CONFIDENTIAL

Peter KilfordPeter KilfordStudy Director, Drug MetabolismStudy Director, Drug Metabolism

Harrogate, UKHarrogate, UK

Plasma Protein Binding: Regulatory Plasma Protein Binding: Regulatory Expectations, Special Considerations and Expectations, Special Considerations and

Translation to Clinical Development Translation to Clinical Development

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Background

Plasma protein binding

– Physico-chemical interaction between a drug and plasma proteins

– Most common proteins in plasma are albumin and alpha 1-acid glycoprotein

– Drugs generally bind to plasma proteins in a reversible manner

Protein + Drug Protein-drug complex

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Background

• Protein binding methods are well documented– Benefits of high throughput equilibrium dialysis systems (HT

dialysis and RED) are well known [1,2]

• Protein binding is often thought of as an “easy, quick win” study with little or no challenges associated with experiments

• Recently, we have seen increased demand for these studies at Covance:– Determination of accurate binding values for highly bound

unlabelled drugs

– Need for accurate protein binding values to support late stage development of a drug

[1] Banker et al. 2003, J. Pharm Sci: [2] Waters et al. 2008, J. Pharm Sci

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Regulatory GuidelinesICH M3 (R2) 2009

Before initiating human trials:- “in vitro” plasma protein binding data for

animals and humans…should be evaluated”

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10

PPB - Techniques

3 main approaches used to determine PPB:– Equilibrium Dialysis

• Spectrum/Dianorm• Harvard 96-well dialysis• HTdialysis• Rapid Equilibrium Dialysis (RED)

– Ultrafiltration• Centrifree tubes etc.

– Ultracentrifugation

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Methodology – Equilibrium Dialysis

Donor (Plasma) Acceptor (Buffer)

Protein + Drug Free drug

Membrane

Advantages

- Easy to perform- Inexpensive- Generally low non-specific

binding (NSB)

Disadvantages

- Drug insolubility in dialysate- Potentially longer dialysis times

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Methodology - Ultrafiltration

Plasma chamber

Membrane

Ultra-filtrate chamber

Centrifugation

Advantages

– Quick and easy to perform– Inexpensive

Disadvantages

– Nonspecific binding (NSB) is common

– May underestimate fu for highly bound drugs

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Traditional Technique

No longer preferred approach

Disadvantages

– Equilibration time (4–16 hours)– Potential for volume shifts

(differences in osmotic pressure)– Setup is time consuming– Limited to 20 dialysis samples / kit– Require relatively large volumes of

plasma

Advantages

– Low Nonspecific Binding (made with Teflon)

– Relatively easy to use

Conventional Equilibrium Dialysis

Spectrum / Dianorm apparatus

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Preferred Technique

Novel Teflon base plate with disposable dialysis cells

• Increased surface area to volume ratio (compared to other ED approaches)

Disadvantages

– Low sample volume (radiolabel)– Challenging for radiolabeled

approaches due to lower sample volumes

Advantages

– Shorter incubation times (2–4 hrs)

– Low Nonspecific Binding

– Easy to use

RED (Rapid Equilibrium Dialysis)

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Standard PPB Experiment ConsiderationsDrug Candidate Plasma StabilityIn All Species≥ 6 hours?

Data Output:% Bound% Free

% Recovered

Equilibrium Time, 1 species 

(e.g. human)

YesYes

YesYes

Can you stabilize?

NoNo

Use ultrafiltrationw/ short spin time

NoNo

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Standard PPB Experiment Compound Requirements

Radiolabelled Compound

– Radiolabel should have sufficient purity (≥98% preferably)

– Are detection methods sensitive enough to allow determination of low free fractions (<1% free)?

– Analysis by LSC and radio HPLC

Non-radiolabelled Compound

– Purity not an issue

– Need to decide on LC/MS method - fully validated or qualified bioanalyticalapproach

– Additional cost implications

– Excellent sensitivity for measuring low levels of free fraction (<1%)

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Standard PPB Experiment LC-MS/MS Analytical Methods

Validated

– Characterization as defined in the regulatory guidance

– Provides absolute analyteconcentration

– Acceptance Criteria ± 15%

– Short & long term stability

– Assess selectivity in matrix from all species

Qualified

– Limited characterization

– Calibration and Quality Control samples prepared using authentic reference standard

– Provides absolute analyteconcentration

– Broader Acceptance Criteria (± 15-20%)

– Short term stability

– Selectivity in one matrix batch blank

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PPB Approaches Throughout Drug Discovery & Development

Screening

Methods

or

Non-qualified

Methods

Discovery PIPreclinical PII PIII Post Approval

Non-GLP Clinical, GLP and Non-GLP

Validated MethodsRodent & Non-rodent Plasma (Unchanged Drug)

Other matrices as appropriate

Metabolites as appropriate?

Human Plasma

Other matrices as appropriate

Metabolites as appropriate?

Additional preclinical species

Other matrices as appropriate

Metabolites as appropriate?

Qualified MethodsSamples from:

• in vitro (e.g. PPB)

• Some mechanistic PK, PK/PD

• Most non-standard matrices (e.g. tissues)

Quantification of metabolites

Image adapted from EBF Presentation at AAPS Nov 2009, courtesy of EBF

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The Relevance of Experimental Conditions

• Investigated a number of different conditions

• However, will focus on control of pH in this section

• pH control either by:

– Increased buffer strength

– Use of CO2 incubator

• Recently, we undertook some work to investigate the change in pH over time in a range of species

• We also further assessed the unbound fraction under different conditions

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pH Control for PPB ExperimentsChange in plasma pH over time

Mouse

Dog

Rat

Rabbit

Human

Time (hours)0 2 4 6

pH

7

7.2

7.4

7.6

7.8

8

8.2

8.4

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Change in plasma pH in presence of 5% CO2

pH Control for PPB Experiments

Mouse

DogRat

RabbitHuman

Time (hours)0 2 4 6

pH

7

7.2

7.4

7.6

7.8

8

8.2

8.4

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pH Control for PPB ExperimentsEffect of pH on Fraction Unbound (Rat)

Testosterone Warfarin Caffeine

Fold

cha

nge

fu +

CO

2 / f

u

0.8

1.0

1.2

1.4

1.6

1.8

2.0

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pH Control for PPB Experiments

• Potential to over estimate binding if pH not controlled

• Recommend that PPB incubations are conducted in a 5% CO2 environment

Summary of findings regarding pH

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Applications for Ex Vivo Samples

• Typically, ex vivo plasma samples are obtained from patients enrolled in clinical studies

– Often from healthy or disease state populations

• Guidance documents detail the expectations & criteria to assess PPB in these ex vivo samples

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Regulatory Guidelines

PK in Patients with Impaired Hepatic Function 2003Fraction unbound assessed in hepaticallyimpaired subjects when compounds

- highly cleared by liver (ER >0.7)- Extensively protein bound (>90%)

PK in Patients with Impaired Hepatic Function 2003Fraction unbound assessed in hepaticallyimpaired subjects when compounds

- highly cleared by liver (ER >0.7)- Extensively protein bound (>90%)- -Measure fu in all subjects (or at least peak and

trough)

PK in Patients with Impaired Renal Function 2010

- Similar to hepatic but not consistent. Fraction unbound assessed

- Extensively protein bound (>80%)- If binding is independent of drug concentration

then single samples may be assessed

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Regulatory GuidelinesPK in Patients with Impaired Hepatic

Function 2005Drug has high protein binding then PK should be described and analysed as unbound drug- General agreement with FDA- Measure fu in all subjects (or at least peak and trough)

PK in Patients with Impaired Renal Function 2004

- Similar to FDA but does not indicate threshold of PPB

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Overall Regulatory Guide• FDA and EMA recommend measurement of unbound concentration

for ex vivo PK studies with renally and hepatically impaired subjects

– Generally measure fu at each time point

– But there are exceptions to this which should be considered when designing experiments

• For other populations (e.g. paediatrics) then an in vitro study may be adequate

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• How do we measure fu in these samples?– Similar to in vitro plasma samples (pre- or post- dose plasma)– Recommend ED in 5% CO2 environment to control pH– Analysis via LC-MS/MS

• What is rationale for measuring fu in diseased populations?– Concentrations of albumin and 1-acid glycoprotein can vary by

up to 50%– These differences can result in altered free fractions– Changes in free fraction may cause subsequent unpredictable

changes in total and unbound exposure• What is the clinical relevance of measuring fu?

– Can assess whether or not dose adjustment is necessary– Can help with interpretation of PK data

Applications for Ex Vivo Samples

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Increase in fu -

Decrease in AUC but

AUCu stays the same.

Result = no clinical significance

Effect of Changes in fu

int

gabs

CL fuDose F F AUC

int

gabsu

CLDose F F AUC

fu AUC AUCu

Oral administration and hepatic clearance

Example 1

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When fu May Be Clinically Relevant

NoYes†Non-Hepatic Cl

NoNoHepatic Cl

PO Administration

NoYes*Non-Hepatic Cl

NoYes*Hepatic Cl

IV Administration

Low Extraction

Ratio

High Extraction

Ratio

* 25/456 drugs meet this criteria† 0/456 drugs meet this criteria

Adapted from Benet and Hoener, 2002, Clin Pharmacol Ther

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Effect of Changes in fu

int

gabs

CL fuDose F F AUC

int

gabsu

CLDose F F AUC

fu AUC AUCu

Oral administration and hepatic clearance

Example 2

Increase in fu + Decrease CLint

AUC stays same but

AUCu increases

Result = possible clinical impact e.g. Salicylate

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Salicylate Kinetics in Hepatic Impairment

[ Total Plasma ] [ Unbound Plasma ]

AUCu

fu

Clearance

Roberts et al. 1983, Eur J Clin Pharmacol

(fu)

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• Plasma samples received from hepatic impaired patients• fraction unbound (fu)

– Normal patients = 0.0045 ± 0.0003 – Severe Hepatic impairment = 0.0109 ± 0.0012 – Approximate 2-fold increase in fu in hepatic-impaired population

PK data– Oral administration– Drug had high hepatic extraction and large Vd– Total exposure (AUC) and Cmax decreased

• Normal = 0.0760 ng/mL• Hepatic-impaired = 0.0392 ng/mL

– t½ prolonged in hepatic-impaired population• Normal = 51.4 hr• Hepatic-impaired = 81.8 hr

Case Study – Drug X

int

gabs

CL fuDose F F AUC

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Case Study – Drug X• Nevertheless, total unbound exposure to drug X did not change

– Normal, AUCu = 3.36 ng·hr/mL– Hepatic-impaired, AUCu = 3.49 ng·hr/mL

• fu has no effect on unbound drug exposure

• Conclusion – For this compound there was no requirement for dose adjustment– Highlights importance of assessing unbound PK parameters

int

gabsu

CLDose F F AUC

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Case Study – Drug Y• Plasma samples received from selective patients (hepatic

impairment)

– Could PPB explain some unexpected observations?

• Fraction unbound (fu) determined

– fu was <10% but variable between individuals

– Albumin and acid glycoprotein levels measured

– fu correlated with changing levels of one of the proteins

• Conclusion– Unlikely to result in dose adjustment

– Information subsequently helped clinical team to interpret initial exposure data

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Case Study – Drug Z• Drug was not extensively bound• Plasma samples obtained from clinical trials (renal & hepatic

impaired)• Fraction unbound (fu) determined• Not surprisingly…

– fu was consistent across all individuals– No effect of disease state on fu – Total protein higher in Renal impaired patients– Albumin noticeably lower and more variable in hepatic impaired

patients

• Conclusion– Unbound concentrations unlikely to be affected

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Summary

• By combining a range of in vitro techniques, we are able to support the scientific requirements and regulatory expectations to accurately assess protein binding prior to Phase I

• Techniques can be applied for measurement of binding from clinical samples

• There is an increasing expectation to quantify fu from clinical samples:

– Driven by guidelines– Helps interpret PK data– Predicts unbound concentrations– May explain adverse events – Important in assessing potential drug-drug interactions