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『창의적 예측경영』『 효율적 내실경영 』
Plasma Protein binding
Chapter 14
2015. 12. 9
Lee, Sang-Hwi
1. Plasma Protein Binding (PPB) Fundamentals
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The PK and PD properties of drugs are largely a function of the reversible
binding of drugs to plasma or serum proteins, such proteins include
albumin(HSA), α1-acid glycoprotein (AGP), lipoproteins, erythrocyte and α, ß‚ and
γ globulins.
Generally, only the unbound drug is available for diffusion or transport across cell
membranes, and for interaction with a pharmacological target (e.g. receptor, ion
channel, transporter, enzyme).
As a result, the extent of plasma protein binding of a drug influences the drug’s
action as well as its distribution and elimination.
# α1-acid glycoprotein
(AGP)
# Human Serum Albumin(HSA)
500~700 μM (35~50 mg/ml)
binds strongly to organic
anions (carboxylic acid, phenols)
Basic and neutral drugs (minor)
~60%
15 μM (0.5~1.0 mg/ml)
Basic drugs (amines)
Hydrophobic drugs (steroids)
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Drug-PPB interaction
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Protein binding is reversible, then a chemical equilibrium will exist
between the bound and unbound states
Protein + drug ⇌ Protein-drug complex
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Electrostatic(strong) and hydrophobic(weak) interaction.
average equilibrium time : 0.02s (rapid)
PPB(high), Dose (high)  The available binding sites on plasma proteins
can be saturated  Toxicity & Side effect (increase)
ex) Plasma albumin 농도는 500~700 uM (35~50 mg/ml) 이므로, 분자량
300인 약물은 max. 180 ug/ml 까지 결합 가능 하며 , 그 이상에서는 포화
현상에 의해 free 한 약물이 증가하며 tissue로 이행량이 증가 하여 독성 유
발 가능. (약제학-서울대출판사)
• Plasma protein concentrations can vary in different disease states or with
age.
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PPB Effects
Fraction unbound in plasma does not always correlate to in vivo PK parameters
If ) Highly bound (> 99%) & tightly bound (slow dissociation)
1)
2)
3)
4)
5)
Retain drug in plasma compartment
Restrict distribution of drug into target tissue (reduce volume of distribution Vd)
Decrease metabolism, clearance, and prolong t½
Limit brain penetration (BBB)
Require higher loading doses
but lower maintenance doses
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14.2.1 Impact of PPB on Distribution (Vd)
PPB can have either a “restrictive” or a “permissive” (nonrestrictive)
effect on drug disposition.
/ fu, tissue (increase)
Vd = Vplasma + Vtissue X (fu, plasma / fu, tissue ) fu, Plasma
V (high)
d
if)
PPB (high), fu, Plasma (low)  Vd (low)
PPB (low), fu, Plasma (high)  Vd (high)
nonspecific binding in tissue (high), fu, tissue (low)  Vd (high)
nonspecific binding in tissue (low), fu, tissue (high)  Vd (low)
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2.2 Effect of PPB on Clearance
High PPB can be “restrictive “or “permissive” of liver extraction.
2.3 Effect of PPB on Pharmacology
1. Pharmacology can be affected by PPB.
2. Enzyme inhibition can be reduced if the compound is bound to plasma proteins.
1) Anti-inflammatory drugs : Acid compound  PPB (high) (> 99% ; 26,6%)
- Plasma binding > tissue binding  NSAIDs have low tissue distribution.
2) Renal/Cardiovascular drug : PPB (high) (>90% ; 51%)
3) CNS drugs : PPB (high) (>90% ; 52%)
4) Chemotherapeutic drug
: Antibiotic, Antiviral, Antifungal, Anticancer drugs
 PPB (low) (< 90% ; 77.2%, >99% ; 8.1%)
Biochemical pharmacology 2002,64,1355-6-
2. PPB Effects : Indication of how changes in key molecular
properties will affect a range of ADMET parameters
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14.3 PPB Case Studies
1. Case I : Bristol-Myers Squibb ; J. Med. Chem. 2008, 51, 5897 (IGF-1R, Anticancer)
2. Case II : Biota Europe Ltd. ; J. Med. Chem. 2010, 53, 3927 (FtsZ, Antibacterial )
3. Case III : Astrazeneca ; BMCL, 2009, 19, 930 (MurI inhibitor , Antibiotic)
4. Case IV : Merck ; BMCL 2010, 20 , 657 (CNS, Alzheimer)
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Case I. Insulin-like Growth Factor-1 Receptor (IGF-1R) inhibitor
RTKs
(Bristol-Myers Squibb : J. Med. Chem. 2008, 51, 5897)
Path A
Cell proliferation
Path B
Apotosis
Cell survival
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To Improve ADME properties (lead optimization)
Activity, Solubility, PPB, CYP3A4
Devoid of PXR transactivation /
CYP3A4 inhibition
(BMS-695735)
Solubility (137 ug/ml)
PPB (86.9%)
CYP3A4 inhibition (26 uM)
(BMS-536924)
Poor solubility (<1 ug/ml)
High PPB (99.9%)
Strong CYP3A4 inhibition (0.05 uM)
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1(if, po : 50mg/kg)
10 (po : 50mg/kg)
# Effective dose (mouse)
0.1 uM x 480 (M.W.) /1000 = 0.048 ug/ml
# Effective dose (mouse)
0.034 uM x 512(M.W.)/1000 = 0.017 ug/ml
# PPB = > 99.9%  0.1% unbound
50 ug/ml (?) x 0.1 / 100 = 0.05 ug /ml
# PPB = 86.9%  13.1% unbound
2.3 ug/ml x 13.1 / 100 = 0.3 ug/ml
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# Safety
1) Genotoxicity Ames (-)
2) CA (-)
3) Noncytotoxic in human hepatocyte
 Compound 1 (100 mg / kg ; ca. MTD dose), AUC 20 uM x h / 20 mg
Therapeutic index value (low)
TGI (93%)
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Case II. Potent inhibitors Bacterial cell division protein FtsZ
FtsZ is a protein encoded by the ftsZ gene that assembles into
a ring at the future site of the septum of bacterial cell division.
This is a prokaryotic homologue to the eukaryotic protein tubulin.
FtsZ has been named after "Filamenting temperature-sensitive
mutant Z"
Molecular Structure of FtsZ
J. Med. Chem. 2010, 53, 3927–3936
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
To Improve pharmaceutical properties
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BA 57%
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J. Med. Chem. 2010, 53, 3927–3936
Z1=C, Z2=C : 8j
Z1=N, Z2=C : 2
Effective dose (0.36 ug/ml)
8J
8J (i.v.: 2 mg/kg)
2
2 (i.v.: 3 mg/kg)
# Effective dose (mouse)
0.25 uM x 354.76 (M.W.) /1000 = 0.089 ug/ml
# Effective dose (mouse)
# PPB = 96.4 %  3.6 % unbound
0.5 ug/ml x 3.6 / 100 = 0.018 ug /ml
# PPB = 85.4 %  14.6% unbound
3.7 ug/ml x 14.6 / 100 = 0.54 ug/ml
1 uM x 355.75(M.W.)/1000 = 0.36 ug/ml
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Case III. Potent and selective inhibitors of
Helicobacter pylori glutamate racemase (MurI)
: Pyridodiazepine amines
Astrazeneca ; BMCL 19 (2009) 930~936
1) MurI is a bacterial cytoplasmic enzyme
that catalyzes the conversion of L-glutamate
to D-glutamate, one of the essential amino
acids in peptidoglycan synthesis.
2) The disruption of peptidoglycan biosynthesis
is lethal to bacteria and therefore inhibitors of
glutamate racemase should be useful as
antibacterials.
3) The murI gene is conserved in all bacterial
species that synthesize peptidoglycan and its
essentiality has been well-demonstrated in a
number of bacteria.
4) The unique biophysical and biochemical
properties of H. pylori MurI relative to the
MurI of other bacteria could allow for the
discovery and development of specific MurI
inhibitors.
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BMCL 19 (2009) 930~936
Improved solubility & Reduced plasma protein binding
IC50 : 1.7 uM
Solubility : 0.5 uM
PPB : 99.7%
IC50 : 2 uM
Solubility : 1365 uM
PPB : 81.8%
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Case IV. Pyridine containing Muscarine1 positive allosteric
modulators with reduced plasma protein binding
Merck : BMCL 20 (2010) 657~661
-Alzheimer’s disease(AD)
Plasma protein binding (lowering)
CNS exposure (enhance)
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14.5 Strategy for PPB in Discovery
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In general, the prospective use of PPB data for predicting in vivo PK and PD in
drug discovery can be misleading.
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Many commercial drugs have high (> 99%) PPB.
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PPB may be restrictive or permissive for penetration into tissues.
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PPB can increase the PK t½ (by keeping the compound in the blood and
restricting clearance), but it also can restrict exposure to the therapeutic target (by
reducing penetration into tissues).
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PPB alone can be either a positive or a negative aspect of a compound.
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However, PPB can be useful, retrospectively, as part of an ensemble of in vitro
diagnostic tests to understand the impact of PPB on PK or pharmacological
effects.
Only when PPB is placed into context with PK parameters can valuable insight be
gained into the disposition of the molecule.
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Effects of structure modification on in vivo exposure.
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Guidance for applying principles of plasma
protein binding in drug discovery
 Advance drug candidate
 Avoid structural modification to reduce the free drug fraction
for plasma protein binding
 Do not use shift assays
 Avoid the trap of total drug concentration and brain/plasma
ratio
 Discover the missing link between pharmacokinetics and
pharmacodynamics
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Methods
Original
New*
Dialysis buffer
PBS containing 100 mM sodium phosphate, 150 mM
sodium chloride
100 mM KPO4 0.6% NaCl pH 7.4
Preparing
samples
DMSO Stock spiking(0.5%이하) (final 10ug/ml)
20mg/ml DMSO stock -> 2mg/ml MeOH
(x10) -> 20 ug/ml(990ul Pla + 10 ul sample
(x100))
Sample volume
Plasma 100 ul
Buffer 300 ul
Plasma 300 ul
Buffer 500 ul
Sampling
S : Plasma 50 ul + blank buffer 50 ul
B : Buffer 50 ul + blank plasma 50 ul
 ACN 100 ul
S : Plasma 10 ul+ blank pla 10 ul + blank
buffer 100 ul
B : buffer 100 ul + blank pla 20 ul
 ACN 100 ul
* : Validation of an Automated HTS PPB assay (BD biosciences, Application Note #474)
 Incubation time 은 4 hr 동일하게 실시.
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PPB : Equilibrium Dialysis assay
20 ug/ml
Buffer
Plasma
500 ul
300ul
Area (*104)
Area (*104)
PPB(%)
compound
IS
Ratio
compound
IS
Ratio
#1
0.00065
139.0
0.00001
16.40
157.0
1.25
99.99955
#2
0.00084
146.0
0.00001
20.80
151.0
1.65
99.99958
#3
0.00064
166.0
0.00000
23.00
154.0
1.79
99.99974
1.57
100.000
173
1.78
87.9%
Mean
1.78
Mean
Recovery(%)
0.00001
25.7
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Problem
1. Would high plasma protein binding of a compound (e.g., 99.9%) and
low dissociation rate (Kd) tend to increase or decrease each of the following,
compared to a compound with moderate plasma protein binding (e.g., 50%) and
moderate dissociation rate (Kd)?
Metabolic clearance
Renal clearance
Tissue concentration
Tissue distribution
Brain penetration
Blood concentration
PK half-life
Pharmacological effect for non-blood stream target
2.
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