CONFIDENTIAL

Peter Kilford Peter Kilford

Study Director, Drug Metabolism Study Director, Drug Metabolism
Harrogate, UK Harrogate, 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
winstudy 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 Guidelines
ICH M3 (R2) 2009
Before initiating human trials:
- in vitroplasma protein binding data for
animals and humansshould 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 (416 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 (24 hrs)
Low Nonspecific Binding
Easy to use
RED (Rapid Equilibrium Dialysis)
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Standard PPB Experiment Considerations
DrugCandidate
PlasmaStability
InAllSpecies
6hours?
DataOutput:
%Bound
%Free
%Recovered
EquilibriumTime,
1species
(e.g.human)
Yes
Yes
Yes
Yes
Canyou
stabilize?
No No
Useultrafiltration
w/shortspintime
No
No
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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 bioanalytical
approach
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 analyte
concentration
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 analyte
concentration
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 PI Preclinical PII PIII
Post
Approval
Non-GLP Clinical, GLP and Non-GLP
Validated Methods
Rodent & 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 Methods
Samples 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 CO
2
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 Experiments
Change in plasma pH over time
Mouse
Dog
Rat
Rabbit
Human
Time (hours)
0 2 4 6
p
H
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% CO
2
pH Control for PPB Experiments
Mouse
Dog
Rat
Rabbit
Human
Time (hours)
0 2 4 6
p
H
7
7.2
7.4
7.6
7.8
8
8.2
8.4
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pH Control for PPB Experiments
Effect of pH on Fraction Unbound (Rat)
Testosterone Warfarin Caffeine
F
o
l
d

c
h
a
n
g
e
f
u

+
C
O
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%
CO
2
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
2003
Fraction unbound assessed in hepatically
impaired subjects when compounds
- highly cleared by liver (ER >0.7)
- Extensively protein bound (>90%)
PK in Patients with Impaired Hepatic Function
2003
Fraction unbound assessed in hepatically
impaired 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 Guidelines
PK in Patients with Impaired Hepatic
Function 2005
Drug 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% CO
2
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
AUC
u
stays the same.
Result = no clinical significance
Effect of Changes in fu
int
g abs
CL fu
Dose F F
AUC

int
g abs
u
CL
Dose F F
AUC

fu AUC AUCu
Oral administration and hepatic clearance
Example 1
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When fu May Be Clinically Relevant
No Yes

Non-Hepatic Cl
No No Hepatic Cl
PO Administration
No Yes* Non-Hepatic Cl
No Yes* 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
g abs
CL fu
Dose F F
AUC

int
g abs
u
CL
Dose F F
AUC

fu AUC AUCu
Oral administration and hepatic clearance
Example 2
Increase in fu + Decrease CL
int
AUC stays same but
AUC
u
increases
Result = possible clinical impact
e.g. Salicylate
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Salicylate Kinetics in Hepatic Impairment
[ Total Plasma ] [ Unbound Plasma ]
AUC
u
fu
Clearance
Roberts et al. 1983, Eur J Clin Pharmacol
(
f
u
)
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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
t

prolonged in hepatic-impaired population

Normal = 51.4 hr
Hepatic-impaired = 81.8 hr
Case Study Drug X
int
g abs
CL fu
Dose 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 nghr/mL
Hepatic-impaired, AUCu = 3.49 nghr/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
g abs
u
CL
Dose 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