bounddrug is not available for excretion into thetissues, leading to a low apparent volume ofdistribution.3. In the absence of extensive first pass meta-bolism or active renal clearance, clearance isdecreased if a compound is highly proteinbound.

4. Saturation of binding sites at increaseddoses will increase the concentration of un-bound drug and may result in nonlinear phar-macokinetics.

The plasma proteins typically involved inthe binding of drug molecules are albumin andα-1 acid glycoprotein. Albumin is the mostabundant plasma protein (concentration in nor-mal human plasma is ∼0.6 mM) with severalbinding sites and accounts for binding of bothacidic and basic compounds. α-1 acid glyco-protein binds primarily basic compounds andis present at a concentration of approximatelybetween 10 and 25 µM in normal humanplasma (Sansom and Evans, 1995). Both iso-lated human proteins are commercially avail-able, allowing binding parameters to be deter-mined.

The concentration, structure, and functionof human plasma proteins may be altered ac-cording to age and disease, resulting in a changein the degree of binding of compounds (Lindupand Orme, 1981). Binding can also be affectedby the presence of competing endogenous com-pounds (Sansom and Evans, 1995). In additionto these inherent variations, the presence ofanother drug may affect plasma protein bind-ing. While the clinical implications of proteinbinding interactions are rarely significant, it isprudent to investigate the potential for interac-tion in vitro and analyze the findings in thecontext of clearance and therapeutic index (the


7.5.8


Plasma Proteins

ratio of the effective dose compared to the toxicdose). An algorithm for determining the clinicalsignificance of potential protein binding dis-placement interactions is presented in Figure7.5.2 (Rolan, 1994).

The protocols described in this unit providesimple methods for determining the binding ofa compound to plasma or serum proteins fromthe full range of toxicological species and man,as well as to isolated proteins. Any of thedescribed techniques can also be used to deter-mine binding parameters and to investigate thepotential for binding interactions with co-ad-ministered compounds. A useful explanation ofthe theory and limitations of equilibrium dialy-sis and ultrafiltration is presented in Sebille etal. (1990).

Critical Parameters

UltrafiltrationThe technique of ultrafiltration (see Basic

Protocol 1) determines only the unbound testarticle concentration. The remaining test articleis assumed to be bound to plasma proteins, not

the apparatus. Thus, the presence of nonspecificbinding will lead to an overestimation of thepercent bound to plasma proteins. It is notadvisable to simply subtract the nonspecificbinding determined in the absence of plasmafrom the percentage plasma protein bound asthe presence of the plasma may affect the affin-ity of the test article for the apparatus. If thenonspecific binding is found to be >5%, analternative technique should be used, such asequilibrium dialysis.

The aim of in vitro measurement is to predictthe situation in the human body following adose of the test article. As such, the experimentshould mimic physiological conditions asclosely as possible. Ensure that the centrifugeand rotor are at 37°C prior to spinning ultrafil-tration units and adjust the pH of the plasma topH 7.4. Variations from these physiologicalconditions may lead to variable and unpre-dictive results, as exemplified by propranolol(Paxton and Calder, 1983).

Ultrafiltration relies on smooth transition ofthe plasma water through the membrane. Theexistence of an air pocket caused by air bubbles

Table 7.5.1 Troubleshooting Guide to Plasma Protein Binding Experiments

Problem Possible cause Solution

Low percentage recovery Excessive nonspecific bindingto the equipment

Investigate alternative methodof determination. Equilibriumdialysis is the method ofpreference for compoundswhich tend to bindnonspecifically.

Degradation of test articleduring experiment

Minimize incubation times. Ifusing equilibrium dialysis

Poor extraction from plasma(equilibrium dialysis)

Investigate alternativeextraction procedures

Deviation from literaturevalues

Alteration in plasma pH Carefully adjust plasma pH to7.4 prior to spiking

Atypical protein concentrationin plasma

Repeat experiment with adifferent batch of plasma

Volume shift duringequilibrium dialysis

Account for volume change incalculations

Compromised membrane orfilter

Inspect membrane or filterand replace if necessary

High variation in results Problems with analyticaltechnique

Optimize analytical technique

Poor mixing of dosing stock Repeat

Equilibrium not achieved(equilibrium dialysis)

Confirm equilibration atdialysis time selected

Compromised membrane orfilter

Inspect membrane or filterand repeat if necessary


7.5.9

Pharmacokinetics

Table 7.5.2 Percentage Binding of a Test Article to Plasma Proteins from Various Species

ReplicateMean protein binding at10 µM (%)

Mean protein binding at100 µM (%)

Buffera

1 5.0 4.02 4.0 1.0Mean 4.5 2.5Mouse

1 69 632 88 68Mean 88 66Dog

1 87 742 84 75Mean 86 75Monkey

1 83 672 85 67Mean 84 67Human

1 81 772 82 77Mean 82 77aPercentage nonspecific binding.

Table 7.5.3 Percentage Binding of a Test Article to Plasma Proteins from Various Speciesa

ReplicateProtein bound at10 µM (%)

Protein bound at50 µM (%)

Recovery at10 µM (%)

Recovery at50 µM (%)

Bufferb

1 NA NA 77 842 NA NA 79 85Mean NA NA 78 85Rat

1 99.9 99.2 100 932 99.6 98.7 100 90Mean 99.7 98.9 100 92Dog

1 98.5 97.6 99 1002 98.4 97.1 100 100Mean 98.5 97.4 100 100Human

1 99.6 98.9 83 1002 99.8 98.2 86 100Mean 99.7 98.5 85 100aAbbreviations: NA, not applicable.bPercentage nonspecific binding.


7.5.10


Plasma Proteins

Is drug interest >90%protein bound

Does the drug have anarrow therapeutic index

What is the hepaticextraction ratio of the drug

Would a transient increasein free drug concentration

be clinically relevant

Clinically significantinteraction not likely

Is the drug given i.v.

Clinically significantinteraction likely.

Perform a clinical studyto quantify effects.

yes

yes

yes

yes

high

low no

no

no

no

Figure 7.5.2 Algorithm for determining the clinical significance of potential protein binding dis-placement interactions.

Pro

tein

bin

ding

(%

)

0

10

20

30

40

50

60

70

80

90

100

Buffer Mouse Rat Dog Monkey Human

Figure 7.5.3 Mean percentage binding of propranolol to plasma proteins from various speciesand nonspecific binding (NSB) to the ultrafiltration apparatus (buffer). Doses are 1 (light gray) and10 µg/µl (dark gray).


7.5.11

Pharmacokinetics

in the unit will hinder this transition. Removeall air bubbles from the ultrafiltration units priorto centrifugation to ensure accuracy.

The production of an ultrafiltrate volume>40% of the plasma volume may cause thebound compound to dissociate due to a shift inthe equilibrium, leading to an underestimationof the percent protein bound (Whitlam andBrown, 1981). If such a large ultrafiltrate vol-ume is observed, the procedure should be re-peated, adjusting the centrifugal force as appro-priate.

The use of radiolabeled test articles easesthe analysis of the ultrafiltrate considerably;however, any test article-related impurities,metabolites, or degradants containing the radi-olabel would be quantified as the test article,leading to an underestimation of the percent-age bound. Thus the purity of the free com-pound should be confirmed by LC/UV orLC/MS to confirm that it is indeed the testarticle that is failing to bind to the plasmaproteins.

Equilibrium dialysisThe dialysis membranes contain preserv-

atives that may interfere with the binding oranalysis of the compounds. Thus, they shouldbe soaked as described in the protocol to equili-brate them to dialysis conditions.

The aim of the in vitro measurement is topredict the situation in the human body follow-ing a dose of the test article, as such, conditionsduring the experiment should mimic physi-ological conditions as closely as possible. Thus,adjust the pH of the plasma to pH 7.4 and ensurethat the dialysis is performed at 73°C. Vari-ations from these physiological conditions maylead to variable and unpredictive results, asexemplified by propranolol (Paxton and Cal-der, 1983).

The aim of the equilibrium dialysis is toreach conditions at which the bound and freetest article are in equilibrium. If the compart-ments are removed prior to reaching equilibra-tion, the percent protein bound will be overes-timated. Thus, the dialysis should be performedfor at least the equilibration time determined inthe initial experiment.

Analysis of the plasma compartment byLC/UV or LC/MS requires extraction from theprotein-rich matrix. If the extraction of the testarticle is variable or incomplete, the calculatedtest article concentration and thus the percentprotein binding will be incorrect. The efficiencyof any plasma extraction should be >90% witha variation of <5% to be acceptable.

A >15% variation in volume recoveredfrom each compartment may affect the finalresults. The experiment should be repeated,measuring the final compartment volumes andaccounting for any differences when calculat-ing percent bound (Tozer et al., 1983).

The use of radiolabeled test articles easesthe analysis of the buffer compartment consid-erably; however, any test article-related impu-rities, metabolites, or degradants containing theradiolabel would be quantified as the test arti-cle. This can lead to an underestimation of thepercent bound. Thus, the purity of the freecompound should be confirmed by LC/UV orLC/MS to confirm that it indeed the test articlethat is failing to bind to the plasma proteins.

Table 7.5.1 presents some common prob-lems with the assays detailed in this unit, alongwith their possible causes and solutions.

Anticipated ResultsThe degree of binding of compounds to

plasma proteins varies greatly—i.e., anywherebetween <1% and >99%. Representative re-sults are presented in this section.

UltrafiltrationThe degree of binding of the representative

compound propranolol to mouse, rat, dog,monkey, and human plasma proteins, and to theultrafiltration equipment is presented in Table7.5.2 and depicted graphically in Figure 7.5.3.The low degree of nonspecific binding to theultrafiltration units (listed as “buffer” in thetable and figure) confirms that the technique ofultrafiltration is suitable for this compound.Binding affinity of propranolol to proteins inmouse, rat, dog, monkey, and human plasma ismoderately high. The decreased percentageprotein binding at the higher propranolol con-centrations (in all but mouse plasma) suggestssaturation of the binding sites.

Equilibrium dialysisThe equilibration profile of a proprietary test

article during dialysis is depicted in Figure7.5.4. The dialysis time subsequently selectedfor determination of plasma protein binding ofthis test article was 4 hr to guarantee that equili-bration was achieved.

Example plasma protein binding resultsgenerated by equilibrium dialysis are presentedin Table 7.5.3 and depicted in Figure 7.5.5.These results illustrate the advantages of bothcompartments being readily available foranalysis following equilibrium dialysis. Thecompound recovery in the absence of plasma


7.5.12


Plasma Proteins

is only 78% at 10 µM and 85% at 50 µM,suggesting that nonspecific binding would bean issue when determining plasma proteinbinding. However, in the presence of plasmathe compound recovery is much improved, theexception being in the presence of humanplasma at 10 µM (Table 7.5.3). The percentageprotein binding determined with the reducedrecovery in human plasma is still meaningful,although the actual concentration of test articlerepresented is 15% of nominal.

Time Considerations

UltrafiltrationThe number of samples that can be investi-

gated in one day depends on the capacity of thecentrifuge rotor (note that only a single concen-tric circle should be used in order to standardizethe centrifugal force on each sample). If thenumber of samples required to fill the rotor isconsidered a batch, then a single batch requires∼1 hr to process to the analytical stage. Ifincubation of additional batches is initiatedonce centrifugation of the previous batch has

0

10

20

30

40

50

60

70

80

90

100

Rat Dog Human

Pro

tein

bin

ding

(%

)

Figure 7.5.5 Mean percentage binding of a test article to plasma proteins from various speciesas determined by equilibrium analysis. Doses are 10 (light gray) and 50 µM (dark gray).

00 1 2 3 4 5 6 7

20

40

60

80

100

120

140

160

Test

art

icle

con

cent

ratio

n (n

g/m

l)

Duration of dialysis (hr)

Figure 7.5.4 Equilibration time of a test article during dialysis of fortified PBS. Concentration ofarticle is measured in both the dosing (circles) and buffer (triangles) compartments.


7.5.13

Pharmacokinetics

commenced, the time required per batch isreduced by 30 min. Nonspecific binding of thetest article to the equipment should be deter-mined prior to protein binding determinations.

Traditional equilibrium dialysisThe initial nonspecific binding and equili-

bration time determination requires 1 hr forset-up and 20 min at each sampling point. Eachdialyzer has a capacity of 20 cells, thus two testarticles can be investigated utilizing a singledialyzer (each test article investigated at fivetime points in duplicate).

The total time required for the plasma pro-tein binding determination is dependent on therequired equilibration time. The set up, includ-ing test article formulation and plasma spiking,requires ∼2 hr, while terminal sampling re-quires ∼1 hr.

96-well-plate equilibrium dialysisSet-up of the 96-well plate requires ∼2 hr

including spiking of the plasma. The dialysislasts 20 hr and terminal sampling requires only20 min.

Literature CitedLindup, W.E. and Orme, M.C. 1981. Plasma protein

binding of drugs. Br. Med. J. 282:212-214.

McEnlay, J.C. and D’Arcy, P.F. 1983. Protein bind-ing displacement interactions and their clinicalimportance. Drugs 25:495-513.

Paxton, J.W and Calder, R.L. 1983. Propranololbinding in serum: Comparison of methods andinvestigation of effects of drug concentration,pH, and temperature. J. Pharmacol. Meth. 10:1-11.

Rolan, P.E. 1994. Plasma protein binding displace-ment interactions – why are they still regarded asclinically important? Br. J. Clin. Pharmacol.37:125-128.

Sansom, L.N. and Evans, A.M. 1995. What is thetrue clinical significance of plasma protein bind-ing displacement interactions? Drug Safety12:227-233.

Sebille, B., Zini, R., Madjar, C.V., Thaud, N., andTillement, J.P. 1990. Separation procedures usedto reveal and follow drug-protein binding. J.Chromatogr. 531:51-77.

Tozer, T.N., Gambertoglio, J.G., Furst, D.E., Avery,D.S., and Holford, N.H.G. 1983. Volume shiftsand protein binding estimates using equilibriumdialysis: Application to prednisolone binding inhumans. J. Pharm. Sci. 72:1442-1446.

Whitlam, J.B. and Brown, K.F. 1981. Ultrafiltrationin serum protein binding determinations. J.Pharm. Sci. 70:146-150.

Contributed by Natasha DowRicerca LLCConcord, Ohio


7.5.14


Plasma Proteins

Methods

Documents

Transcript of Methods