Protein binding rohit bharti

PROTEIN BINDING

R O H I T B H A R T I

M . P H A R M . 1 S T Y R .

C H A N N A B A S W E S H W A R P H A R M A C Y C O L L E G E , L A T U R .

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Protein Binding

Passive (not active) transport of drugs across biological membranes is influenced by protein binding.

Binding may occur - with plasma proteins or - with non-specific tissue proteins in addition to the drug’s receptors.

***Only drug that is not bound to proteins (i.e., free or unbound drug) can diffuse across membranes

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Protein binding

not only affects the activity of the drug (bound = inactive) But also can influence its distribution from one compartment

to another. This is particularly true with respect to glomerular filtration

and passive transport.

Free + Protein Bound Drug Drug

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Plasma Extracellular water

Plasma protein Tissue proteindrug

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Plasma Proteins bindings

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The fraction of total drug in plasma that is bound is determined by

the drug concentration,

its affinity for the binding sites, and

the number of binding sites.

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Binding of drug to proteins may:

Facilitate the distribution of drugs

Inactivate the drug by not enabling a sufficient concentration of free drug to develop at a receptor site

Retard the excretion of a drug

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Extensive plasma protein binding will cause more drug to stay in the central blood compartment. Therefore drugs which bind strongly to plasma protein tend to have lower volumes of distribution.

these plasma proteins, albumin, which comprises 50 % of the total proteins binds the widest range of drugs.

Acidic drugs commonly bind to albumin, while basic drugs often bind to alpha1-acid glycoproteinsand lipoproteins.

Many endogenous substances, steroids, vitamins, and metal ions are bound to globulins.

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HUMAN SERUM ALBUMIN

Is the most important protein that binds to drug molecule due to its high concentration compared with other proteins ( mol.wt. 65,000 )

It binds both acidic and basic

Constitute 5% of the total plasma

Both endogenous compounds such as fatty acids, bilirubin & tryptophan as well as drugs bind to HSA

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Albumin

Expansion of vascular compartment

Increased catabolism

Decreased synthesis

Hemorrhage / exudative losses,

Renal/gut losses

Increased capillary permeability

Decreased lymphatic return

Serumalbumin

concentrations

• Infusion of plasma or albumin

• Decreased catabolism

• Increased synthesis (e.g. insulin)

• Contraction of vascular compartment (e.g. vasoconstrictors)

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Albumin

Has 2 main binding sites with high constant of association (104-106 M-1)

The sites are charged positively and bind mainly anionic molecules Ex: NSAIDs

Carboxylic acids bind to site II (hydrophylic forces)

Non-carboxylic acid (enol derivative): site I

Oxicam : both sites (the warfarine site)

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NSAID’s, sulphonamides, phenytoin

Benzodiazepines, ibuprofen, cloxacillin, fatty acids medium chain

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∂1-ACIDGLYCOPROTEIN

Also known as orosomucoid (∂1-globulin)

(mol.wt. 44,000)

Binds to numerous drugs

Have greater affinity for basic than acidic drugs molecules

Binds only basic and highly lipophilic drugs

It binds to a drugs like imipramine, amitriptyline, lidoacine, propranolol, quinidine etc.

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LIPOPROTEINS

Plasma conc. Is much less than HAS, AAG.

It binds to lipophilic drugs because of their high lipid content.

Mol wt. varies from 2 lakhs to 34 lakhs depending on their chemical composition.

Binding of drugs to lipoproteins is non-competitive because of no specific and non specific binding sites.

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CLASSIFICATION OF LIPOPROTEINS

1. Chylomicrons (least dense & largest in size)

2. Very low density liporoteins ( VLDL )

3. Low density lipoproteins ( LDL )

4. High density lipoproteins

The main physiological role of lipoproteins is circulation of lipids to tissues through the blood.

Also play an important role in the transport of drugs to tissues.

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Drug binding to lipoproteins

Non-restrictive

Involves lipid solubilization of the drug into the lipidic core of the lipoproteins and/or interaction with the surface phospholipids

Because of multiple binding possibilities drug such as imipramine and propanolol show a relatively constant free fraction in plasma

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Things to remember:

Many drugs bind to the same receptor site but drugs with higher affinity will replace those drugs with lower affinity by competition

Only free and unbound drugs exert therapeutic effect by interacting with receptors

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Drugs may bind to protein through:

Hydrophobic Interaction Proposed by Kauzmann

tendency to develop of hydrophobic molecules or parts of molecules to avoid water because they are not readily accommodated in the H-bond structure of water

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Drugs may bind to protein through:

Self-Association Some drug may self dissociate to form dimers, trimers or

aggregates of larger size

Dimers or trimers - is a reaction product of two or three identical molecules

May affect solubility, diffusion, transport, therapeutic action of drugs

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Protein binding is determined by:

Dialysis

Ultracentrifugation

Ultrafiltration

Sephadex-gel filtration

Molecular filtration

Electrophoresis

Agar plate test

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The Pharmacokinetic Importance of Protein Binding

Drug-protein binding influences the distribution equilibrium of the drug

Plasma proteins exert a buffer and transport function in the distribution process

Only free and unbound drug acts can leave the circulatory system and diffuse into the tissue

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Disease and Protein Binding

Protein binding will be affected by the presence of diseases

Drugs showing Decrease Extent of Protein Binding

in the following diseases:

LIVER RENAL

Dapsone

Diazepam

Morphine

Phenytoin

Prednisolone

Quinidine

Tolbutamide

Triamterene

Barbiturates Salicylates

Cardiac Glycosides Sulfonamides

Chlordiazepoxide Triamterene

Clofibrate

Diazepam

Diazoxide

Furosemide

Morphine

Phenylbutazone

Phenytoin 24

Disease and Protein Binding

When drugs bind to protein, Albumin concentration is reduced

The exchange of proteins between plasma and interstitial compartment (normally proceeds at a rate of 5% plasma protein per our) will be hampered.

The diffusion of plasma the to interstitial fluid is increased by:

Inflammatory process

Pregnancy

use of oral contraceptives

Diabetes

Septic shock

Pulmonary Edema

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Is there often displacement of drug from the binding site?

No For a substantial displacement to take place, the

displacer must occupy most of the available binding site thereby lowering the binding site available to the primary drug

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Drug plasma protein binding

Expressed in % or by fu (free fraction)

>90% = highly bound

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The free fraction : fu

fu = =free concentration

total concentration

Cfree

Ctot

Definition:

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Ctot is a function of Cfree , not the inverse

Distribution

Action

Elimination Interaction

F FBmax

Kd Alb.

Measured total concentration

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The free, the Bound &

the total concentration

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CK

CB C

D free

freemaxbound

The bound concentrationCbound

Cfree

Bmax

KD

• The bound concentration

Bmax : maximal concentration ofbinding sites

– proportionnal to plasma protein concentration

KD : free drug concentration corresponding to half maximal binding– inversely proportional to drug affinity for the protein binding

Bmax/2

Ctot is a function of Cfree

Ctot = Cfree + Cbind

Ctot = Cfree + Bmax x Cfree

Kd + Cfree

Dependent variable Parameters Independent variable controlled by Clfree

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Relationship between fu, the Free and the bound concentrations

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binding- 34

Dmax

Du

KB

K f

Effect of modifications of plasma proteinconcentration (Bmax) on fu

Plasma protein increase Bmax is increased

fu is increased

fu is decreased

Plasma protein decrease Bmax is decreased

binding- 35

Algorithm for determining clinical significance of potential binding displacement interaction

Is drug of interest >90% protein bound?

Yes

Does the drug have a narrow therapeutic index ?

Yes

What is the hepatic extraction ratio of the drug ?

High

Is the drug given IV?

Clinically significant interaction likely. Perform a clinical study to quantify effects

Clinically significant interaction not likely

Would a transient increase in free drug concentration be clinically relevant ?

no

no

low

no

no

Yes

Yes