『창의적 예측경영 』『 효율적 내실경영 』

Plasma Protein binding

2015. 12. 9Lee, Sang-Hwi

Chapter 14

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.

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# Human Serum Albumin(HSA)500~700 μM (35~50 mg/ml) binds strongly to organic anions (carboxylic acid, phenols)Basic and neutral drugs (minor)

# α1-acid glycoprotein (AGP)15 μM (0.5~1.0 mg/ml)Basic drugs (amines)Hydrophobic drugs (steroids)

~60%

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. • 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 300 인 약물은 max. 180 ug/ml 까지 결합 가능 하며 , 그 이상에서는 포화 현상에 의해 free 한 약물이 증가하며 tissue 로 이행량이 증가 하여 독성 유발 가능 . ( 약제학 - 서울대출판사 )

• Plasma protein concentrations can vary in different disease states or with age.

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PPB Effects

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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

Fraction unbound in plasma does not always correlate to in vivo PK parameters

14.2.1 Impact of PPB on Distribution (Vd)

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PPB can have either a “restrictive” or a “permissive” (nonrestrictive) effect on drug disposition. Vd = Vplasma + Vtissue X (fu, plasma / fu, tissue )

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)

fu, Plasma / fu, tissue (increase) Vd (high)

2.2 Effect of PPB on Clearance

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High PPB can be “restrictive “or “permissive” of liver extraction.

2.3 Effect of PPB on Pharmacology1. 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

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

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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

(Bristol-Myers Squibb : J. Med. Chem. 2008, 51, 5897)

Cell proliferation Apotosis Cell survival

RTKs

Path A

Path B

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(BMS-536924)Poor solubility (<1 ug/ml)High PPB (99.9%)Strong CYP3A4 inhibition (0.05 uM)

(BMS-695735)Solubility (137 ug/ml)PPB (86.9%)CYP3A4 inhibition (26 uM)

To Improve ADME properties (lead optimization)

Devoid of PXR transactivation / CYP3A4 inhibition

Activity, Solubility, PPB, CYP3A4

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# Effective dose (mouse) 0.034 uM x 512(M.W.)/1000 = 0.017 ug/ml

# PPB = 86.9% 13.1% unbound 2.3 ug/ml x 13.1 / 100 = 0.3 ug/ml

# Effective dose (mouse) 0.1 uM x 480 (M.W.) /1000 = 0.048 ug/ml

# PPB = > 99.9% 0.1% unbound 50 ug/ml (?) x 0.1 / 100 = 0.05 ug /ml

1(if, po : 50mg/kg) 10 (po : 50mg/kg)

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TGI (93%)

# 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)

14J. Med. Chem. 2010, 53, 3927–3936

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

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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 : 8jZ1=N, Z2=C : 2

# Effective dose (mouse) 1 uM x 355.75(M.W.)/1000 = 0.36 ug/ml

# PPB = 85.4 % 14.6% unbound 3.7 ug/ml x 14.6 / 100 = 0.54 ug/ml

# Effective dose (mouse) 0.25 uM x 354.76 (M.W.) /1000 = 0.089 ug/ml

# PPB = 96.4 % 3.6 % unbound 0.5 ug/ml x 3.6 / 100 = 0.018 ug /ml

8J (i.v.: 2 mg/kg) 2 (i.v.: 3 mg/kg)

Effective dose (0.36 ug/ml)

8J 2

Case III. Potent and selective inhibitors of Helicobacter pylori glutamate racemase (MurI) : Pyridodiazepine amines

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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

• 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.

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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

SamplingS : Plasma 50 ul + blank buffer 50 ulB : Buffer 50 ul + blank plasma 50 ul ACN 100 ul

S : Plasma 10 ul+ blank pla 10 ul + blank buffer 100 ulB : buffer 100 ul + blank pla 20 ul ACN 100 ul

Incubation time 은 4 hr 동일하게 실시 .

* : Validation of an Automated HTS PPB assay (BD biosciences, Application Note #474)

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20 ug/ml Buffer Plasma

PPB(%)

　 500 ul 300ul 　

　 Area (*104) 　 Area (*104) 　

　 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

Mean 0.00001 　 　 1.57 100.000

Recovery(%) 　 25.7 173 1.78 87.9%

　 　 　 　　 Mean 1.78 　

PPB : Equilibrium Dialysis assay

Problem

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Metabolic clearanceRenal clearanceTissue concentrationTissue distributionBrain penetration

Blood concentration PK half-lifePharmacological effect for non-blood stream target

2.

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)?