BAHAN AJAR UNTUK TOPIK MINGGU KE-3

Topic 3 : Drug Protein Binding

MATA KULIAH :

FARMAKOKINETIKA

Disusun oleh PJMK :

M.M. Farida Lanawati Darsono, S.Si.,M.Sc

Semester Genap
Tahun Akademik : 2021-2022

Universitas Katolik Widya Mandala Surabaya

Fakultas Farmasi

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 Topic 3 : Drug Protein Binding
Ikatan O-P
 Merupakan suatu ikatan yg terbentuk dari hasil interaksi obat dg protein di dalam plasma
 Merupakan suatu ikatan yg menghasilkan kompleks makromolekul

Sifat ikatan O-P

a. Reversibel:
* ikatan kinia yg lemah (ikt. Hidrogen/gaya van der waals)
* contoh : gol asam amino --- penyusun rantai protein
b. Irreversibel:
* ikatan kimia yg kuay (ikt. Kovalen)
* timbul toksisitas
* contoh :
a. jangka pendek : parasetamol --- hepatotoksisitas
b. jangka panjang : parasetamol – karsinogenik kimia

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 Protein binding generally refers to the binding of a drug to proteins in blood plasma.
 The interaction can also be between the drug and tissue membranes, red blood cells, and
other components of the blood.
 The amount of drug bound to protein determines how effective the drug is in the body.
 The bound drug is kept in the blood stream while the unbound components of the drug may
be metabolized or extracted, making them the active part of the drug. So, if a drug is 95%
bound to a binding protein and 5% is free, that means that 5% of the drug is active in the
system and causing pharmacological effects.
 Protein binding is often reversible and thus creates a chemical equilibrium, in which the
chemical reaction can go backward and forward with no net change in reactants and products.
 This means that a cell that is effective at extracting the unbound drug may extract more of the
drug as it disassociates in the course of achieving equilibrium.
 The equation for reversible protein binding is: Protein + drug ⇌ Protein-drug complex
 The proteins commonly involved with protein binding are albumin, lipoproteins, and al-
glycoprotein.
 A protein is a chain of amino acids joined by peptide bonds.
 Acidic drugs will tend to bind to albumin, which is basic and basic drugs will primarily bind
to al-glycoprotein, which is acidic.
 Acidic drugs may also bind to lipoproteins if the albumin is saturated. Lipoprotein binding is
not binding in the strict sense of the term; it is closer to dissolving and is common in lipid
soluble drugs.
 A drug that binds to tissue often binds to melanin-rich tissue or DNA.
 The amount of protein binding and the fraction unbound, written as the concentration of
unbound drug over the total concentration of the drug, depends on several factors.
 It is determined by the drug’s affinity for the protein, the concentration of the binding protein,
and the concentration of the drug relative to the binding protein.

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 This is important when considering other medications that a patient might be on because
certain proteins may already be saturated, which would affect the amount of free drug and
possibly change the desired pharmacologic effects.
 For example, if drug A saturated a certain binding protein and then drug B was not able to
bind to that protein, then there would be a higher concentration of unbound drug B. Drug B
could also competitively displace drug A from the binding protein, thus raising the unbound
fraction of drug A.
 This process happens fairly quickly, in minutes to hours, and both scenarios could have
adverse effects. Many drugs, however, have different binding proteins, different binding sites
on a protein, or are not present in high enough relative concentration to saturate the proteins,
and so do not compete with the other drug or drugs in use.
 Likewise, the ability of the body to extract the drug can affect the drug’s clearance into the
body.
 Renal failure and liver disease often negatively impact the body’s ability to extract the
unbound drug.
 For these reasons, it is important to consider previous medical issues, the total concentration
of the drug, the unbound fraction of the drug, and any other medications a patient may be
taking.
 So, if a drug is 95% bound to a binding protein and 5% is free, that means that 5% of the
drug is active in the system and causing pharmacological effects.
 Protein binding is often reversible and thus creates a chemical equilibrium, in which the
chemical reaction can go backward and forward with no net change in reactants and products.
 In addition to being a unique structure, a bacterial protein also has the ability to bind with
other proteins.
 Protein binding involves the formation of very strong links between two different proteins.
(http://www.wisegeek.com/what-is-protein-binding.htm)

Membrane Proteins
 An enzyme called protein kinase C is another interior peripheral membrane protein.
 It initiates signaling pathways inside the cell.
 Peripheral membrane proteins do not interact with the non-polar region of the cell
membrane.
 Both structurally and functionally, they are integral parts of the membranes of cells.
 Each integral membrane protein molecule has an intricate relationship with the membrane
within which it is situated.
 Various routes are used to administer drugs but in most cases drugs reach their site of action
via the systemic circulation.
 Once within the circulation a drug is clearly not confined to its intended site of action.
 Instead it is distributed widely throughout the body.
 At this point you may have anticipated some important questions:
 How is the drug carried in the blood? Is the entire drug free to exert an effect?
 Can the presence of one drug in the circulation affect another?
 To begin to understand the answer to these questions we need first of all to think about
plasma proteins
(http://www.nottingham.ac.uk/nmp/sonet/rlos/bioproc/plasma_proteins/6.html)

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Plasma Proteins
 Suppose we take a sample of blood and mix it in a tube with an anticoagulant to stop it
clotting, before spinning it in a centrifuge.
What now will our sample look like?
 The cellular components will have sunk to the bottom of the tube – they form about 45% of
the sample.
 The remaining 55% is the liquid we call plasma.
What does plasma consist of?
 Most of it is water – about 92%, whilst plasma proteins form about 7%.
 The remaining 1% is other dissolved solutes such as inorganic ions.

Types of Plasma Protein

 Most, but not all, plasma proteins are manufactured in the liver including albumins.
 These are the most abundant and form about 60% of all plasma proteins.
 They contribute to osmotic pressure, help to control water balance and are involved in the
transport of substances in blood including drugs.
 Globulins (globular proteins) form 35% of the whole and include antibodies, whilst others
have transport functions.
 Approximately 4% of plasma proteins, such as fibrinogen, have a clotting function whilst the
remaining less than 1% are regulatory such as metabolic enzymes

Drugs and Plasma Proteins

 The main influence of plasma proteins on drugs is in their distribution.
 The most important plasma proteins in this context are albumin, acid-glycoprotein and beta-
globulin.
 Once a drug has been absorbed into the circulation it may become attached (we say bound) to
plasma proteins.
 However this binding is rapidly reversible and non-specific – that is many drugs may bind to
the same protein.
 It is important to recognise that plasma proteins do not represent target tissues and drug
binding produces no physiological effect.
 Drug–plasma protein binding forms a "reservoir" of drug, but only the free (unbound) drug is
available to the tissues to exert a therapeutic

Jenis protein yg terikat dg obat

 Albumin
 α1-acid glycoprotein (AAGP)
 lipoprotein

albumin
 Disintesi di hati
 BM= 65.000-69.000
 Terdistribusi di plasma dan cairan ekstraseluler
 T ½ eliminasi = 17-18 hari
 Konsentrasi normal : 3,5 – 5,5 % atau 4,5 mg/dL
 Fungsi :
1. mengatur tekanan osmotis darah
2. trasnpor komponen endogenous dan exogenous (ex: free fatty acid, bilirubin, hormon)

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 Bersifat selektif
 Terdiri dari 6 tempat ikatan
* 2 --- mengikat kuat --- asam lemah
* 2 --- mengikat bilirubin
* 1 --- mengikat obat (site 1)
* 1 --- mengikat obat (site 2)
 Memiliki pH = (-) ve charge
 Mengikat obat yg bersifat asam lemah
 Low binding afinity & high binding capacity

AAGP
 Is an acute phase reaction which has one binding site selective for basic drug
 BM = 44.000
 Konsentrasi dalam plasma = 0,4 – 1%
 Terutama mengikat obat yg bersifat basa (kationik)
 Level konsentrasi AAGP akan mengikat pada kondisi:
a. trauma
b. kehamilan
c. myocardial infraction
d. chronic RF
e. malignancy

lipoprotein
 Merupakan komplek makromolekul antara lemak dg protein
 Berdasarkan “density” dan “pemisahan secara ultrasentrifuse” terdiri dari:
1. VLDL
2. LDL
3. HDL --- good protein
 Berfungsi transpor lemak plasma dan ikatan obat (jika albumin jenuh) --- siklosporin,
trigliserida, kolesterol
 Memiliki “non polar lipid core” :
1. ester kolesterol & trigliserida
2. fosfolipid & free kolesterol
3. apo-lipoprotein
 Binds (terikat) highly with lipoprotein drug
 Lipoprotein level changes depend on fasted or fed state
 Competition binding with albumin

RBCs (Red Blood Cells)

 Mengikat kedua komponen endogenous & exogenous
 Meliputi 45 % dr volume darah
 Tidak terlalu berpengaruh pada Vd

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Cara evaluasi OP
 Tujuan : untuk mengetahui sejauh mana ikatan obat dengan protein yg terjadi
 Umumnya secara : in vitro
 Perangkat yg diperlukan :
a. instrumen
b. protein yg dimurnikan ---albumin
c. membran semipermiabel
 Metode penentuan :
a. langsung : uv-vis, NMR, dll
b. dialisis : ultracentrifugation
 Keuntungan : pengukuran lebih mudah, hemat waktu, peralatan umum
 Kerugian : biaya mahal

Faktor yg mempengaruhi OP
1. Obat :
* Sifat fisikokimia
* [C] total dalam tubuh
2. Afinitas obat terhadap protein:
* tetapan asosiasi (ka)
3. Interaksi obat:
* kompetisi : obat vs zat lain
* perubahan protein (sbg substrat)
4. Patofisiologis pasien:
* kelainan liver atau ginjal, dll
5. Protein

Factor affecting drug protein binding

1. factor relating to the drug
a) Physicochemical characteristic of drug
b) Concentration of drug in the body
c) Affinity of drug for a particular componant
2. factor relating to the protein and other binding componant
a) Physicochemical characteristic of the protein or binding componant
b) Concentration of protein or binding componant
c) Num. Of binding site on the binding site
3. drug interation
4. patient related factor

Drug related factor

 Physicochemical characteristics of drug
 Protein binding is directly related to lipophilicity
lipophilicity = the extent of binding
 e.g. The slow absorption of cloxacilin in compression to ampicillin after i.m. Injection is
attributes to its higher lipophilicity it binding 95% letter binding 20% to protein
 Highly lipophilic thiopental tend to lacalized in adipose tissue .
 Anionic or acidic drug like . Penicillin , sulfonamide bind more to HSA
 Cationic or basic drug like . Imepramine alprenolol bind to AAG

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CONCENTRATION OF DRUG IN THE BODY
 The extent of drug- protein binding can change with both change in drug and protein
concentration
 The con. of drug that binding HSA does not have much of an influence as the thereuptic
concentration of any drug is insufficient to saturate it
Eg. Thereuptic concentration of lidocaine can saturate AAG with which it binding as the
con. Of AAG is much less in compression to that of HSA in blood

DRUG PROTEIN / TISSUE AFFINITY

 Lidocaine have greater affinity for AAG than HSA
 Digoxin have greater affinity for protein of cardiac muscle than skeleton muscles or
plasma

Protein or tissue related factor

Physicochemical properity of protein / binding componant – lipoprotein or adipose tissue
tend to bind lipophilic drug by dissolving them to lipid core .
The physiological pH determine the presence of anionic or cationic group on the albumin
molecule to bind a verity of drug
Concentration of protein / binding componant
Mostly all drug bind to albumin b/c it present a higher concentration than other protein
Number of binding sites on the protein
Albumin has a large number of binding site as compare to other protein and is a high capacity
binding component

Drug interaction
a. Competition b/w drug for binding site (displacement interaction )
When two or more drug present to the same site , competition b/w them for interaction with
same binding site . If one of the drug (A) is bound to such a site , then administration of the
another drug (B) having high affinity for same binding site result in displacement of drugs
(A) from its binding site. This type of interaction is known as displacement interaction.
Wher drug (A) here is called as the displaced drug and drug (B) as the displacer .
Eg. Phenylbutazone displace warferin and sulfonamide fron its binding site
b. Competition b/w drug and normal body constituent

The free fatty acids are interact to with a number of drug that bind primarily to HSA . When
free fatty acid level is increase in several condition – fasting , - pathologic – diabeties ,
myocardial infraction , alcohol abstinence – the fatty acid which also bind to albumin influence
binding of several drug
binding – diazepam
- propanolol
binding - warferin
Acidic drug like – sod. Salicilate , sod . Benzoate , sulfonamide displace bilirubin from its
albumin binding site result in neonate it cross to BBB and precipitate toxicity (kernicterus )

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Hal-hal yg harus diperhatikan dalam evaluasi OP
 Kondisi setimbang antara obat terikat & obat bebas dapat dipertahankan
 Metode yg dipakai harus valid untuk rentang konsentrasi yg cukup besar
 Kontaminasi & denaturasi protein yg akan dipakai tidak terjadi
 Metode yg dipakai harus mempertimbangkan : pH, [C] ionik dr media, donan efek
 Metode tsb dapat mendeteksi ikatan OP yg bersifat reversibel dan irreversibel
 Hasil mencerminkan kondisi :”in vivo”

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