1. Plasma Protein binding
Chapter 14
Lee, Sang-Hwi

2. 1. Plasma Protein Binding (PPB) Fundamentals
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.
# Human Serum Albumin(HSA)
500~700 uM (35~50 mg/ml)
binds strongly to organic
anions (RCOOH, Phenols)
Basic / neutral drugs (minor)
# α1-acid glycoprotein (AGP)
15 uM (0.5~1.0 mg/ml)
Basic drugs (amines)
Hydrophobic drugs (steroids)
~ 60 %

3. Drug-PPB interaction
Protein binding is reversible, then a chemical equilibrium will exist between the bound and unbound states
Protein + drug ⇌ Protein-drug complex
Electrostatic(strong) and hydrophobic(weak) interaction.
an 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 유발 가능. (약제학-서울대출판사)
Plasma protein concentrations can vary in different disease states or with age.

4. PPB Effects
Fraction unbound in plasma does not always correlate to in vivo PK parameters
If ) Highly bound (> 99%) & tightly bound (slow dissociation)
1) Retain drug in plasma compartment
2) Restrict distribution of drug into target tissue (reduce volume of distribution Vd)
3) Decrease metabolism, clearance, and prolong t½
4) Limit brain penetration (BBB)
5) Require higher loading doses
but lower maintenance doses

5. 14.2.1 Impact of PPB on Distribution (Vd)
PPB can have either a “restrictive” or a “permissive” (nonrestrictive) effect on drug disposition.
Vd = Vplasma + Vtissue X (fu, plasma / fu, tissue )
fu, Plasma / fu, tissue (increase)
 Vd (high)
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)

6. 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

7. 2. PPB Effects : Indication of how changes in key molecular properties will affect a range of ADMET parameters

9. 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)

10. Case I. Insulin-like Growth Factor-1 Receptor (IGF-1R) inhibitor
RTKs
(Bristol-Myers Squibb : J. Med. Chem. 2008, 51, 5897)
Path B
Path A
Cell proliferation Apotosis Cell survival

11. To Improve ADME properties (lead optimization)
Activity, Solubility, PPB, CYP3A4
(BMS )
Solubility (137 ug/ml)
PPB (86.9%)
CYP3A4 inhibition (26 uM)
Devoid of PXR transactivation /
CYP3A4 inhibition
(BMS )
Poor solubility (<1 ug/ml)
High PPB (99.9%)
Strong CYP3A4 inhibition (0.05 uM)

12. 1(if, po : 50mg/kg)
10 (po : 50mg/kg)
# Effective dose (mouse)
0.1 uM x 480 (M.W.) /1000 = ug/ml
# PPB = > 99.9%  0.1% unbound
50 ug/ml (?) x 0.1 / 100 = ug /ml
# Effective dose (mouse)
0.034 uM x 512(M.W.)/1000 = ug/ml
# PPB = 86.9%  13.1% unbound
2.3 ug/ml x 13.1 / 100 = 0.3 ug/ml

13. # Safety
1) Genotoxicity Ames (-)
2) CA (-)
3) Noncytotoxic in human hepatocyte
TGI (93%)
Compound 1 (100 mg / kg ; ca. MTD dose), AUC 20 uM x h / 20 mg
Therapeutic index value (low)

14. 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

15.  To Improve pharmaceutical properties

16. BA 57%

17. Effective dose (0.36 ug/ml)
J. Med. Chem. 2010, 53, 3927–3936
Z1=C, Z2=C : 8j
Z1=N, Z2=C : 2
Effective dose (0.36 ug/ml) 8J 2
2 (i.v.: 3 mg/kg)
8J (i.v.: 2 mg/kg)
# Effective dose (mouse)
0.25 uM x (M.W.) /1000 = ug/ml
# PPB = 96.4 %  3.6 % unbound
0.5 ug/ml x 3.6 / 100 = ug /ml
# Effective dose (mouse)
1 uM x (M.W.)/1000 = 0.36 ug/ml
# PPB = 85.4 %  14.6% unbound
3.7 ug/ml x 14.6 / 100 = ug/ml

18. 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.

19. BMCL 19 (2009) 930~936
Improved solubility & Reduced plasma protein binding
IC50 : 2 uM
Solubility : 1365 uM
PPB : 81.8%
IC50 : 1.7 uM
Solubility : 0.5 uM
PPB : 99.7%

23. 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)

26. 14.5 Strategy for PPB in Discovery
In general, the prospective use of PPB data for predicting in vivo PK and PD in drug discovery can be misleading.
Many commercial drugs have high (> 99%) PPB.
PPB may be restrictive or permissive for penetration into tissues.
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).
PPB alone can be either a positive or a negative aspect of a compound.
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.

27. Effects of structure modification on in vivo exposure.

28. 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

31. Methods Original New* Incubation time 은 4 hr 동일하게 실시. 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
* : Validation of an Automated HTS PPB assay (BD biosciences, Application Note #474)
Incubation time 은 4 hr 동일하게 실시.

32. PPB : Equilibrium Dialysis assay
20 ug/ml
Buffer
Plasma
PPB(%)
500 ul
300ul　
Area (*104)
compound IS
Ratio #1
139.0
16.40
157.0
1.25 #2
146.0
20.80
151.0
1.65 #3
166.0
23.00
154.0
1.79
Mean
1.57
Recovery(%)
25.7
173
1.78
87.9%

33. Problem
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.