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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 .
1
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
2
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
3
4
Plasma Extracellular water
Plasma protein Tissue proteindrug
5
Plasma Proteins bindings
6
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.
7
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
8
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.
9
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
10
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)
11
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
13
∂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.
14
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.
15
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.
16
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
20
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
21
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
23
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
25
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
26
Drug plasma protein binding
Expressed in % or by fu (free fraction)
>90% = highly bound
27
The free fraction : fu
fu = =free concentration
total concentration
Cfree
Ctot
Definition:
28
Ctot is a function of Cfree , not the inverse
Distribution
Action
Elimination Interaction
F FBmax
Kd Alb.
Measured total concentration
29
The free, the Bound &
the total concentration
30
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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
32
Relationship between fu, the Free and the bound concentrations
33
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